Abstracts for the Twelfth Annual Meeting of the Society ... · Melissa Millard,1 Markus Müschen,2 and Anat Erdreich-Epstein2; 1Pediatrics, ... Abstracts for the twelfth Annual Meeting

Abstracts for the

Twelfth Annual Meeting

of the Society for

Neuro-Oncology

November 15 –18, 2007

Copyright 2007 by the Society for Neuro-Oncology

468 Neuro-Oncology ■ OctOber 2007

ANGIOGENESIS

AN-01. GLIOMA ENDOTHELIAL MARKER CHARACTERIZATION IN BRAIN TUMOR ANGIOGENESISEleanor Carson-Walter,1 Yang Liu,1 Bethany Winans,1 Eveline Shue,2 and Kevin Walter1; 1Neurosurgery, University of Rochester, Rochester, NY, USA; 2Neurosurgery, University of Pittsburgh, Pittsburgh, PA, USA

Malignant brain tumors, particularly glioblastoma (GBM), are char-acterized by profound angiogenesis, disruption of the blood brain barrier, and vasogenic edema. These alterations provide opportunities for targeting the tumor vasculature. Using Serial Analysis of Gene Expression (SAGE) performed on microvascular endothelial cells isolated from fresh surgical brain tumor specimens, we identified a subset of 21 genes termed glioma endothelial markers (GEMS), which were selectively up-regulated in GBM microvasculature. We confirmed the endothelial induction and localization of one of these markers, PV-1, at the RNA and protein level and demon-strated its responsiveness to VEGF signaling. Using PV-1 as a model, we now expand our characterization of GEMS expression and regulation in brain tumors. RT-PCR and real-time PCR were used to screen a panel of 12 clinical specimens, including 5 GBMs, 5 adenocarcinoma brain metas-tases (mets), and 2 non-neoplastic cortex samples for all 21 putative GEMS. From our original list of SAGE derived GEMS, we were able to demon-strate upregulation of MMP14, COL6A2, COL1A1, COL3A1, HSP, G2, LAMC3, PLXNA2, CXCR7, MG50 and TEM1 in GBM and brain mets, while showing little to no expression in non-neoplastic brain. Additionally, one GEM, ANXA3, while induced strongly in only 1/5 GBMs, was present in 5/5 mets. We demonstrated that GEMS expression is conserved across species by using RT-PCR of intracranial U87MG GBM xenografts in athy-mic mice. Endothelial localization of gene expression was confirmed using ISH or IH. GEMS expression in vitro was characterized using HMVEC cells treated with pro-angiogenic growth factors, including VEGF and SF/HGF and a clinically relevant glucocorticoid, dexamethasone. As many of our target GEMS are cell surface or extracellular proteins, they provide attrac-tive targets for clinical intervention. Further characterization of the human and murine expression profile, molecular regulation, and drug responsive-ness of these genes will aid in the development of such protocols.

AN-02. ABL KINASE PARTICIPATES IN APOPTOSIS INDUCED BY DISRUPTION OF THE ACTIN CYTOSKELETON IN HUMAN BRAIN MICROVASCULAR ENDOTHELIAL CELLSMelissa Millard,1 Markus Müschen,2 and Anat Erdreich-Epstein2; 1Pediatrics, Childrens Hospital Los Angeles, CA, USA; 2Pediatrics, Childrens Hospital Los Angeles, University of Southern California, Los Angeles, CA, USA

To investigate whether endothelial apoptosis on the non-integrin bind-ing matrix poly-L-lysine (PLL) was due to the lack of integrin ligation or the inability of cells to spread we plated human brain microvascular endothe-lial cells (HBMEC) on vitronectin and treated them with latrunculin B, a naturally occurring marine toxin that specifically binds actin, inhibits its polymerization, disrupts microfilament organization, and reverses cell spreading. In presence of latrunculin B (50–500 nM) HBMEC transiently rounded, but remained attached. Phalloidin stain showed disruption of actin filaments coincident with the loss of cell spreading within 15 min. Interestingly, the morphological changes, including the changes in actin organization, completely resolved by 18h. Despite this recovery of normal morphology, latrunculin B induced dose-dependent apoptosis in 4 indepen-dent isolates of HBMEC (but not in 6 adherent and non-adherent tumor cell lines examined; PARP cleavage, western blot). The apoptosis was inhibited by caspase inhibitors (zBOC-FMK, zVAD-FMK) without change in the transient loss of cell spreading. c-Abl integrates multiple signals to coordinate F-actin dynamics, while F-Actin itself has inhibitory effect on Abl kinase activity. Interestingly, the Abl inhibitor, STI-571 (5–10 mM; Gleevec) effectively inhibited HBMEC apoptosis induced by latrunculin B, suggesting requirement for Abl kinase activity in mediating this apoptosis. Supporting this, apoptosis induced by latrunculin B was associated with

cleavage of Abl. Interestingly, apoptosis induced by the integrin avb3/avb5 inhibitor, RGDfV, that is associated with changes in cell morphology, was also suppressed by STI-571, suggesting that RGDfV-induced apoptosis was at least in part mediated via Abl kinase. Taken together, these findings sug-gest that transient disruption of the actin cytoskeleton is a pro-apoptotic stimulus in endothelial cells and that this apoptosis involves Abl kinase activation. These findings also suggest a possible (and surprising) role for STI-571 in protection of endothelial cells from Abl-mediated apoptosis, that may contribute to blood vessel normalization in anti-angiogenic therapy in cancer.

AN-03. PLASMINOGEN KRINGLE 5 INDUCES APOPTOSIS OF BRAIN MICROVESSEL ENDOTHELIAL CELLS: SENSITIZATION BY RADIATION AND REQUIREMENT FOR GRP78Braden McFarland,1 Jerry Stewart,2 Robert Nordal,3 Don Davidson,4 Jack Henkin,4 and Candece Gladson2; 1Neurobiology, University of Alabama at Birmingham, Birmingham, AL, USA; 2Pathology, University of Alabama at Birmingham, Birmingham, AL, USA; 3Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL, USA; 4Abbott Laboratories, Abbott Park, IL, USA

Recombinant plasminogen kringle 5 (rK5) has been shown to induce apoptosis of dermal microvessel endothelial cells (MvEC) (Davidson et al., 2005). As we are interested in anti-angiogenic therapy for malignant astrocytoma tumors and radiation is a standard early treatment modality, we investigated the pro-apoptotic effect of rK5 on brain MvEC plus/minus prior irradiation. We found that rK5 treatment of brain MvEC induced apoptosis in a dose- and time-dependent manner, measured as the cleavage of caspases-7 or 3, and prior irradiation significantly sensitized the cells to the pro-apoptotic effect. The pro-apoptotic effect of rK5 required expres-sion of glucose regulated protein 78 (GRP78) based on blocking studies with an antibody directed toward GRP78, as well as the downregulation of GRP78 with small interfering (si) RNA. Our findings have potential appli-cation as a new therapy, as we also demonstrate that expression of GRP78 protein is upregulated on brain MvEC in malignant astrocytoma tumor samples as compared to the normal brain, and immunoblotting studies con-firmed the upregulation of GRP78 in the tumor samples. These data suggest that irradiation sensitizes brain MvEC to the pro-apoptotic effect of rK5 and this signal requires GRP78.

AN-04. OSTEOPONTIN (OPN) EXPRESSION CORRELATES WITH GLIOMA AGGRESSIVENESSSamira Guccione,1 Taichang Jang,2 Yi-Shan Yang,1 Hongbin Cao,1 Terri Haddix,1 Michael Glantz,3 Quynh-Thu Le,1 Lawrence Recht,1 and Griffith Harsh1; 1Stanford University, Stanford, CA, USA; 2University of Massachusetts Medical School (Worcester), Worcester, MA, USA; 3Dept of Oncology, University of Utah Medical Center, UT, USA

Although numerous reports have correlated OPN expression either at mRNA or protein levels with systemic tumor aggressiveness, it remains unclear to what extent its levels vary in primary glial neoplasms. To address this, we first assessed OPN expression in a panel of paraffin embedded tumor samples using immunohistochemistry (IHC) and noted robust albeit heterogeneous expression only in glioblastoma (GBM). We next assessed a series of CSF specimens obtained from patients with gliomas and found elevated expression in GBM relative to non-GBM glial tumors. Finally, we noted an increase in OPN mRNA levels in biopsy samples obtained from contrast enhanced tumoral areas relative to non-contrast enhanced areas in GBM patients. Our findings are therefore similar to those reported in vari-ous systemic cancers in which OPN levels are highest in the most aggressive tumor samples and suggest a possible role for this molecule as a biomarker in this disease.

N e u r o - o N c o l o g y

Abstracts

Abstracts for the twelfth Annual Meeting of the Society for Neuro-Oncology (SNO)

Neuro-Oncology ■ OctOber 2007 469

AN-05. BONE MARROW-DERIVED MMP-9–EXPRESSING HEMATOPOIETIC CELLS ARE CRITICAL CONTRIBUTORS TO GLIOBLASTOMA ANGIOGENESISKan Lu,1 Rose Du,1 Claudia Petritsch,1 Hanqiu Song,1 Scott VandenBerg,2 Zena Werb,1 and Gabriele Bergers1; 1University of California, San Francisco, San Francisco, CA, USA; 2Department of Pathology, University of California, San Francisco, San Francisco, CA, USA

Glioblastoma multiforme (GBM) is characterized by hypoxic and necrotic tumor cores that in turn make it one of the most angiogenic tumors. While it was previously thought that only preexisting endothe-lial cells could be activated to sprout a new vascular network, emerging evidence now suggests that tumors are also capable of recruiting a hetero-geneous population of bone marrow-derived (BMD) progenitor and acces-sory cells to facilitate vessel formation in a process called adult vasculo-genesis. Using an orthotopic model of genetically engineered mouse GBMs coupled with GFP bone marrow transplantation, we show that these highly angiogenic tumors recruit a substantial number of GFP1 BMD cells. In contrast, tumors deficient in HIF-1 were non-angiogenic and demonstrated a dramatic reduction in BM recruitment. FACS analysis of the GFP1 BMD cells revealed that they included endothelial and pericyte progenitors, but the majority of them were CD451 hematopoietic cells expressing matrix-metalloproteinase (MMP)-9. Interestingly, MMP-9–deficient GBMs in MMP-9–deficient host animals were still capable of recruiting CD451 BMD cells, but yet were also non-angiogenic. However, when these mice and tumors were supplemented with GFP1CD451 BMD cells from MMP-9–expressing wild-type animals, tumor angiogenesis was restored in areas of the tumor where BMD cells were recruited. These results suggest that the MMP-9–expressing CD451 population of BMD cells plays a critical role in the angiogenic response. Furthermore, they support the process of vasculogenesis as an important contributor to tumor angiogenesis that may represent new therapeutic targets.

AN-06. IDENTIFICATION OF GLIOMA NEOVASCULATURE-SPECIFIC PROTEINS BY PROTEOMICS TECHNIQUES—AND PRACTICAL APPLICATIONS THEREOFJohan Kros,1 Dana Mustafa,1 Pingpin Zheng,1 Peter Sillevis Smitt,2 and Theo Luider2; 1Pathology, Erasmus Medical Center, Rotterdam, Netherlands; 2Neurology, Erasmus Medical Center, Rotterdam, Netherlands

Recently, we have specifically identified low-molecular caldesmon (l-CaD) in the CSF of gliomas patients by 2-dimensional electrophoresis and were able to validate the results by immunohistochemistry to glioma tissue specimens. It appeared that the blood levels of l-CaD are significantly raised in glioma patients and we are currently testing this potential serum marker in a clinical setting. It appeared that specific expression of l-CaD was not confined to vascular structures, but was also seen in individual cells and cell clusters in gliomas. These cells were identified as endothelial precursor cells (EPCs) and activated endothelial cells (ECs). The levels of these cell fractions appeared to be significantly increased in the peripheral blood of glioma patients, indicating that there is a significant contribu-tion of vasculogenesis to neovascularization in gliomas. In order to find more proteins specifically expressed in either vasculogenesis or angiogen-esis in glial tumorigenesis, we applied state-of-the-art proteomic techniques (MALDI-MS/MS and FTMS) to glioma tissue samples and found some additional proteins specifically expressed in the hypertrophied microvas-culature of the tumors. Currently, we are characterizing the cells in which these proteins are expressed, hypothesize on their specific function, and try to test them as markers for angiogenesis (and possibly tumor activity) in the serum of glioma patients.

AN-07. COMBINED TEMOZOLOMIDE (TMZ) AND ANTI-ANGIOGENIC THERAPY OF GLIOMAS: A CAPRICIOUS COCKTAIL?Pieter Wesseling,1 An Claes,1 Cathy Maass,1 Judith Jeuken,1 Arend Heerschap,2 and William Leenders1; 1Pathology, Radboud Univ. Nijmegen Med. Ctr., Nijmegen, Netherlands; 2Radiology, Radboud Univ. Nijmegen Med. Ctr., Nijmegen, Netherlands

The marked angiogenic reponse in glioblastomas (GBMs) and the promising results of application of angiogenesis inhibitors (AIs) for other cancers have led to the first clinical trials with AIs (both antibodies and tyrosine kinase inhibitors/TKIs) in GBM patients. However, it is presently unclear how various AIs exactly affect the GBM microvasculature and what the optimal combination is of AIs with other therapeutic modalities. The aim of this research is to establish in genotypically and phenotypically

relevant, orthotopic animal models the optimal therapeutic regimen for combination of AIs with the alkylating agent temozolomide (TMZ). Like human GBMs, our orthotopic human GBM E98 xenograft model in nude mice consistently shows a combination of compact and diffuse infiltrative growth in the brain parenchyma. Using this model, we first investigated the effect of monotherapy with vandetanib (ZD6474), a TKI with specific-ity towards VEGF receptor 2, EGFR and RET. This treatment resulted in significantly increased hypoxia in compact, angiogenesis-dependent areas, but not in diffuse infiltrative areas. This can be explained by the extensive incorporation of the pre-existent brain microvascular network in this lat-ter region. Combination therapy with vandetanib and TMZ in this E98 model (known with hypermethylation of the MGMT promotor) resulted in delayed tumor growth, whereas monotherapy of TMZ often led to complete eradication of the tumor. A possible explanation of this phenomenon is that anti-angiogenic therapy leads to “normalization” of the blood brain bar-rier, thereby decreasing the availability of TMZ in the tumor tissue. Also in the U87 orthotopic GBM model in nude mice (characterized by compact, expansive growth), combination therapy with TMZ and vandetanib did not result in better tumor control than TMZ monotherapy. Importantly, our parallel MR imaging studies (with and without Gd-DTPA as contrast agent) in the E98 and U87 models suggest that AI induced normalization of the brain tumor microvasculature may very well lead to overestimation of the effect of anti-angiogenic therapy. Based on this animal experimen-tal work we conclude that the application of combined chemo- and anti- angiogenic therapy for human glioma patients should be performed with caution. Optimalization of dosage and scheduling of AIs in combina-tion with other therapeutic modalities will help to augment rather than antagonize the response to chemotherapy. Systematic preclinical studies in relevant, orthotopic animal models may facilitate the identification of the optimal therapeutic regimens. (This research is supported by Dutch Cancer Society grant KUN 2003–2975.)

AN-08. VASCULAR NORMALIZATION CAN LIMIT INFLAMMATION TO ENHANCES ONCOLYTIC VIRUS THERAPYKazuhiko Kurozumi,1 Jayson Hardcastle,1 Roopa Thakur,1 Ming Yang,2 Gregory Christoforidis,2 William Carson,3 E. Antonio Chiocca,1 and Balveen Kaur1; 1Neurological Surgery, The Ohio State University, Columbus, OH, USA; 2Radiology, The Ohio State University, Columbus, OH, USA; 3Surgery, The Ohio State University, Columbus, OH, USA

Glioblastoma multiforme is a complex tissue consisting of neoplastic, stromal, endothelial, and pericytic cells embedded in a complex extracel-lular matrix. The complex interplay between cancer cells and their stromal resonse is crucial in determining the fate of a tumor and its response to treatment. Oncolytic virus (OV) therapy is a radical biological strategy being tested in several clinical trials in patients with malignant astrocy-tomas. OVs are viruses that can infect and replicate selectively within tumor cells, but not in normal non-neoplastic cells. Detailed elucidation of the response of tumor environment to therapy with OV has been neglected. Given the highly significant role played by tumor blood vessels in deter-mining the tumors progression and response to therapy we evaluated the effect of OV treatment on tumor vasculature. Our results have indicated a four-fold increase in vascular leakage upon infection with OV compared to injection with PBS (control). To evaluate if vascular hyperpermeability was a consequences of host immune responses to infection, we investigated changes in inflammatory response with Quantitative Real Time PCR array for inflammatory genes. Analysis of OV-induced changes in 84 cytokine and chemokine receptors revealed a significant induction of 44 genes in this category suggesting that increased vessel permeability was the result of endothelial cell activation in response to inflammation induced by OV infection of tumor. Finally, we show that vascular normalization achieved by pretreatment with a single dose of cyclic RGD peptide (cRGD; a known anti-angiogenic agent and antagonist of integrins) suppressed OV-induced vascular leakage, suppressed CD45-positive leucocyte infiltration in tumor tissue, and significantly increased OV propagation and survival of rats with brain tumors. Transcript profiling of inflammatory gene changes between OV and OV 1cRGD peptide, also revealed a reduction in OV induced induc-tion of host inflammatory genes. In conclusion, anti-inflammation achieved by vascular normalization enhanced the therapeutic efficacy of OV.



AN-09. ADULT HUMAN MESENCHYMAL STEM CELLS CONTRIBUTE TO NEOANGIOGENESIS OF MALIGNANT GLIOMAChristian Schichor,1 Benjamin Korte,2 Fabian Trillsch,2 Roland Goldbrunner,2 and Jörg-Christian Tonn2; 1Neurosurgical Department, University of Munich, Munich, Germany; 2Germany

In this study, we evaluated the use of adult human mesenchymal stem cells (hMSC) as potential vehicles in the context of cell-based stategies for the treatment of residual tumor following surgical resection. The cells were in vitro found to be readily incorporated into growing endothelial tubes in tube formation assays using endothelial cells derived from malignant gliomas. In vivo, hMSC showed to be actively integrated into tumor vessels in implanted human gliomas (U373) in immune-deficient rats. The use of the endothelial specific Tie2 promoter/enhancer in engineered MSC dem-onstrated the selective activation of reporter gene in the context of MSC differentiation while being recruited into the neoangiogenetic vasculature of the glioma. This is the first report about hMSC, contributing to neoangio-genetic vasculature of gliomas in vitro as well as in vivo. The results suggest that human mesenchymal stem cells may provide a ready source of cells for the generation of a genetically modified cellular vector directly targeting neoangiogenetic vasculature of gliomas.

CELL BIOLOGY

CB-01. A NOVEL BRAIN TUMOR SUPPRESSIVE MECHANISM FOR P53: PARACRINE INDUCTION OF APOPTOSIS THROUGH GALECTIN-3 SECRETIONFatima Khwaja,1 Abdessamad Zerrouqi,2 Narra Devi,3 and Erwin Van Meir2; 1Departments of Neurosurgery, Hematology/Oncology, Winship Cancer Institute, GA, USA; 2Emory University, Atlanta, GA, USA; 3Department of Neurosurgery, Winship Cancer Institute, Atlanta, GA, USA

P53 is a transcription factor that regulates the expression of a large number of proteins, some of which are critical effectors of its tumor sup-pressive activities. In the present study we demonstrate the existence of a novel mechanism whereby the tumor suppressor p53 can control tumor growth. We provide evidence that p53 expressing cells can induce cell death in bystander cells through p53 controlled release of galectin-3 (Gal-3), a 31 kDa b-galactoside-binding pro-apoptotic factor. Galectin-3 is not a direct target of wt-p53; rather its secretion is facilitated by p53 transcrip-tional activation of TSAP6, a key mediator of the non-traditional secretory pathway. We demonstrated the biological importance of p53 controlled galectin-3 secretion by showing that it inhibits anchorage-independent tumor cell growth in vitro and strongly reduces glioma formation in vivo. In conclusion, our data demonstrate that p53 can exert cell-extrinsic control over tumor growth through a novel mechanism leading to increased secre-tion of galectin-3.

CB-02. TARGETING MOLECULAR MOTORS PROVIDES A NEW WAY OF BLOCKING GLIOMA INVASION AND ANGIOGENESISSteven Rosenfeld,1 Christopher Beadle,2 Jennifer Kharani,3 and Peter Cannoll3; 1Columbia University, New York, NY, USA; 2Neurology, Columbia University, New York, NY, USA; 3NY, USA

Molecular motors are a class of enzymes that convert the energy ATP hydrolysis into movement, and they drive a number of cellular processes that are central to maintaining the malignant phenotype. These include chromosome congression and separation, as occurs in mitosis; maintenance of cell tension and shape, as occurs in the formation of elongated blood ves-sels from endothelial cells; and cell motility, as occurs during the process of tumor invasion. Nonetheless, relatively little attention has been paid to the role of molecular motors in driving the malignant phenotype, in part because of a lack of reliable inhibitors that could be applied in in vitro and in pre-clinical models of tumor growth and invasion. This situation has recently changed with the development of several high affinity inhibi-tors of two groups of molecular motors—myosin II and kinesin spindle protein—and we have utilized these inhibitors to examine the role of these motors in shaping the invasive behavior of malignant gliomas. Our previous work had shown that migration of malignant glioma cells in brain occurs in two steps—protrusion of a thin filopodium, followed by a contraction of the cell posterior that propels the nucleus forward. Using blebbistatin, a specific inhibitor of myosin II, we now show that this motor is specifi-cally responsible for forward movement of the cell posterior, including the nucleus, and we have confirmed these findings through the use of RNAi sup-pression of myosin II synthesis. Furthermore, we find that myosin II is abso-

lutely required for movement of glioma cells through a three dimensional, mechanically-constrained matrix, while it is unnecessary for movement on an unconstrained, 2-dimensional surface, such as a cover slip. This suggests that myosin II is specifically required to generating the intracellular forces needed for pushing gliomas through the tightly packed extracellular space that characterizes the brain. We have also examined the role of kinesin spindle protein (KSP) in maintaining the malignant phenotype. This work has been possible by the recent discovery of several high affinity inhibitors of KSP, one of which is in phase I and II trials in patients with solid malig-nancies. Although KSP was originally described to function in maintaining the mitotic spindle, we have found that its inhibition by monastrol and S-trityl cysteine completely blocks glioma migration in Transwell cham-bers. We also found that these inhibitors completely block formation of endothelial tubes in vitro. Our studies demonstrate that at least two groups of molecular motors are absolutely necessary for powering glioma invasion, and that at least one is also needed for angiogenesis. The recent development of high-affinity inhibitors of these motors, at least one of which is being investigated in phase I and II trials, implies that an anti-molecular motor targeting strategy for the treatment of gliomas is possible. We are now inves-tigating this further in pre-clinical models of gliomagenesis.

CB-03. CREATION AND EXPRESSION OF SPARC-GFP DELETION MUTANTS IN THE HUMAN U87 GLIOMA CELL LINEHeather McClung1 and Sandra Rempel2; 1Pharmacology, Wayne State University, Detroit, MI, USA; 2Henry Ford Hospital, Detroit, MI, USA

Secreted Protein Acidic Rich in Cysteine (SPARC) is highly expressed in all grades of astrocytoma. SPARC increases cell motility and slows cell cycle progression in normal cells. Similarly, in gliomas, SPARC increases tumor invasion, while suppressing tumor growth. It is thought that these functions may be independent, but it is not known how SPARC carries out these functions. SPARC is composed of an N-terminal acidic domain, a follistatin-like domain, containing an Epidermal Growth Factor (EGF)-like module, and a C-terminal extracellular calcium binding (EC) domain. The overall goals of this study are to determine whether the acidic domain or the EGF-like module are involved in the changes in cell attachment and motil-ity as well as the anti-proliferative effects of SPARC in gliomas. Deletion mutant constructs, lacking either the acidic domain or the EGF-like module, were created from a parental vector encoding for SPARC with a C-terminal Green Fluorescent Protein (GFP) tag. U87 cells were stably transfected with GFP vector, wild-type SPARC-GFP, or either of the deletion mutants. Clones were selected and expanded. Expression and intracellular localiza-tion were determined by fluorescence imaging. The expression levels and secretion of each of the constructs were determined by Western blot analy-sis. Like wildtype SPARC, the intracellular localization for both constructs is perinuclear and both mutants are secreted and run at the expected size on Western blot, which indicates that they are being expressed and processed normally. Preliminary studies by confocal microscopy indicate that the cells expressing the deletion mutants have an altered, more spread morphology in comparison to wildtype SPARC-GFP–expressing cells. Interestingly, differences in cell morphology exist between the cells expressing the two deletion mutants, suggesting domain-specific effects. Effects on cell prolif-eration were determined by cell counting. Ninety-six hours after plating equal numbers of cells, SPARC-GFP-expressing cells were less proliferative (6.1 3 105 62.0 3 104) than GFP-expressing cells (7.7 3 105 62.1 3 104 [p 5 0.001]). Cells expressing the acidic domain deletion and cells express-ing the EGF-like module deletion were less proliferative (4.7 3 105 62.8 3 104 and 4.5 3 105 63.9 3 104, respectively) than wildtype SPARC-GFP (p , 0.02) and control cells (p , 0.001), suggesting that both domains are important in the regulation of cell proliferation. In conclusion, the cells expressing the deletion mutants will serve as tools to examine the roles of these two domains within SPARC in altering adhesion, cytoskeletal struc-ture, motility, proliferation and the signaling pathways which accompany these cellular functions.

CB-04. EGFR AND PTEN MODULATE TISSUE FACTOR EXPRESSION IN GLIOBLASTOMA THROUGH AP-1 TRANSCRIPTIONAL ACTIVITYYuan Rong, Carol Tucker-Burden, Vladmir E Belozerov, Donald L Durden, Erwin G Van Meir, and Daniel J Brat; Winship Cancer Institute, Atlanta, GA, USA

Glioblastoma (GBM) is a highly malignant, rapidly progressive astrocy-toma that is distiguished pathologically from lower grade tumors by necro-sis and microvascular hyperplasia. Both hypoxia and pseudopalisading necrosis are critical for angiogenesis and accelerated growth. Vaso-occlusive and prothrombotic mechanisms could initiate or propagate this cascade by causing hypoxia and necrosis. Tissue factor (TF), the main cellular initiator



of coagulation is over-expressed in GBMs and likely favors thrombosis. PTEN loss and EGFR amplification/mutation are two genetic alterations seen in GBMs but not in lower grade astrocytomas that could be responsible for elevated TF levels. The most frequent EGFR mutation in GBM involves deletion of exon 2–7 resulting in a constitutively active form EGFRvIII. In the current study, we found that overexpression of EGFR or EGFRvIII in U87MG cells caused increased basal TF expression in vitro. Stimula-tion of EGFR with EGF led to upregulation of TF in a dose-dependent manner. To determine whether activation of specific transcription factors (AP-1, NFkappaB, and Egr-1) were responsible for this effect, we performed luciferase reporter assays coupled with individual promoter binding sites (AP-1, NFkappaB, and Egr-1); with the full length wt TF promoter; and with AP-1 or NFkappaB deleted forms of the TF promoter. We found that EGFR-mediated TF upregulation depended strongly on AP-1 transcriptional activity. AP-1 promoter activity following EGFR activation was associated with increased p-c-Jun and p-JNK levels and could be attenuated by the Jun-N-Terminal Kinase (JNK) inhibitor SP600125. Decreased AP-1 activity and EGFR stimulated TF expression were also noted following exposure to the PI3-K inhibitor LY294002 and the mTOR inhibitor Rapamycin, suggesting that the PI3-k/Akt/mTOR pathway also influences TF expression through a similar final common pathway. This was further supported by the finding that the reintroduction of PTEN into U87MG cells by lentiviral infection led to reduced EGFR-mediated TF expression and decreased levels of AP-1 dependent transcription. In conclusion, we found that EGFR upregulates TF expression in GBM cells through activation of AP-1 transcription via both MAPK/JNK and PI3-K/Akt/mTOR signaling pathways.

CB-05. FRA-1 TRANSACTIVATES JUNB IN RESPONSE TO ONCOGENIC ACTIVATION OR STRESS IN GBM CELLSWaldemar Debinski,1 William Brown,2 and Denise Gibo3; 1Neurosurgery, Wake Forest University, Winston-Salem, NC, USA; 2Radiologic Sciences, Wake Forest University, Winston-Salem, NC, USA; 3Neurosurgery, Wake Forest University School of Medicine, Winston-Salem, NC, USA

Growing evidence suggests that Fos-related antigen 1 (Fra-1), an AP-1 transcription factor, may play an important role in maintenance/progression of Glioblastoma multiforme (GBM). Fra-1 is controlled by the signaling pathways of oncogenic receptors, such as epidermal growth factor receptor (EGFR) and is one of a few factors found to be regulated by EGFRvIII in GBM cells. In the current study, we used for the first time both siRNA and shRNA approaches to examine the effect of fra-1 knock downs on GBM cells over-expressing Fra-1. Furthermore, we found previously that ectopic Fra-1 causes an increase in the expression of genes for AP-1 factors junB, junD and the angiogenic VEGF, among several others, in GBM cells; we thus analyzed proteins expression and co-immunoprecipitated (IP) with Fra-1, and vice-versa. We also analyzed in more detail the phosphorylation status of Fra-1 in GBM cells. Finally, we examined the neo-vasculature of U-87 MG tumors in which Fra-1 was either knocked down or further upregulated. When using fra-1 siRNA, we found that G48 and U-251 MG GBM cells had their Fra-1 levels diminished by almost 100%; a similar result was obtained with fra-1 shRNA. This was accompanied by very prominent changes in cell morphology that included an almost complete retraction of cellular processes and cell rounding. We next found that ectopic Fra-1 not only causes an increase in junB gene expression, but it augments the levels of immunoreactive JunB protein in several GBM cell lines. We found that the expression of JunB, but not that of c-Jun, JunD, or ERK increased in response to EGF signaling similarly to Fra-1, with immunoreactive JunB up-regulated in parallel to an increase in Fra-1. Importantly, this was followed by an avid pairing between Fra-1 and JunB, as documented by reversed co-IP experiments, and not between Fra-1 and other AP-1 transcription factors. Furthermore, we showed that Fra-1 is highly phosphorylated in glioma cells with serine residues primarily undergoing this process. We now find that Fra-1 is phosphorylated when forming a complex with JunB. Interestingly, there is a subtle shift of Fra-1 immunoreactive protein band in Western blotting due to an increase in the phosphorylation state in response to either oncogenic signaling or stress of irradiation. This is independent of the reactive oxygen species (ROS) formation. Using collagen IV staining of vascular basement membranes, we examined the vascular patterns of tumors in which Fra-1 was altered. The U-87 MG[Fra-1(-)] tumors grew much slower and the vessels were of dramatically lesser density, larger and shorter when compared to controls. Thus, Fra-1 is a phosphorylated factor that transactivates JunB with which it makes AP-1 pairs preferentially in GBM cells, as recently confirmed in other cancer model systems (EMBO J. 26:1878–90, 2007). Moreover, Fra-1 takes part in a control of GBM cells architecture that might be relevant to the invasive and/or migratory nature of these cells and affects prominently GBM neo-vasculature.

CB-06. TIE2 MEDIATES ADHESION-DEPENDENT CELL SURVIVAL IN GLIOMASVanesa Martin, Dan Liu, Jing Xu, Juan Fueyo, and Candelaria Gomez-Manzano; Department of Neuro-Oncology, U.T. M.D. Anderson Cancer Center, Houston, TX, USA

Abnormal expression and function of tyrosine kinase receptors is one of the most common genetic events in malignant gliomas. In previous stud-ies, our group identified the ectopic expression of Tie2 receptor (originally described as endothelial cell specific) in non-stromal cells within glioma tumors. Thus, we found that surgical glioblastoma specimens contained a subpopulation of Tie21/CD31– and Tie21/GFAP1 cells, suggesting that Tie2 is indeed expressed outside the vascular compartment of gliomas. The levels of expression appeared to be associated to the progression from lower to higher grade tumors. Tie2 activity in glioma cells was correlated with an increase in cell adhesion to extracellular matrix components by upregula-tion of integrin beta-1 expression. Here, we report an increase in glioma cell adhesion to endothelial cells (ECs) in Ang1–stimulated U87 cells (Tie2 posi-tive) that was downregulated in cells treated with Tie2 siRNA. Similarly, by using an isogenic system in which Tie2 was cloned into U251 glioma cells (Tie2 negative), we corroborated the role of Tie2 in glioma-to-endothelial cell adhesion. The increase in cell adhesion was calcium-dependent, as was further demonstrated by the upregulation in glioma cells of calcium- dependent adhesion molecules like integrin beta-1 and N-cadherin. We also showed that Tie2–expressing glioma cells, attached to ECs or to extracellu-lar matrix components, were more resistant to cell death induced by chemo-therapeutic agents. MTT and Annexin V binding studies reveled that Tie2 activity increases the resistance to apoptosis-induced cell death. Interest-ingly, Ang1 treatment induced Tie2 phosphorylation and activation of the Ras/MAPK survival pathway. These results, together with the reported fact that malignant gliomas express high levels of Ang1, suggest the existence of an autocrine loop that modulates glioma-to-extracellular matrix and glioma-to-endothelial cell adhesion. Finally, these newly described Tie2–mediated biological functions buttress the tenet of a vascular tumor niche in malignant gliomas and may have a critical role in the chemorresistance of these tumors.

CB-07. EPIDERMAL GROWTH FACTOR RECEPTOR ACTIVATION RESULTS IN ENHANCED CYCLOOXYGENASE-2 EXPRESSION THROUGH P38 MITOGEN-ACTIVATED PROTEIN KINASE-DEPENDENT ACTIVATION OF THE SP1/SP3 TRANSCRIPTION FACTORS IN HUMAN GLIOMASKaiming Xu and Hui-Kuo Shu; Radiation Oncology, Emory University, Atlanta, GA, USA

Expression of cyclooxygenase-2 (COX-2) has been linked to many cancers and may contribute to malignant phenotypes including enhanced proliferation, angiogenesis and resistance to cytoxic therapies. Malignant gliomas are highly aggressive brain tumors that display many of these characteristics. One prominent molecular abnormality discovered in these astrocytic brain tumors is alteration of epidermal growth factor receptor (EGFR) through gene amplification and/or mutation resulting in excessive signaling from this receptor. We found that EGF-mediated stimulation of EGFR tyrosine kinase in human glioma cell lines induces expression of both COX-2 mRNA and protein. The p38 mitogen-activated protein kinase (p38–MAPK) pathway was a strong downstream factor in this activation with inhibition of this pathway leading to strong suppression of COX-2 induction. The p38–MAPK pathway can activate the Sp1/Sp3 transcription factors and this appears necessary for EGFR-dependent transactivation of the COX-2 promoter. Analysis of COX-2 promoter/luciferase constructs revealed that transcriptional activation of the COX-2 promoter by EGFR requires the Sp1 binding site located at -245/-240. Furthermore, Sp1/Sp3 binding to this site in the promoter is enhanced by EGFR activation both in vitro and in vivo. Enhanced DNA binding by Sp1/Sp3 requires p38–MAPK activity and correlates with increased phosphorylation of the Sp1 transcrip-tion factor. Thus, EGFR activation in malignant gliomas can transcrip-tionally activate COX-2 expression in a process that requires p38–MAPK and Sp1/Sp3. Finally, treatment of glioma cell lines with prostaglandin E2 (PGE2), the predominant product of COX-2 activity, results in increased vascular endothelial growth factor expression thus potentially linking eleva-tions in COX-2 expression with tumor angiogenesis in malignant gliomas.



CB-08. DOWNREGULATION OF ENDOGENOUS FIP200 PROTEIN INHIBITS CELL PROLIFERATION AND INDUCES APOPTOSIS OF IMMORTALIZED ASTROCYTES AND GLIOBLASTOMA CELLSDongyan Wang,1 Jerry Stewart,1 Braden McFarland,2 G. Gillespie,2 Louis Nabors,2 Russell Pieper,3 Jun-Lin Guan,4 and Candece Gladson2; 1Pathology, University of Alabama at Birmingham, Birmingham, AL, USA; 2University of Alabama at Birmingham, Birmingham, AL, USA; 3University of California, San Francisco, San Francisco, CA, USA; 4MI, USA

FIP200 is a novel protein that was initially identified as a focal adhesion kinase (FAK) family interacting protein. Recent genetic studies indicated the FIP200–null mouse is an embryonic lethal and immunohistochemi-cal studies of the embryo showed increased apoptosis in the liver and heart, suggesting FIP200 promotes cell survival (Gan et al., J. Cell Biol. 2006;175(1):121–133). We investigated the role of endogenous FIP200 pro-tein in the survival and proliferation of immortalized human astrocytes and of glioblastoma cells by downregulating FIP200 protein with siRNA and examining the effect on cell function. Significant downregulation of FIP200 protein (. 90% decrease) was achieved with two different siRNAs as compared to a control siRNA. The downregulation of FIP200 protein resulted in an increased activity of Pyk2, a FAK family member, while FAK activity and protein level remained unchanged. Downregulation of FIP200 resulted in an inhibition of cell proliferation in both cell types propagated as monolayers in complete media. Furthermore, the downregulation of FIP200 induced apoptosis in both cell types propagated in complete media, and measured as an increased cleavage of caspase-7 (an effector caspase). In other cell types, investigators have shown the overexpression of Pyk2 can promote apoptosis (Xiong and Parsons, J. Cell Biol. 1997, 139: 529–539). These findings are likely of physiologic relevance as we detected FIP200 protein by western blot analysis of lysates from normal brain and malignant glioma (anaplastic astrocytoma and glioblastoma) biopsy samples. We are currently examining the cellular localization of FIP200 in normal brain and glioblastoma tumor samples. In summary, our data suggest that (1) FIP200 is necessary for the survival of immortalized astrocytes and glioblastoma cells in the conditions of our experiments; (2) the increased Pyk2 activity observed with the downregulation of FIP200 may promote apoptosis; and (3) FIP200 could be a potential therapeutic target in glioblastoma tumors.

CB-09. REGULATION AND FUNCTION OF N-CADHERIN POST-TRANSLATIONAL CLEAVAGE IN GLIOBLASTOMA CELLSZachary Kohutek and Isa Hussaini; University of Virginia, Charlottesville, VA, USA

Glioblastomas (GBMs) are highly aggressive astrocytic tumors that are capable of invading extensively throughout the brain, due in large part to their secretion of pro-invasive proteases such as the matrix metallopro-teinases (MMPs) and the related family of a disintegrin and metallopro-teinases (ADAMs). These proteases are characterized by their ability to cleave extracellular matrix components, but they have also been shown to be capable of processing many cell surface protein substrates. One such sub-strate is N-cadherin, a homophilic cell-cell adhesion glycoprotein that plays important roles in tissue morphogenesis, as well as tumor cell adhesion and metastasis in a wide range of tumor types. Expression of N-cadherin is increased in many cancers, yet in glioblastomas no consensus has yet been reached on the role of N-cadherin expression in promoting tumor invasion. In addition, the biological significance of metalloproteinase-initiated cleav-age of most cell surface proteins has not yet been investigated in astrocytic tumors. In this study we investigated the mechanism and role of MMP-mediated post-translational processing of N-cadherin in glioblastomas. We found that N-cadherin cleavage was increased in glioblastoma cell lines compared to normal astrocytes, and that this shedding process was induced by the addition of various tumor-promoting growth factors such as epider-mal growth factor (EGF) and the protein kinase C (PKC) activator phorbol 12–myristate 13–acetate (PMA). In addition, we found that PMA-stimulated N-cadherin cleavage in U-1242 and U-251 glioblastoma cells was blocked by pharmacological inhibitors and shRNA directed against PKC-alpha and PKC-delta, and that these pathways seemed to converge on the pro-tease ADAM-10, the activity of which was required for both basal and PMA-induced cleavage of N-cadherin in glioblastoma cells. Furthermore, functional studies using blocking antibodies indicated that N-cadherin may regulate glioblastoma cell motility in vitro. In summary, we conclude that increased N-cadherin cleavage in glioblastoma cells is regulated via a PKC-ADAM cascade, and that this regulated cleavage of N-cadherin may be important in mediating aspects of glioblastoma pathophysiology, includ-ing cell motility and adhesion.

CB-10. FOCAL ADHESION KINASE AND ATP-CITRATE SYNTHASE ARE POTENTIAL SHP-2 SUBSTRATES IN EGFRVIII-EXPRESSING HUMAN GLIOBLASTOMA CELLSYi Zhan and Donald O’Rourke; University of Pennsylvania School of Medicine, Philadelphia, PA, USA

SHP-2 is a non-transmembrane protein tyrosine phosphatase (PTP) and positively regulates mitogenic signals in receptor tyrosine kinase pathways. The oncoprotein EGFRvIII is commonly mutated in primary human glio-blastomas (GBMs) that arise de novo rather than from precursor lesions. EGFRvIII enhances GBM transformation, cell survival and confesses a poor progress. Previously we have shown that EGFRvIII is strongly coupled to the SHP-2 PTPase, EGFRvIII requires SHP-2 for GBM transformation and the EGFR/SHP-2 forms a constitutively activated complex in GBM cells. But the role of SHP-2 in EGFRvIII transformation is unknown. The iden-tification of SHP-2 physiological substrates in distinct cell types may pro-vide insight into mechanisms of EGFRvIII transformation. Previous studies have shown that SHP-2 substrates include EGFR and Gab1. SHP-2 can also interact with other molecules, such as platelet/endothelial cell adhe-sion molecule-1 (PECAM-1), signal-regulatory protein (SIRP), platelet-derived growth factor receptor (PDGFR) and insulin receptor substrate-1. Recently, alpha-catenin has been identified as SHP-2 substrate by using a SHP-2 double mutant (SHP-2 DM) that “traps” potential substrates in co-immunoprecipitation studies and modulation of alpha-catenin tyrosine phosphorylation by SHP-2 effects cell transformation. We have previously reported that p130 and p150 proteins serve as possible SHP-2 substrates in GBM cells by using the SHP-2 DM trapping mutant. In EGFRvIII-expressing GBM cells, the p130 and p150 proteins specifically interacted with the SHP-2 DM. This interaction was blocked by the EGFR-specific tyrosine kinase inhibitor AG1478, indicating that this interaction was EGFR kinase-dependent. Here, we report the identification of p150 by mass spectroscopy (MS) as EGFRvIII. This result further suggests that SHP-2 is recruited to the EGFRvIII transformation complex upon constitutive EGFRvIII kinase acti-vation. MS analysis of p130 showed that there are two potential candidates for this SHP-2 binding partners: focal adhesion kinase (FAK) and ATP-citrate synthase. The SHP-2 binding interaction to FAK and/or ATP-citrate synthase is also EGFR kinase-dependent. Both proteins have been shown to be actively involved in cell migration and transformation. Identification of these proteins using specific antibodies in GBM cells and human glioma samples suggests that the EGFRvIII/SHP-2 complex formation with FAK and/or ATP-citrate synthase is physiologic. Further examination of their interaction should reveal the mechanisms of EGFRvIII transformation.

CB-11. PTEN HAS TUMOR PROMOTING PROPERTIES IN THE SETTING OF GAIN-OF-FUNCTION P53 MUTATIONSYunqing Li,1 Fadila Guessous,1 Opeyemi Ibidapo,1 Lauren Fuller,1 Elizabeth Johnson,1 Bachchu Lal,2 Isa Hussaini,1 John Laterra,3 David Schiff,1 and Roger Abounader1; 1University of Virginia, Charlottesville, VA, USA; 2Department of Neurology, Kennedy Krieger Institute, MD, USA; 3Johns Hopkins University, Baltimore, MD, USA

We show, for the first time, that the tumor suppressor PTEN can have tumor promoting properties. We find that PTEN induces tumor cell prolif-eration, inhibits tumor cell death and increases in vivo xenograft growth in glioma cells harboring specific p53 mutations. We demonstrate that PTEN acquires these unexpected properties by enhancing the protein levels of gain-of-function mutant p53 (mut-p53). Gain-of-function p53 mutants possess oncogenic functions such as the transcriptional activation of genes that promote various malignancy parameters. We find that PTEN restora-tion to PTEN-null glioma cells harboring mut-p53 at codons 273 and 248 (U373 and SNB19) leads to induction of G1/S cell cycle progression and cell proliferation and to inhibition of cell death. PTEN expression reduces the G1/G0 fraction from 68.0 62.4% to 53.8 61.1% (n 5 8 ; p ,0.05) in U373 and from 50.7% 61.1% to 42.6 61.3% (n 5 5 ; p , 0.05) in SNB19 cells. PTEN restoration to PTEN-null glioma cells expressing wild-type (wt)-p53 (U87 and A172) inhibits G1/S progression as expected from a tumor sup-pressor. The cell cycle changes induced by PTEN translate into consistent changes in cell proliferation. PTEN significantly induces cell proliferation in U373 and SNB19 cells and inhibits cell proliferation in U87 and A172 cells. Similarly, PTEN reduces the chemotherapy-induced dead cell frac-tion from 27.5 63.4% to 8.7 63.4% (n 5 3 ; p , 0.05) in U373 cells and from 56.3 61% to 23.3 64% (n 5 3 ; p , 0.05) in SNB19 cells but sig-nificantly increases cell death in U87 and A172 cells. Conversely, PTEN inhibition in cells expressing PTEN and mut-p53 (U1242 and T98G) leads to inhibition of cell proliferation and inhibition of in vivo tumor growth. ShRNA-mediated inhibition of PTEN expression in U1242 cells that har-bor a gain-of-function mutation of p53 at codon 175 leads to a significant inhibition of in vivo xenograft growth. While control cells generated intrac-ranial xenografts with a crossectional area of 155.6 3 103 66.9 3 103 mm2, PTEN knock-down cells generated xenografts with a crossectional area of 43.9 3 103 611.1 3 103 mm2 (n 5 20 ; p , 0.01). We demonstrate the dependency of the tumor promoting effects of PTEN on mut-p53 by



showing that knock-down of mut-p53 expression inhibits or reverses the oncogenic effects of PTEN. Inhibition of mut-p53 expression by siRNA or shRNA leads to partial inhibition of PTEN-induced cell cycle progres-sion, complete inhibition of PTEN-induced cell proliferation and reversal of PTEN-induced cell survival. Mechanistically, we demonstrate that PTEN expression enhances mut-p53 protein levels via inhibition of mut-p53 degra-dation by Mdm2 and possibly also via direct protein binding. We first show that PTEN expression leads to a significant increase in mut-p53 protein lev-els. We then show that PTEN regulates the levels and nuclear/cytoplasmic localization of Mdm2 and that Mdm2 regulates mut-p53 levels. We also show that PTEN directly binds and stabilizes mut-p53 protein. Altogether, we therefore demonstrate that PTEN can acquire oncogenic properties by enhancing gain-of-function mut-p53 via inhibition of Mdm2 and direct binding. These findings describe a novel and intriguing function of PTEN and have important implications for experimental and therapeutic strategies that aim at manipulating PTEN or p53 in human tumors including gliomas. They suggest that the mutational status of PTEN and p53 should be consid-ered in order to achieve favorable therapeutic outcomes. The findings also provide an explanation for the low frequency of simultaneous mutations of PTEN and p53 in human cancer and gliomas.

CB-12. DEVELOPMENT OF A NOVEL CELL-BASED IMAGING AND QUANTITATION ASSAY FOR ENDOSOMAL TRAFFICKING ANALYSIS OF MUTANT EGFRs PREVALENT IN GBMsAaron Gajadhar and A. Guha; Arthur and Sonia Labatt Brain Tumor Research Center, University of Toronto, Toronto, Ontario, Canada

Aberrant signaling through Epidermal Growth Factor Receptor (EGFR) mutants, notably EGFRvIII and EGFRc958, is prevalent in GBMs. We pos-tulate that a component of this oncogenic signaling by mutant EGFRs is due to delayed internalization kinetics and/or altered endocytic trafficking, resulting in aberrant receptor recycling and oncogenic activation compared to activated wild-type(wt) EGFR. In this study, we report the development and utility of an automated, cell-based fluorescent imaging assay to detect and quantify the presence of fluorescently labeled EGFRs in the endocytic compartment. This Cellomics KineticScan based assay, allows analysis of the internalization kinetics and receptor endosomal trafficking routes of the EGFRvIII and EGFRc958 mutants. In our methodology, EGFRwt/vIII/and c958 isoforms have been fused to GFP tags to create functional chimera. These constructs were then transfected into CHO cells, having no endog-enous EGFR expression, and utilized for assay processing. Here we describe a series of assay development experiments to optimize the automated acqui-sition and quantitation of cellular images. These include protocol param-eter settings important for imaging and image processing including object identification, image exposure settings, separation of plasma membrane and endosomal compartment regions, and the use of gating thresholds criti-cal for cell sorting and subpopulation analysis. Preliminary results using EGFRwt-GFP demonstrates the selectivity of the assay to discern between surface bound plasma-membrane receptor and bright peri-nuclear staining typical of internalized receptor in the endosomal compartment following EGF stimulation. Furthermore, as proof-of-principle for validating the assay algorithm, we can detect and quantify an accumulation of EGFRwt in the endocytic compartment in a time-dependent manner, as has previously been shown by traditional biochemical analysis. Forthcoming, experiments will compare the internalization rates of the EGFRvIII and EGFRc958 with that of the EGFRwt. Additionally, this assay platform will be utilized to deter-mine whether the EGFRvIII and EGFRc958 may have a tendency towards a recycling route over a lysosomal route. This aberrant feature could allow sustained aberrant signaling, and a mechanism for altering the strength and duration of oncogenic signaling in GBMs. Taken together, these results demonstrate the potential utility of a Cellomics based internalization and endosomal trafficking assay for efficiently exploring downregulation char-acteristics of EGFR isoforms prevalent in GBM. In addition, this study pro-vides an example of a fully automated, robust screening platform allowing high-resolution, multi-parameter imaging and quantitation on whole cells.

CB-13. REGULATION OF MITOCHONDRIAL APOPTOSIS BY ENDOPHILIN B1 IN GLIOMAKhatri Latha, Vaibhav Chumbalkar, and Oliver Bogler; University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Bax interacting factor (Bif-1)/Endophilin B1 is a Bax binding part-ner that promotes the Bax conformational change and insertion into the mitochondrial membrane and sensitizes hematopoietic cells to apoptosis in response to stimuli that trigger mitochondrial mediated apoptosis. Down-regulation of this protein or overexpression of endophilin B1 lacking the NH(2)-terminal lipid-modifying domain causes striking alterations of the mitochondrial distribution and morphology. Our preliminary data show

that in glioma cells, transfection of Bif-1 alone is sufficient to induce apop-tosis, while HeLa cells in our experiments, and hematopoietic cells, in published reports, require an additional trigger of apoptosis. This suggests that glioma cells are particularly sensitive to Bif-1 mediated apoptosis and that intervention in it might have significant translational potential. Bif-1 induced apoptosis can be functionally antagonized by Bcl-2 and BclXl, and enhanced by the endophilin binding partners Alix and SETA/CIN85. We are comparing the Bif-1 interactome in HeLa and glioma cells in an attempt to understand the apoptosis biology of glioma cells.

CB-14. DEREGULATED NOTCH PATHWAY INDUCES TETRAPLOIDY IN MENINGIOMAGilson Baia,1 Stefano Stifani,2 Michael McDermott,1 and Anita Lal1; 1Neurosurgery Dept., University of California, San Francisco, San Francisco, CA, USA; 2Center for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada

Meningiomas are the second most common brain tumor and a con-siderable cause of morbidity and mortality because of their location and because of existence of variants with malignant characteristics. The molec-ular mechanisms underlying meningioma tumorigenesis, except for the loss of NF2 gene, are poorly understood. Previously, we had shown that several components of the Notch signaling pathway are deregulated in meningioma tumors. Specific Notch function is dependent on the cellular context and the particular homologs expressed. Here we show that exogenous expression of the Notch signaling effector, HES1, induces tetraploidy in three different meningioma cell lines. This altered ploidy is associated with nuclear atypia, the appearance of micronuclei and mitotic spindle abnormalities. Expres-sion of both activated NOTCH1 and NOTCH2 receptors produces the same phenotype. Tetraploidy often leads to aneuploidy and consequently, to enhanced tumorigenicity. HES1–expressing SF3061 cells were sorted into diploid and tetraploid fractions. Spectral Karyotyping (SKY) analy-sis revealed that tetraploid cells develop a greater number of chromosomal translocations over time in culture, when compared to vector control cells. Thus, the HES1 induced tetraploidy was resulting in aneuploidy. Overex-pression of HES1 suppresses apoptosis in response to staurosporine and enhances cell survival in vitro. Together, these observations point to a role for the Notch signaling pathway in enhancing chromosomal instability and tumorigenicity in meningiomas.

CB-15. GATA4 EXPRESSION IN THE NORMAL CNS AND ITS FREQUENT LOSS IN HUMAN GLIOMASSameer Agnihotri, Deepak Kamnasaran, Cynthia Hawkins, and A. Guha; University of Toronto, Toronto, Ontario, Canada

The GATA family of transcription factors consist of six members and recognize the consensus DNA binding motif (A/T)-GATA-(A/G). GATA transcription factors regulate a myriad of biological functions including organogenesis, differentiation, lineage commitment, proliferation and apoptosis. GATA family members have also been implicated as tumor sup-pressor genes (TSG) in several human cancers, including our recent work on GATA6 in murine and human malignant gliomas. The role and function of GATA4 in the human and mouse normal and pathological nervous system is unknown. Our overall objective was to characterize the expression of GATA4 in the human and mouse CNS and to determine whether it is a candidate TSG in human GBMs. Normal brain specimens were evaluated for GATA4 expression by immunohistochemistry (IHC) in adult human and murine brains of varying developmental ages. IHC analysis demon-strated ubiquitous expression in a variety of CNS cells including neurons, glia (astrocyte, oligodendrocyte, ependymocytes and choroid plexus epi-thelium) and the brain endothelium. Isolated normal murine and human astrocytes expressed GATA4 by Western blot analysis, whereas GATA4 expression was lost in human GBM cells lines and GBM operative samples. Tissue microarray (TMA) of varying glioma subtypes demonstrated GATA4 loss in a large percentage of human GBMs, but variable degrees of loss in samples of gliosarcomas, low-grade astrocytomas, low- and high-grade oli-godendrogliomas. GATA4 siRNA mediated gene silencing in predisposed non-transformed astrocytes harboring oncogenic ras or null for p53 signifi-cantly increased their proliferation ability. Conversely, re-introduction of GATA4 expression in transformed human and murine GBM cells, shifted the cells from a S/G2M to a G1 phase, with an increase in apoptosis. Col-lectively, these results are suggestive of GATA4 possessing tumor suppressor functions. Our data is the first to demonstrate a GATA4 expression profile in the CNS and implicate GATA4 as a TSG in murine and human malignant gliomas. Current studies are focused on: Mutational analysis to investigate how GATA4 function is lost in gliomas; transformation assays to determine if loss of GATA4 in normal astrocytes can initiate transformation of nor-mal glial cells and if GATA4 over expression in transformed glial cells can revert transformation. The significance of this study is to better understand



gliomagenesis and identify potential downstream targets of GATA4 that may serve as therapeutic treatments.

CB-16. ANALYSIS OF EGFRVIII SIGNALING IN GLIOMAYeohyeon Hwang, Vaibhav Chumbalkar, Khatri Latha, and Oliver Bogler; University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

EGFRvIII, a deletion mutant which occurs in about 40% of glioblas-tomas, is a potent oncogene that promotes growth and survival of cancer cells. The signaling of EGFRvIII is ligand-independent, does not involve receptor dimerization, is low intensity which allows it to evade the normal mechanisms of internalization and degradation by the endocytic machinery, and hence is persistent. To determine whether EGFRvIII’s oncogenic signal is dependent on its low intensity we are attempting to force dimerization by creating receptor chimeras with variants of the FKBP12 protein that are brought together by rapamycin-derivatives. Using this model, as well as constitutively expressing glioma cell lines with different PTEN background we are also examining the signaling pathways activated by EGFRvIII using quantitative multidimensional chromatography of peptides followed by tandem mass spectrometry to identify phosphoproteins and phosphoryla-tion sites.

CB-17. AKT1 AND 2 MEDIATE MALIGNANT FUNCTION IN GBMAnna Joy,1 Mitsitoshi Nakada,2 Sonya Seif-Naraghi,2 Satoko Nakada,2 Jessica Rennert,2 Christian Beaudry,2 Jeanne Sasse,2 Nhan Tran,2 Shannon Fortin,2 Luigi Mariani,3 Jean Zenklusen,4 Howard Fine,4 Burt G. Feuerstein,1 and Michael Berens2; 1Neurology, St. Josephs Hospital and Medical Center, Phoenix, AZ, USA; 2Translational Genomics Research Institute, Phoenix, AZ, USA; 3AE (Europe); 4National Institutes of Health, Bethesda, MD, USA

The Akt pathway has been implicated in progression of astrocytic tumors. In order to refine the Akt pathway as a therapeutic target in glio-blastoma multiforme (GBM) we determined activated Akt isoform(s) that mediate cellular behavior, and are associated with clinical parameters. GBM tissue contains predominantly activated (ser473 phosphorylated) Akt2, some Akt1, but little detectable Akt3 relative to normal brain. Expression of Akt1 and 2 message correlates directly with tumor grade and inversely with survival. In contrast, Akt3 message correlates inversely with tumor grade and directly with long term survival. siRNA knockdown experiments suggest that Akt 2 promotes migration and survival, while Akt3 promotes apoptosis and plays no role in migration. Taken together, the data indicate that Akt3 function differs from Akt1 and 2 function, and support a role for Akt1 and 2 in the malignant behavior of glioma cells. Selective inhibition of Akt1 and 2, or downstream effectors specific to Akt1 and 2 may be more effective and less toxic than general inhibition of Akt or PI3K. Supported by NIH/NINDS R01 NS042262 (MB), NIH/NINDS R21 NS043446 (MB) and the Barrow Neurological Foundation (BGF).

CB-18. ALIX PHOSPHORYLATION BY P38 DURING MITOTIC ARREST INHIBITS MULTIPLE PARTNER PROTEIN INTERACTIONSRob Dejournett,1 Ryuji Kobayashi,2 Mark Bedford,3 Jian Kuang,4 and Oliver Bogler1; 1University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Department of Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3Science Park, University of Texas M.D. Anderson Cancer Center, Smithville, TX, USA; 4Department of Experimental Theraputics, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Alix is a 95 kDa scaffold or adaptor protein which promotes growth arrest of HeLa cells and suppresses tumor formation in nude mice. Signifi-cantly for brain tumors, Alix is known to regulate EGFR endocytosis; the mechanism of this regulation is unclear but likely involves post-translational modifications. We previously demonstrated that the Xenopus homolog of Alix, Xp95, is phosphorylated during progesterone-induced meiotic matu-ration of Xenopus oocytes, a model of M-phase induction. To further char-acterize phosphorylation of Xp95, we analyzed purified, phosphorylated Xp95 by mass spectrometry, and found two phospho-residues within the proline-rich domain (PRD), one of which is T745. T745 is within the con-served binding site to SETA/CIN85/SH3KBP1, an adaptor protein involved in EGFR endocytosis. Xp95 phosphorylation at T745 was shown to inhibit SETA interaction: Xp95 from immature but not mature oocytes can bind

SETA in vitro, and a phospho-T745 peptide of Xp95 shows reduced binding to recombinant SETA. As an additional method to identify phosphorylation sites, we subcloned Xp95 and found that amino acids 706–786 were neces-sary and sufficient for the phosphorylation-dependant gel-shift. To extend these results to mammalian cells, we examined mitotic arrested HeLa cells and found that deletion of a similar region of Alix (aa 717–784) blocked the gel-shift of Alix. We also identified p38 stress-activated kinase (SAK) as a candidate kinase for T745 phosphorylation, using phospho-T745 specific antibodies. Using recombinant active p38, we found that not only does Alix T745 phosphorylation inhibit binding to SETA, but also to the Alix part-ners PLCgamma, and Endophilin B1, all of which are involved with EGFR signal transduction and endocytosis. Together, these findings indicate that Xp95/Alix is phosphorylated within the PRD during M phase induction and EGFR endocytosis, and that this phosphorylation regulates interaction with binding partners. These results suggest a novel mechanism of blocking pro-tein trafficking during mitosis via phosphorylation of Alix by p38 SAK.

CB-19. NF2 TUMOR SUPPRESSOR FUNCTION ACTS THROUGH THE HIPPO PATHWAY IN HUMAN MENINGIOMASKatharine Striedinger,1 David Gutmann,2 and Anita Lal1; 1University of California, San Francisco, San Francisco, CA, USA; 2Washington University in St. Louis, St. Louis, MO, USA

Neurofibromatosis type-2 (NF2) is a cancer predisposition syndrome caused by mutations in the NF2 gene, and is characterized by the occur-rence of meningiomas. Thus loss of NF2 and its product merlin are critical for the development of meningiomas. In this study we explored the molecu-lar mechanisms by which NF2 acts as a tumor suppressor in immortalized human meningioma cell lines. NF2 expression was stably suppressed by RNA interference in merlin positive meningiomas. Also we introduce wild type NF2 under a tetracycline inducible expression system in a malignant merlin negative meningioma cell line. As expected, loss of contact depen-dent inhibition, anchor independent growth and increased proliferation was observed when NF2 was silenced. The function of merlin as a negative regu-lator of growth is conserved in Drosophila where it has recently been shown to act upstream of the Hippo signaling pathway, regulating cell proliferation and apoptosis. We hypothesized that merlin could exert its tumor suppres-sor function through mammalian homologue of hippo pathway in humans. To test this hypothesis, we analyzed the expression of different components of the hippo pathway in the presence or absence of merlin. At the tran-scriptional level we found lower levels of MST2 and LATS in cells that lack NF2. At the protein level we found that YAP, a transcriptional activator and the effector protein of the hippo pathway, is inactive in the presence of merlin and inversely, merlin absence correlated with activation of YAP resulting in the transcription of genes involved in suppression of apoptosis and increased cell proliferation. Our data suggests that merlin signal via hippo is conserved in humans. Functional strategies to test the involvement of merlin in the hippo pathway are currently being developed.

CB-20. DIFFERENTIAL APAF-1 LEVELS ALLOW CYTOCHROME C TO INDUCE APOPTOSIS IN BRAIN TUMORS BUT NOT IN NORMAL NEURAL TISSUESCarrie Johnson,1 Yolanda Huang,2 Amanda Parrish,2 Robert Wechsler-Reya,3 Mohanish Deshmukh,4 and Sally Kornbluth2; 1Pharmacology and Cancer Biology, Duke University, Durham, NC, USA; 2NC, USA; 3Duke University, Durham, NC, USA; 4University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Brain tumors are typically resistant to conventional chemotherapeutics, most of which initiate the apoptotic pathway upstream of mitochondrial release of cytochrome c. In this study, we demonstrate that directly activat-ing apoptosis downstream of the mitochondria, with cytosolic cytochrome c, kills brain tumor cells but not normal brain tissue. Specifically, we show that cytosolic cytochrome c is sufficient to induce apoptosis in glioblas-toma and medulloblastoma cell lines. In contrast, primary neurons from the cerebellum and cortex are remarkably resistant to cytosolic cytochrome c. Importantly, tumor tissue from mouse models of both glioblastoma and medulloblastoma display hypersensitivity to cytochrome c when compared to surrounding non-neoplastic brain tissue. This differential sensitivity to cytochrome c is attributed to high Apaf-1 levels in the tumor tissue as com-pared to low or undetectable Apaf-1 levels in the non-neoplastic brain tis-sue. Together, these results demonstrate an unexpected sensitivity of both adult and pediatric brain tumors to direct post-mitochondrial induction of apoptosis. Moreover, they raise the possibility that this phenomenon could be exploited therapeutically to selectively kill brain cancer cells while spar-ing the surrounding brain parenchyma.



CB-21. EXPRESSION OF SRC FAMILY KINASES IN HUMAN DERIVED GLIOMA CELL LINES INDUCES MITOCHONDRIAL LOCALIZATION OF EGFRVIIITerrance Johns,1 Anna Cvrljevic,2 Amelia Johnston,3 Vino Pillay,2 and Nick Hoogenraad4; 1Oncogenic Signalling, Ludwig Insitute for Cancer Research, Melbourne, Victoria, Australia; 2Ludwig Institute for Cancer Research, Victoria, Australia; 3LaTrobe University, Victoria, Australia; 4La Trobe University, Bundoora, Victoria, Australia

The most common mutation of the epidermal growth factor receptor (EGFR) in glioma is the EGFRvIII. This mutant has a specific deletion between exons 2–7 of the EGFR gene, which leads to the elimination of 267 amino acids from the extracellular domain and the insertion of a novel gly-cine at the fusion junction. While the EGFRvIII is unable to bind any known ligand it displays a low level of constitutive activation, which is enhanced by the inability of the receptor to undergo down-regulation. Glioma cell lines expressing the EGFRvIII contain a large intracellular pool of the receptor. Given that mutated proteins are often processed more slowly, and that this large pool of the EGFRvIII predominately contains high mannose glycosyla-tion typical of endoplasmic reticulum (ER) localization, it has been assumed this intracellular pool would be co-located with the ER or golgi apparatus. However, using careful confocal microscopy analysis and biochemical tech-niques we could only detect small amounts of the EGFRvIII associated with these organelles in U87MG human glioma cells. Interestingly, these initial studies suggested co-localization of EGFRvIII with mitochondria. Further-more, we showed that EGFRvIII in these cells is phosphorylated at tyrosine 845 in a src family kinase dependent manner. Since it has been recently shown that src over-expression enhances the localization of the wild type EGFR to the mitochondria, through tyrosine 845 phosphorylation, we co-transected U87MG cells expressing the EGFRvIII with a constitutively acti-vated src. Co-expression of these molecules dramatically enhanced mito-chondria localization of the EGFRvIII as determined with both microscopic and biochemical techniques. Dasatinib, an inhibitor of src family kinases, completely prevented the mitochondria localization of EGFRvIII in this cell line. As the mitochondria located EGFRvIII was fully glycosylated and constitutively active, its mitochondrial localization could be related to its tumorgenicity. Indeed, its interaction with potential mitochondria targets will be reported. Finally, a large pool of functionally significant EGFR may explain the resistance of this molecule to some EGFR therapeutics; a pos-sibility we are currently exploring in xenografts models in nude mice.

CB-22. A NOVEL MOLECULAR MECHANISM FOR CHEMORESISTANCE IN GLIAL TUMORS: NF-KAPPA B–MEDIATED EXPRESSION OF MGMTIris Lavon,1 Dana Fuchs,1 Daniel Zrihan,1 Gilat Efroni,1 Bracha Zelikovitch,1 Yakov Fellig,2 and Tali Siegal1; 1Gaffin Center for Neuro-Oncology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel; 2Department of Pathology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel

Glial tumors are the most common primary CNS neoplasms. The out-come for high grade tumors is poor, with life expectancy of only about one year for patients with gllioblastoma multiforme (GBM). Standard therapy includes various combinations of radiotherapy and chemotherapy. Chemotherapy is largely based on alkylating agents such as temozolomide or nitrosurea. O6–Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that protects cells against alkylating insults. The level of MGMT cellular expression correlates with tumor’s chemoresis-tance of patients with GBM. The objective was to identify the molecular mechanisms that regulates MGMT expression. We have discovered two putative NF-kappaB binding sites within the MGMT promoter region and were able to demonstrate that the transcription factor NF-kappaB plays a major role in the regulation of MGMT, a function not recognized before. Forced expression of the NF-kappaB subunit p65 in HEK293 cells induced an increase in MGMT expression while addition of the NF-kappaB super repressor DNIkappaB completely abrogated the induction. We also found a significant correlation between the extent of NF-kappaB activation and MGMT expression in the glioma cell lines and the human glial tumors tested. This correlation was independent of MGMT promoter methylation. We also showed that cell lines with forced expression of p65 or those with high constitutive NF-kappaB activity are less sensitive to nitrosourea treat-ment. Suppression of MGMT activity in these cells (with O6-benzylguanine) completely abolishes the chemoresistance acquired by NF-kappaB. Based on our results we propose a model of novel molecular mechanism in which NF-kappaB mediates chemoresistance through induction of MGMT. According to this simplified model the majority (81%) of gliomas that bear high constitutive NF-kappaB activity should exhibit augmented MGMT expression. Following treatment with alkylating agents, MGMT removes the methyl adducts from the DNA and the DNA is therefore restored. Our results are of potential clinical significance as several therapeutic inhibi-tors that block NF-kappaB activation are under development. Our findings

indicate that such inhibitors would be of great clinical value for sensitizing MGMT-positive expressing cells to alkylating chemotherapeutic treatment and may assist in overcoming chemoresistance.

CB-23. DIVERSE GENETIC BACKGROUNDS DETERMINES EGFR MEDIATED RESPONSE TO ERLOTINIB IN GBM CELL LINESSebila Kratovac,1 Angel Ayuso-Sacido,1 and John Boockvar2; 1Weill Medical College of Cornell, New York, NY, USA; 2Neurosurgery, Weill Medical College of Cornell, Neurosurgery Department, New York, NY, USA

Gliobastoma multiforme (GBM) is an incurable primary brain tumor of the central nervous system. The epidermal growth factor receptor (EGFR) gene is amplified or mutated (EGFRvIII) in 30%–50% of human GBM. Erlotinib is an EGFR/EGFRvIII inhibitor used in the treatment of some human can-cers, and has been shown to be active in GBM with the overexpressed/mutated EGFR. In order to test the effectiveness of erlotinib on GBM cells with diverse genetic backgrounds, we used U87 and LN229 GBM cell lines to generate EGFR- or EGFRvIII-overexpressing mutants. Because increased migration and invasiveness are key characteristic of GBM, we carried out chemotaxis and invasiveness assays with parental/wtEGFR /EGFRvIII over expressing GBM cell lines. We found that erlotinib inhibited EGF-mediated chemotaxis and invasion in U87wtEGFR and LN229wtEGFR and that the level of erlotinib inhibition differed in the two cell lines. We also character-ized the signals downstream of EGFR and observed significant differences between the two cell lines in their levels of PTEN expression. In light of our findings, we propose that the differences in EGFR downstream signaling, as well as other genetic alterations such as the deletion of PTEN, could be responsible for the differential response to erlotinib. We therefore con-clude that personalizing erlotinib treatment based on patient GBM levels of EGFR and PTEN may help to identify targeted subgroups of patients whose tumors may show reduced brain tumor cell dispersal.

CB-24. RIP1, AN INFLAMMATION INDUCED PROGNOSTIC MARKER IN GLIOBLASTOMASeongmi Park,1 Deepti Ramnarain,1 Kimmo Hatanpaa,2 Bruce Mickey,3 Guanghua Xiao,4 Krishna Puttarparthi,1 Christopher Madden,3 and Amyn Habib1; 1Neurology, University of Texas Southwestern Medical Center at Dallas, TX, USA; 2Pathology, University of Texas Southwestern Medical Center at Dallas, TX, USA; 3Neurosurgery, University of Texas Southwestern Medical Center at Dallas, TX, USA; 4Clinical Sciences, University of Texas Southwestern Medical Center at Dallas, TX, USA

Activation of inflammatory and immune responses has powerful effects on cell cycle progression and cancer. In this study, we show that the recep-tor interacting protein (RIP, RIP1), a critical mediator of inflammation and stress induced NF-kappa B activation, also strongly activates the PI3K-Akt pathway, inhibits p27Kip1 and regulates cell cycle progression. Increased expression of RIP1 is sufficient to activate both the NF-kappa B and PI3K-Akt pathways which are known to be activated in glioblastoma (GBM), the most common adult malignant brain tumor. RIP1 is overexpressed in about 50% of GBM but not in lower grade glioma and increased RIP1 expres-sion confers an ominous prognosis in GBM. The level of RIP1 correlates with activation of Akt in GBM. Importantly, silencing RIP1 in glioma cells inhibits their proliferation. We demonstrate that inflammation powerfully augments RIP1 level in models of CNS inflammation and in inflamma-tion induced cancer. Thus, RIP1 may represent a novel oncogenic protein upregulated by inflammation rather than genetic alteration in cancer. We propose a new model linking inflammation and cancer in which the inflammatory tumor microenvironment induces expression of an oncogenic protein (RIP1) that, in turn, drives the malignant phenotype and confers chemoresistance in a cell autonomous manner by constitutive activation of key pro-survival pathways. Our data also suggest that persistent NF-kappa B activation in inflammatory states may result, in part, from inflammation induced increases in levels of upstream activators.



CB-25. EXPRESSION AND FUNCTIONAL SIGNIFICANCE OF INHIBITORS OF APOPTOSIS PROTEINS (IAPS) IN HUMAN GLIOMASJoydeep Mukherjee,1 Amparo Wolf,2 and Abhijit Guha3; 1Cell Biology, Arthur and Sonia Labatt Brain Tumor Research Center, The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada; 2Arthur and Sonia Labatt Brain Tumor Research Center, The Hospital for Sick Children Research Institute, Ontario, Canada; 3Neurosurgery and Cell Biology, Toronto Western Hospital and Hospital for Sick Children Research Institute, Toronto, Ontario, Canada

Inhibitors of Apoptosis Proteins (IAPs) are a highly conserved family of proteins, which inhibit the final common extrinsic or intrinsic apoptotic signaling mediated by activated caspase3. Aberrations in cell-survival sig-nals is integral to the transformation and therapeutic resistance in cancer, including human gliomas. The regional expression profile and associated functional relevance of IAPs in glioma is largely unknown and is the subject of our experiments. We examined the expressional levels of the four major IAPs (cIAP1, cIAP2, XIAP and Survivin) in low- and high-grade human glioma specimens. Regional differences in IAPs, between the “center-pseudopalisading” and “periphery-infiltrating” areas, was investigated by Real-Time qRT-PCR on RNA obtained by Laser Capture Microdissected (LCM) GBM cells from these two regions. The qRT-PCR results were vali-dated by immunohistochemistry (IHC) on corresponding paraffin embed-ded sections. In-vitro experiments with human GBM cell lines with siRNA knockdown of relevant IAPs were undertaken to investigate the functional relevance. cIAP1 was the only IAP member with increased expression in low-grade astrocytomas compared to normal brain, while all the IAPs examined were differentially expressed in GBMs. cIAP2 was increased in the “periphery-infiltrating” compared to the “center-pseudopalisading” GBM cells. In contrast, XIAP and Survivin was increased in the “pseudo-palisading” vs. “infiltrating” GBM cells. To determine if increased expres-sion of XIAP and Survivin maybe contributing to the apoptosis resistance phenotype of the pseudopalisading cells, siRNA knockdown of XIAP and Survivin was undertaken. Knock down of both XIAP and Survivin rendered the GBM cells more susceptible to hypoxia and chemotherapy induced apoptosis, without any alteration in proliferation. Results from overexpres-sion studies and in vivo experiments with shRNA mediated knockdown are currently pending. We hypothesize that aberrant increased IAPs, especially XIAP and Survivin by pseudopalisading GBM cells, plays an important role in cell-survival and thereby therapeutic resistance and recurrence. Modula-tion of these IAPs may directly decrease glioma growth or indirectly render them more sensitive to radiation and/or chemotherapy.

CB-26. THE WARBURG EFFECT AND TUMOR CELL SURVIVAL IN HUMAN GBMAmparo Wolf,1 Joydeep Mukherjee,1 and Abhijit Guha2; 1Arthur & Sonia Labatt Brain Tumor Center, Hospital for Sick Children, Toronto, Ontario, Canada; 2Neurosurgery and Cell Biology, Toronto Western Hospital and Hospital for Sick Children Research Institute, Toronto, Ontario, Canada

GBMs are resistant to apoptosis induced by the hypoxic microenviron-ment and standard therapies including radiation and chemotherapy. We postulate that the Warburg effect, a preferential glycolytic phenotype of tumor cells even under aerobic conditions, plays a role in these aberrant pro-survival signals. In this study we quantitatively examined the expres-sion profile of hypoxia-related glycolytic genes within pathologically- and MRI-defined “center” and “periphery” of GBMs. We hypothesize that expression of hypoxia-induced glycolytic genes, particularly hexokinase 2 (HK2), favors cell survival and modulates resistance to tumor cell apop-tosis by inhibiting the intrinsic mitochondrial apoptotic pathway. GBM patients underwent conventional T1–weighted contrast-enhanced MRI and MR spectroscopy studies on a 3.0T GE scanner, prior to stereotactic sampling (formalin and frozen) from regions which were T1–Gad enhanc-ing (“center”) and T2-positive, T1-Gad negative (“periphery”). Real-time qRT-PCR was performed to quantify regional gene expression of glyco-lytic genes including HK2. In vitro functional studies were performed in U87 and U373 GBM cell lines grown in normoxic (21% pO2) and hypoxic (,1% pO2) conditions, transfected with HK2 siRNA followed by measure-ment of cell proliferation (BrdU), apoptosis (activated caspase 3/7, TUNEL, cytochrome c release) and viability (MTS assay). There exists a differential expression profile of glycolytic enzymes between the hypoxic center and relatively normoxic periphery of GBMs. Under hypoxic conditions, there is increased expression of HK2 at the mitochondrial membrane in GBM cells. In vitro HK2 knockdown led to decreased cell survival and increased apoptosis via the intrinsic mitochondrial pathway, as seen by increased mitochondrial release of cytochrome-C. Increased expression of HK2 in the center of GBMs promotes cell survival and confers resistance to apoptosis, as confirmed by in vitro studies. In vivo intracranial xenograft studies with

injection of HK2–shRNA are currently being performed. HK2 and possibly other glycolytic enzymes may provide a target for enhanced therapeutic responsiveness thereby improving prognosis of patients with GBMs.

CB-27. A NOVEL PROGNOSTIC SUBGROUP OF GLIOBLASTOMA WITH ACTIVATED HOX DOMAINS THAT ARE REVERSIBLY REGULATED THROUGH THE PI3K PATHWAYBruno Costa,1 Justin Smith,1 Ying Chen,2 Heidi Phillips,3 Kenneth Aldape,4 Giuseppe Zardo,5 Janice Nigro,6 C. David James,2 Jane Fridlyand,2 and Joseph F. Costello1; 1University of California, San Francisco, San Francisco, CA, USA; 2CA, USA; 3SSF, CA, USA; 4University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 5Italy; 6Bergen, Norway

Glioblastoma (GBM) is the most common and aggressive primary brain tumor and existing therapy regimens are often ineffective. The methylation status of MGMT promoter region is currently the best molecular marker of prognosis in GBM patients treated with radiation and Temozolomide. However, MGMT is not the sole prognostic determinant and does not completely define patient outcome. HOX genes encode transcription fac-tors that play crucial roles in normal development and have been shown to be altered in different tumor types. Considering the very limited number of studies characterizing the importance of HOXA genes in gliomagenesis, we investigated the relevance of HOXA genes in GBM and their potential as prognostic molecular markers. A cluster of HOXA genes was aberrantly expressed in GBM cell lines and primary tumors, but not in lower grade gliomas. Notably, expression of HOXA9 was associated with shorter over-all and progression-free survival for GBM patients, in two independent sets of tumors. Furthermore, univariate and multivariate survival analy-ses showed that combined assessment of HOXA9 expression and MGMT methylation provides a better prognostication tool for GBMs than MGMT alone. In fact, a subset of GBM patients whose tumors have a methylated MGMT promoter do not exhibit the expected favorable outcome; HOXA9 expression identified a subset of patients who, despite expectedly favorable MGMT methylation, showed very poor outcomes. In GBM xenografts, aberrant overexpression of HOXA9 was associated with PTEN gene inac-tivation. Indeed, pharmacological manipulations of the PI3K pathway sug-gested that the mechanism of aberrant HOXA gene activation involves epi-genetic alterations of these loci that are dependent on alterations of the PI3K pathway. In conclusion, we found that a subset of the most highly malignant GBMs show a chromosomal domain of gene activation encompassing the HOXA gene cluster, where aberrant HOXA9 activation seems to be a novel molecular marker of prognosis for GBM that could improve MGMT-based predictions. These findings may help to stratify GBM patients for specific therapies. Overall, our data support the hypothesis that whole gene clusters along a chromosomal region may be activated in human tumors, and that epigenetic alterations, namely modifications at the histone tails as a result of PI3K pathway dysfunction, may be one of the underlying mechanisms affecting these aberrant expression profiles. Studies to clarify the functional roles of HOXA9 in GBM are under way.

CB-28. HSP90 ALPHA RECRUITS FLIP-S TO THE DEATH-INDUCING SIGNALING COMPLEX AND CONTRIBUTES TO TRAIL RESISTANCE IN HUMAN GLIOMAAmith Panner; University of California, San Francisco, San Francisco, CA, USA

Heat-shock protein 90 (HSP90) is a molecular chaperone that contrib-utes to the proper folding and stability of target proteins. Because HSP90 has been suggested to interact with FLIP-S, the key regulator of TNF-alpha-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in glioma cells, we examined the role HSP90 played in controlling TRAIL response. HSP90 alpha was found to associate with FLIP-S in resting cells in a manner dependent on the ATP-binding N-terminal domain of HSP90 alpha. Fol-lowing TRAIL exposure, HSP90 alpha and the client FLIP-S protein were recruited to the death-inducing signaling complex (DISC). siRNA-mediated suppression of HSP90 alpha did not alter total cellular levels of FLIP-S, but rather inhibited the recruitment FLIP-S and other anti-apoptotic proteins such as RIP and FLIP-L to the DISC, and sensitized otherwise resistant glioma cells to TRAIL-induced apoptosis. These results show that HSP90 alpha, by localizing FLIP-S to the DISC, plays a key role in the resistance of tumor cells to TRAIL, and perhaps other pro-apoptotic agents. The results also define a novel means of apoptotic control by HSP90 alpha that may in turn help explain the global anti-apoptotic effects of this protein.



CB-29. PTEN-MEDIATED AUTOPHAGY IS INDEPENDENT OF MTOR INHIBITION AND AMPK ACTIVATION IN GBM CELLSJuinn-Lin Liu, Zhenyu Mao, Tiffany Lafortune, Ta-Jen Liu, Jeannine Garnett, Dimpy Koul, and W.K. Alfred Yung; Neuro-Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Induction of autophagy has been developed as an alternative thera-peutic regimen for tumors that are defective in apoptotic pathway. mTOR plays a pivotal role in negative regulation of autophagy in many cell types. However, the regulation of autophagy appears very complex. For instance, inhibition of PI3K by pan inhibitors, such as wortmanin or LY294002, actually suppresses autophagy despite that mTOR is a downstream effec-tor of PI3K class I cascade. It is due to that the key component for initia-tion of autophagic cell death, PI3K class III, is also inhibited by pan PI3K inhibitors. On the other hand, activated AMPK has been demonstrated to directly inhibit mTOR by phosphorylation or indirectly through activat-ing TSC2. Theoretically activation of AMPK should also impose the same effect as mTOR inhibitors in inducing autophagic cell death. However, AMPK activators do not necessarily stimulate autophagy depending on the cell type. Our lab focuses on characterizing the biologic function of PTEN in the nucleus. We found that nuclear PTEN induced G1 arrest and tumor suppression without inhibiting Akt. However, nuclear PTEN could still suppress mTOR and S6K activities possibly through activation of AMPK. Thus, although wild-type PTEN has been shown to be capable of inducing autophagy, the exact mechanisms are far from being full understood. In this study, we utilized the ecdysone-inducible system in U251HF cells to dissect the molecular mechanisms involved in PTEN-mediated autophagy. Upon induction of ponasterone A in 1% serum for 48 hours, both wild-type and nuclear PTEN induced significant autophagy in U251HF cells (more than 40%). By contrast, treatment of pIND vector-transfected U251HF (VgRxR) parental cells with inhibitors to PI3K (wortmanin), or mTOR (RAD001 and rapamycin), and activators for AMPK (AICAR and metformin) only induced minimum autophagy (less than 10%), even though mTOR activ-ity was suppressed. Furthermore, AMPK inhibitor, compound C, did not block nuclear PTEN-induced autophagy. Altogether our data strongly sug-gest that both wild-type and nuclear PTEN-induced autophagy is indepen-dent of mTOR inhibition and AMPK activation. Our preliminary results revealed no marked difference in autophagic signaling except that JNK/SAPK is activated by wild-type and nuclear PTEN. Further characterization is underway to verify if PTEN-mediated autophagic programmed cell death is dependent on JNK/SAPK pathway and to elucidate the mechanisms by which PTEN activates JNK/SAPK.

CB-30. METHYLATION PROFILING DIFFERENTIATES SECONDARY GBM AND LOW-GRADE ASTROCYTOMAShichun Zheng,1 Zachary Morrison,1 Andres Houseman,2 Joe Patoka,1 Christian Ramos,1 Sean McBride,1 Daphne Haas-Kogan,1 Michael Prados,1 David Stokoe,1 Susan Chang,1 Margaret Wrensch,1 Joseph Wiemels,1 Mitchel Berger,1 and John Wiencke1; 1University of California, San Francisco, San Francisco, CA, USA; 2University of Massachusetts Lowell, Lowell, MA, USA

Epigenetic alterations including gene promoter DNA hypermethylation can lead to transcriptional inactivation of glioma tumor suppressor genes and thus promote tumorigenesis. The pattern of gene targets affected by aberrant DNA methylation could indicate the lineage and cell of origin of gliomas. We hypothesize that glioma subtypes/grades may have distinct epigenetic characteristics that could be used to create molecular subgroups that may help to define etiologic subgroups and provide prognostic tools for assessing patient response to therapy. 201 glioma tumor samples (primary glioblastoma GBM 40, secondary GBM 11, recurrent GBM 12, AS II 30, AS III 9, OA II 26, OA III 10, OD II 23, adult ependymoma 16, pediatric ependymoma 16, other pediatric brain tumors 6), 13 normal brain tissues and 6 GBM cell lines were analyzed for methylation at 12 glioma-related gene loci (PEG3, HOXA9, SLIT2, MGMT, PTEN, RASSF1A, RFX1, MAGEA1, TMS1, EMP3, SOCS1, and ZNF342). Genomic DNA was iso-lated from fresh frozen tissue samples obtained from UCSF Brain Tumor Tissue Bank, and bisulfite treated with Chemicon CpGenome DNA Conver-sion Kit. DNA methylation profiling using quantitative methylation-specific polymerase chain reaction (Q-MSP) was used to assess methylation status. Exploratory analyses using random effects models revealed similarities among methylation levels that grouped secondary GBM with low-grade gliomas and separately from primary or recurrent GBM. There were highly statistically significant differences among the different grades and types of glioma with respect to methylation levels and genes affected by differential methylation (likelihood ratio test; p , 0.0001). Bayesian model averaging techniques suggest that specific genes appeared to be methylated among different glioma subgroups. RASSF1A and RFX1 were common targets of differential methylation in secondary GBM and low-grade glioma whereas HOXA9 was characteristic of primary and recurrent GBM. Overall methy-lation levels were highest for most putative tumor suppressor genes among

low-grade gliomas and secondary GBMs. Several primary GBMs were out-liers and demonstrated methylation patterns similar to low grade gliomas and secondary GBMs. Hypomethylation of MAGE1A and the imprinted PEG3 were most common in primary and recurrent GBM. Work is in prog-ress to correlate these changes with the TP53 mutation and EGFR amplifica-tion status of these tumors. Methylation profiling using a relatively small panel of informative loci indicated clustering of gene methylation events that were distinct among major subtypes of glioma. Most dramatically the low-grade gliomas demonstrated the highest levels of hypermethylation and secondary GBM contained methylation levels similar to the low-grade tumors. These results suggest that significant epigenetic features of low-grade glioma are preserved in cells as they undergo other changes during their evolution toward aggressive high-grade tumors. Methylation pro-files should prove useful in defining molecular subtypes and in stratifying patients for etiologic and clinical outcome studies.

CB-31. IMATINIB MESYLATE (STI571, GLEEVEC) UP-REGULATES THE ACTIVATION OF P42/P44MAPK AND CORRELATES WITH THE ENHANCED PROLIFERATION IN MALIGNANT GLIOMA CELLSTan XQ,1 Dong YC,1 Ren H,1 Xu HW,1 Jiang Tao,2 Rainov NG3,4; 1Harbin Medical University, Harbin, China; 2Beijing Neurosurgery Institute, Beijing, China; 3Klinikum Augsburg, Augsburg, Germany; 4Martin-Luther-University Halle-Wittenberg, Germany

The aim of this study is to investigate the effect on downstream sig-naling pathways under the tyrosine kinase inhibitor, imatinib mesylate (STI571, Gleevec) treatment targeted against PDGFR in human malig-nant glioma cells. RT-PCR and real time PCR were employed to investi-gate the expression of PDGF ligands and cognate receptors, as well as the dual phosphatases MKP-1 and MKP-3, which were downstream of p42/p44MAPK , at mRNA level in tested malignant glioma cells; time-coursed Western blotting was employed to detect the correlated protein expression. MTT assays were performed to examine the glioma cell growth inhibition under imatinib treatment, alone or in combination with MAPK protein kinase inhibitor PD098059. Immunoblot-based differential protein kinase screening was performed on U87MG glioma cells with or without treat-ment of imatinib. 10 mM or above imatinib can completely or partial inhibit glioma cell growth. Western blots verified the inhibition of PDGFRb phos-phorylation in U87MG cells. Time-course data revealed that the imatinib treatment, unrelated to serum and growth factor stimulation, resulted in the significantly enhanced phosphorylation of the p42/p44MAPK, which is downstream of imatinib target PDGFR, as early as 15 min and maintained constitutively activated after 24 h of imatinib treatment in both T98G and U87MG malignant glioma cells. Two correlated multi-immunoblot analysis indicated that treatment with imatinib further confirmed with each other the significant activation of the p42/p44MAPK in U87MG cells. Combina-tion of imatinib with PD098059 treatment significantly reduced imatinib resistance used alone at its lower concentrations. Results from real-time PCR indicated the altered expression of MKP-1 and MKP-3 at different time points related to the p42/p44MAPK phosphorylation. Dose-dependent growth inhibition was obtained in malignant glioma cell culture under the treatment of imatinib. However, the enhanced activation of p42/p44MAPK cascades upon application of imatinib in glioma culture may indicate the intrinsic complexity and evolution of glioma cells under RTK inhibitor treatment. Further experiments are highly warranted to investigate down-stream of PDGFR, the involvement of other cell signaling network under imatinib treatment in cell culture as well as animal experiments, with the goal to better understand the biological feature of the cell signaling trans-duction circuits in malignant glioma cells and highlight better strategies for targeted therapy against malignant glioma.



EPIDEMIOLOGY

EP-01. CNS HODGKIN’S LYMPHOMA: AN INTERNATIONAL PRIMARY CNS LYMPHOMA COLLABORATIVE GROUP (IPCG) REPORT OF 13 CASESElizabeth Gerstner,1 Lauren Abrey,2 David Schiff,3 Andrés Ferreri,4 Andrew Lister,5 Silva Montoto,5 Richard Tsang,6 Eckhard Thiel,7 Francesc Graus,8 Nancy Harris,1 and Tracy Batchelor1; 1Massachusetts General Hospital, Boston, MA, USA; 2Memorial Sloan-Kettering Cancer Center, NY, USA; 3University of Virginia, Charlottesville, VA, USA; 4San Raffaele H Scientific Institute, Italy; 5St. Bartholomew’s Hospital, London, United Kingdom; 6Princess Margaret Hospital, Toronto, Ontario, Canada; 7Charité-Campus Benjamin Franklin, Berlin, Germany; 8Hospital Clinic, Barcelona, Spain.

Hodgkin’s lymphoma (HL) involves the central nervous system (CNS) in 0.2%–0.5% of all cases. Most published reports consist of single cases. We retrospectively collected data on 13 patients with parenchymal CNS involvement of HL (CNS-HL) treated at 8 IPCG centers from 1972–2007. All 13 patients had classical HL: 6 classic NOS, 5 nodular sclerosis, and 2 mixed cellularity. Ten of 13 patients had histological confirmation of CNS-HL by biopsy, resection, or autopsy and 3 patients had neuroimaging findings consistent with secondary CNS-HL. Only 1 patient had primary CNS-HL. In 4 patients, CNS-HL was discovered simultaneously with systemic HL. In 8 patients, CNS-HL appeared an average of 37.1 months (4–189 months) after discovery of systemic HL. Three of these 8 were in “systemic” CR when CNS-HL was discovered. Patients presented with a variety of symptoms including weakness (4/13), altered mental status (3/13), seizure (3/13), and pain/sensory symptoms (3/13). Other symptoms included headache, cranial nerve palsy, tremor, memory loss, and fatigue. Lumbar puncture was performed in 8 patients. One patient had “atypical” cells, 5/8 had elevated protein and 2/8 had leptomeningeal tumor based on autopsy or MRI. Nine patients had solitary brain lesions involving the parietal lobe (2), frontal lobe (2), temporal lobe (2), frontal/temporal lobes (1), clivus/cavernous sinus (1), and cerebellum (1). Multifocal disease was found in 4 patients, 2 of whom had brain, spinal cord, and leptomeningeal disease. Four patients were treated with chemotherapy and radiation resulting in 2 PRs and 2 CRs; 4 received chemotherapy alone resulting in 1 CR, 2 PD, and 1 unknown response; 4 received radiation alone (3 WBRT, 1 stereotactic radiosurgery) resulting in 3 CRs and 1 PR; and 1 patient had an unknown treatment history. In addition, 3 patients were given intrathecal chemother-apy with methotrexate or cytarabine and 2 patients received PBSCT. Radio-graphic response to any type of treatment was observed in 9/11 patients. Six patients have died and the median overall survival (OS) is 65.11 months (12–235 months) after initial diagnosis of HL (either systemic or CNS) and 20.21 months (2.2–39.6 months) after diagnosis of CNS-HL. Four patients died from progressive HL, 1 patient died of an unrelated stroke in CR, and 1 patient died of a pulmonary embolism with evidence of systemic and CNS-HL at the time of autopsy. Of the 4 patients who were diagnosed with simultaneous CNS-HL and systemic HL, 1 has died of progressive HL. Of the 8 patients who were diagnosed with CNS-HL after previous diagnosis of systemic HL, 5 have died and the median OS is 75.1 months. CNS-HL is rare and our series of 13 patients is the largest one reported to date. CNS-HL can affect any part of the CNS and occurs either in the setting of widespread disease (5 patients had stage IV disease) or when disease is well controlled (3 patients were in systemic CR). Although there is no established standard of care for CNS-HL, 9/11 treated patients achieved a radiographic response and long-term survival is possible.

EP-02. TREATMENT PATTERNS AND PROGNOSIS IN PATIENTS WITH HUMAN IMMUNODEFICIENCY VIRUS (HIV) AND PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA (PCNSL)Teri Nguyen, Katherine Panageas, Elena Elkin, Lisa DeAngelis, and Lauren Abrey; Memorial Sloan-Kettering Cancer Center, New York, NY, USA

The incidence of HIV associated PCNSL has decreased in the era of highly active anti-retroviral therapy (HAART). As other complications of HIV infection have become less prevalent with improved immune reconsti-tution, non-Hodgkin lymphoma (NHL) has become a more common AIDS defining illness. Using SEER cancer registry data linked with Medicare claims, we identified PCNSL cases diagnosed from 1994–2002. Presence of HIV/AIDS was determined from a previously validated claims-based case finding algorithm, and from information on underlying cause of death. Treatment was defined as radiation therapy (RT) alone, chemotherapy (CTX) alone, combined CTX and RT, or no treatment based on Medicare claims in the six months following diagnosis of PCNSL. We assessed the effects of comorbidity, year of diagnosis, and sociodemographic character-istics on the odds of receiving treatment. We also calculated survival for the cohort by treatment, age, and year of PCNSL diagnosis. We identified

184 patients with both HIV and PCNSL, of whom 162 (88%) were age , 50 years, 173 (94%) were male, 108 (59%) were white, 71% (39%) were black, 158 (86%) were unmarried at the time of PCNSL diagnosis, and 178 (97%) lived in metropolitan areas. Eighty-five patients (46%) were treated with RT alone, 18 (10%) received RT and CTX, 8 (4%) received CTX alone, and 73 (40%) receive no treatment in the first 6 months following PCNSL diagnosis. We did not observe significant time trends in treatment patterns, and no sociodemographic factors were significantly associated with receipt of any treatment. Among those treated, receipt of CTX was significantly associated with female gender (OR 18.08, CI 2.1–158, p , .01), and white race (OR 3.39, 95% CI 1.09–10.54, p 5 .03). The overall median survival was 2 months (95% CI, 2–3). The median length of follow-up for survivors was 33 months. Receipt of any treatment and age ,50 years did not affect survival. In unadjusted analysis, there was a significant improvement in survival for patients diagnosed with PCNSL after 1996 (p 5 .028). In this population-based cohort of people with PCNSL and HIV, only 60% of patients received any treatment for their PCNSL. Despite improved treat-ment for both HIV and PCNSL over the past decade, survival for patients with both diseases remains dismal. Age is not a prognostic factor for this population as it is for immunocompetent patients with PCNSL. The use of HAART may have more impact on the course of disease than any other variable, including treatment with XRT and CTX.

EP-03. PATTERNS OF CARE AND OUTCOMES AMONG ELDERLY INDIVIDUALS WITH PRIMARY MALIGNANT ASTROCYTOMAJill Barnholtz-Sloan,1 Vonetta Williams,2 John Maldonado,2 Heather Stockwell,3 Marc Chamberlain,2 and Andrew Sloan1; 1Case Western Reserve University, Cleveland, OH, USA; 2H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, USA; 3University of South Florida, Tampa, FL, USA

To evaluate the association between age at diagnosis, patterns of care, and outcome among elderly individuals with an anaplastic astrocytoma (AA) and glioblastoma (GBM) 1,753 individuals with primary GBM and 205 individuals with primary AA 66 years and older (diagnosed 6/91–12/99) were identified from the Surveillance, Epidemiology and End Results (SEER) database and linked to Medicare information. All individuals had the same Medicare coverage for 6 months before and 12 months after diag-nosis to gather pre-diagnosis co-morbidity and post-diagnosis treatment information. Odds of undergoing various combinations of treatments and associations with outcome were calculated by tumor type and age and adjusted by various predictors. Age was not associated with treatment dif-ferences in individuals with AA. Very aged (751) individuals with GBM were more likely to have biopsy only (OR 5 2.53, 95% CI [1.78, 3.59]), surgery only (OR 5 1.47, 95% CI [1.15, 1.87]), or biopsy and radiation (OR 5 1.39, 95% CI [1.07, 1.82]) and less likely to receive multi-modality therapies. Regardless of age or histology, those treated with biopsy only had decreased survival. Individuals with GBM, who had surgery only or biopsy and radiation had worse prognosis compared to individuals treated with surgery and radiation. There were no differences in survival by histol-ogy. Very aged individuals with malignant astrocytomas were more likely to receive limited treatment (most pronounced in individuals with GBM). Survival variation correlated with treatment combinations. These findings suggest that age is associated with non-receipt of effective therapies and hence worse prognosis, in clinical neuro-oncology.

EP-04. SYMPTOM IMPROVEMENT IN PATIENTS TREATED FOR RECURRENT OR PROGRESSIVE LOW-GRADE GLIOMAErin Dunbar and Lawrence Kleinberg; Johns Hopkins University, Baltimore, MD, USA

Randomized studies demonstrate no difference in overall survival in low-grade glioma (LGG) patients treated with upfront versus delayed radiation therapy (RT) following initial surgery. At the time of recurrence/progression (R/P), the impact of RT and surgery (S) on symptoms is less clear and no consensus on management exists. This retrospective study was conducted to assess symptom control after treatment for R/P LGG. 647 patients treated for R/P LGG from 12/01/1996 through 12/31/2006 at Johns Hopkins University (JHU) underwent IRB-approved review. Inclu-sion criteria included LGG at R/P, age . 18, receipt of only RT or S at R/P, and all care at JHU. Indications for treatment and both 1 and 6 month patient reports of symptom severity, scored as “improvement,” “worsen-ing,” or “stability” were recorded. 108 patients met inclusion criteria. Aver-age age was 39 years and 55 were female (51%). The median follow-up was 3,290 days (range 1,130–6,731). Eighty-four patients (78%) received S and 13 (12%) received RT. Indications for treatment included symptom progression alone (54 [50%]), both symptom and radiographic progression ([35 [32%]) and radiographic progression alone (18 [(17%]). Common pro-



gressive symptoms included seizure in 70 (65%), focal neurologic deficit in 57 (53%), and headache in 15 (14%). First, those treated for symptomatic progression alone were evaluated for symptomatic improvement. Of the 52 patients treated with S, 32 (62%) reported improvement, 14 (27%) worsen-ing and 6 (12%) stability at 1 month and 14 (27%) improvement, 2 (4%) worsening, and 22 (42%) stability at 6 months. Of the 2 patients treated with RT, 2 (100%) reported improvement at 1 month and stability at 6 months. Second, those treated for both symptomatic and radiographic pro-gression were evaluated for symptomatic improvement. Of the 28 patients receiving S, 17 (61%) reported improvement, 5 (18%) worsening, and 4 (14%) stability at 1 month and 3 (11%) improvement, 1 (4%) worsening, and 12 (43%) stability at 6 months. Of the 7 patients receiving RT, 5 (71%) reported improvement, 1 (14%) and worsening at 1 month and 3 (43%) improvement and 2 (29%) stability at 6 months. Third, those treated for radiographic progression alone were evaluated for symptomatic improve-ment. Of the 4 patients receiving RT, 4 (100%) reported improvement at 1 month and 3 (75%) improvement and 1 (25%) stability at 6 months. Of the 14 patients receiving S, 1 (7%) reported improvement, 3 (21%) worsening, and 6 (43%) at 1 month and 3 (21%) improvement and 9 (64%) stability at 6 months. The most common indication for treatment for R/P LGG was symptom progression, usually seizures. There was a high chance of symp-tom improvement at both 1 and 6 months with either RT or S. There was a slightly higher chance of worsening within the 1st month and a slightly lower chance of improvement beyond the 1st month in those who received S. Our results support the current practice of delaying therapy until R/P. Prospective studies comparing the natural and iatrogenic symptoms of both R/P and treatment are needed.

EP-05. RACIAL AND SOCIOECONOMIC DISPARITIES IN ENROLLMENT IN CTEP-SPONSORED BRAIN TUMOR THERAPEUTIC TRIALSFred Barker and William Curry; Neurosurgical Service, Massachusetts General Hospital, Boston, MA, USA

Patients who enroll in clinical trials for cancer treatment represent a minority of persons potentially eligible for such trials. We used demo-graphic information on brain tumor clinical trial participants in compari-son with population controls to determine patient-related factors favor-ing trial enrollment. Demographic information on 13,539 participants in CTEP-sponsored brain tumor therapeutic trials, 1996–2005, compared to population-based controls from SEER, CBTRUS and Nationwide Inpatient Sample (NIS) databases. 57% of patients were adults; a wide variety of histologies were eligible for enrollment in 208 protocols. Of 3407 adult glioblastoma patients, 43% were treated at diagnosis (less than 3% of all adults diagnosed with GM in the United States during this time period), 50% entered trials at recurrence and 7% enrolled in trials open to both stages. 2.2% of all GM patients enrolled were black and 1.9% were His-panic. For GM patients treated at diagnosis, trial patients had median age 55 yr and 63% were male (SEER: median age 64, 58% male). Compar-ing GM trial participants (enrollment at diagnosis) to age-matched SEER controls, the odds ratio for participation for black patients compared to whites was 0.38 (95% CI 0.26–0.56, p , 0.001). For other glial histolo-gies, ORs for enrollment for black patients compared to whites (CBTRUS data) were 0.2 to 0.8. Similar disparities in enrollment were found for adult Hispanic glioma patients. Compared to NIS patients who underwent biopsy or resection of malignant primary brain tumors, in adults age less than 65 yr, rates of private insurance were similar between GM at-diagnosis trial patients (79%) and population controls (76%), but rates of Medicaid and self-pay were sharply lower in trial patients (Medicaid: 2.4% vs. 10.1%; self-pay: 2.5% vs. 4.8%). For medulloblastoma trials, ORs for participation were 0.81 for black patients and 0.56 for Hispanic patients (compared to CBTRUS data). For all trials, off-study reasons that threatened trial valid-ity (loss to follow-up, refusal of follow-up, toxicity/side effects, “other”) were no more common in black or Hispanic patients (5.9%, 6.1%) than in non-Hispanic whites (8.8%). These off-study reasons were recorded in 10.3% of private insurance patients, 5.3% of Medicaid patients and 4.7% of self-pay patients. Adults enrolled in CTEP-sponsored glioma trials were younger and more likely to be male than population controls. We found disproportionately low enrollment in trials by black and Hispanic patients and by those with Medicaid insurance or self-pay status.

EP-06. NOONAN SYNDROME ASSOCIATED WITH PRIMARY NEUROGLIAL TUMOR OF THE CENTRAL NERVOUS SYSTEMCourtney Sherman,1 Ignacio Gonzales,1 Afshan Ali-Nazir,2 Jonathan Finlay,3 and Girish Dhall4; 1Children’s Hospital Los Angeles, Los Angeles, CA, USA; 2The Children’s Cancer Alliance and Center for Blood Disorders, Fountain Valley, CA, USA; 3University of Southern California, Los Angeles, CA, USA; 4Pediatric Hematology-Oncology, Children’s Hospital Los Angeles, Los Angeles, CA, USA

Noonan Syndrome (NS) is an autosomal dominant condition with vari-able phenotypic expression and presence of dominant mutations in the pro-tein tyrosine phosphatase nonreceptor type 11 (PTPN11) gene in approxi-mately 50% of cases. We describe a case of a six year old male patient with NS and primary neuroglial tumor of the central nervous system (CNS). The patient was diagnosed with NS shortly after birth because in addition to fitting clinical criteria for NS, including facial dysmorphism, congenital heart defects, skeletal abnormalities and developmental delay, genetic anal-ysis revealed a heterozygous single base change of A to G in exon 3 of the PTPN11 gene. The patient presented with headaches, vomiting, episodes of dizziness and altered mental status after falling out of his bed in May 2006. A computed tomography (CT) scan of the head and magnetic resonance imaging (MRI) scan of the brain revealed an infiltrative enhancing mass involving the suprasellar cisterns, sella turcica and hypothalamus. Addi-tionally, diffuse leptomeningeal disease was present around the brainstem and cerebellum extending down to the spinal cord. In June 2006, a frame-less stereotactic biopsy of the suprasellar mass was performed. Histopatho-logical analysis revealed a hypocellular tumor whose cells were fairly uni-form with round to oval nuclei, finely granular chromatin and mild nuclear pleomorphism. The cytoplasm was found to form long tapering processes. Focal myxoid change was noted in the background with small microcyst formation. The tumor was irregularly partitioned by fibrovascular septae. Numerous blood vessels were observed; however, endothelial proliferation and glomeruloid formations were not present. Mitoses, Rosenthal fibers, necrosis and calcifications were absent. On immunohistochemistry, tumor cells were diffusely positive for glial markers such as glial fibrillary acidic protein (GFAP) and neuronal markers such as synaptophysin and protein gene product (PGP) whereas Ki-67 positivity was observed in approximately 2% of tumor cells. In conjunction with the imaging studies, this histopatho-logical analysis was most consistent with a diagnosis of leptomeningeally disseminated low-grade neuroglial tumor arising in the hypothalamic/chiasmatic region. A regimen of oral temozolomide (200mg/ m2/day) was initiated, prescribed daily for 5 days and repeated every four weeks. While associations between NS and cancers have been described, an association between NS and primary neuroglial tumors specifically has not been recog-nized. A review of the literature provides only two reported cases of tumors of glial origin in patients with NS. In addition, the patient had a germ line mutation in the PTPN11 gene, which is located on chromosome 12 (12q24) and encodes the non-receptor protein tyrosine phosphatase (PTP) SHP-2. This mutation is predicted to change the standard asparagine codon (AAC) to an aspartic acid codon (GAC) at amino acid position 58 of the protein (abbreviated Asn58Asp). Identification of this Asn58Asp mutation in the PTPN11 gene is consistent with the diagnosis of autosomal dominant NS. While this particular mutation has previously been described in NS, it has yet to be specifically associated with cancer pathology and thus, it may or may not be related to the primary neuroglial tumor present in our patient.

EP-07. NEUROLOGIC MANIFESTATIONS OF MAFFUCCI’S SYNDROME: CASE REPORT AND LITERATURE REVIEWTara Morrison1 and Gregory Vorona2; 1Neurology, Drexel University, Philadelphia, PA, USA; 2Drexel University, Philadelphia, PA, USA

Maffucci’s Syndrome is a rare condition characterized by multiple car-tilaginous masses and cutaneous hemangiomata. Only 14 previous cases of Maffucci’s Syndrome complicated by intracranial chondrosarcoma have been reported in the literature, with minimal description of the neurological presentation of these patients. We report a case of 52 year old female with a twenty-three year history of chondrosarcoma located anterior to the right temporal lobe. Her symptoms initially presented as “polyp” in her right ear and hearing loss, and she developed a right facial paralysis after her third surgery. With tumor progression the patient also developed worsening ambulation, increasing diplopia due to a right 6th nerve palsy, right tongue atrophy, and mild dysphagia. The patient ultimately received radiotherapy, with stabilization of tumor size and neurological symptoms at 31 months follow-up. Review of the literature found 14 cases of Maffucci’s Syndrome with skull base chondrosarcomas. The male: female ration was 4:10. The mean age at presentation was 34, range 5 18 to 52, median 5 32. The average lapse between initial symptom presentation and diagnosis of chon-drosarcoma was 33 months. The most common presenting symptom was diplopia, overwhelming due to a 6th nerve palsy. Headache and changes in facial sensation were also very common initial symptoms. In contrast, the most common presentation of relapse after therapy were changes in vision.



We report a woman with Maffucci’s Syndrome complicated by intracranial chondrosarcoma and compare her initial neurological symptoms to that of previous patients described by the literature. Our patient is unique in that she did not demonstrate any of the most common presentations (diplopia, changes in facial sensation, or headache) when she first was diagnosed. Additionally, our patient is the only recorded to develop progressive 12th nerve palsy (tongue wasting and deviation). These findings reiterate the importance of performing a comprehensive neurological examination and workup in patients presenting with rare medical disorders.

EP-08. GLIOBLASTOMA IN CALIFORNIA 2000–2004: A PRELIMINARY STATE-WIDE POPULATION-BASED DESCRIPTIONRudolph Schrot1 and Monica Brown2; 1University of California, Davis, Sacramento, CA, USA; 2Sutter General Hospital, CA, USA

Epidemiologic studies of primary brain tumors are limited by small sample sizes and sampling bias. The California Cancer Registry (CCR) provides a robust source of epidemiologic data within a populous and demographically diverse geographic area. We report here a preliminary epidemiologic description of glioblastoma multiforme for the entire state of California from 2000–2004. Data for invasive glioblastoma in California were obtained from the CCR and were analyzed by sex, age, race/ethnicity, urban/rural status by Rural Urban Commuting Area (RUCA) codes, and socioeconomic status (n 5 4,455). Incidence rates were age-adjusted to the 2000 U.S. population. Denominator data for rates were derived from the California Department of Finance population estimates. The age-adjusted incidence rate (AAIR) of glioblastoma across all groups was 3.1 cases per 100,000. Our preliminary analyses showed more cases were male (58.8%) with AAIR by sex of 3.9 for males and 2.3 for females per 100,000. Peak age range for diagnosis was 50–79 years old, with a median age of 63. Age-specific incidence rates revealed a sharp peak in the 7th decade (15.6 cases per 100,000) which was fairly consistent across racial and gender subcat-egories, although differing in magnitude. The vast majority of cases were non-Hispanic white (75.5%); followed by Hispanic at 15.6% and Asian/Pacific Islander comprising 5.3% of cases. The AAIR was highest for male non-Hispanic whites (4.8) and lowest for female Asian/Pacific Islanders (1.0). Nearly all cases were from patients residing in urban areas of Cali-fornia (92.4%) at time of diagnosis, regardless of race/ethnicity (91.1% of non-Hispanic white, and 95.6% of Hispanic cases). About half of the cases were from higher SES level patients(51.1%); this same pattern was seen for non-Hispanic white and Asian/Pacific Islander cases (56.8% and 55.7%, respectively), but not for Hispanic and African American cases (32.5% and 26.1%, respectively). Discussion: The incidence of glioblastoma in Califor-nia shows unique and distinct distribution patterns. Cases were associated with being male, non-Hispanic white, older, and residing in an urban area. We have reported here the first statistical description of glioblastoma in California using the CCR. By leveraging the diversity and high popula-tion of California, the CCR allows for meaningful statistical description of glioblastoma incidence among diverse demographic groups. We anticipate a more comprehensive description of primary central nervous system tumors using the CCR.

EP-09. INCIDENCE, RADIOGRAPHIC, AND CLINICOPATHOLOGICAL FEATURES OF ATYPICAL MENINGIOMA: A 7–YEAR EXPERIENCESamuel Crosby,1 Michael Edgeworth,2 Kathleen Egan,3 and Reid Thompson4; 1Vanderbilt University School of Medicine, Nashville, TN, USA; 2Department of Neurology, Vanderbilt University Medical Center, Nashville, TN, USA; 3H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA; 4Department of Neurosurgery, Vanderbilt University Medical Center, Nashville, TN, USA

Atypical meningiomas (WHO II) comprise about 5%–10% of all men-ingiomas, have a worse prognosis and require a more aggressive manage-ment approach than typical meningiomas (WHO I). The histopathologi-cal criteria that designate meningiomas as atypical vary greatly and are complicated. In this study, we examined the presentation, demographics, histopathological characteristics, radiographic characteristics, and treat-ment courses of all atypical meningiomas diagnosed over a 7-year period at a large referral center in Tennessee. We retrospectively examined data from the medical records of all patients pathologically diagnosed with menin-gioma at Vanderbilt University Medical Center between 2000 and 2006. A total of 196 meningiomas were identified, of which 33 were atypical (17%). Of these patients, 22 were female (67%) and 11 were male (33%); 29 were Caucasian (87.9%), 3 African American (9%), and 1 Hispanic (3%). The mean age was 51.4 years (range: 20 years–80 years); 10 of these patients (30%) had a history of cancer other than meningioma (medulloblastoma [2], neurofibroma, ALL, pineal region tumor, lymphoma, retinoblastoma,

prostate, melanoma, and breast) with 5 receiving prior cranial irradiation. One patient had neurofibromatosis type 2 and another had retinoblastoma. Common symptoms at presentation were focal deficits in 22 patients (67%), headaches in 14 (42%), seizures in 10 (30%), and hydrocephalus in 1 (3%). Histologically, the average MIB-labeling rate of those reported was 13.9 68.5% (n 5 11) with 6 others described as “brisk.” The average mitotic rate reported was 3.8 62.0 per 10 hpf (n 5 13). On examination of the pathology report, they were characterized by a small cell component in 17 (52%), hypercellularity in 13 (39%), sheeting in 13 (39%), brain inva-sion in 9 (27%), EMA staining in 8 (24%), prominent nucleoli in 6 (18%), positive vimentin stain in 4 (12%), and chorioid features in 3 (9%). Radio-graphically, 26 (78%) were solitary and 7 (21%) were multifocal. Other radiographic features included mass effect on ventricle size in 20 (61%), heterogeneous enhancement in 15 (45%), edema in 15 (45%), midline shift in 12 (36%), and radiologic necrosis in 5 (15%). Treatment involved surgery with gross total resection in 28 (85%) or subtotal resection in 5 (15%). Adjuvant treatments included radiotherapy (n 5 7), embolization (n 5 5), and hydroxyurea (n 5 4). In our experience the prevalence of atypical meningioma at VUMC was slightly higher (17%) than what is typically reported in the literature (5%–10%). Of the atypical meningioma cases, two-thirds were women, consistent with the predilection of this disease in females. Approximately one third had a prior history of cancer (30%), half of which had received previous ionizing radiation to the head, both known risk factors for meningioma. The most common histological characteristics reported were small cell component, hypercellularity, sheeting, and brain invasion, all features known to be less common in benign meningiomas (WHO I). Most common radiographic features reported were mass effect on ventricle size, heterogeneous enhancement, edema, and midline shift, all of which suggest a larger and more aggressive tumor. A planned multi-center case-control study will investigate risk factors for these tumors and other histological subtypes of meningioma. Supported in part by grant M01 RR-00095 from the National Center for Research Resources, National Institutes of Health.

EP-10. CREATION OF A RETROSPECTIVE SEARCHABLE NEUROPATHOLOGIC DATABASE FROM PRINT ARCHIVES AT TORONTO’S UNIVERSITY HEALTH NETWORKSidney Croul,1 Sepehr Ehsani,2 Andrea Bernstein,3 Donald Winter,2 Fred Gentili,2 Sylvia Asa,4 and Tim-Rasmus Kiehl2; 1Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; 2Toronto, Ontario, Canada; 3Halifax, Nova Scotia, Canada; 4University of Toronto, Toronto, Ontario, Canada

University Health Network (UHN), in its capacity in providing neuro-oncologic care, now utilizes a laboratory information system (LIS) which was instituted in September 2001. For the 75 years preceding the LIS, more than 50,000 reports exist in paper format. High-throughput automated scanning of the paper archives was employed to add the most recent 30 years of paper records (30,000 neuropathology specimens) to the LIS. The searchable Portable Document Format (PDF) files generated from the scans were filtered through a multi-tiered process driven by Java computer pro-grams that selected relevant patient and diagnostic information. A second series of programs queried the neuropathologist-assigned diagnoses and successfully converted these to the standardized WHO format. This was achieved with a master list of key site and diagnostic terms, and prioritiza-tion rules that were determined on a trial and error basis. Categorization, verification, and consolidation were completed within three months and on a small budget.

EP-11. POLYMORPHISMS IN IL4R GENE RELATED TO SURVIVAL IN GLIOMA PATIENTSMichael Scheurer, E. Amirian, Yumei Cao, Randa El-Zein, Victor A. Levin, and Melissa Bondy; University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Although malignant gliomas are the most common type of primary brain tumor in adults, there is a lack of definitive information in the epidemi-ologic literature about the prognostic factors that influence patient survival. Median survival time for glioblastoma, the most malignant glial neoplasm, is approximately one year, and 90% of patients die within three years after diagnosis. Previous literature and preliminary analyses in our group pro-vide some evidence that atopic diseases, IgE levels, and inflammatory gene polymorphisms may be associated with risk of developing glioblastomas. Few studies, however, examined these as prognostic factors in gliomas as a group. We, therefore, designed the current study to investigate the affects of certain inflammatory gene single nucleotide polymorphisms (SNPs) on survival by treatment and histology (high grade vs. anaplastic/low grade). Using a population of newly-diagnosed adult glioma cases (n 5 694) identi-fied between 2001 and 2006 from Harris County, Texas, we examined



SNPs of nine interleukin genes. Preliminary analyses were performed using Kaplan-Meier curves to examine differences in survival by genotype for each gene. The interleukin-4 receptor (IL4R) gene was chosen for further analysis by Cox proportional hazards regression. The association between five IL4R SNPs and mortality hazard was examined controlling for age at diagnosis, sex, extent of surgical resection, and chemotherapy. Interaction terms were used to explore possible effect modification between radiation therapy and the IL4R SNPs. Among high-grade glioma cases, rs1805016 (TT vs. GT/GG) was significantly protective against mortality (HR: 0.59; 95% CI: 0.40–0.88). Furthermore, significant interactions were found between radiation therapy and rs1805011, rs1805015, and rs1805012, indicating that the effect of radiation therapy may differ depending on the patient’s genotype. In the presence of radiation therapy, these SNPs appear to confer increased hazard; whereas the SNPs seem to have a protective effect when no radiation treatment is given. These findings indicate that polymorphisms in inflammation pathways may play an important role in overall survival of glioma patients. In addition, a patient’s genotype may be of importance when considering glioma treatment options. Further research on the polymorphisms of the IL4R gene and their effects on the prognosis of glioma patients is warranted.

EP-12. WORLD-WIDE INCIDENCE OF PRIMARY MALIGNANT AND NON-MALIGNANT BRAIN TUMORSCarol Kruchko,1 Jennifer Propp,2 Kate Schellinger,2 and Bridget McCarthy2; 1CBTRUS, Hinsdale, IL, USA; 2Division of Epidemiology, University of Illinois at Chicago, Chicago, IL, USA

In order to contribute to the general knowledge of the international scope of brain tumors, CBTRUS has assessed the incidence, prevalence, and mortality of malignant primary brain tumors and the incidence of non-malignant primary brain tumors in the world. Brain and other nervous system tumors (International Classification of Disease, 10th revision codes C70–C72) were the neoplasms of interest. GLOBOCAN 2002 software (made available by the World Health Organization’s International Agency for Research on Cancer) was utilized to compute incidence, mortality, and prevalence statistics for malignant tumors. CBTRUS developed statisti-cal models based on country level or geographical region tumor registry statistics and population demographics to calculate non-malignant tumor incidence. Rates were calculated per 100,000 and age-standardized to the World Standard. It is estimated that the incidence of primary malignant brain tumors is 3.7 per 100,000 per annum for males and 2.6 per 100,000 per annum for females in 2002. This represents an estimated 108,277 males and 81,305 females who were diagnosed with a primary malignant brain tumor in 2002, an overall total of 189,582 individuals. Rates differed among geographic regions and between more and less developed countries. CBTRUS has calculated a preliminary estimate of 157,833 newly diagnosed non-malignant brain tumors per annum for 2002 (males: n 5 58,851; females: n 5 98,982). The preliminary overall rate for non-malignant brain tumors was estimated to be 2.6 per 100,000 (males: 2.0 per 100,000; females: 3.1 per 100,000). World-wide mortality rates for primary malig-nant brain tumors in 2002 are estimated to be 2.8 in males (n 5 80,084) and 2.0 in females (n 5 61,639), with variation by geographic region. The partial prevalence (1-, 3-, and 5-year prevalent cases) of primary malignant brain tumors among males was 50,736, 113,995, and 157,689, respectively; among females it was 37,726, 85,366, and 118,875, respectively. The partial prevalence (1-, 3-, and 5-year prevalent cases) combines the annual num-ber of new cases and the corresponding probability of survival by time. Non-malignant brain tumors are often as devastating as malignant brain tumors but are not routinely collected by cancer surveillance organizations world-wide, making it difficult to obtain an accurate estimate of incidence. Even statistics for malignant tumors may be conservative. Not all countries have well developed cancer registries in which accurate statistics are kept, and the incidence and prevalence in many less developed countries could be underestimated because of limited access to diagnosis and care. On the other hand, differences in incidence and prevalence could be due to differ-ences among population characteristics, including age distributions, racial/ethnic differences or differences in exposures to risk factors, in different geographical regions. The misclassification of some metastatic cancers as primary cancers may also influence the reliability of the mortality statis-tics. The potential reasons for the differences in incidence, prevalence, and mortality around the world are manyfold and will be further elucidated and explored.

EP-13. REGIONAL DIFFERENCES IN HIGH-GRADE GLIOMA IN CANADAJoseph Megyesi1 and Cyril DeSilva2; 1Clinical Neurological Sciences (Neurosurgery), Society for Neuro-Oncology, London, Ontario, Canada; 2Clinical Neurological Sciences (Neurosurgery), University of Western Ontario, London, Ontario, Canada

Canada is diverse with regional differences in ethnicity, diet and envi-ronment. We investigated geographical variation in high grade glioma between St. John’s, Newfoundland (STJ) and London, Ontario (LON). STJ is in a relatively pristine, maratime environment while LON is more industrialized and located in the heartland. Patients treated for high grade glioma (HGG) at LON and STJ between 1998 and 2004 were identified from office charts. Charts were reviewed for patient demographics and pre-senting symptoms and data analyzed using the chi-square test. The chart review produced 137 patients: 73 from LON and 64 from STJ. The LON patients consisted of 46 males and 27 males with a mean age of 63.1 years. The STJ patients consisted of 43 males and 21 females with a mean age of 59.3 years. There was no significant difference in mean age. However, sig-nificantly more patients were under 60 in STJ compared to LON (p 5 0.02). In addition chi-square analysis revealed STJ and LON patients presented differently (p 5 0.03). The former tended to present with headache, symp-toms of increased intracranial pressure and seizures while the latter tended to have focal neurological deficits. It appears that there are regional differ-ences in the clinical features of HGG. Additional work should investigate regional differences with the aim of identifying factors responsible.

EP-14. AN EPIDEMIOLOGICAL STUDY OF BRAIN CANCER MORTALITY AND INCIDENCE AMONG A COHORT OF JET ENGINE MANUFACTURING WORKERS: YEAR 5 STUDY UPDATEGary Marsh,1 Jeanine Buchanich,1 Frank Lieberman,2 Ada Youk,1 Zb Bornemann,1 Nurtan Esmen,3 Steven Lacey,3 Kathleen Kennedy,3 and Roger Hancock3; 1Department of Biostatistics, University of Pittsburgh, Pittsburgh, PA, USA; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 3University of Illinois at Chicago, Chicago, IL, USA

In 2002, the University of Pittsburgh undertook a 7-year exploratory historical cohort study and nested case-control study to investigate a sus-pected cluster of malignant brain cancer at a jet engine manufacturing plant in North Haven, CT. A preliminary comparative cancer incidence analy-sis conducted by the Connecticut Department of Health was inconclusive. Our cohort includes more than 235,000 former and current employees with work experience since 1952 in one or more eight manufacturing facilities in the Hartford, CT area. The total and cause-specific mortality experience of the cohort will be examined from 1952 to 2004 and compared with the standard populations of the U.S., State of Connecticut, and the Connecticut regional county area. Also, the benign and malignant brain cancer experi-ence of the cohort will be evaluated from 1976 to 2004 and compared to national and regional brain cancer incidence rates. Each identified case of benign and malignant brain cancer will be matched on age, sex and year of birth to a cohort member without brain cancer at the diagnosis date of the case. The case-control study will enable the collection of data on potential risk factors for brain cancer and co-exposures unavailable from existing record sources, and if the participation rate is adequate, will afford a com-prehensive and focused evaluation of brain cancer occurrence in relation to demographic, work history and occupational exposures while controlling for potential confounding factors. Case-control data collection includes a structured telephone interview and the acquisition of medical records and tissue specimens from cases of malignant glioblastoma multiforme (GBM). We plan to use microdissection-based genotyping of the tumor specimens obtained with the appropriate consent from subjects to determine if these tumors have similar or different mutational profiles than sporadic high grade GBMs. The target number for our pilot genetic study is 20 subjects who were younger than 70 years old at time of diagnosis. Our investiga-tion is complemented by a companion exposure assessment project at the University of Illinois at Chicago that will characterize the historical work practices and exposures that occurred in each study plant. We will use the work history and exposure data in our cohort and case-control studies to examine the relationship between brain cancer mortality and incidence and the past working environment of the plants. Our study is the largest and most comprehensive study of its kind ever conducted to investigate envi-ronmental agents possibly causally related to development of benign and malignant brain cancers. This poster will present and discuss study progress and plans as of the end of the fifth project year.



EXPERIMENTAL THERAPEUTICS

ET-01. MOLECULAR IMAGING AND THERAPY PLATFORM: PRE-SELECTING RESPONDING TUMORS TO ANTIANGIOGENIC THERAPYSamira Guccione,1 Yi-Shan Yang,1 and Steven Choi2; 1Stanford University, Stanford, CA, USA; 2CA, USA

The ability to screen patient responders to a chemotherapeutic agent prior to administration of a drug is of significant value. We have developed an integrin-targeted drug delivery system that can target tumor vessels that have upregulated expression of the integrin avb3. This platform delivery system can also be used for imaging in many clinical imaging modalities including MRI, PET, and SPECT. We have used this platform to image and deliver genes to the tumor vessels in the rat GBM tumor model. Here we will present the correlation between screening responding tumors using MRI prior to treatment. The difference in therapeutic efficacy will be described in these tumor models. Glioblastoma model U87, was used in nude mice to evaluate temporal changes in T1- and T2-weighted images in MRI using integrin-targeted nanoparticles containing chelated Gadolinium as T1 MRI contrast agent. The treatment group received intravenous injections of integrin-targeted nanoparticles containing a mutated Raf gene that lead to extensive apoptosis of the tumor vessels. Control animals were shame treated with saline. Pixel intensities were measured and positively correlated to therapeutic efficacy. GBM and their tumor models are highly vascular neoplasms that likely respond well to the antiangiogenic therapy like the one we have developed here. Tumors were significantly reduced in size (.95%) and did not recur, even 6 months after treatment completion. Contrary to the GBM model, the SCCVII tumor (model for head and neck cancer) is not very vascular and did not respond well to this anti-angiogenic treatment. molecular imaging (integrin-targeted imaging) using this platform agent as an MRI contrast agent was predictive of therapeutic efficacy. Here we introduce a novel anti-angiogenic agent that can also be used as a molecu-lar imaging probe to predict responding tumors from those that will not respond.

ET-02. FK506 BINDING PROTEIN (FKBP5) MEDIATES GLIOMA CELL GROWTH AND SENSITIVITY TO RAPAMYCIN TREATMENT THROUGH REGULATING AKT AND NF-KB SIGNAL TRANSDUCTIONWei Jiang,1 Simona Cazacu,1 Cunli Xiang,1 Jean Zenklusen,2 Howard Fine,2 Michael Berebs,3 Brock Armstrong,4 Chaya Brodie,1 and Tom Mikkelsen1; 1Henry Ford Hospital, Detroit, MI, USA; 2National Institutes of Health, Bethesda, MD, USA; 3TGen, Phoenix, AZ, USA

FK506 binding protein 5 (FKBP5 or FKBP51), an immunophilin, belongs to the family of peptidyl prolyl cis/trans isomerases, functions together with chaperones to help protein folding. Using glioma cDNA microarray analysis, we found that FKBP5 was overexpressed in glioma tumors and correlated with overall survival. This finding was further validated by real-time RT-PCR and Western blot analysis. We then employed both RNAi to knock-down FKBP5 expression and cDNA transfection to overexpress FKBP5 to examine the roles of FKBP5 in glioma cells. Cell growth analysis showed that cell proliferation was suppressed after FKBP5 expression was inhibited for 5 days but was enhanced by FKBP5 overexpression. In addi-tion, rapamycin-resistant glioma cells, both PTEN positive and negative, were synergistically sensitive to rapamycin after FKBP5 was knocked down, but the response of glioma cell to rapamycin treatment was suppressed when FKBP5 was overexpressed in glioma cells, suggesting that FKBP5 mediated glioma cell response to mTOR inihibitor treatment and that the effect of FKBP5 on the cell response to mTOR inhibitor was PTEN-independent. To verify the function of FKBP5 in glioma cell signaling, we employed West-ern blot to analyze the phosphorylation of IkappaBalpha, NF-kappaB and AKT, and EMSA to study the DNA binding ability of NF-kB after FKBP5 expression was regulated by siRNA transfection or overexpression. Western blot analysis showed the expression level of phosphorylated NF-kB and AKT was regulated by FKBP5, and that the expression of IkappaBalpha was increased in FKBP5-depleted cells and decreased in FKBP5-overex-pressed cells. Moreover, EMSA data showed that overexpression of FKBP5 stimulated DNA:NF-kB binding ability. These results together suggest that FKBP5 involves in AKT and NF-kB pathway activation in glioma cells. In conclusion, our study demonstrates that FKBP5 plays an important role in glioma growth and chemoresistance via regulating signal transduction of AKT and NF-kB pathway.

ET-03. PHENYLACETYLGLUTAMINE (PG) AND PHENYLACETATE (PN) INTERACT ADDITIVELY TO PRODUCE DETACHMENT-INDUCED APOPTOSIS/ANOIKIS IN GLIOBLASTOMA CELLSSonali Patil, S.R. Burzynski, Emilia Mrowczynski, and Kzystof Grela; Burzynski Research Institute, Stafford, TX, USA

Phenylacetylglutamine (PG) and phenylacetate (PN) are major compo-nents of antineoplastons A-10 and AS2-1. These formulations are currently in advanced clinical trials for the treatment of primary brain tumors. Phe-nylacetate has been examined independently in the past by other research-ers as a potential anti-tumor agent in glioblastoma, medulloblastoma and hematological, breast, pancreatic, prostate, and thyroid malignancies. We have investigated the anti-proliferative effects of PG and PN used separately and in combination in U87 human glioblastoma cells. Though this is the cell line we have focused our attention on, we also have briefly examined the effects of these agents on BT20, DAOY, and U373 cells. Our data on anti-proliferative effect of PG and PN show that the two drugs have a constant relative potency, (R) with PN being 5 times more potent than PG. This feature has allowed us to study the effect of fixed ratio combinations of the two drugs in isobolographic analysis. We present here evidence to show that PG and PN when used in combination are additive at lower doses. These combinatorial doses are in the range of therapeutic significance. In median effect analysis, the Combination Index, CI is . 1. We have observed a dose dependant induction of detachment in cells treated with PG or PN. We have been able to demonstrate that PG causes anoikis or detachment-induced apoptosis in glioblastoma cells using TUNEL as well as Annexin V staining. Our studies indicate that PG enters the U87 cells via glutamine channels and also significantly inhibits the uptake of glutamine by cells. This may be an important feature of its mechanism of action since cancer cells are highly dependant on glutamine. Deprivation of intracellular glutamine could be a key factor in growth inhibition caused by PG. Investigation of the cell cycle by propidium iodide staining and fluocytometry shows an increased number of cells in the sub-diploid stage upon treatment with PG and/or PN, indica-tive of apoptosis. However there does not appear to be any effect on the cell cycle itself. We have conducted a total human gene array screen using the Affymetrix Human Genome plus 2.0 oligonucleotide arrays, for genes regulated by PG and a combination of PG and PN. The gene TXNIP was up-regulated almost 5-fold with PG, and over 120-fold with a combination of PG and PN. Other interesting genes that are significantly up-regulated are CLDND1, ATF3, CASP5, TP53, TRIB3, and UNC5B. Genes that were down-regulated include AKT2, ASPM, and CDCA8. Based on the results of this study we are currently investigating the possible effect of these antine-oplastons on the redox system involving thioredoxin, cell division (borea-lin, ASPM), apoptosis (caspase 5, p53, netrin receptor), and AKT pathway (AKT2, TRB3).

ET-04. IN VITRO EFFECTS OF CETUXIMAB ON EGFR AND DOWNSTREAM MEDIATORSBenedikte Hasselbalch,1 Marie T. Stockhausen,1 Ulrik Lassen,2 and Hans Skovgaard Poulsen1; 1Radiation Biology, Rigshospitalet, Copenhagen, Denmark; 2Department of Oncology, Rigshospitalet, Copenhagen, Denmark

Treatment outcome of Glioblastoma multiforme (GBM) remains poor despite multimodal therapies. Current standard care for newly diagnosed GBM is surgical resection, followed by radiotherapy plus concomitant and adjuvant chemotherapy with temozolomide. Median survival receiving this therapy is 14,6 month. Between 30%–60% of these patients will experi-ence relapse within 6 month of standard therapy. In case of early relapse, treatment options are limited and systematic trials investigating tumor response to new drugs and the mechanisms involved have to be performed. Primary GBMs, are frequently associated with amplification and/or muta-tions of the epidermal growth factor receptor (EGFR). EGFR is involved in regulation of cell proliferation, growth, survival and motility. Two of the pathways downstream of EGFR are the Ras/Raf/ERK and PI3K/AKT path-ways. Inactivating mutations of the tumor suppressor gene PTEN occurs in approximately 15%–60% of all GBM and contributes to an abnormally high activity of the PI3K/AKT pathway. Aim: Investigating the effects of cetuximab on human glioblastoma cell lines: U87MG, U87MGvIII (express the EGFR type III deletion mutant, EGFRvIII), U118MG, U373MG, and SKMG3 in comparison with the human head and neck cell line, HN5. Inhibition of EGFR signaling in vitro with increasing concentrations of cetuximab (Erbitux) with or without the addition of EGF showed moder-ate effects on GBM cell viability. In comparison, HN5 with known EGFR amplification and wild type PTEN is sensitive to increasing concentrations of cetuximab. However, in the presence of EGF, almost no effect on HN5 cell viability was observed. Western blotting experiments showed that EGF is capable of inducing phosphorylation of EGFR in the U87MG, U87vII-IMG and SKMG3 cell lines, all of which have mutated PTEN and that this could be inhibited by increasing concentrations of cetuximab. Both ERK and AKT were phosphorylated in the presence of EGF, however limited or



no effect was observed from treatment with cetuximab. The Same results were observed for HN5. Furthermore Western blotting experiments showed that low basal phosphorylation of EGFR, ERK and AKT was present in the glioblastoma cell lines in the absence of EGF, and these phosphoryla-tions were insensitive to cetuximab. In contrast, HN5 showed decreased phosphorylation of EGFR as well as abolished phosphorylation of ERK and AKT with increasing concentrations of cetuximab. Discussion: These results suggest that EGFR inhibition of the glioblastoma cell lines U87MG, U87vIII, SKMG3 by cetuximab does not affect cell viability even when phosphorylation of EGFR is inhibited. This might be explained by the sustained activity of the downstream mediators AKT and ERK. Further-more our results indicate that in the presence of EGF, HN5 cells maintain their viability through the Ras/Raf/ERK and PI3K/AKT pathways even at high concentrations of cetuximab. This might be due to competition between EGF and cetuximab, which also could explain the lack of effect of cetuximab on cell viability. Our work demonstrates that high expression of EGFR is not predictive for response to cetuximab and PTEN mutation and/or inactivation could explain the resistance to cetuximab. These results emphasize the importance for individualized patient therapy and combined anticancer treatment.

ET-05. THE “YING AND YANG” OF MODULATING ENDOPLASMIC RETICULUM STRESS IN GLIOMA THERAPYThomas Chen1; 1University of Southern California, Los Angeles, CA, USA

For the past two years, our group has examined whether modulation of endoplasmic reticulum stress (ERS) may be useful in the treatment of malig-nant gliomas. ERS is induced when a tumor cell is exposed to external stress such as hypoxia, chemotherapy, or radiation therapy. One cellular protec-tive mechanism is induction of GRP78, an ER molecular chaperone and key regulator of the unfolded protein response (UPR). GRP78 is expressed at low levels in normal adult brain, but is significantly elevated in malignant glioma tissue specimens and human malignant glioma cell lines. Glioblas-toma cell lines treated with siRNA to GRP78 (siGRP78) have significantly lowered resistance to temozolomide (TMZ), as established by colony survival assays. Glioma cell lines treated with (-)-epigallocatechin gallate (EGCG), a major component of green tea which can bind to intracellular GRP78, also had increased chemosensitivity to TMZ. Conversely, over-expression of GRP78 via gene transfer, confers resistance to TMZ induced cell death. These results identify a novel chemoresistance mechanism in malignant gliomas, and demonstrate that GRP78, in addition to being a potential prognostic marker for malignant brain tumors, is a novel chemo-sensitizing target. Besides inhibition of ERS chaperones such as GRP78 to increase glioma chemosensitivity, we have also recently demonstrated that small molecules such as dimethyl-celecoxib (DMC) and protease inhibitors (ie nelfinavir, atazanavir) induce ERS to the extent that the glioma cell is no longer to able to mount a sufficient protective response, resulting in apop-tosis. We have demonstrated that DMC, nelfinavir, and atazanavir decrease cell culture viability and survival in glioblastoma cell lines. We also demon-strate that these drugs induce a variety of ERS markers, including GRP78, CHOP, and Caspase 4. Transfection with siGRP78 further sensitizes glioma cells to killing by DMC, nelfinavir, and atazanavir, while inhibition of cas-pase 4 prevents drug-induced apoptosis. Treatment with nelfinavir leads to aggresome formation and accumulation of polyubiquitinated proteins, demonstrating proteasome inhibition. These effects can be demonstrated in vivo, as glioma xenografts treated with all three drugs demonstrate a robust induction of the proapoptotic marker CHOP. Our data demonstrates that modulation of ERS, by either preventing glioma cells from mounting a nor-mal protective response (i.e., via GRP78), or inducing so much ERS, that it overcomes the normal protective UPR, is a novel mechanism for induction of chemosensitization in malignant gliomas.

ET-06. BEVACIZUMAB AND/OR CARBOPLATIN IN A NOVEL HUMAN NUDE RAT GLIOMA MODEL: EFFICACY AND DYNAMIC MAGNETIC RESONANCE IMAGINGKristoph Jahnke,1 Leslie Muldoon,2 Csanad Varallyay,2 Seth Lewin,2 and Edward Neuwelt2; 1Neurology and Medicine, Oregon Health and Science University, Portland, OR, USA; 2Neurology, Oregon Health and Science University, Portland, OR, USA

The purpose of this study was to evaluate the efficacy of bevacizumab and/or carboplatin in a nude rat human glioma xenograft model and the impact of this regimen on dynamic magnetic resonance imaging (MRI). The care and use of animals was approved by the institutional Animal Care and Use Committee and were supervised by the Oregon Health and Science Uni-versity (OHSU), Department of Animal Care. Male athymic nude rats (rnu/rnu) received intracerebral injection of UW28 human glioma cells. MRI scans to confirm tumor were done on the day of treatment (day 7–10 after

implantation) and one week later. Rats were randomized to four groups: (1) untreated controls (n 5 9); (2) bevacizumab 10 mg/kg intravenous (n 5 6); (3) carboplatin 200 mg/ m2 intravenous (n 5 6); and (4) bevacizumab plus carboplatin (n 5 6). Dynamic MRI for the evaluation of cerebral blood volume and blood-brain barrier permeability was done before and 24 hours after treatment with bevacizumab (n 5 3) and compared to controls (n 5 3). Rats were followed for survival. Tumor growth was found in 90% of inoculated animals, with tumors mainly located in the caudate nucleus and cortex. Median overall survival was: (1) controls, 16 days (range, 10–22); (2) bevacizumab, 23 days (17–30); (3) carboplatin, 22 days (19–24); (4) bevacizumab plus carboplatin, 36 days (31–39). Dynamic MRI showed that cerebral blood volume was reduced by bevacizumab treatment. Post-gadolinium T1 weighted MRI signal intensity was less pronounced after treatment with bevacizumab as compared to the baseline scan, suggesting tumor permeability was reduced. The UW28 glioma model yielded consis-tent and reproducible results. Carboplatin plus bevacizumab was effective and seemed superior to bevacizumab or carboplatin monotherapy, suggest-ing that besides bevacizumab effects on tumor vasculature, actual tumor cell death contributes to the efficacy of this regimen. The promising survival data warrant future clinical trials using bevacizumab plus carboplatin in glioblastoma.

ET-07. CONVECTION-ENHANCED DELIVERY (CED) OF LIPOSOMAL CPT-11 FOR IMAGE-GUIDED BRAIN TUMOR TREATMENT IN CANINE SPONTANEOUS GLIOMASPeter Dickinson,1 Richard LeCouteur,1 Robert Higgins,1 John Bringas,2 John Park,3 Charles Noble,4 Tracy McKnight,3 Mitchel S. Berger,3 and Krystof Bankiewicz2; 1University of California, Davis, Davis, CA, USA; 2Neurosurgery, Brain Tumor Research Center, University of California, San Francisco, CA, USA; 3University of California, San Francisco, San Francisco, CA, USA; 4Hermes Biosciences Inc., CA, USA

We hypothesized that newly developed nanoparticle agents can be used to treat brain tumors, via systemic administration or CED with MRI-guid-ance. For systemic treatment, nanoliposomal CPT-11 is a novel liposome-based nanoparticle featuring highly stable drug encapsulation in a long circulating carrier. In the rat intracranial U87 tumor xenograft model, i.v. treatment with nanoliposomal CPT-11 produced 13-fold higher drug expo-sure in tumors based on tissue AUC than free CPT-11. Systemic treatment with nanoliposomal CPT-11 resulted in significantly improved survival, including apparent cures in some animals, as compared with free CPT-11. Based on these results, nanoliposomal CPT-11 is proceeding to Phase I clinical testing in advanced brain tumor patients. For CED, prior stud-ies in rodents, dogs and primates established the feasibility of MRI-based monitoring of CED of liposomal Gd. To evaluate this as an image-guided brain tumor treatment, we investigated the delivery, safety and efficacy of liposome-based nanoparticles containing gadolinium and CPT-11 follow-ing CED into spontaneous canine gliomas. In this veterinary trial, 4 canine patients with biopsy confirmed gliomas (1 astrocytoma III, 2 astrocytomas II, 1 oligodendroglioma) were infused with liposomes containing gadoteri-dol (1.85 mM) and CPT-11 (48.2 mg/ml). CED was monitored in real time by sequential MRI to assess localization and volume of distribution. Suc-cessful intratumoral delivery of nanoliposomal CPT-11 was demonstrated in all animals by MRI, although significant variability was observed and appeared related to catheter placement, rate of infusion, presence of necrosis and location in relation to ventricular and subarachnoid spaces. Infusions of up to 777 ml were feasible over 3.75 h using 1–3 catheters and infusion rates up to 4 ml/min. A majority of infusions were limited by eventual leakage into ventricular or subarachnoid spaces within 4 hours as captured by MRI. Efficacy of this treatment was clearly observed, with decreases in tumor volume of up to 80% on MRI; also, all dogs showed improvement in clini-cal signs following treatment. Anti-tumor effects appeared to correlate with MRI-based delineation of extent and localization of infusions. Host toxic-ity was minimal on serial neurological evaluations. Mild CSF lymphocytic pleocytosis was documented following several of the infusions. Postmortem histopathologic analysis in 2 dogs was correlated with in-life MRI data. Significant differences were seen between infused and non-infused tumor tissue, including presence of frank tumor necrosis and histologic evidence of treatment effect in infused areas, including decreased MIB-1 labeling index. No tissue injury was observed in surrounding normal brain. These studies indicate that MRI-guided CED infusion of liposome-based agents into spontaneous canine gliomas is feasible, well-tolerated and efficacious. The findings emphasize the role of MRI monitoring of therapeutic CED infusions to optimize delivery parameters. Canine spontaneous gliomas are a unique experimental paradigm that is highly translational to human tumors. Because CED of liposomal CPT-11 has marked anti-tumor activ-ity in this clinical model, we conclude that the approach warrants further investigation for the treatment of human brain tumors.



ET-08. IN VIVO EFFICACY OF HERPES SIMPLEX VIRUS ICP0 IN COMBINATION WITH RADIATION FOR THE TREATMENT OF INTRACRANIAL GLIOBLASTOMA AFTER CONVECTION-ENHANCED DELIVERYCostas Hadjipanayis,1 Wendy Fellows-Mayle,2 and Neal DeLuca2; 1University of Pittsburgh, Pittsburgh, PA, USA; 2PA, USA

The herpes simplex virus 1 (HSV-1) infected cell protein 0 (ICP0) is an E3 ubiquitin ligase with various effects on cell metabolism including cell cycle arrest, DNA repair inhibition, transactivation of gene expression, histone deacetylase (HDAC) regulation, and inhibition of the interferon response. Convection-enhanced delivery (CED) of either the ICP0-producing HSV-1 mutant, d106, or the recombinant HSV-1 mutant, d109, devoid of all viral genome expression, was performed to determine the in vivo efficacy of ICP0 in combination with ionizing radiation (IR) in the treatment of glioblastoma multiforme (GBM). Intracranial U87-MG human GBM xenografts were established in athymic nude mice. Animal survival was determined after mice were randomized (groups of ten) to undergo intracranial CED of either the replication-defective d106 or d109 viruses or Hanks’ balanced salt solu-tion (HBSS), prior to a single session of whole-brain irradiation (0 or 10 Gy). Mice were randomly sacrificed during an 80-day observation period, post tumor implantation, to determine virus brain distribution and persistence, viral xenograft infection, and intracranial tumor burden. Hematoxylin/eosin staining and immunohistochemistry for enhanced green fluorescent protein (EGFP) were performed on brain sections. Median survival for ani-mals that underwent treatment with HBSS alone, d109 alone, d106 alone, HBSS1IR, d1091IR, and d1061IR, was 28, 35, 41, 39, 39, and 68 days (p , 0.01) respectively. Infection of GBM xenografts and the adjacent brain was found in 9 of 12 (75%) animals after CED of d106 that were randomly sacrificed on days 7 and 9 post tumor implantation. Persistent intracere-bral viral infection was found up to 17 days after d106 CED. Complete tumor regression was found in 10 of 17 (58%) animals sacrificed up to 21 days post tumor implantation that underwent d106 CED and whole-brain irradiation. In vivo ICP0 production after intratumoral d106 CED resulted in a significant increase in animal survival and a tumoricidal effect when combined with whole-brain irradiation. CED of the ICP0-producing d106 mutant allows for distribution of HSV-1 in human GBM xenografts and persistent viral infection. ICP0 is a multifunctional protein that may serve as a novel gene therapy candidate.

ET-09. SAGOPILONE (ZK-EPO), A POTENT NOVEL EPOTHILONE, IS EFFECTIVE IN MODELS OF HUMAN GLIOMA AND BRAIN METASTASESStefanie Hammer,1 Iduna Fichtner,2 Johannes Merk,3 Rosemarie B Lichtner,1 Andrea Rotgeri,1 Ulrich Klar,1 Lars Breimer,4 and Jens Hoffmann1; 1TRG Oncology, Bayer Schering Pharma AG, Berlin, Germany; 2Berlin-Buch, Experimental Pharmacology and Oncology, Berlin, Germany; 3Evangelische Lungenklinik, Berlin, Germany; 4Bayer Healthcare Pharma, Montville, NJ, USA

The resistance of human brain tumors and metastases to most chemo-therapeutic drugs is a significant clinical problem, and is largely due to the inability of systemic therapies to cross the blood-brain barrier, partly because of multidrug resistance (MDR) pumping systems, and also due to the development of MDR in the tumors themselves. Sagopilone (ZK-EPO), the first fully synthetic epothilone in clinical development, has shown sig-nificant in vitro and in vivo antitumor activity compared with paclitaxel and other standard agents in a wide range of tumor models. Sagopilone is not recognized by cellular efflux mechanisms and maintains its activity even in MDR tumor models. Based on these characteristics, the antitumor efficacy of sagopilone has been examined in models of human brain tumors and brain metastases associated with breast cancer. U373 and U87 glioma cells were orthotopically implanted into the right hemisphere of nude mice (8–10 mice/group), which were then treated with 2 3 9 mg/kg i.v. sagopilone or 5 3 8 mg/kg i.p. paclitaxel 2 days after tumor transplantation, modeling an adjuvant treatment schedule. MDA-MB-435 breast cancer cells were orthotopically implanted into the right hemisphere of female nude mice. Treatment groups of 10 mice received 10 mg/kg sagopilone (i.v., days 3 and 14), 8 mg/kg paclitaxel (i.p., days 3–7), or vehicle only. The free access of sagopilone to the brain is demonstrated by an AUCbrain:AUCplasma ratio of 0.8, 40 minutes after an i.v. application to scid mice, compared with a ratio of almost 0 for paclitaxel. Sagopilone shows strong antitumor activity in vivo in the orthotopic implanted glioma models U373 and U87, with sagopilone inducing a complete response in 89% (n 5 9) and 60% (n 5 10) of animals, respectively, compared with 0% of animals treated with paclitaxel for both models. No neurotoxic side effects were observed. In the human breast cancer model MDA-MB-435, sagopilone significantly inhibited brain tumor growth, with a mean tumor volume at 25 days of 0.4 60.3 mm3, compared to 2.2 6 1.2 mm3 with paclitaxel treatment (p 5 0.0007) and 2.5 6 1.3 mm3 in untreated controls. The ability of sagopi-lone to cross the blood-brain barrier translates into potent activity against human glioma models and brain metastases associated with human breast

cancer models in vivo. Results suggest that sagopilone could be a potential therapeutic option for the treatment of human brain tumors or for breast cancer brain metastases, especially as an adjuvant therapy. Sagopilone is currently undergoing clinical trials in patients with glioblastoma, and breast, lung, ovarian, and prostate cancer.

ET-10. P53 INHIBITION INCREASES TEMOZOLOMIDE RESPONSIVENESS OF GLIOBLASTOMA XENOGRAFTSEduard Dinca, Ramona Voicu, Michael Prados, Mitchel Berger, and C. David James; Dept. Neurological Surgery, Brain Tumor Research Center, University of California, San Francisco, San Francisco, CA, USA

The use of temozolomide (TMZ) for treating newly diagnosed glioblas-toma multiforme (GBM) has, to a large extent, become standard of care in clinical neuro-oncology practice. There is, however, substantial variation between tumors in response to TMZ. Here we have investigated the role of p53 in contributing to TMZ response, using patient-derived, serially-propagated GBM xenografts. Inhibition of wild-type p53 activity using 2 different approaches (RNA interference and chemical inhibition with a pifithrin alpha derivative, PFT), sensitizes both MGMT-methylated (TMZ sensitive) and MGMT non-methylated (TMZ resistant) GBM to temozolo-mide in vitro. The same treatments applied to a p53–null GBM xenograft did not alter TMZ-responsiveness in vitro. Parallel in vivo studies, using an orthotopic xenograft model of GBM, enabled for bioluminescence imaging, are in progress and the results of these will reported at the meeting. The in vitro results available at this time indicate that wt p53 activity may actually protect tumors from the cytotoxic effects of TMZ.

ET-11. H-FERRITIN siRNA DELIVERED BY CATIONIC LIPOSOME INCREASES THE EFFICACY OF CHEMOTHERAPY FOR TREATING GLIOMAXiaoli Liu,1 A.B. Madhankumar,1 Jonas Sheehan,1 Becky Slagle-Webb,2 Michael McCauley,2 Qing X. Yang,2 and James R. Connor1; 1Neurosurgery, Pennsylvania State University, Hershey Medical Center, Hershey, PA, USA; 2PA, USA

The goal of this project is to increase the efficacy of chemotherapy for treating glioma by silencing H-ferritin gene using small interfering RNA (siRNA) delivered through cationic liposomes. Two widely used chemother-apeutic agents for treating glioma is carmustine (BCNU) or temodar that alkylates DNA and disrupts DNA synthesis. Frequently, gliomas are resis-tant to BCNU and Temodar, limiting the efficacy of chemotherapy treat-ment. Ferritin, as an iron storage protein, has been shown to be increased in a variety of tumor tissues. In tumor cells, ferritin can be found in the nucleus, where it has the function of protecting DNA from damage. We hypothesize that H-ferritin gene silencing may increase the sensitivity of chemotherapeu-tic agents (BCNU or Temodar) for treating gliomas. We delivered H-ferritin siRNA through cationic liposomes that is nonimmunogenic. The toxicity of the liposomes was evaluated in two human astrocytoma cell lines that are U251 and CCF-STTG1 (ATCC #1718). MTS/PMS assay confirmed that the liposomes used are non-toxic. The complex of siRNA with liposome was confirmed using fluorescence labeled siRNA on a 1% agarose gel. H-ferritin protein expression was effectively decreased in U251 and CCF-STTG1 cell lines as demonstrated by Western blot. The cytotoxicity assay showed that H-ferritin siRNA inhibits U251 and C282Y cell proliferation rate in the presence of BCNU and temodar. In an in vivo model, using a subcutaneous glioma tumor, the presence of siRNA for H-ferritin delivered by liposome increased the rate of decline of the tumor size in the presence of BCNU and temodar. In conclusion, our data demonstrate that silencing the H-ferritin gene appears to be an effective way to increase chemotherapeutic sensitiv-ity. As importantly, we have demonstrated that siRNA can be delivered effectively by targeted cationic liposomes.



ET-12. SYNERGISTIC ACTION OF A POLYNUCLEAR PLATINUM COMPOUND AND PI3–K INHIBITOR ON MALIGNANT GLIOMA CELL LINESHo-Shin Gwak,1 Vaibhav Chumbalkar,2 Yeo-Hyeon Hwang,1 Khatri Latha,1 Rob Dejournett,2 Nicholas Farrell,3 Garth Powis,4 and Oliver Bogler1; 1University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA; 2Neurosurgery, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3Virginia Commonwealth University, VA, USA; 4Department of Developmental Therapeutics, UT MD Anderson Cancer Center, TX, USA

Chemotherapy for malignant glioma has shown limited patient benefit owing to low rates of response, profound side effects with higher dose regi-mens and frequent resistance. To overcome this difficulty, we are testing novel polynuclear platinum compound (PPC), BBR3610, which have shown effectiveness in the nanomolar range, in combination with a PI3-K inhibi-tor. Three cell lines of different p53 and PTEN status (U87, LNZ308 and LN229) were treated with BBR3610 at concentrations around the IC50 (1–5 nM for colony forming assay, 10–100 nM for proliferation inhibition assay) and the PI3-K inhibitor, PX-866, at a dose effective for block the kinase with negligible killing effect. The combination index (CI) was calculated to define drug synergism, using CalcuSyn software. All combination tested showed synergism (CI , 0.9) at moderate (CI 5 0.4–0.7) to strong (CI , 0.3) level in all 3 cell lines. We then examined the mechanism of action of the observed synergism, by cell cycle analysis using FACS, apoptosis assay by annexin-V FITC binding and autophagy by transient GFP-LC3 transfec-tion followed by direct observation of autophagic granules. The cell cycle analysis showed a combination of the G2/M arrest typical for BBR3610 and G1 arrest for PX-866. In addition, the percentage of Annexin V binding cells was increased significantly (p , 0.05) from less than 10% for BBR3610 alone to 30%–50% for the combination with PX-866 depending on the cell line. In contrast, the number of cells with GFP-LC3 granules, indicative of autophagy, was not significantly increased by the combination treatment. Consistent with these data, we observed an increase in PARP cleavage, but not in the ratio of LC3-II to LC3-I. Our results suggest that the potent chemotherapeutic action of BBR3610 can be enhanced by combination with a PI3-K inhibitor, by forcing malignant glioma cells into apoptotic cell death.

ET-13. MRI AND BIOLOGICAL ASSESSMENT OF INTRATUMORAL HEMORRHAGES IN EXPERIMENTAL GLIOBLASTOMA TREATED WITH LOW AND HIGH DOSES OF SUNITINIBSophie De Boüard,1 Simon Roussel,2 Emmanuèle Lechapt-Zalcman,2 Edwige Petit,2 Sophie Krieger,3 Jérôme Toutain,2 Romaric Saulnier,2 Pascal Gauduchon,3 Myriam Bernaudin,2 and Jean-Sébastien Guillamo4; 1GRECAN, Caen, France; 2UMR-CNRS 6185, France; 3GRECAN, France; 4CERVOxy Group Hypoxia and Cerebrovascular Pathophysiology, UMR-CNRS 6185, Caen, France

Sunitinib is an antiangiogenic multitargeted tyrosine kinase inhibitor. Despite a potential risk of intratumoral necrotic microhemorrhages, we recently showed that sunitinib had antitumor effects in orthotopic glioblas-toma (GBM) in mice. Objective: The aim of this study was to assess factors associated with sunitinib-related hemorrhages and erythropoietin (EPO) levels which has been recently shown to be up-regulated by VEGF inhibi-tors. Two doses (“low” 20mg/kg/d and “high” 80mg/kg/d, oral adminis-trations) were used in experimental orthotopic 9L rats and normal rats. Animals were monitored with neurological exams and MRI to evaluate tumor growth and hemorrhages (T2 and T2* sequences in a 7T scanner). Blood samples were obtained for hemogram and EPO levels. Only the low dose was associated with 33% increase in survival time compared to the high dose and control. However, low and high dose groups had similar effects in tumor size and neurological scores compared to control animals. There was a dose-dependent moderate increase in tumoral microhemor-rhages seen in T2* sequences and histology in sunitinib-treated animals. Monitoring of blood samples revealed a dose-dependent leucopenia and thrombopenia whereas red blood cells remained stable. Furthermore, EPO levels were increased in a dose- and time-dependent manner. Low dose of sunitinib, as used in human, is associated with antitumor efficacy and safety in experimental GBM. Toxicity of high dose of sunitinib may be related to intratumoral microhemorrhages and other systemic toxicities. Moreover, this study is in favor of EPO blood levels as a surrogate marker of sunitinib activity.

ET-14. RAMBO (RAPID ANTI-ANGIOGENESIS MEDIATED BY ONCOLYTIC VIRUS): A NOVEL ONCOLYTIC VIRUS DESIGNED TO TARGET GLIOMAS AND THEIR MICROENVIRONMENTJayson Hardcastle, Kazuhiko Kurozumi, Martin Sayers, Yoshinaga Saeki, E. Antonio Chiocca, and Kaur Balveen; Department of Neurological Surgery, Ohio State University Medical Center, Columbus, OH, USA

Like most solid tumors, glioblastoma multiforme (GBM) is also a com-plex tissue comprised of cancerous and normal stromal cells embedded in a complicated extracellular matrix (ECM). The intricate and continu-ous interplay between cancer cells and their microenvironment dictates a tumor’s progression as well as response to therapy. We have been investi-gating the changes induced in the microenvironment of tumors implanted in rat brains in response to therapy with oncolytic virus (OV). We have found that OV administration triggers secretion of a very angiogenic ECM, which causes residual disease to be much more angiogenic and aggressive than the primary untreated tumor. These results underscore the importance of treating both neoplastic cells and the tumor microenvironment. Here, we describe a novel “dually armed” OV (RAMBO, rapid anti-angiogenesis mediated by oncolytic virus) that lyses tumors and secretes an ECM that is not conducive to growth of new blood vessels. RAMBO is a herpes simplex virus-1 (HSV-1)–derived OV designed to replicate specifically in tumors and to express Vasculostatin, a novel anti-angiogenic agent under the gov-ernance of an immediate early (IE4/5) viral promoter. This engineering facilitates early and robust transgene expression following infection com-pared to the limited expression of transgene achieved using cytomegalovirus (CMV) promoter. Vasculostatin is a naturally secreted proteolytic fragment of brain angiogenesis inhibitor 1 (BAI1) that is primarily expressed in nor-mal brain. We have recently shown that it is not expressed in a majority of human GBM specimens and glioma cell lines (Kaur et al., 2003). Further expression of Vasculostatin has a potent anti-angiogenic and -tumorigenic effect in vivo (Kaur et al 2005). Secreted Vasculostatin has been identified by western blot analysis of infected glioma cells as soon as 4 hours post infection. More significantly, over-expression of secreted Vasculostatin does not interfere with the ability of OV to replicate and lyse glioma cells in vitro. Further, the functionality of the secreted Vasculostatin was con-firmed in a directed in vivo angiogenesis assay (DIVAA™, Trevigen). Finally treatment of mice bearing intracerebral (U87EGFR human glioma cells) tumors with RAMBO demonstrated a 30% increase in the number of mice showing complete response (50% survivors, n 5 10/group) compared to mice treated with the control rHSVQ OV (20% survivors, n 5 10/group). This is the first study describing the therapeutic efficacy of Vasculostatin delivery in established tumors. (Recipient of the 2007 OSUMC Research Day Travel Award.)

ET-15. IDENTIFICATION OF CYR61 IN CEREBROSPINAL FLUID AS A BIOMARKER FOR EVALUATING OV EFFICACY IN BRAIN TUMORSKazuhiko Kurozumi, Jayson Hardcastle, Roopa Thakur, Joshua Shroll, Akihiro Otsuki, E. Antonio Chiocca, and Balveen Kaur; Neurological Surgery, The Ohio State University, Columbus, OH, USA

Although clinical trials of oncolytic virus (OV) therapy for malignant gliomas have demonstrated the safety of this approach, high expectations of efficacy remain unmet. To enhance OV efficacy, a correlation linking the level of intratumoral OV over time and patient outcome/response is neces-sary. However, such a correlation is currently not feasible due to the lack of available biomarkers that faithfully reflect OV activity in tumors. Recent studies have demonstrated that transient immune suppression of animals by cyclophosphamide (CPA) treatment reduces viral clearance by host innate immune responses and permits better oncolysis. We exploited the increased viral activity seen in CPA treated animals to identify secreted protein which can be utilized as biomarkers for OV activity. We performed transcriptional profiling of experimental rat brain tumors treated with OV, in PBS or CPA treated animals. Total RNA from brain tumor tissues, extracted 12 and 72 hours after OV injection, from CPA treated or untreated animals was hybridized to an Affymetrix rat genome arrays 230 2.0 representing 28,000 rat cDNAs. To identify changes in secreted proteins we searched probe sets in affymetrix database: http://www.affymetrix.com/analysis/netaffx for extra cellular component in GO Cellular Component Description. To mini-mize direct immunosuppressive effects of CPA, genes with known function in host immune responses were excluded. We identified a total of ten genes showing greater than 2 fold up regulation in CPA treated animals. From this profiling we selected CYR61 for further evaluation as its levels were consistent with predicted levels of OV in each group (12 hours and 72 hours after OV treatment in PBS or CPA treated animals). Quantitative Real time PCR verification further revealed a significant linear correlation (Pearsons coefficients, p , 0.05) between OV and CYR61 levels in tumor tissue of treated and untreated animals. Given this linear correlation we postulated that cyr61 induction was a direct effect of OV infection of glioma cells. We



tested this by evaluating changes in CYR61 mRNA in vitro and uncovered a significant and early induction of CYR61 in glioma cells infected with OV. This induction was observed in glioma cells infected with multiple HSV-1 derived OVs, and in multiple glioma cell lines. We have also confirmed this induction to hold true in primary human brain tumor biopsy derived neu-rospheres infected with OV. Further western blot and Immunofluorescent staining of OV infected glioma cells in vitro and in vivo revealed increased CYR61 protein expression. Finally we tested levels of CYR61 protein in cerebrospinal fluid of rats with brain tumors treated with OV to evaluate the significance of using CYR61 induction as a potential biomarker for OV therapy. In summary these results have uncovered the early and significant induction of CYR61 upon OV infection of rat gliomas and indicate the potential of using its presence in CSF as a biomarker for OV activity in brain tumors.

ET-16. CILENGITIDE AND SYNERGY WITH RADIATIONTom Mikkelsen,1 Kevin Nelson,1 Steven Brown,1 Simon Goodman,2 Chaya Brodie,1 and James Ewing3; 1Henry Ford Hospital, Detroit, MI, USA; 2Merck KGaA, Germany; 3Detroit, MI, USA

Brain tumor growth and invasion are dependent on the infiltrating ability of tumor cells, a phenotype which also is associated with a rela-tive resistance to therapy, including the up-regulation of genes conferring resistance to apoptosis. Invasion, therefore, has become an active target for drug discovery. Anti-motility or anti-invasion therapy has the potential to restore the sensitivity of infiltrating glioma cells to therapeutic induction of cell death using radiation and/or chemotherapy. Several agents, including cilengitide (aka EMD121974, (cyclo (Arg-Gly-Asp-D-Phe-[N-Met]-Val) a cyclic RGD-binding peptide), target the alphavß3 and alphavß5 integrins, which is upregulated in glioma tumor and neovascular endothelial cells. Blocking this integrin signal inhibits glioma cell migration and initiates a process of tumor and neovascular endothelial cell death. Cilengitide is currently in clinical trial, and initial efficacy studies using it as a single agent suggest activity. Dose-response determinations and the assessment of treatment efficacy with such agents are problematic, though, primar-ily owing to the lack of validated surrogate endpoints, making issues like dose and schedule finding problematic. Mechanistically, infiltrating glioma cells upregulate genes, which render them relatively resistant to apoptosis from conventional modalities like radiation and chemotherapy. Since anti-motility agents have shown little efficacy when used as single agents, we embarked on pre-clinical studies and suggest that cilengitide may exert an improved therapeutic effect when combined with other agents which induce apoptosis. We have generated striking data that cilengitide exerts synergy with radiation in an animal model of glioblastoma, but with very specific timing requirements. We have undertaken studies to optimize this efficacy and further refine the molecular pathways responsible. This work will fur-ther enhance this glioma therapy, which will inform the next generation of clinical trials with this agent. We have also used non-invasive techniques of imaging which will also allow the optimization and mechanism studies to occur more quickly using these surrogates, which will also be translated to the clinic.

ET-17. TARGETING HYALURONAN INTERACTIONS IN AN ORTHOTOPIC SPINAL CORD GLIOMA MODELAnne Gilg, Sandra Tye, Bryan Toole, and Bernard L Maria; Medical University of South Carolina, Charleston, SC, USA

Malignant behaviors of cancer cells are influenced by the autocrine effect of hyaluronan/CD44 interactions with receptor tyrosine kinases, down-stream cell survival pathways such as the phosphoinositide 3–kinase/Akt pathway, and on the expression of ATP-binding cassette (ABC) drug efflux transporters. In this study, we evaluated the potential of small hyaluronan oligomers, which antagonize the activity of endogenous hyaluronan, to suppress tumor growth in a rat spinal cord model of glioma. Glioma cells engrafted into the rat spinal cord show patterns of dispersal that strongly resemble malignant human gliomas, including invasion of Sherer’s struc-tures: white matter, subpial, and hyaluronan (HA)-rich perineuronal nets. Even though spinal cord gliomas account for less than 10% of CNS gliomas, our animal model provides an effective way to focus on the propensity of intramedullary spinal gliomas to aggressively invade the CNS. Because of the sharp grey matter/white matter interface and the preponderance of longitudinally arranged tracts in the cord, glioma cells rapidly invade the white matter and within 3 to 4 weeks of engraftment, are detected in the lower brainstem. This model is also attractive because functional behavioral measures developed to evaluate spinal cord function can be used as markers of tumor growth and segmental spinal cord dysfunction. Finally, the model permits testing agents whether by oral, systemic, intrathecal, or direct infu-sion into, rostral, or caudal to the invading intramedullary tumors. Reduc-

tion of tumor size by single intratumoral injection of hyaluronan oligomers was associated with decreased proliferation (Ki67), increased apoptosis (TUNEL), and down-regulated activation of Akt and expression of breast cancer resistance protein (BCRP/ABCG2) in C6 rat gliomas in vivo. Double labeling immunohistochemistry for tumor cells (beta-galactosidase express-ing) revealed that BCRP positive cells were not in fact tumor cells, but rep-resented a distinct population of host cells concentrated primarily around blood vessels within the tumor. These BCRP-positive cells double labeled with nestin, a marker of neural progenitor cells. In addition, some BCRP-positive cells double labeled with CD45, a marker of cells of hematopoi-etic origin. These results suggest that the effects of hyaluronan oligomers on BCRP expression in gliomas in vivo were primarily due to suppression of the recruitment of BCRP-positive progenitor-like cells to the tumors. Thus, glioma-derived hyaluronan not only increases key cellular signaling pathways that mediate malignant behaviors, but it probably also enhances recruitment of neural and extraneural cellular elements that promote growth and invasion of tumors in the CNS. The use of non-toxic and non-immunogenic hyaluronan oligomers may potentiate current therapies for the most malignant gliomas and, for lower grade tumors, enable radiation dose reduction to preserve neurological function. In summary, the safety of hyaluronan oligomers and their effects on multiple molecular pathways that govern glioma growth, invasiveness and drug resistance make them attractive for therapeutic development.

ET-18. ANTI-GLIOMA EFFECTS OF PROTEIN KINASE INHIBITORS THAT SIMULTANEOUSLY BLOCK INVASION AND PROLIFERATIONShante Williams, Michal Nowicki, Jakub Godlewski, E. Antonio Chiocca, and Sean Lawler; Department of Neurological Surgery, Ohio State University Medical Center, Columbus, OH, USA

In order to effectively treat invasive gliomas, therapeutic strategies must target both the proliferative and invading portions of the tumor. In our pre-vious studies we used pharmacologic agents and siRNA to identify GSK-3 (glycogen synthase kinase-3) as a potential anti-invasive therapeutic target in glioma cells. A large number of small molecule GSK-3 inhibitors are known and available commercially. These compounds have a broad range of potency and selectivity. For example, many GSK-3 inhibitors also inhibit members of the related CDK (cell cycle dependent protein kinase) family. CDKs are critical in driving cell proliferation, are known to be upregulated in glioma and are well known as anti-tumor therapeutic targets. These observations suggest that a single agent with overlapping specificities for GSK-3 and CDKs could simultaneously block both cell proliferation and invasion, an attractive combination for glioma. In this study we report data from a panel of inhibitors from the paullone and indirubin families, which all inhibit GSK-3 in vitro in the low nanomolar range, but have highly vari-able IC50 values for CDK1 inhibition. This panel of compounds was ini-tially examined in invasion assays using U87 cells which revealed that they all block glioma cell invasion in an in vitro spheroid assay system at similar very low micromolar concentrations, in several glioma cell lines. However, wide variability was observed in cell proliferation assays, which correlated well with the IC50 of CDK inhibition. The use of these inhibitors presents the attractive possibility of a beneficial combinatorial effect due to their overlapping inhibitory properties. Ongoing studies will assess the effects of dual CDK and GSK-3 inhibition in invasive intracranial glioma animal models.

ET-19. IN VIVO CHARACTERIZATION OF THE BLOOD-BRAIN AND BLOOD-TUMOR BARRIER IN ORTHOTOPIC XENOGRAFT TUMORS DERIVED FROM ADULT AND PEDIATRIC MALIGNANT GLIOMASJianjun Li,1 Gerald Grant,2 Charles Pegram,1 and Darell Bigner2; 1Pathology, Duke University, Durham, NC, USA; 2Duke University, Durham, NC, USA

A better preclinical understanding of blood-brain (BBB) and blood-tumor barrier (BTB) is critical not only to model the drug delivery but also to interpret the treatment response of malignant brain tumors following sys-temic therapy. The spatial heterogeneity of the BTB was characterized for 5 xenograft malignant gliomas implanted intracranially. Quantitative per-meability was calculated for intracranially implanted human glioblastoma xenograft tumors derived from 4 adult and 1 pediatric patient, respectively (12A7, 256, 270 and 6B47, and 456). Xenograft tumors were stereotacti-cally implanted in immune compromised mice. Quantitative autoradiogra-phy was used to measure the regional permeability expressed as Ki (ml/g.min) following intravenous infusion of 14C labeled amino-iso-butyric acid (14C-AIB; MW 103 Da) as a radiotracer. All 5 intracranial GBM xenograft tumors uniformly displayed a higher permeability than the brain surround-



ing the tumor. There was marked spatial heterogeneity of blood-tumor bar-rier permeability within each tumor. A gradient in permeability was seen from the tumor center (highest Ki), which was not always the geometric cen-ter, towards the tumor perimeter (lowest Ki). The BTB area with Ki . 0.02 ml/g.min corresponded to the tumor area as determined with immunohis-tochemistry. When the 5 xenograft tumors were compared independently, the 270 xenograft tumor was characterized by the highest permeability to 14C AIB while 456 the lowest. BTB permeability increased directly with tumor maximal section area (mm2) in 12A7 (r 5 0.84), 6B47 (r 5 0.46) , and 256 ( r 5 0.82). Intracranial tumors also consistently displayed a lower quantitative permeability than the BTB permeability obtained in the same xenograft tumors implanted subcutaneously. Each brain tumor xenograft was characterized by a unique signature of blood-brain and blood-tumor barrier function which was highly dependent on tumor size and the local tumor microenvironment. Future preclinical testing of new candidate tumor targets and BTB permeability modulation will depend on a better character-ization of the blood-brain and blood-tumor barrier in both xenograft and transgenic models of malignant gliomas.

ET-20. HISTONE DEACETYLASE INHIBITORS AUGMENT ANTITUMOR EFFECTS OF HERPES SIMPLEX VIRUS-BASED ONCOLYTIC VIRUSESAkihiro Otsuki, Ankita Patel, Masataka Suzuki, Kazue Kasai, E. Antonio Chiocca, and Yoshinaga Saeki; Dardinger Laboratory for Neuro-Oncology and Neurosciences, The Ohio State University Medical Center, Columbus, OH, USA

Replication-conditional viral mutants (oncolytic viruses) represent a promising therapeutic alternative for patients with malignant gliomas, and these viruses have been increasingly studied in both preclinical and clinical settings. Recent research, including ours, indicates that human glioma xeno-graft lines and freshly derived primary glioma cells are often more resistant to oncolytic viruses than are glioma cell lines that have been maintained for a long time in serum-containing monolayer culture. Several lines of primary human glioma cells have been derived from clinical glioma specimens and tested for their responsiveness to interferon (IFN)-beta; many of these were revealed to be more responsive than conventional glioma cell lines. The treatment with IFN-beta significantly reduced propagation of oncolytic herpes simplex viruses (HSV) in the primary glioma cells. In vivo admin-istration of oncolytic viruses is believed to induce rapid activation of host innate immune responses, presumably by activating phagocytic cells and the prominent production of type I IFNs by these phagocytes. Such responses are thought then to impede efficient replication and spread of oncolytic viruses in tumors in vivo. Histone deacetylase inhibitors (HDACIs) are a promising new class of antineoplastic agents known to upregulate expres-sion of exogenous genes and inhibit induction of IFN-stimulated genes. We hypothesized that treatment with HDACIs could enhance gene expression from oncolytic HSV and inhibit IFN-mediated innate antiviral responses in tumor cells, thus improving the therapeutic efficacy of oncolytic HSV. As expected, initial transduction of oncolytic HSV was significantly improved after treatment with valproic acid (VPA), a well-characterized HDACI. VPA also inhibited induction of several IFN-stimulated genes in HSV-infected glioma cells and improved viral propagation even in glioma cells treated with IFN-beta. These results suggest that HDACIs are useful pharmaco-logical modifiers that enhance the antitumor effects of oncolytic HSV in malignant gliomas.

ET-21. ALKYLADENINE-DNA GLYCOSYLASE PROMOTES GLIOMA RESISTANCE TO TEMOZOLOMIDE-INDUCED N-METHYLPURINESJohn Silber, Michael Bobola, Nolan Smith, and Douglas Kolstoe; Neurological Surgery, University of Washington, Seattle, WA, USA

The large majority of alkylator-induced base changes in DNA occur at the ring nitrogens of purines. Alkyladenine-DNA glycosylase (AAG) is a repair activity that initiates removal of N-alkylpurines by cleaving the glycosylic bond between the altered base and deoxyribose yielding an abasic site. We have recently reported (Bobola et al., Human Glioma Sensitivity to the Sequence-Specific Alkylating Agent Methyl-Lexitropsin, Clin Cancer Res 13;612–20, 2007) that AAG promotes resistance in glioma cells to methyl-lexitropsin, a sequence specific methylating agent that almost exclusively produces the documented cytotoxic lesion 3-methyladenine. Importantly, 3-methyladenine comprises approximately 10% of the base adducts produced by temozolomide (TMZ). To obtain evidence that AAG-mediated removal of 3-methyladenine and other N-methylpurines promotes glioma resistance to TMZ, we determined the effect of suppressing AAG activity with antisense oligonucleotides on the TMZ sensitivity in a panel of human glioma cell lines. Suppressing AAG activity ~2-fold was accom-panied by an ~2-fold reduction in the TMZ dose required to reduce survival

to 10% in all lines examined. Importantly, potentiation of cytotoxicity was accompanied by a comparable reduction in the abundance of abasic sites, suggesting that enhanced lethality reflected the persistence of N3–meA in DNA. Increased killing accompanying suppression of AAG was observed in the absence of O6-methyguanine-DNA methyltransferase, demonstrat-ing that failure to repair 3-methyladenine and other N-methylpurines was solely responsible for greater sensitivity. Antisense treatment also sensitized mismatch repair deficient lines, suggesting that AAG as a potential tar-get for anti-resistance therapies to overcome acquired resistance to TMZ. Interestingly, suppression of AAG activity was accompanied by increased sensitivity to the chloroethylator BCNU, an agent that does not alkylate at the N3 position of adenine. This finding is consistent with reports that 3MAG recognizes a wide range of altered purines as substrates and sug-gests that unrepaired chloroethyl purines or their hydroxyethyl derivatives, contribute to the cytotoxicity of chloroethylating agents in gliomas. An ongoing analysis in malignant glioma tissue has revealed that AAG activity is significantly associated with MGMT activity (r 5 0.635; p < 0.007), suggesting that the greater responsiveness of MGMT deficient gliomas to TMZ may reflect inefficient repair of N-methylpurines as well as failure to remove O6-methylguanine.

ET-22. EFFECTS OF ANTI-EGFR TREATMENT ON MOLECULAR ALTERATIONS EXHIBITED BY PRIMARY GBM OF DIFFERENT SENSITIVITIESHetal Bhanushali, Sharon Longo, and Gregory Canute; Department of Neurosurgery, SUNY Upstate Medical University, Syracuse, NY, USA

We examined the effect of the anti-epidermal growth factor receptor (EGFR) antibody, Erbitux, on Glioblastoma Multiforme (GBM) with dif-ferential EGFR expression. Erbitux binds the extracellular domain of the EGF receptor and inhibits receptor activation. EGFR overepxression and/or amplification is found in about 40%–50% of primary GBM tumors. The clinical response using EGFR-based therapies has not been predictable. Therefore, we sought to find additional molecular determinants for GBM responsiveness to anti-EGFR treatment. The purpose of this study was to look at the key molecules of the survival pathways like phosphatidyl-inositol 3-kinase (PI3K/Akt) and mitogen-activated protein kinase (MAPK/ERK) which are commonly activated by EGFR stimulation. The expression of the key molecules was assessed in primary GBM lines with differential EGFR expression before and after Erbitux treatment. Flow cytometry analysis was used to determine the EGFR levels. Two GBM lines expressed high EGFR and two expressed low EGF receptor levels. Erbitux produced dose-dependent cytotoxicity in high EGFR expressing GBM as opposed to very little or no cytotoxicity in low EGFR expressing GBM lines. Cell cycle analysis by flow cytometry demonstrated increased apoptosis in high EGFR expressing GBM lines and no change in cell cycle in low EGFR expressing GBM upon exposure to Erbitux. Immunoblotting revealed that low EGFR expressing GBM were associated with high pAkt and pErk expression in untreated lysates and sustained those active levels even after Erbitux treatment in vitro. Immunoblots of the high EGFR expressing GBM demonstrated very low levels of pAkt and pErk expression before and after Erbitux treatment in vitro. High PTEN expression was observed in high EGFR expressing GBM while low PTEN expression was found in low EGFR expressing GBM. This data suggests that Erbitux sensitive GBM have EGFR amplifi-cation and overexpression combined with high PTEN expression and low activated levels of Akt and ERK. In contrast, GBM resistant to Erbitux have low EGFR and PTEN expression with high levels of active Akt and ERK.

ET-23. THE EFFECTS OF CANNABINOID COMBINATION TREATMENTS ON HUMAN GLIOBLASTOMA CELL PROLIFERATION, INVASION AND SURVIVALSean McAllister,1 Jahan Marcu,2 Rigel Christian,2 Liliana Soroceanu,2 Garret Yount,2 and Pierre-Yves Desprez-Yves2; 1Cancer Research, California Pacific Medical Center Research Institute, San Francisco, CA, USA; 2California Pacific Medical Center Research Institute, San Francisco, CA, USA

Delta-9–THC, the major psychoactive constituent in Cannabis sativa, has been shown to an effective inhibitor of GBM growth in vitro and in vivo. It has been suggested that non-psychoactive cannabinoids, compounds that do not interact efficiently with cannabinoid 1 (CB1) and CB2 receptors, can enhance the actions of delta-9–THC. Using multiple human glioblastoma multiforme (GBM) cells lines and primary cultures derived from human malignant tumors, the anticancer properties of non-psychoactive can-nabinoids were compared to synthetic and natural CB1 and CB2 agonists including delta-9–THC. Overall we determined that the non-psychoactive cannabinoid, cannabidiol (CBD), was most potent at inhibiting cell pro-liferation and inducing apoptosis in multiple cell lines. CBD was also able to inhibit the invasiveness of GBM cells. We therefore propose to test the



activity of delta-9–THC in combination with CBD and determine the effects of the combination on GBM cell proliferation, invasion, and survival. To determine the positive and negative aspects of constituent interaction, mul-tiple different concentration combinations of delta-9–THC and CBD will be assessed in a 2 3 2 design. Using this model, we will be able to determine if the combination of constituents can produce additive or greater than addi-tive (synergistic) effects. Antagonist or less than additive effects can also be assessed using this method. The signal transduction mechanisms associ-ated with the combination treatments will also be studied. We hypothesize that the combination of cannabinoid constituents will be more active than delta-9–THC alone.

ET-24. P53 STATUS AS A MOLECULAR DETERMINANT OF CHEMOSENSITIVITY IN SHORT-TERM CULTURES DERIVED FROM MALIGNANT ASTROCYTOMASally Ashmore,1 Tracy Warr,2 Katherine Karakoula,2 Samantha Ward,2 Blanca Suarez-Merino,2 Sarah Brown,3 Montri Luxsuwong,2 Philip Warren,4 David Thomas,2 and John Darling5; 1Institute of Neurology, London, United Kingdom; 2Institute of Neurology, London, AE (Europe), United Kingdom; 3University of Wolverhampton, Wolverhampton, AE (Europe), United Kingdom; 4University of Wolverhampton, AE (Europe); 5University of Wolverhampton, Wolverhampton, United Kingdom

We have previously demonstrated a relationship between the presence of a mutation of the p53 gene and sensitivity to CCNU and vincristine (VCR) but not doxorubicin (DOX) in a panel of 17 short-term cultures (passage level , 15) derived from human malignant astrocytoma. In order to understand the molecular mechanisms that might be responsible for this, we have examined the p53 codon 72 status of these cell lines together with the methylation status of the p53 and p73 genes, MDM2 expression and methylation of the MGMT promoter and expression of MGMT protein. Eight cultures (47%) harbored p53 mutations and one culture without a p53 mutation exhibited aberrant expression of wild-type p53 protein. Eight of nine of these cultures (89%, seven with mutations and one with dysregu-lated wild-type p53) were sensitive to CCNU and VCR. There did not seem to be a consistent relationship between p53 methylation, p73 methylation or MDM2 expression and sensitivity to CCNU although two cultures with methylated p53 were sensitive to CCNU in the absence of a p53 mutation. One culture that was resistant to CCNU despite harboring a p53 mutation was extremely sensitive to both VCR and DOX and was the only culture to possess a Pro72P polymorphism. 11/17 cultures (65%) were essentially negative for nuclear MGMT protein expression. Of these, only two had methylated MGMT gene promoters and one had a partially methylated promoter. In the six cultures that exhibited high levels of MGMT expres-sion, five had unmethylated MGMT promoters and one displayed a par-tially methylated MGMT promoter. Of the nine cultures with aberrant p53 genes, seven did not express high levels of nuclear MGMT protein but had unmethylated MGMT promoters. These data demonstrate that aberrant p53 gene status resulting from either mutation or p53 methylation correlates with low MGMT expression and with in-vitro sensitivity to CCNU and that expression of the Pro72P polymorphic variant of p53 abrogates the sensitiv-ity to CCNU usually associated with p53 mutation.

ET-25. POLYNUCLEAR PLATINUM COMPOUNDS INDUCE AUTOPHAGY IN GLIOMAVaibhav Chumbalkar,1 Keishi Fujiwara,1 Ho-Shin Gwak,1 Yeo Hyeon Hwang,1 Yasuko Kondo,1 Nicholas Farrell,2 and Oliver Bogler1; 1University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Virginia Commonwealth University, VA, USA

There is an ongoing urgent need for novel therapies for gliomas, mainly because of the low median survival rate with current modalities. Lately much emphasis has been on targeting signal transduction pathways. But until such therapies result in new standard of care, the rationale of studying the drugs whose effectiveness is not dependent on differentially expressed or mutated molecular targets, remains strong. BBR3610, a polynuclear platinum compound (PPC) is one such promising compound. Previously our group has observed that BBR3610 has improved efficacy as compared to cisplatin in vitro as well as in vivo. It was also observed that BBR3610 does not induce apoptosis and instead induces G2M arrest in short-term experi-ments. Autophagy has recently been shown to be a major mechanism of action for several chemotherapeutic agents, prompting an in depth study of its role in response to BBR3610. We used glioma cell lines LNZ308 and U87 and found induction of autophagy at therapeutic levels of BBR3610. For this we looked for the formation of acidic vesicles by Acridine Orange which showed time and dose dependent increase. Analysis of the more specific marker of autophagy LC3, both by the formation of punctuate pattern of GFP-LC3 and by increase in the LC3-II isoform on western blot, indicated that BBR3610 induced autophagy. In vivo, intracranial U87 tumor xeno-

graft treated with BBR3610 also showed significant induction of autophagy. Blocking of the autophagy pathway by silencing of ATG5, which is an essen-tial gene in autophagy induction, resulted in the attenuation of autophagy upon treatment with BBR3610, further confirming autophagy induction by the drug. Interestingly, when autophagy was blocked we observed only a modest increase in the apoptosis and overall increase in cell survival sug-gesting autophagy is the primary mechanism of action of PPCs in suppress-ing glioma growth. To study markers of response and resistance to PPCs, we have generated a BBR3610-resistant variant of LNZ308 by treating it with sublethal incremental doses of BBR3610 over time. We intend to identify markers of resistance by comparing the proteome of this resistant line with the parental cells. We will use a quantitative multidimensional chroma-tography of peptides followed by tandem mass spectrometry to identify proteins which are overexpressed in resistant cell lines, and use the same approach to identify proteins that change upon treatment with BBR3610 to identify markers of response. These markers will be validated in models, and developed with the hope that they may be used to guide translation of these promising agents.

ET-26. IDENTIFICATION OF THE SRC TYROSINE KINASE FYN AS A NOVEL DASATINIB-SENSITIVE MOLECULAR TARGET IN GLIOBLASTOMAKan Lu,1 Shaojun Zhu,2 Eduard Dinca,1 Ramon Felciano,3 Jeremy Caldwell,4 Steve Horvath,2 Timothy Cloughesy,2 Stanley Nelson,2 Gabriele Bergers,1 David James,1 and Paul Mischel5; 1University of California, San Francisco, San Francisco, CA, USA; 2University of California, Los Angeles, CA, USA; 3Ingenuity Systems, Inc., CA, USA; 4Genomics Institute of the Novartis Research Foundation, CA, USA; 5Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA, USA

Glioblastoma is the most common malignant primary brain tumor and among the most lethal cancers. Recent clinical trial results indicate that glioblastoma patients can benefit significantly from molecularly targeted therapies, therefore the identification of new pathway targets, particularly those for which safe and effective inhibitors are available, is a top prior-ity. The dual Src/Abl kinase inhibitor dasatinib (BMS-354825) has proven effective in treating chronic myeloid leukemia, however it has yet to be thoroughly investigated for activity against solid tumors. Here, we have examined gene expression data from glioblastoma clinical samples using a comprehensive molecular interaction network approach to identify the Src-family tyrosine kinase Fyn as a previously unrecognized target in glio-blastoma. We show that Fyn is overexpressed and persistently phosphory-lated in independent glioblastoma patient sample sets. Reduction of Fyn expression significantly inhibited tumor cell invasion and proliferation, whereas Fyn overexpression promoted these activities. Similar results were observed with the related Src-family kinase Lyn. Glioblastoma cells were highly sensitive to dasatinib in vitro, demonstrating dramatic inhibition of tumor cell invasion and proliferation concomitant with inhibition of Fyn phosphorylation. In an orthotopic, bioluminescence-enabled glioblastoma xenograft mouse model, dasatinib significantly prolonged survival and reduced tumor luminescence at clinically achievable doses. Collectively, these results identify the Src-family kinases, particularly Fyn and Lyn, as compelling targets for therapy and suggest that dasatinib may have promis-ing clinical efficacy for the treatment of glioblastoma.

ET-27. A NOVEL HIF INHIBITOR BLOCKS GLIOMA GROWTH THROUGH A NON-CANONICAL MODULATION OF THE HSP90 PATHWAYVladimir E Belozerov,1 Taku Narita,2 Jiyoung Mun,3 Rita Noronha,4 Hui Mao,5 Saroja Devi,6 Hui Wang,7 Don Hill,7 Carlos Moreno,8 Mark Goodman,5 Kyriakos Nicolaou,4 Ruiwen Zhang,7 Suazette Reid,9 Binghe Wang,9 and Erwin Van Meir1; 1Department of Neurosurgery, Emory University, Atlanta, GA, USA; 2Department of Neurosurgery & Hematology/Oncology, Winship Cancer Institute, Atlanta, GA, USA; 3Department of Radiology, Winship Cancer Institute, Atlanta, GA, USA; 4Department of Chemistry, Scripps Research Institute, La Jolla, CA, USA; 5Emory University, Atlanta, GA, USA; 6Department of Neurosurgery, Winship Cancer Institute, Atlanta, GA, USA; 7Department of Pharmacology, University of Alabama at Birmingham, Birmingham, AL, USA; 8Department of Pathology, Emory University, Atlanta, GA, USA; 9Department of Chemistry, Georgia State University, Atlanta, GA, USA

Hypoxia is a frequent characteristic of solid tumors, that appears as the tumor mass outgrows the existing vascular supply. The presence of hypoxia in solid tumors strongly contributes to their malignant phenotype by increasing angiogenesis, migration, clonal selection of mutations in anti-apoptotic genes (e.g., in TP53 or Ras), and maintaining the undifferentiated



state of cancer stem cells. Furthermore, the hypoxic areas of solid tumors are resistant to traditional chemo- and radio-therapies. Altogether, the prop-erties of hypoxic tumor cells make them an important therapeutic target. A unifying property of these cells is the expression of HIF-1, a key transcrip-tion factor that coordinates adaptive responses to hypoxia, providing cancer cells the means to survive and proliferate under hypoxic conditions. We and others have shown that disrupting HIF-1 function significantly inhibits tumor growth, highlighting the importance of developing small molecules that could antagonize the HIF-1 pathway. To identify specific small mol-ecule inhibitors of HIF-1, we screened a combinatorial library of natural product-like compounds. KCN1, a lead inhibitor identified in this screen, potently inhibits HIF-1 activity in various cancer cell lines (IC50 4mM), while exerting minimal effects on the levels of HIF-1b, other short-lived pro-teins, or control proteins. To probe the molecular mechanism of this novel inhibitor, we compared the changes in global gene expression caused by KCN1 to those caused by a variety of previously characterized drugs using the Connectivity Map database. The gene expression signature of KCN1 was found to be similar to those produced by known inhibitors of Hsp90. Biochemical analyses confirmed that KCN1 modulates the Hsp90 function by inhibiting the association of the chaperone with a model client protein. Interestingly, KCN1 does not inhibit the enzymatic activity of Hsp90 via direct binding to the ATPase pocket in a manner similar to established Hsp90 inhibitors (e.g., geldanamycin and radicicol). Rather, the drug alters the availability of Hsp90 to its clients, including HIF-1, by changing the subcellular distribution of the chaperone. Detailed experimental evidence supporting this novel mechanism will be presented. In an effort to translate KCN1 to the clinic we evaluated its biodistribution, toxicity and anti-tumor efficacy in animal models. Pharmacokinetic experiments demonstrated the biodistribution of KCN1 and its accumulation in an orthotopic brain tumor. Pharmacodynamic measurements evidenced that HIF-1 signaling was inhib-ited in subcutaneous tumor xenografts. Systemic administration of KCN1 in nu/nu mice harboring sc human glioblastoma xenografts evidenced strong anti-tumor effects in the absence of noticeable toxic effects. These data sug-gest that KCN1 is a promising new agent for the treatment of gliomas in a clinical trial.

ET-28. CELLULAR AND IN VIVO ACTIVITY OF A NEW PI3K INHIBITOR-PX866 FOR TREATMENT OF HUMAN GLIOBLASTOMADimpy Koul,1 Ruijun Shen,2 Jennifer Edge,2 Yasuko Kondo,2 Douglas Webb,2 James Bankson,2 Sabrina Ronen,2 D. Lynn Kirkpatrick,3 Garth Powis,4 and W.K. Alfred Yung2; 1Neuro-Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3ProlX Pharmaceuticals, Tucson, AZ, USA; 4Department of Developmental Therapeutics, UT MD Anderson Cancer Center, Houston, TX, USA

The phosphatidylinositol-3–kinase (PI-3–kinase)/PTEN/Akt pathway is a prerequisite for a wide spectrum of cancers, either via the acquisition of activating mutations in the PI3K catalytic subunit itself, or via loss of PTEN. In this respect class 1 PI3Ks represents well-validated molecules for tar-geted drug discovery. We demonstrate, herein, that a newly developed PI3K inhibitor PX866, a wortmannin analog with selectivity for p110 a, b, and (ProlX Pharmaceuticals), effectively inhibits signaling through the PI3K/Akt cascade in a set of glioma cells inhibiting various PI3K signaling components such as Akt, p70S6K1, TSC-2, and pS6. PX866 produces growth inhibitory activities in glioma cell lines with IC50 ranging from 4–8 mM. PX866 did not induce apoptotic cell death where as it did induce autophagy- type-2 programmed cell death in U87 glioma cells in a dose dependent manner. In vivo experiment with U87 SC xenograft demonstrated an 84% growth inhibition after 4 weeks treatment at an oral dose of 2.5mg/kg in a qod schedule. PX-866 increased the median survival of animals with i.c.U87 tumors from 31 to 38 days with inhibition of p-AKT and p-S6 as shown by IHC and reverse phase lysate array. To develop a non-invasive method to assess biological activity of PX866, magnetic resonance spectroscopy (MRS) was performed to determine molecular response to PX-866 in the U87 model and tumor volume was assessed by T2 MRI sequence. Tumor volumes dropped on average from 20611 mm3 in control animals (n 5 10) to 5 64 mm3 in PX-866 treated animals (n 5 10, p , 0.002). To assess the potential of MRS-detectable metabolic changes as noninvasive biomarkers of response to PX-866, we performed localized MRS in control and treated animals. Spectra obtained from normal contra-lateral brain differed from spectra of untreated tumors in choline to NAA (Cho/NAA) ratio. Cho/NAA ratio was 0.8 60.1 in contra-lateral brain and increased to 1.7 60.4 (n 5 10; p , 0.002) in the untreated gliomas. PX-886 treatment did not affect the spectra recorded from normal brain. In contrast, spectra from the tumor region indicated that Cho/NAA in PX-866–treated tumors was significantly lower by 30% compared to untreated tumors at 1.2 60.5 (n 5 10; p , 0.02). Our findings demonstrate that PX-866–treatment results in MRS-detect-able metabolic changes indicating a partial normalization of the metabolic profile of treated tumors. Taken together, these data demonstrate that PI3K inhibitor PX866 has significant activity in signal inhibition, cell cycle arrest,

growth inhibition and autophagy in human glioblastoma in vitro and in vivo. In addition MRS can be used to non-invasively monitor the molecular effect of PX-866 in gliomas in vivo which further confirm the value of MRS in monitoring early molecular response to this PI3K inhibitor in patients in vivo affirming that PI3K/Akt pathway is a highly specific molecular target for molecular therapeutics development for glioblastoma and other cancers with aberrant PTEN/PI3K expression.

ET-29. COMBINED ANTI-TUMOR EFFECT OF BEZ235 WITH RAD001 AND SCH66336Ta-Jen Liu,1 Tiffany Lafortune,2 Dimpy Koul,2 Sauveur-Michel Maira,3 Frédéric Stauffer,3 Carlos Garcia-Echevrria,3 and W.K. Alfred Yung1; 1University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Neuro-Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3Novartis Pharma AG, Basel, Switzerland

Early phase I/II clinical trials using small molecular inhibitors target-ing a single genetic alteration yielded limited success. The complex genetic abnormality in human gliomas such as an amplification of EGFR, mutation and/or deletion of tumor suppressor gene PTEN, and elevated Ras-GTPase activity is the hallmark in glioma progression. As a result multiple signaling pathways including that of PI3K, Akt, and MAPK are constitutively acti-vated rendering the futility of a single agent therapy. Therefore, therapeu-tic efficacy for target inhibition of multiple signal nodes warrants in-depth assessment. As a single agent, BEZ235, a dual PI3K and mTOR inhibitor, treatment resulted in multifaceted anti-tumor effect including G1 cell cycle arrest, inhibition of VEGF expression and autophagy; a type II programmed cell death. Furthermore, in vivo experiment with U87 IC model demon-strated oral administration of BEZ235 significantly prolonged survival of xenograft animals in a dose-dependent manner (p 5 0.026) without weight loss, suggesting the effectiveness and safety of BEZ235. We examine the anti-proliferative effect of BEZ235 combining either with RAD001, a spe-cific mTOR inhibitor, or with SCH66336, a farnesyl transferase inhibitor compared to single agent alone. The effect of treatment on glioma cell pro-liferation was assessed using the sulforhodamine B (SRB) assay. To assess if combined treatment results in, antagonistic, additive, or synergistic out-come, we employed a combination index (CI) analysis using the Calcusyn software from Biosoft, UK. Our results demonstrate that the CI values for either BEZ235/RAD001 or BEZ235/SCH66336 combination were , 1 indicative of synergistic effect. Western blot analyses were performed to examine the expression of the p-Akt, p-S6K1, p-S6, and p-4EBP1, and p-Erk1/2 following the treatments as an indication for the signal pertur-bation. Combination treatment of both BEZ235/RAD001 and BEZ235/SCH66336 greatly inhibited the expression of phospho-S6 as compared to corresponding single treatment suggesting that inhibition of multiple signal nodes lead to enhanced attenuation of translational machinery and anti- proliferative effect. In vivo studies of the combination treatment are under-way. In summary, BEZ235 is an active dual PI3K/mTOR inhibitor however when combined with inhibition of a parallel signaling pathway such as Ras/Raf/MAPK with SCH66336 they exerted synergistic anti-tumor effect. Pos-itive outcome from the in vivo experiments will pave way for development of future clinical trial for combination therapy.

ET-30. NON-LEAKY, REGULATED GUTLESS ADENOVIRAL VECTORS MEDIATED INDUCIBLE TRANSGENE EXPRESSION IN THE BRAIN IN VIVO AND IN DOG AND HUMAN GLIOMA CELLSWeidong Xiong,1 Marianela Candolfi,1 Kurt Kroeger,1 Mariana Puntel,1 Donna Palmer,2 Phillip Ng,2 Pedro Lowenstein,1 and Maria Castro1; 1Gene Therapeutics Research Institute, Cedars-Sinai Medical Center and UCLA, Los Angeles, CA, USA; 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA

In view of recent advances in gene therapy technologies, the use of regu-latable, non-leaky expression systems is becoming a critical requisite to safe and effective clinical gene therapy. Glioblastoma multiforme (GBM) is a dev-astating brain tumor for which there is no cure. Although serotype 5 adeno-viral (Ads) have been used successfully in clinical trials for GBM (Immonen et al., Mol Ther., 2004), anti-viral vector immune responses limit the clinical efficacy of first generation Ads. In the present work, we have engineered a novel regulatable, non-leaky system consisting of a tetracycline-dependent reverse-transactivator, rtTA2S-M2, in combination with Tet-repressor, tTSKid, under the control of the murine cytomegalovirus (mCMV) pro-moter, encoded within high capacity, helper-dependent adenovirus vec-tors (HC-Adv). We developed two HC-Adv encoding the b-galactosidase (HC-Ad-mTetON-bgal) and the therapeutic transgene, i.e., human soluble Flt3L (HC-Ad-mTetON-hsFlt3L), under the control of the regulatable TetON system driven by the mCMV promoter. We assessed b-Galactosidase (bgal) expression from HC-Ad-mTetON-bgal within the brain. HC-Ad-mTetON-bgal vector was injected into the striatum at 1 3 107 blue forming



units (BFU). Twenty-fours prior to injection, the rats were given drinking water with 2 mg/ml doxycycline (DOX). At the appropriate time points, rats were perfused and brains were homogenized for bgal enzyme activity assay, or post-fixed in 4% PFA for immunohistochemistry. Our in vivo data demonstrate that stringent regulation kinetics of bgal expression with co-localization of bgal within MAP-2 (neurons) and GFAP (astrocytes) immu-noreactive cells in the striatum. Non-leaky, regulated, long-term transduc-tion in the brain was attained even in the presence of a systemic immune response to Ads as would be encountered in the clinic. We also assessed inducibility, leakiness of expression of our HC-Ad in mouse GL26 cells, rat CNS1 cells, J3T dog glioma cells, established human glioma cell lines (U251, U87), and primary glioma cell cultures from intra-operative biopsies (IN859, IN2045) infected with either HC-Ad-mTetON-bgal or HC-Ad-mTetON-hsFlt3L (5,000VP/cell) and incubated with or without 1 mg/ml DOX for 72 h. Transgene expression levels were determined by immuno-cytochemistry and bgal enzymatic activity assay, or hsFlt3L concentration (ELISA). High level transgene expression was achieved in all the human glioma cells, and in rat CNS-1 cells. The expression of Flt3L was tightly regulated by DOX. hsFlt3L expressed from HC-mTetON-hsFlt3L was effi-ciently released by all the GBM cells. The levels of hsFlt3L in the presence of DOX were around 5–60 nmol/ml, while in the absence of DOX they were undetectable (p , 0.001). Our results in vivo and in vitro demonstrate that the rtTA2S-M2 transactivator in conjunction with IRES and tTSkid transcriptional silencer displays strong and stringent induction kinetics with negligible basal activity in the uninduced state. Taken together, our data strongly support the use of tightly regulated high-capacity adenoviral vectors in gene therapy approaches for the treatment of human GBM. Supported by NIH/NINDS Grant 1R01 NS44556.01, Minority Supplement NS445561; 1R21-NSO54143.01; 1UO1 NS052465.01to M.G.C.; NIH/NINDS Grants 1 RO1 NS 054193.01; RO1 NS 42893.01; U54 NS045309–01, and 1R21 NS047298-01 to P.R.L.

ET-31. HIGH-CAPACITY GUTLESS ADENOVIRAL VECTORS MEDIATE INDUCIBLE, NON-LEAKY EXPRESSION OF PSEUDOMONAS EXOTOXIN FUSED TO MUTATED IL-13Weidong Xiong, Marianela Candolfi, Kurt Kroeger, Mariana Puntel, Pedro Lowenstein, and Maria Castro; Gene Therapeutics Research Institute, Cedars-Sinai Medical Center and UCLA, Los Angeles, CA, USA

Glioblastoma (GBM) expresses IL-13a2R, not present in normal brain. The fusion of mutated IL-13 (muIL-13) to Pseudomonas exotoxin (PE) generates a chimeric cytotoxin that proved effective in preclinical models (Debinski et al., Nat Biotechol. 1998) The wild type form of human IL13 fused to PE has been tested in GBM clinical trials (Kunwar et al., J Clin Oncol. 2007; Kioi et al., Technol Cancer Res Treat. 2006). Since the half life of the toxin in vivo is short and the wild type human IL13-PE is not the optimum target for GBM, we wished to test the hypothesis that by expressing the mutated form of IL13 fused to PE from a high capacity gut-less Ad vector, we would develop an improved therapy for GBM. Further, GBMs usually recur, therefore, long term, stable and regulated delivery of muIL13-PE within the tumor mass constitutes an attractive therapeu-tic approach. We developed a high capacity adenoviral (HC-Ad) plasmid expressing PE fused to muIL-13 with high affinity for the glioma-associated IL-13a2R and negligible binding to the physiological IL13/IL4R. We con-structed HC-Ad plasmid; pSTK-mCMV-TetON-muIL4–TRE-muIL13–PE encoding domain II (cell entry and toxin activation) and domain III (cyto-toxicity) of PE linked to mutated IL13 (i.e., muIL-13). Constructs encode a mutated IL-4 that binds to IL13/IL4R to reduce any putative binding and concomitant toxicity of muIL-13–PE to normal cells. Expression is driven by the regulatable bidirectional TRE promoter, which is activated by a doxycycline (Dox)-dependent transactivator (TetON). To make this system non-leaky, we also encoded a trans-repressor that inhibits expression in the absence of Dox. During vector production, transgenes can also be expressed in the producer cells, we therefore rescued HC-Ads encoding muIL-13–PE toxin in EF2 expressing 293 cells that are resistant to the toxin. Producer cells were stably transfected with pCI-neo-EF2, which encodes the gene for ADP ribozilation-resistant elongating factor-2 (EF2). HC-Ad vectors were generated using 5mg of HC-Ad plasmid DNA that was linearized and trans-fected into EF2–producer cells. Transfection of 293 and COS-7 cells with pSTK-mCMV-TetON-muIL4–TRE-muIL13–PE resulted in Dox-dependent transgene expression, as determined by immunocytochemistry of muIL-4, muIL-13 and PE. Secretion of muIL-4 and muIL-13 was assessed by ELISA. Transgene expression was negligible without Dox. 293 and COS-7 cell via-bility was unaffected by pSTK-mCMV-TetON-muIL4–TRE-muIL13–PE; however, their conditioned media (secreted muIL13–PE) induced cytotox-icity in IL-13a2R expressing U251 and IN859 human glioma cells. We conclude that muIL-13–PE secreted from HC-Ad infected cells will be taken up by neighboring GBM cells which express the IL-13Ra2, constituting a powerful regulated, non-leaky targeted approach. Since HC-Ad vectors can sustain long term gene expression even in the presence of a systemic anti-Ad immune response (Thomas et al., Proc Natl Acad Sci. USA, 2000) as would

be encountered in patients, they are excellent candidates for implement-ing this approach in GBM clinical trials. Supported by NIH/NINDS 1R01 NS44556.01, Minority Supplement NS445561; 1R21-NSO54143.01; 1UO1 NS052465.01to M.G.C.; 1 RO1 NS 054193.01; RO1 NS 42893.01; U54 NS045309-01, and 1R21 NS047298-01 to P.R.L.

ET-32. TARGETED CANCER-GENE THERAPY USING A HIF-DEPENDENT ONCOLYTIC ADENOVIRUS ARMED WITH INTERLEUKIN-4Michele Kyle,1 Zhaobin Zhang,2 Eric Sandberg,2 Narra Devi,2 Daniel J Brat,3 Zhiheng Xu,4 Mourad Tighiouart,4 Jeffrey Olson,2 Erwin Van Meir,2 and Dawn Post1; 1Dept. of Neurosurgery, SUNY Upstate Medical University, Syracuse, NY, USA; 2Dept. of Neurosurgery, Emory University, Atlanta, GA, USA; 3Dept. of Pathology, Emory University, Atlanta, GA, USA; 4Biostatistics Research and Informatics, Emory University, Atlanta, GA, USA

Hypoxic/HIF-active tumor cells are an important therapeutic target for which there are no approved treatments. These tumor cells are a viable and aggressive subpopulation associated with increased patient mortality, tumor growth, malignancy, and therapeutic resistance. We previously devel-oped an oncolytic adenovirus (HYPR-Ad) for the specific killing of hypoxic/HIF-active tumor cells. We then created a second-generation HYPR-Ad armed with an interleukin-4 gene (HYPR-Ad-IL4) by placing the Ad E1A viral replication and IL-4 genes under the regulation of a bidirectional hypoxia/HIF-responsive promoter. The IL-4 cytokine was chosen for its potent anti-tumor activity including the induction of a host anti-tumor immune response and anti-angiogenic activity. In culture, HYPR-Ad-IL4 exhibits hypoxia-dependent regulation of E1A and IL-4 protein expression, viral replication, and cytolysis in a panel of brain tumor and normal cell lines. HYPR-Ad-IL4 treatment of established s.c. human glioma xenografts by intratumoral injection results in rapid and maintained tumor regres-sion with the same potency as that of wild-type dl309–Ad. HYPR-Ad-IL4 treated tumors display extensive necrosis, fibrosis, and widespread viral replication. Additionally, these tumors contained a distinctive leukocyte infiltrate and prominent hypoxia. The anti-tumor efficacy of HYPR-Ad-IL4 was next evaluated using orthotopic human glioma xenograft models. In Study 1, LN229 glioblastoma cells were pre-infected in culture with HYPR-Ad-IL4, dl309–Ad, or PBS and then injected intracranially into nu/nu mice. The average survival of mice in the PBS, dl309–Ad, and HYPR-Ad-IL4 groups was 44.4, 114.6, and 112.7 days, respectively. In Study 2, LN229 tumors were established intracerebrally in nu/nu mice using a guide-screw system. Two weeks later mice were injected intratumorally with HYPR- Ad-IL4, dl309–Ad, or PBS. Treatment was once/week for 3 weeks. The average survival of mice in the PBS, dl309–Ad, and HYPR-Ad-IL4 groups was 29, 38, and 40 days, respectively. We are currently refining the virus injection dose and schedule to improve the therapeutic response and will also evaluate a host anti-tumor immune response and toxicity to surround-ing normal brain tissue. The use of an oncolytic Ad that locally delivers IL-4 to tumors is novel and we expect that HYPR-Ad-IL4 will have broad therapeutic use for all solid tumors that have hypoxia or active HIF, regard-less of tissue origin or genetic alterations.

ET-33. CHRONIC CONVECTION-ENHANCED DELIVERY OF TOPOTECAN PROVIDES SURVIVAL BENEFIT IN A RAT GLIOMA MODELKim Lopez,1 Marcela Assanah,1 Peter Canoll,2 and Jeffrey Bruce1; 1Dept. of Neurological Surgery, Columbia University Medical Center, New York, NY, USA; 2Pathology, Columbia University Medical Center, New York, NY, USA

Early-phase clinical studies have shown the efficacy of convection- enhanced delivery (CED) of various agents in patients with glioblastoma multiforme (GBM). Although nearly all studies involved short-term infu-sion of agents, chronic delivery could maximize drug distribution and pro-vide a more physiologically sound strategy for long-term tumor control. Based on our success with CED of topotecan in rat glioma models as well as in a Phase I clinical trial, we investigated the safety and efficacy of chronic CED in a novel rat glioma model that utilizes glial progenitor cells. PDGFB-GFP retrovirus was injected into the subcortical white matter of adult rats to form GBMs by targeting glial progenitor cells. At 7 days post-injection (DPI), 4 groups commenced 7-day CED of PBS, 7-day CED of 136 mM topotecan (short-term, high-dose group; 136 mM 5 maximum tolerated dose), 21-day CED of 136 mM topotecan (long-term, high dose), or 7-day CED of 136 mM topotecan followed by 14-day CED of 13.6 mM topotecan (long-term, high-to-low dose). A separate cohort of animals (n 5 6) was sacrificed at 7 DPI to document presence of tumors prior to treatment. Sur-vival data was analyzed via Kaplan-Meier method with log-rank test. Tissue was analyzed via histopathology and immunohistochemistry. All animals



sacrificed at 7 DPI had small, histologically evident tumors. PBS-treated animals uniformly died of large GBM-like lesions and had a median survival of 16 days (n 5 6) vs. 58 days (n 5 5) for animals given short-term 136 mM topotecan infusion (p , 0.001). Three of five animals in the long-term, high dose group died within 4 days of the 2nd pump implantation (16–18 DPI). These 3 animals were also observed to be seizing for a few hours soon after the 2nd pump implantation. Histologic analysis of these brains revealed massive necrosis at the treatment site with no identifiable tumor cells. There are 2 long-term survivors in this group (80 DPI). One of six animals in the long-term, high-to-low dose group died at 27 DPI (6 days after implanta-tion of the 2nd pump containing 13.6 mM topotecan). Histology revealed a bacterial abscess at the treatment site with no identifiable tumor. This group contains 5 long-term survivors (68 DPI). Short-term CED of high-dose topo-tecan provided a significant survival advantage and extending treatment beyond 7 days provides an even greater advantage. However, a continuous infusion of high-dose topotecan was poorly tolerated with 60% of animals succumbing to drug toxicity. An alternative dosing schedule, which starts with the maximum tolerated dose but is maintained chronically with a lower dose, provides the same survival advantage but with significantly less drug-induced morbidity and mortality. Chronic, maintenance convection-enhanced delivery of topotecan is effective in our rat glioma model and warrants further investigation in clinical trials.

ET-34. TARGETED OPENING OF THE BLOOD-BRAIN BARRIER BY PHOTODYNAMIC THERAPYHenry Hirschberg,1 Marlon Mathews,2 Qian Peng,3 and Steen Madsen4; 1University of California, Irvine, Irvine, CA, USA; 2Neurological Surgery, University of California, Irvine, Orange, CA, USA; 3The Norwegian Radium Hospital, Oslo, Norway; 4University of Nevada, Las Vegas, NV, USA

Eradication of infiltrating glioma cells poses a significant clinical chal-lenge that is unlikely to be solved using conventional treatment regimens. This is due to the fact that these migratory cells are protected by the blood-brain barrier (BBB) which prevents the delivery of chemotherapeutic agents or photosensitizers. We have evaluated the ability of aminolevulinic acid (ALA) mediated photodynamic therapy (PDT) to selectively open the BBB. This will permit access of chemotherapeutic agents to brain tumor cells, but limit their penetration into normal brain remote from the site of illu-mination. ALA-PDT was performed on non tumor bearing inbred Fisher rats at increasing fluence levels. Post-contrast T1 MRI scanning was used to monitor the degree of BBB disruption which can be inferred from the intensity and size of the contrast agent visualized. Contrast agents of several molecular weights were employed. F98 tumor cells were also implanted into the brains of some animals following PDT and chemotherapeutic agents were thereafter administered. PDT at fluence levels of 9 J showed no impact on the BBB barrier. In contrast at a fluence of 17–26 J significant contrast was observed. No effect on the BBB was observed if 26 J of light were given in the absence of ALA. The BBB was found restored 72 h after PDT. The ability of the F98 cells to form tumors is being studied following PDT and chemotherapy for various agents and fluence levels. PDT was highly effec-tive in opening the BBB in a limited region of the brain. The degradation of the BBB appeared 1–2 h following treatment and was temporary in nature lasting for an interval of 48–72 h.

ET-35. MENINGIOMA GROWTH INHIBITION BY COMBINATION HYDROXYUREA AND CALCIUM CHANNEL ANTAGONIST USING A NOVEL INTRACRANIAL MENINGIOMA LUCIFERASE-EXPRESSING MOUSE MODELRandy Jensen, Issac Elam, Bryan Ragel, and David Gillespie; Neurosurgery, Huntsman Cancer Institute, Salt Lake City, UT, USA

We have demonstrated that combining hydroxyurea (HU) and calcium channel antagonists results in significantly growth inhibition of malignant meningioma growth in a subcutanaeous flank model. Demonstrating this effect in intracranial model is necessary before moving to human clinical trials. We have developed meningioma cell lines that constitutively express luciferase, making it relatively easily to test the efficacy of these agents on intracranial meningioma growth. A reporter plasmid under the control of a modified thymidine kinase promoter that consistently expresses luciferase was stably transfected into primary and malignant meningioma cell lines. These tumor cell lines were injected stereotactically into frontal convexity and skull base locations. These cells were treated with HU and/or either diltiazem or verapamil. The activity of the tumors was assessed weekly by measuring the luciferase activity using an IVIS imaging system. Selected ani-mals were also imaged with magnetic resonance (MR) imaging to correlate with the luciferase imaging. After six weeks the mouse brains containing the tumors were harvested. Tumor sections were analyzed using electron, light microscopy, and immunohistochemistry for EMA, vimentin, MIB proliferative index. and vascular density. Luciferase activity was correlated

with increased tumor growth over time and with the MR imaging studies. Intracranial tumors were histologically consistent with meningiomas by light, electron microscopy and immunohistochemistry. Preliminary find-ings with the intracranial model shows decreased luciferase activity with combination therapy compared to treatment with HU alone. We also dem-onstrated a decrease in the MIB-1 ratio and a decrease in microvascular density. Meningioma cells can be stably transfected with a constitutively active luciferase reporter. This provides a reliable model for measuring of intracranial meningioma growth that potentially could be used for assess-ment of various treatment modalities. The addition of diltiazem or vera-pamil to HU augments meningioma growth inhibition in vivo by decreasing proliferation and microvascular density. These results suggest a possible role for these drugs as an additional adjuvant therapy for recurrent or unresect-able meningiomas.

ET-36. DELTA-24–RGD IN COMBINATION WITH RAD001 INDUCES AUTOPHAGIC CELL DEATH AND 80% LONG-TERM SURVIVORSMarta Alonso,1 Candelaria Gomez-Manzano,1 Hong Jiang,1 Jing Xu,1 Yuji Piao,1 Tomohisa Yokoyama,1 Seiji Kondo,1 W.K. Alfred Yung,1 and Juan Fueyo2; 1University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Neouro-Oncology, U.T. M.D. Anderson Cancer Center, Houston, TX, USA

In this study we sought to determine whether the oncolytic adenovirus Delta-24–RGD in combination with Everolimus (RAD001) would result in an enhanced anti-glioma effect in vivo. The in vitro cytotoxic effect and rep-lication properties of Delta-24–RGD alone or in combination with RAD001 were assessed in U87 MG glioma cells. Cell cycle profiles were assessed by flow cytometry. Autophagy was evaluated by analysis of acidic vesicu-lar organelles and ATG5 levels. Athymic mice bearing glioma xenografts received a single intratumoral injection of Delta-24–RGD and/or RAD001. MRI and pathologic examination of the brain were used to assess the exis-tence and characteristics of tumors and the anti-glioma effect. Expression of adenoviral proteins and ATG5 were used to assess the replication of Delta-24–RGD, and the induction of autophagy in vivo. Viability assays showed that Delta-24–RGD antitumoral activity is synergistically enhanced by combination with RAD001. Interestingly, combination treatment of Delta-24–RGD with RAD001 induced autophagy in vitro. In vivo we showed that Delta-24–RGD improved survival of tumor-bearing animals in a dose- dependent manner. Of significance, RAD001 enhanced the antiglioma effect of Delta-24–RGD and resulted in 80% long-term survivors. Immu-nostaining of the treated tumors showed the upregulation of ATG5 in the treated tumors indicating the therapeutic induction of autophagy. This is the first report showing that Delta-24–RGD plus RAD001 causes autophagic cell death, and dramatically increases survival of glioma-bearing animals. Our data support the further testing of this multimodal therapy in a Phase I clinical trial.

ET-37. TRANSFECTION OF SCHWANN CELLS WITH A RADIATION-INDUCIBLE PROMOTER EXPRESSING TNF- RESULTS IN CELL DEATH AFTER RADIATION TREATMENT IN VITROMatthew Rendel,1 Ania Pollack,1 and Hinrich Staecker2; 1Neurosurgery, University of Kansas Medical Center, Kansas City, KS, USA; 2Otolaryngology, University of Kansas Medical Center, Kansas City, KS, USA

Vestibular schwannomas are histologically benign vestibular nerve tumors commonly associated with Neurofibromatosis Type 2 (NF2). How-ever, because of their location, vestibular schwannomas can cause signifi-cant neurological deficit secondary to mass effect. Stereotactic radiosurgery (SRS) can be effective treatment when the tumors are of small size, but in large tumors carries increased risk of morbidity with damage to vital structures of the brain and spinal cord. Additionally, SRS as a therapeutic strategy aims to prevent tumor growth and does not result in lysis of tumor cells. Therefore, tumor bulk remains. Tumor Necrosis Factor-a (TNF-a) is directly apoptotic and necrotic to some tumor cell types. In Schwann cells, TNF-a has been shown to up-regulate apoptotic pathways, resulting in some cell death, but not necessarily be completely toxic. We have utilized an adenovirus vector expressing TNF-a driven by a radiation-inducible promoter to examine effects of TNF-a on Schwann cells after radiation exposure. Recent work has examined the response of primary cultures of human schwannomas to radiation alone and combined therapy with the TNF-a expressing adenovector followed by low-dose radiation. After suc-cessfully culturing human Schwannoma cells, the cells were then transfected with the adenovirus carrying TNF-a. The tissue cultures were then treated with a 2- or 5-Gray (Gy) radiation dose. Cell survival was calculated. Cell counts after viral treatment, prior to radiation treatment, showed cell loss secondary to transfection alone, with a cell survival of 53%–83%. Radia-



tion treatment resulted in significant lysis of schwannoma cells in vitro, with near total cell loss (cell survival of 0%–8%). Similar results were obtained with 2- and 5-Gy doses. This experimental data lays the groundwork for the clinical implication that, with gene therapy, vestibular schwannomas may be treated with the TNF-a vector prior to radiation treatment. With the use of the radiation-inducible promoter, the potential toxicity of TNF-a to non-radiated tissue should be limited. Previous studies have shown that an adenovirus carrier can successfully deliver genetic material to the ves-tibular nerve after injection through the round window of the inner ear. Additionally, gene transfection has been successfully accomplished by direct intratumoral injection of viral vectors with limited systemic toxicity. With successful vector delivery, lower radiation doses perhaps then may be utilized with increased effect and fewer side effects. Results also suggest that significant tumor size reduction may occur secondary to tumor lysis. Presently, we are developing methods to examine the effects of retarget-ing vectors specifically to schwannomas and testing the response of com-bined TNF-a and radiation therapy in an in vivo model of schwannomas/ neurofibromas. This treatment approach is potentially rapidly translatable to human clinical trials since a similar TNF-a producing vector has already been successfully used in a variety of ongoing human clinical trials for solid tumors (TNFerade, Genvec Inc.).

ET-38. BIODEGRADABLE MICROSPHERES LOADED WITH GLEEVEC (STI571) INHIBIT THE GROWTH OF HUMAN GLIOMA TUMORS IN AN ORTHOTOPIC MOUSE MODELLata G Menon,1 Seung-Ki Kim,2 Ofra Benny,3 Peter Black,4 Marcelle Machluf,5 and Rona Carroll1; 1Neurosurgery, Brigham and Womens Hospital, Boston, MA, USA; 2Department of Neurosurgery, Seoul National University Hospital, South Korea; 3Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Israel; 4Neurosurgery, Dana-Farber/Brigham and Women’s Cancer Center, Boston, MA, USA; 5Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, Israel

Glioblastomas (GBM) are lethal tumors with a 12–18 month median survival despite aggressive treatment. In an effort to develop new thera-peutic strategies to treat GBM, we have designed poly (lactic-co-glycolic acid (PLGA) microparticles, which deliver STI571 (GLEEVEC, provided by Novartis), a small molecule kinase inhibitor. STI571 targets the activated Abl oncoprotein and certain members of the subgroup III receptor tyrosine kinase family, including the receptors for PDGF. Over expression and /or activation of PDGF receptors plays a prominent role in the progression to glioblastoma. This suggests that PDGF receptors might be a therapeutic target for glioblastoma. The local continuous release of STI571 at the tumor site overcomes many obstacles associated with systemic delivery, and elimi-nates the need for daily administration and prolongs treatment efficiency. Our studies demonstrate the potential and therapeutic efficacy of STI71– loaded microspheres in mouse models of glioma. Polymeric microspheres were prepared from various compositions of poly (lactic-co-glycolic) acid (PLGA) and loaded with STI571 in concentrating ranging from 0.25%-0.5% w/w STI571 release profiles and biological activity were confirmed both in vitro & in vivo. Microspheres were prelabeled with 6-coumarin to locate in the tumor bed by immunoflourescence. The human glioblastoma cell line U87–MG and murine glioblastoma cell line GL261 were used for all studies. Therapeutic efficacy of PLGA-STI571 microspheres was exam-ined in two subcutaneous and an orthotopic human glioblastoma xenograft model. In vitro, STI571 released from PLGA microspheres remained bio-logically active leading to a decrease in cell proliferation and cell viability. In addition, the levels of phophorylated -PDGFR-b in GL261 and U87–MG cells treated with STI571 were reduced compared to the control. In vivo, a significant inhibition of subcutaneous glioma tumor growth was observed by a single local injection of PLGA microspheres loaded with a low concen-tration of STI571 (88% for U87 and 79% for GL261). Intracranial adminis-tration of PLGA-STI571 led to a 79% reduction in U87MG tumor volume. MRI was successfully implemented for monitoring the dynamic changes of tumor growth and inhibition in response to the slow release treatment of the polymeric microspheres. Immunohistochemical analysis demonstrated a marked decrease in proliferation index and tumor vessel density in the subcutaneous model, and induction of apoptosis in the intracranial model. This is the first study to demonstrate the therapeutic efficacy of the local delivery of STI571 using a polymeric delivery system. Our results indicate that polymeric microspheres are a powerful therapeutic delivery system, which allows the administration of drugs directly to the tumor site, leading to significant tumor regression. Moreover, this system offers an improved alternative to the current therapies by reducing drug doses, and using single administration. In the future this system might be used in combination with other anti-cancer drugs in order to further improve efficacy.

ET-39. SAFETY OF ERLOTINIB FOLLOWING CONVECTION-ENHANCED DELIVERY TO RODENT NAIVE BRAIN: IMPLICATIONS FOR HUMAN GLIOBLASTOMA THERAPYIoannis Karampelas,1 Eleni Souliopoulos,1 John Boockvar,1 Mark Edgar,2 and Mark Souweidane1; 1Neurological Surgery, Weill Medical College of Cornell University, New York, NY, USA; 2Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

The epidermal growth factor receptor (EGFR) is genomically ampli-fied and/or overexpressed in 40%–50% of human glioblastoma tumors. Erlotinib is a small molecule EGFR tyrosine kinase inhibitor that has shown strong blockade both of the wild type and one mutant variant, EGFRvIII. This agent has yet to be tested with respect to toxicity or efficacy following convection enhanced delivery (CED), a method of direct drug delivery that distributes therapeutics to the brain interstitial space. Here, we describe our initial results of toxicity of Erlotinib delivered by CED to the rodent naïve brain. Naïve nude rats underwent CED of escalating concentrations of erlotinib (n 5 3/concentration level; 0.5 mmol/L, 1.0 mmol/L, 2.0 mmol/L, and 5.0 mmol/L, vol. 5 6 mL) at 0.1 ml/min. To investigate potential toxic-ity the animals were given daily gross neurological exams. All animals were euthanized 2 weeks post-infusion via an i.p. injection of sodium pentobar-bital. Histopathologic sections were assessed for microscopic evidence of injury or reactive changes. All animals tolerated CED of erlotinib without surgical morbidity. No clinical neurologic deficits were noted. No histo-pathological changes, other than those associated with cannula insertion, were detected upon evaluation by a blinded neuropathologist. Local tissue toxicity of erlotinib following its administration via CED in the rat striatum at concentrations with known in-vitro efficacy was non-detectable. These promising preliminary data encourage us to investigate additional param-eters of erlotinib-based CED (e.g., dose optimization) in glioma therapy. CED may be an ideal method for the distribution of potent chemotherapeu-tic agents such as erlotinib to tumors of the CNS.

ET-40. PRE-CLINICAL EVALUATION OF ERLOTINIB AND 13–CIS-RETINOIC ACID FOR THE TREATMENT OF MALIGNANT GLIOMASWill R. Voelzke, Jason T. Graves, M. Brian Hemphill, Denise M. Gibo, Waldemar Debinski, W. Jeffrey Petty, and Glenn Lesser; Wake Forest University, Winston-Salem, NC, USA

As single agents, erlotinib and 13–cis-retinoic acid (CRA) have estab-lished clinical activity for the treatment of malignant gliomas. Both inhibit epidermal growth factor receptor (EGFR) signaling and repress cyclin D1 protein expression through distinct mechanisms in various epithelial cancers, such as aerodigestive tract cancers. A clinical trial combining erlotinib with a retinoid has demonstrated promising results for the treat-ment of aerodigestive tract cancers. To evaluate a similar strategy for treat-ing malignant gliomas, the growth inhibitory activity of erlotinib and CRA, alone or in combination, was studied in glioblastoma multiforme (GBM) cell lines. Expression of a novel retinoic acid receptor beta isoform, RAR beta1 ,́ that has been linked to retinoid sensitivity was studied in a panel of human GBM cell lines (SNB19, T98G, U-138 MG, and U-251 MG) using an isoform-specific antibody. Cell viability was measured before and after treatment with pharmacologically achievable dosages of erlotinib alone (1, 3, and 5 mM), CRA alone (2 mM), or the combination. Cyclin D1 and EGFR protein expression was also assessed before and after treatment with each drug alone or the combination. The majority of GBM cell lines abundantly expressed the RAR beta1 protein. CRA treatment alone at a dosage of 2 mM led to statistically significant growth suppression in T98G cells (21%, p 5 0.029) with a non-significant trend to decreased proliferation in each of the other cell lines. Treatment with erlotinib as a single agent did not significantly repress proliferation in any of the examined cell lines over the examined dose range (up to 5 mM). However, co-treatment with CRA at 2 mM and erlotinib at 5 mM led to statistically significant growth suppression in each of the examined cell lines (SNB19: 65%, p 5 0.049; T98G: 37%, p 5 0.008; U-138 MG: 65%, p 5 0.012; and U-251 MG: 36%, p 5 0.022). Cooperative repression of cyclin D1 protein was also observed at the same dosages. Combining erlotinib and CRA leads to cooperative repression of GBM cellular proliferation and repression of the critical downstream tar-get, cyclin D1. A phase I clinical trial combining these drugs in patients with relapsed, malignant gliomas is currently in development. RAR beta1 expression will be measured in tumor tissue as a potential predictive marker to select patients for this treatment.



ET-41. PHOSPHODIESTERASE TYPE 4 DRIVES BRAIN TUMOR GROWTH IN VIVOPatricia Goldhoff, Nicole Warrington, David Limbrick, Andrew Hope, Mark Woerner, Erin Jackson, David Piwnica-Worms, and Joshua Rubin; Washington University School of Medicine, St. Louis, MO, USA

As outcomes from malignant brain tumors continue to be limited by poor survival and treatment-related toxicity, novel approaches to cure are essential. Recent studies from our laboratory identified low levels of intrac-ellular cAMP as necessary for brain tumor growth in vivo and cAMP elevat-ing drugs such as the phosphodiesterase type 4 (PDE4) inhibitor Rolipram as potent anti-tumor agents. These findings suggested that PDE4 is an important therapeutic target; therefore we investigated the role of PDE4 in human brain tumors. We found that PDE4 is widely expressed in brain tumors including in astrocytomas of all grades, medulloblastomas of all histological subtypes and oligodendrogliomas. Overexpression of PDE4 in U87 glioblastoma and Daoy medulloblastoma cells resulted in accelerated tumor growth in vitro and in vivo. Furthermore, five-month survival curves for mice bearing intracranial xenografts of U87 glioblastoma cells treated with 30 Gy conformal radiation therapy (XRT, 6 3 5 Gy fractions), temo-zolomide (TMZ, 21 mg/kg/day 3 5 days/month), Rolipram (Rol, 5 mg/kg/day) or combinations thereof, revealed that Rol had equal efficacy to XRT alone or in combination with TMZ. Moreover, Rol enhanced sur-vival when added to combined XRT and TMZ. Insight into the biology of treatment responses was garnered from parallel bioluminescence imaging that indicated tumor growth was slowed by Rol and TMZ, but transiently arrested by XRT. Subsequently, tumors were observed to regain exponential growth prior to mice succumbing to their disease. Conversely, when Rol was added to the combination of TMZ and XRT, there was no recovery from the growth arrest and tumors were observed to regress. Together these data indicate that PDE4 is widely expressed in brain tumors where it stimulates their growth. Further, these results demonstrate that addition of Rol to cur-rent combinatorial therapies can target brain tumor PDE4 and may provide greater disease control and prolonged survival.

ET-42. CD74 IS PREFERENTIALLY OVEREXPRESSED IN SHORT CULTURED CELLS FROM TEMOZOLOMIDE-RESISTANT GLIOBLASTOMA XENOGRAFTS AND THE EXPRESSION IN PATIENT SAMPLES IS ASSOCIATED WITH POOR OVERALL SURVIVALGaspar Kitange, Brett Carlson, Mark Schroeder, Bruce Morlan, Paul Decker, Karla Ballman, Caterina Giannini, and Jann Sarkaria; Mayo Foundation for Medical Education and Research, Rochester, MN, USA

The methylating agent temozolomide (TMZ) has demonstrated signifi-cant cytotoxicity against glioma, and is currently used for initial treatment of glioblastoma (GBM). Over-expression of O6-methyguanine-DNA-meth-yltransferase (MGMT) is an important determinant of TMZ resistance but the expression of MGMT is not absolutely associated with TMZ responsive-ness. We have developed a panel of GBM xenografts from patient tumors, and similar to clinical results, we have identified at least one xenograft line (GBM14) that is sensitive to TMZ despite an unmethylated MGMT pro-moter. To identify other gene products that are involved in TMZ resistance and sensitivity, we evaluated the gene expression in 2 xenograft lines that are sensitive to TMZ (GBM12 and 14) or resistant to TMZ (GBM 43 and 44) before and in after TMZ treatment. Global gene expression was analyzed with microarray technology using the Affymetrix Human Genome U133 Plus 2.0 GeneChip Set, covering more than 54,000 gene transcripts, cor-responding to almost 22,000 known genes. Differentially expressed genes were ranked according to the p-values and only the first 200 high-ranked probes (genes) were considered for further analysis. Hundred-twenty of the high-ranked 200 probes were overexpressed and 80 probes were underex-pressed in the resistant relative to sensitive cells before exposure to TMZ. Among the overexpressed gene probes in the TMZ resistant cells was MGMT and CD74, which is a membrane receptor of macrophage migra-tion inhibitory factor (MIF). Evaluation of CD74 expression by RT-PCR revealed that CD74 is expressed in 7 (30%) of 21 glioblastoma xenograft lines and in 4 (27%) of 15 established glioblastoma cell lines. Immunohis-tochemical evaluation of CD74 expression revealed strong expression in 8 (25%) of 32 glioblastoma patient samples, and high CD74 expression was associated with poor overall survival in this limited patient cohort (median survival 316 vs. 476 days; Log-rank p 5 0.05). Our findings confirm the importance of MGMT in the development of resistance against TMZ and also identify CD74 and other gene products as potential modulators of treat-ment response.

ET-43. NON-LEAKY, INDUCIBLE ADENOVIRAL-MEDIATED DELIVERY OF A MUTATED FORM OF IL-13 (IL13.E13.K) FUSED TO PSEUDOMONAS EXOTOXIN INDUCES REGRESSION OF INTRACRANIAL HUMAN GLIOBLASTOMA XENOGRAFTSMarianela Candolfi,1 Weidong Xiong,1 Akm Muhammad,1 Mariana Puntel,2 Chunyan Liu,1 Kurt Kroeger,2 Sonali Mondkar,1 Ronald Rodriguez,3 Waldemar Debinski,4 Pedro Lowenstein,2 and Maria Castro2; 1Cedars Sinai Medical Center-UCLA, Los Angeles, CA, USA; 2Gene Therapeutics Research Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA; 3Department of Urology, Medical Oncology, Radiation Oncology and Molecular Radiation Sciences, Viral Oncology, Cellu, Johns Hopkins University School of Medicine, MD, USA; 4Brain Tumor Center of Excellence, Wake Forest University, NC, USA

Glioblastomas (GBMs) overexpress IL13alpha2R, which is absent in the normal brain. Attempts to target this receptor and treat recurrent GBM in human patients have used native human IL-13 fused to Pseudomonas exotoxin A (PE), hIL-13–PE38QQR, or Cintredekin Besudotox, adminis-tered locally as protein formulation (Kunwar et al., J Clin Oncol. 2007 (25): 837–844). We wished to test the hypothesis that adenovirus-mediated expression of the mutated form of human IL13 (IL13.E13K) fused to PE, which has been previously shown to target specifically human GBM (Nat Biotechnol. 1998 16(5):449–453), would result in increased survival and tumor regression in human xenograft GBM models. We developed an ade-novirus vector (Ad) expressing a chimeric toxin (muIL-13–PE) composed of PE fused to a mutated form of hIL13 (IL13.E13.K, muIL-13). muIL13 binds to IL13alpha2R with high affinity and it has negligible binding for the physiological IL13/IL4R, which is present in several areas of the brain and can be targeted by the wild type hIL13–PE. We constructed the therapeutic Ad-muIL4–TRE-muIL13–PE that expresses the cytotoxin muIL-13–PE; specific for GBM cells, and, as an extra safety feature, a mutated IL-4 (IL4.Y124D, muIL-4) that blocks the binding of IL13 to the physiological IL13/IL4 receptor (Int J Oncol. 1999 15(3): 481–486). We also constructed a control vector that expresses muIL-4 and muIL-13 (i.e., Ad-muIL4–TRE-muIL13). Transgene expression is driven by the bidirectional TRE pro-moter, which is activated by the transactivator (TetON, expressed within Ad-TetON), in the presence of the inducer (i.e., DOX). We also encoded the tetracycline controlled transcriptional silencer, tTSKid, which inhibits basal levels of gene expression from the tetracycline inducible promoter. Induc-ible expression of muIL-4, as well as muIL-13–PE toxin was detected by immunocytochemistry in COS-7 and human GBM cells. Cell viability was reduced by 70% when the human GBM cells were incubated in the presence of Ad-muIL4–TRE-muIL13–PE in the “ON” state (Dox1), but remained unaffected in the “OFF” state, indicating that the expression of the chi-meric toxin can be tightly regulated. Ad-muIL4–TRE-muIL13–PE did not affect the viability of COS-7 cells, which do not express the IL13 alpha2 receptor. These results suggest that the cytotoxic effect of the chimeric is specific to glioma cells expressing IL13alpha2R and does not target the physiological IL13/IL4R expressed in control COS-7 cells. We also admin-istered Ad-muIL4–TRE-muIL13–PE1Ad-TetON in the striatum of nude mice, which were fed chow containing Dox to activate transgene expres-sion. muIL4, muIL13 and muIL13–PE toxin were detected in the mouse brain 7 days after the intracranial injection, with no toxicity, as determined by Nissl staining and immunocytochemistry of CD3 (T cells) and F4/80 (macrophages). Also, we administered Ads intratumorally into intracranial human U251 GBM xenografts in nude mice. Ad-muIL4–TRE-muIL13–PE significantly increased the survival of tumor-bearing mice when compared to saline-treated or mice treated with the control virus or with mice treated with the hIL13–PE used in the human GBM trial. All Ad-muIL4–TRE-muIL13–PE–treated mice survived for over 100 days after tumor implanta-tion. Our results suggest that regulated adenoviral delivery of muIL13–PE toxin to glioblastomas will lead to specific Dox-dependent cytotoxicity in IL-13alpha2R expressing-GBM cells and not to the surrounding non-neoplastic brain. Supported by National Institutes of Health/National Institute of Neurological Disorders & Stroke (NIH/NINDS) Grant 1R01 NS44556.01, Minority Supplement NS445561; 1R21–NSO54143.01; 1UO1 NS052465.01 to M.G.C.; NIH/NINDS Grants 1 RO1 NS 054193.01; RO1 NS 42893.01; U54 NS045309–01, and 1R21 NS047298–01 to P.R.L. M.C. is supported by NIH/NINDS 1F32 NS058156.01.

ET-44. RECEPTOR-TARGETED NANOVESICLE DELIVERY OF DOXORUBICIN TO INTRACRANIAL BRAIN TUMORSA.B. Madhankumar,1 Becky Slagle-Webb,1 Xinsheng Wang,1 Patti Zimmerman,2 Qing X. Yang,2 Jonas M. Sheehan,1 and James R. Connor1; 1Department of Neurosurgery, Pennsylvania State University College of Medicine, Hershey, PA, USA; 2Department of Radiology, Pennsylvania State University College of Medicine, Hershey, PA, USA

In our previous study we demonstrated that liposomes when conjugated to interleukin-13 (IL-13) will target and deliver chemotherapeutics to a sub-cutaneous glioma tumor model in mice much more effectively than conven-tional unconjugated liposomes. Based on this observation, we developed



intracranial brain tumor model in nude mice using human U87 glioma cells. Mice were administered unconjugated and conjugated liposomes carrying doxorubicin at a concentration of 15 mg/kg body weight subcutaneously to the tumor bearing mice. The tumor size was monitored weekly using MRI contrast. The survival and the weight of the mice were also documented. There is a pronounced reduction in the tumor volume in the mice adminis-tered IL-13 conjugated liposomes compared to those receiving the uncon-jugated liposomes. The difference becomes most pronounced 3 weeks after the tumor xenograft. The co-localization of liposomes with doxorubicin in the tumor was demonstrated by taking advantage of the select expres-sion of IL-13RA2 on the tumor cells and the endogenous fluorescence of doxorubicin. A group of mice were administered with IL-13 conjugated or unconjugated liposomes carrying doxorubicin. After 48 h the animals were sacrificed and the brain was dissected. Immunohistochemistry was performed for IL-13RA2 receptors in a section of the brain that included the tumor. Alexia flour conjugated secondary antibody (for mouse anti-human IL-13RA2) and liposomal doxorubicin (intrinsic red fluorescence) were co-localized on the tumor sections as observed by fluorescence microscopy indicating the selective delivery of doxorubicin through conjugated lipo-somes to the glioma tumors in a receptor specific manner. Finally, a model of the blood-brain barrier was used to demonstrate that the nanovesicles can cross this barrier intact. This approach is necessary to show the efficacy of this targeting platform for tumors in which the BBB is not yet compromised and as a potential use of the nanovesicle system as a surveillance mechanism to prevent recurrence. Using a Bovine-retinal endothelial cell culture model, we demonstrated the nanovesicles could be transcytosed through the model and maintain their toxicity to astrocytoma cells without harming the integ-rity of the BBB. These data show that IL-13 targeted nanovesicles could be a viable option for the treatment of brain tumors.

ET-45. A NOVEL, POTENT, AND SPECIFIC EPHRINA1–BASED CYTOTOXIN AGAINST EPHA2 RECEPTOR-EXPRESSING TUMOR CELLSJill Wykosky, Denise Gibo, and Waldemar Debinski; Neurosurgery, Wake Forest University School of Medicine, Winston-Salem, NC, USA

We previously demonstrated that the EphA2 receptor tyrosine kinase is an attractive molecular target in Glioblastoma multiforme (GBM), as it is over-expressed in a vast majority of patients and in multiple GBM tumor compartments, including tumor cells, neovasculature, and invading cells, but not normal brain (Mol Cancer Res. 2005 3:541–51). The expression of this receptor has been linked to poor survival of patients with GBM, as well as of patients with breast, prostate, lung, ovarian, esophageal, and renal cell carcinoma. Recombinant cytotoxins consisting of a ligand targeted to a tumor-specific molecule and a bacterial toxin derivative are emerging as a way to improve the outcome of patients with GBM. Being that EphA2 is an internalized receptor that is over-expressed in GBM, we have generated a novel EphA2-targeted conjugate cytotoxin composed of ephrinA1, a ligand for EphA2, and a genetically modified bacterial toxin, Pseudomonas exo-toxin A (PE38QQR). EphrinA1-Fc, a recombinant homodimeric form of the ephrinA1 ligand (R&D Systems), was chemically conjugated to PE38QQR (made in house) through the use of the two hetero-bifunctional cross-linking reagents succinimidyl trans-4–(maleimidylmethyl)-cyclohexane-1–carboxy-late (SMCC) and succinimydyl 3–(2-pyridyldithio)propionate (SPDP) (Cancer Res. 52:5379–5385, 1992). These cross-linking reagents react with free lysine residues of the protein chains, forming a stable thioether bond between the proteins. The major conjugate product consisted of one ephrinA1-Fc linked to one PE38QQR; other minor forms obtained from the reaction consisted of two to four PE38QQR molecules conjugated to ephrinA1-Fc. EphrinA1–PE38QQR exhibited extremely potent and dose-dependent killing of GBM cells over-expressing the EphA2 receptor in cell viability assays and clonogenic survival assays, with an average IC50 of ~10 pM. The conjugate was also effective in killing breast and prostate cancer cells that over-express EphA2. The cytotoxic effect of ephrinA1-PE38QQR was specific, since it was completely neutralized by an excess of ephrinA1 ligand. Moreover, normal human endothelial cells, breast cancer cells, and glioma cells that do not over-express EphA2 were not susceptible to the cytotoxin. This is the first attempt to create a cytotoxic therapy based on any of the ephrin ligands of either class, A or B. EphrinA1-PE38QQR is potent and specific, and forms the basis for the further clinical development of ephrinA1-based cytotoxins in the treatment of patients with GBM and other EphA2-expressing malignancies.

ET-46. RE-OXYGENATION OF HYPOXIC GLIOBLASTOMA MULTIFORME CELLS POTENTIATES KILLING EFFECT OF INTERLEUKIN 13–BASED CYTOTOXINS THROUGH RE-ESTABLISHMENT AND FURTHER UP-REGULATION OF IL13 RECEPTOR ALPHA2Tiefu Liu,1 Jaiozhong Cai,1 Denise Gibo,1 and Waldemar Debinski2; 1Neurosurgery, Wake Forest University School of Medicine, Winston-Salem, NC, USA; 2Wake Forest University School of Medicine, Winston-Salem, NC, USA

Glioblastoma multiforme (GBM), a high-grade glioma of poor progno-sis, is typically hypo-oxygenated in many areas of the tumor in spite of a very well developed neovasculature. Hypoxia has been considered one of the main factors determining solid tumors resistance to both radiation and che-motherapy. Recently, molecular targeted therapy using recombinant cyto-toxins containing bacterial toxins has demonstrated potential to improve the outcome of patients with GBM. We have previously generated several candidate drugs in a form of interleukin 13 (IL13)-based cytotoxin contain-ing derivatives of Pseudomonas exotoxin A (e.g., IL13-PE38QQR), diphthe-ria toxin (DT-IL13) or mutated IL13-based fusion toxin (e.g., DT-IL13QM). An IL13–based cytotoxin that is specifically directed towards a tumor- associated receptor expressed in large numbers in GBM, IL13Ralpha2 (IL13R), has yet to enter the clinic. Here, we investigated how hypoxia, a frequent pathobiological phenomenon occurring in all GBM patients, influ-ences the response of GBM cells to the IL13-cytotoxin. Thus, we analyzed the effect of hypoxia on GBM cells susceptibility to the killing by IL13 cytotoxins, the levels of immunoreactive IL13R and subsequent changes in these two parameters in cells returned to normoxic conditions. The experiments were performed in an Invivo2 (Ruskinn, Inc.) hypoxia work-station, and controlled by analyzing expression levels of hypoxia-inducible factor-1 alpha (HIF-1alpha). The cytotoxic activity of IL13-PE38QQR or DT-IL13QM on GBM cells under hypoxic conditions was significantly less potent when compared with that on normoxic cells. In U-251 MG cells, the IC50s (normoxia/hypoxia) of IL13-PE38QQR and DT-IL13QM were 259/466 and 8/74 pM, respectively. The U-251 MG, A-172 MG or SNB-19 GBM cells had HIF-1alpha protein induced after 2 hrs of hypoxia, with peak expression reaching at 6 hrs, which declined during the next 24 hrs and became undetectable after 48 hrs of hypoxia. We did not detect any changes in immunoreactive IL13R levels during the first 6 hrs, but we observed its large decrease after 24 hrs of hypoxia treatment. Importantly, when all three GBM cells were transferred back to normoxic conditions after 24 hrs of hypoxia, IL13R levels steadily increased during 2 hrs to 48 hrs recovery time while HIF-1alpha remained undetectable. In parallel to an increased IL13R expression, the sensitivity of U-251 MG cells to IL13-PE38QQR, DT-IL13 or DT-IL13QM increased 10 fold after hypoxic cells were placed back into normoxic conditions for 4 hrs, 6 hrs or 24 hrs when compared to cells maintained continuously in normoxia. Similar results have been obtained in A-172 MG GBM cells. Thus, our study demonstrates that hypoxia has a profound effect on the action of an IL13-based cyto-toxin in GBM cells that may be in part related to the levels of expression of IL13R. Furthermore, IL13R levels rebound under normoxic conditions with tendency to over-express the receptor at higher than background levels, which is associated with better responsiveness to IL13 cytotoxins. Our data suggest that a previously shown beneficial effect of normalizing solid tumor oxygenation/blood flow on chemotherapy may have its application in the treatment of GBM using IL13-based recombinant cytotoxins.

ET-47. PRECLINICAL AGAROSE COLONY FORMATION ASSAY DEVELOP DFMO-BASED DRUG COMBINATIONSYoshinori Kajiwara,1 Diane D. Liu,2 J. Jack Lee,2 and Victor A. Levin1; 1Neuro-Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Biostatistics, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Chemotherapy for gliomas remains limited because of a plethora of effective drug and drug combinations. Of current interest is alpha-diflu-oromethylornithine (DFMO, eflornithine) that irreversibly inactivates ornithine decarboxylase and the production of the polyamine putresecine. Polyamines have essential roles in DNA synthesis, transcription, and protein synthesis. Furthermore, DFMO has proven clinical efficacy against high-grade gliomas alone and in combination with PCV (procarbazine, CCNU, vincristine). While the interaction between nitrosoureas and DFMO has been widely studied, DFMO in combination with other cytotoxic, biolog-ics, and signal transduction inhibitors is less defined. To establish new two-drug combinations for DFMO, we developed a 3-dimensional aga-rose colony formation assay using GelCount™, a new cell colony counter for gels and soft agars. GelCount™ scans culture plates, counts colonies, and measures the diameter (mm) and density (OD) of the 3-dimensional colonies. The GelCount™ apparatus allows repetitive plate counting over time and through different conditions. We used U251MG, SNB19, and LNZ308 glioma cell lines and MiaPaCa pancreas and SW480 colon cell lines. 1,200 cells/ml were seeded into 12-well plates in DMEM-10% FBS



agarose gel. We studied DFMO, carboplatin, and SAHA, a histone deacety-lase inhibitor, over a 3-log dose range as single agents and in combination. We approximated area under the curve (AUC) as the sum of volumes (mm2 3 OD) in each plate, to calculate IC50 values (Prism 5 software). We report studies of five cell lines with different growth characteristics. Adenocarci-noma colonies were recognized with GelCount™ scanning at 3–4 days, while it took 6 to 7 days to detect glioma colonies. The growth rate of MiaPaCa and SW480 cells was rapid and 100 colonies could be counted in 5–6 days; glioma lines slower and required 9–10 days to count 100 colo-nies. Based on each colony growth curve, we added the first drug on day 2 (adenocarcinoma lines) or day 6 (glioma lines) and the second drug 48 hours later. Colonies were counted 3, 5, and 7 days after the second drug. Alteration of colony AUC was observed after 3 days. Reliable log dose ver-sus AUC curves were observed for all drugs studied. For single agents we found, based on IC50 values (mM 6SD), that DFMO was more effective against LNZ308 (48 631) and MiaPaCa (37 622) than SNB19 (237 694), U251MG (112 637) and SW480 (190 694) colonies. In comparison, glioma colonies were more sensitive to carboplatin than adenocarcinoma colonies with IC50 values of SNB19 (3.0 61.8), U251MG (2.1 61.5), LNZ308 (4.7 65.0), MiaPiaCa (21.5 619.3), and SW480 (28.4 619.2). SAHA was effec-tive in SNB19 (2.6 61.4), U251MG (4.4 62.9) and LNZ308 (1.2 61.3) glioma colonies and MiaPaCa (1.6 60.9) and SW480 (2.7 60.2) adenocar-cinoma colonies. In combination studies, DFMO followed by carboplatin produced additive to synergistic effects in high-dose combinations at high colony densities. DFMO before SAHA also produced additive to synergistic effects but independent of colony density, while the combination of SAHA before DFMO was only effective at in high-dose combinations. We present an efficient preclinical methodology to develop drug combinations using GelCount™ that are more quantitative than traditional colony assays that rely on staining and visual recognition of colony size and number.

ET-48. PRECLINICAL STUDY OF BEVACIZUMAB AND BORTEZOMIB COMBINATION IN MALIGNANT GLIOMADaniela Bota, Jill Maxwell, Stephen Keir, Nancy Bullock, Krystle Horne, James Vredenburgh, and Henry Friedman; Duke University, Durham, NC, USA

There is an unmet clinical need for the therapy of recurrent malignant. Novel therapies are developed in an attempt to target specific molecular mechanisms involved in abnormal signaling and resistance to apoptosis, and to overcome resistance to traditional chemotherapy drugs, such as Temozolomide. The proteasome is one of the key regulators of cell func-tion, and proteasome inhibition leads to apoptotic cell death in a number of malignant cell lines by inactivation of survival protein nuclear factor kB (NF- kB), increased activity of p53 and Bax protein and accumulation of cyclin-dependent kinase inhibitors. Proteasome inhibition reduces angiogen-esis by decreasing VEGF and IL-6 production by the endothelial cells, and modulates hypoxia by regulating HIF-1a expression. Vascular endothelial growth factor (VEGF) is present on the cell surface and around malignant gliomas. Glioma cells, critical in driving proliferation and invasion, secrete VEGF in order to stimulate angiogenesis, and the invasive phenotype can be effectively suppressed by VEGF inhibitors. Our hypothesis is that a drug combination targeting both proteolysis and angiogenesis should enhance our ability to affect the two main driving forces of tumor proliferation: the glioma cells by augmenting growth arrest, oxidative damage and apopto-sis, and the vascular compartment by blocking VEGF at multiple levels: circulation, endothelial cell production and stem cell secretion. We first examined the response of temozolomide resistant malignant glioma cells (H80-OTR) as well as of the sensitive parent line (H80-P) to a combination of the proteasome inhibitor Bortezomib and the humanized monoclonal antibody to VEGF Bevacizumab. Both cell lines showed high sensitivity to low doses of Bortezomib (10-8M to 10-6M), with complete loss of protea-some activity 24 hours after the treatment. In comparison with the H80-P cells, the H80–OTR cells were not affected by low-dose Bortezomib after 4 hours, but robust cell death was seen in both cell lines at 24 and 48 hours. Treatment with Bevacizumab alone (0.5 mg/ml) had a mild and transient effect in the H80-P line, and no cell death induction in the H80-OTR cells. However, the addition of Bevacizumab to Bortezomib lead to increased apoptosis and impaired proliferation in the H80-P cells, and complete loss of cell viability by MTT assay at 48 hours in the H80-OTR line. Adminis-tration of Bortezomib and Bevacizumab was also studied in athymic mice carrying intracranial malignant glioma xenografts (D245-MG). Both intra-peritoneal and intravenous administration of the combination therapy for 4 weeks lead to a survival advantage of 10 days (p 5 0.038 i.p. and 0.006 i.v.), while administration of either drug alone didn’t influence survival. These data indicate that the combination of Bortezomib and Bevacizumab shows promise in preclinical models. Future work is needed to further define the mechanisms of interaction between the two drugs at a molecular level. A clinical trial based on these preliminary results is in progress.

ET-49. STRUCTURAL REQUIREMENTS OF EPHRINA1 FOR THE EFFECTIVE TARGETING OF EPHA2 RECEPTORS IN GLIOMASEnzo Palma, Jill Wykosky, and Waldemar Debinski; Neurosurgery, Wake Forest University, Winston-Salem, NC, USA

We have demonstrated the over-expression of EphA2 receptor tyrosine kinase in a vast majority of patients with high-grade gliomas relative to normal brain tissue, making EphA2 a suitable target for the delivery of diagnostic and therapeutic agents. EphrinA1, a high-affinity ligand for EphA2, binds, activates, and internalizes EphA2 and these properties have been recently exploited in our laboratory to produce a potent anti-glioma ephrinA1-cytotoxin conjugate. We have found that although ephrinA1 is believed to be a GPI-anchored membrane protein that clusters and activates the EphA2 receptor via the formation of higher-order multimeric complexes, this protein is released from glioma cells in a soluble monomeric form and is able to bind and activate EphA2 as such. Moreover, structural analyses of homologous ephrinA5, a member of the ephrinA family, revealed that the receptor binding site is confined to the GH loop connecting the G and H beta strands, a region that is highly conserved across all ephrinA proteins (H-QRFTPFTLGKEFKE-OH). Thus, to understand the structural require-ments necessary to generate clinically relevant ephrinA1-based diagnostic and therapeutic agents, we have produced soluble monomeric ephrinA1 and tested its ability to bind and activate EphA2. In addition, we have started a site-directed mutagenesis analysis of the ephrinA1 GH loop in order to uncover critical residues involved in EphA2 binding. Soluble monomeric ephrinA1 was produced as a cleavage product of a recombinant dimeric ephrinA1-Fc chimera whose ephrinA1 and Fc domains are linked by a Fac-tor Xa peptidyl substrate. We have also purified monomeric ephrinA1 from the cell media of U-251 MG glioma cells expressing ectopic ephrinA1 by size exclusion chromatography using FPLC system. These soluble isoforms of ephrinA1 were able to trigger typical phenotypic changes in U-251 MG glioma cells and neuronal cone growth collapse at similar concentrations as recombinant dimer of ephrinA1–Fc, indicating that covalent dimerization of ephrinA1 is not necessary to bind and activate the EphA2 receptor. In addi-tion, GH loop point mutants of ephrinA1 generated by alanine-scanning mutagenesis of F, T, P, F, T, L, E, and F residues revealed critical amino acid residues responsible for the functional binding of ephrinA1 to EphA2 in U-251 MG glioma cells. Thus, our studies directly document the ability of soluble monomeric ephrinA1 to bind and activate EphA2 in U-251 MG glioma cells and that the location of the EphA2 binding site on ephrinA1 is confined to its conserved GH loop. Together, these findings delineate important structural requirements of ephrinA1 for the effective targeting of EphA2 receptors and for the development of optimal, clinically applicable ligands for the diagnosis and treatment of malignant gliomas.

ET-50. X-RAY IRRADIATION–INDUCED RADIORESISTANT CELL LINE (MGR2R) AND ITS BIOLOGYZhong-Ping Chen; Sun Yat-sen University, Guangzhou, China, China

Radioresistance is the most important problem for successful radio-therapy for gliomas. In order to explore the possible mechanisms of radiore-sistance, a paired sensitive/resistant cell line should be very useful. Human glioma cell line MGR2 was irradiated at interval 2 Gy 3 times, 5 Gy 2 times and had been cultured 5–8 weeks after irradiation each time. After 11 months, the subline was named MGR2R. The survival fraction at 2 Gy(SF2) of MGR2 was 0.208, while of MGR2R was 0.478 (t 5 –6.062 CP 50.040). Doubling time of MGR2 was 3.6 days, while MGR2R was 4.8 days. The distribution of G1 phase by serum-starvation synchronization of MGR2 and MGR2R were 54.8% and 64.7%. There was significant variation in distribution of cell cycle before synchronization and that of synchronization after 36 h without serum culture. Twenty four hours after loss of synchroni-zation by cultured with 10% serum, the distribution of G1 phase in MGR2 and MRG2R were 35.9% and 53.7%, respectively. The rate of entering next phase from G1 phase in MGR2R was slower than that of parent cells. The distributions of cell cycle were significantly different in MGR2R and MGR2. There was significant correlation between SF2 and G1 phase trans-formation (r 5 –0.579, p , 0.01) in these cell lines. After high-dose X-ray intervals irradiation, radiosensitive human gloma cell line MGR2 has been successfully induced to be relatively radioresistant subline (MGR2R).



ET-51. ONCOLYTIC ADENOVIRUS DELTA-24–RGD INDUCES EFFICACIOUS ANTIGLIOMA ACTIVITY IN BRAIN TUMOR STEM CELL MODELSHong Jiang, Candelaria Gomez-Manzano, Tomohisa Yokoyama, Marta Alonso, Seiji Kondo, Jing Xu, B. Nebiyou Bekele, Howard Colman, Frederick Lang, and Juan Fueyo; University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

The newly identified brain tumor stem cells (BTSCs) are critical to the development and maintenance of malignant gliomas. Eradication of the stem-cell compartment of a brain tumor may therefore be essential to achieve long-lasting remission after treatment. In this study, we examined the thera-peutic potential of an oncolytic adenovirus, Delta-24–RGD, targeted to the abnormal p16INK4/Rb pathway in BTSCs. Clonogenic neurosphere- forming BTSCs from post-surgery glioblastoma specimens expressed high levels of adenoviral receptors and allowed for infection and replication of Delta-24–RGD in an Rb-dependent manner. None of the four BTSC lines tested exhibited resistance to adenoviral infection and virus-induced cell death. Interestingly, Delta-24–RGD infection induced autophagic cell death as indicated by accumulation of Atg5 and LC3–II proteins, autophagic vacuoles shown by ultrastructural studies, and an increase of the acidic vesicular organelles in the cells. Treatment of BTSC-derived xenografts with Delta-24–RGD significantly improved the survival of glioma-bearing mice (p , 0.001) and resulted in 25% of asymptomatic long-term survi-vors. Analyses of treated tumors showed that Atg5 expression colocalized with viral fiber protein and delineated a wave-front of autophagic cells that neatly circumscribed areas of virally induced necrosis. Our results show for the first time that BTSCs are susceptible of adenovirus-mediated cell death via autophagy in vitro and in vivo. The in vivo assessment of adenovirus-induced autophagy may be of extraordinary relevance for the monitoring of oncolytic adenoviral efficacy in future clinical trials.

ET-52. ONCOLYTIC ADENOVIRUSES DOWNMODULATE MGMT INCREASING SENSIBILITY OF BRAIN TUMOR STEM CELLS TO TEMOZOLOMIDE: A MECHANISTIC STUDYMarta Alonso, Candelaria Gomez-Manzano, Jing Xu, Yuji Piao, Hong Jiang, W.K. Alfred Yung, Frederick Lang, and Juan Fueyo; University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Recently, several groups have described the existence of a cancer stem cell population in human brain tumors. These population is a preferred therapeutic target since has been proposed to be a possible source of cancer resilience to conventional anti-cancer therapies. Currently, temozolomide (TMZ) constitutes standard treatment for many patients with malignant glioma. Because temozolomide generates only partial responses, due to overexpression of MGMT, glioblastoma treatment requires multimodal therapy. Oncolytic adenoviruses designed to replicate in and destroy tumor cells selectively represent a promising new therapeutic strategy that could improve the outcome of this malignancy. We hypothesize that TMZ can be successfully combined with Delta24-RGD, an oncolytic adenovirus, resulting in an enhanced cytotoxic effect against the brain tumor stem cell population (BTSCs). NSC-2 and NSC-11 brain tumor stem cell lines were isolated and characterized from brain tumor specimens. Our data showed that Delta24-RGD induced a robust cytotoxic effect on BTSCs that was further enhanced when combined with TMZ. This cytotoxic effect was greater in NSC-2 cells. Interestingly, examination of basal MGMT expres-sion in these cell lines showed high levels of MGMT in NSC-2 versus no expression in NSC-11 cell line. Importantly, treatment with temozolomide further increased MGMT levels in NSC-2 and triggered the expression of the enzyme in NSC-11 indicating a possible resistance to the treatment. Moreover, Delta24-RGD infection was able to override the G2/M arrest (. 70% of the cells) induced by TMZ treatment thus, rendering BTSCs more sensitive to cell death. Importantly, Delta24-RGD infection resulted in an E1A-mediated donwmodulation of MGMT expression at RNA level, possibly due to E1A inactivation of p300/CBP function. This work repre-sents the first evidence of successfully targeting BTSCs with an oncolytic virus alone or in combination with chemotherapy. Combination treatment of Delta24-RGD with TMZ resulted in enhanced antitumor effect in BTSCs through abrogation of DNA repair mechanism. These data deserved further in vivo testing since might constitute important criteria for the selection of patients for future clinical trials involving the combination of Delta24-RGD and TMZ.

ET-53. HUMAN MESENCHYMAL STEM CELLS AS CELLULAR DELIVERY VEHICLES OF ONCOLYTIC MYXOMA VIRUS TO BRAIN TUMORSDongqin Zhu,1 Darnell Josiah,1 Adam Studebaker,2 Grant McFadden,3 and Hugo Caldas1; 1Department of Neurosurgery and Brain Tumor Center of Excellence, Wake Forest University School of Medicine, Winston-Salem, NC, USA; 2Center for Childhood Cancer, Columbus Children’s Research Institute, Columbus, OH, USA; 3Department of Molecular Genetics & Microbiology, University of Florida, Gainesville, FL, USA

Human mesenchymal stem cells (hMSCs) are a fibroblastic cell popula-tion commonly isolated from the bone marrow. These cells are characterized by an enhanced self-renewal potential, and the ability to undergo multilin-eage differentiation. Recent evidence suggests that hMSCs selectively home to tumors and contribute to the formation of tumor-associated stroma. The use of hMSCs as cellular delivery vehicles, particularly to deliver anticancer agents directly into the tumor microenvironment where these cells selec-tively engraft and participate in tumor stroma development, is an emerging attractive therapeutic strategy. We hypothesize that hMSCs can be used to deliver myxoma virus, a promising oncolytic virus that suffers from poor biodistribution, to brain cancer cells and experimental brain tumors. Myxoma virus, is a rabbit-specific poxvirus with previously demonstrated oncolytic properties against human experimental brain tumors. hMSCs isolated from distinct donors were infected with increasing multiplicities of infection of vmyxGFP (myxoma virus variant expressing green fluorescent protein) and found to sustain productive viral replication with no significant impact on hMSC cell viability. Further, we performed 2-dimensional and 3-dimensional co-culture assays with “naive” red-fluorescent glioblastoma multiforme (GBM) cell lines at increasing co-culture ratios and observed successful cross-infection of GBM cells from the hMSCs, concomitant with the induction of cell death exclusively in GBM cells. The migration ability of myxoma infected hMSCs towards GBM conditioned media was found to be parallel with that of non-infected hMSCs, suggesting myxoma virus infection and replication do not alter the affinity and migration ability of hMSCs to brain tumors. Preliminary in vivo studies in subcutaneous and intracranial GBM xenografts demonstrate that myxoma virus can be suc-cessfully delivered by hMSCs to the tumor bed when injected at distant sites from the tumor location. In conclusion, our data suggests hMSCs are a promising new cellular delivery vehicle for in situ delivery of oncolytic viruses, like myxoma virus, to brain tumors and future studies to validate the therapeutic efficacy of this strategy in intracranial xenograft models are currently underway.

ET-54. CONVECTION-ENHANCED DELIVERY OF ADENOVIRAL VECTORSSander Idema,1 Martine Lamfers,2 Victor Van Beusechem,3 David Noske,1 Clemens Dirven,2 and W. Peter Vandertop1; 1Neurosurgery, VU University Medical Center, Amsterdam, Netherlands; 2Neurosurgery, Erasmus Medical Center, Rotterdam, Netherlands; 3Oncology, VU University Medical Center, Amsterdam, Netherlands

Convection-enhanced delivery (CED) of Adenoviral vectors or onco-lytic viruses theoretically offers the potential of widespread transgene activ-ity in the brain, or the targeting of tumorcells in large areas compared to direct bolus injection. Failure of CED however could cause severe toxicity due to high local concentrations of viral particles and inflammatory reac-tions. Contrasting results have been obtained when infusing or convecting Adenoviruses in the brain. Theoretically, CED is only succesfull when equal doses delivered in increasing volumes (Vi) cause an increasing volume of distribution (Vd) where the ratio Vd/Vi would ideally be 5, based on the volume of the interstitial space. Using a fixed infusion rate and an opti-mized infusion catheter, we convected 8 3 107 infectious units (iu) AdGFP, expressing the green fluorescent protein, in 0.5% albumin, in the striatum and corpus callosum of Wistar rats. Transgene expression was determined after 3 days in serial slides and total Vd was determined and compared to Vi. In the striatum, Vd was not determined by the Vi, but by the infused dose, suggesting diffusion or receptor saturation rather than convection. In the Corpus Callosum Vd related to Vi, showing an increase in Vd with increasing Vi. The ratio Vd/Vi decreased with increasing Vd, most probably due to the preferential CED fluid pathways connecting to gray matter where adenoviral CED is limited. This observation was confirmed by co-infusion of trypan blue. Monocrystalline iron oxide nanoparticles (MIONs, reso-vist™) the size of adenoviruses were infused or co-infused, and appeared to predict the distribution of transgene expression. Therefore these MRI-contrast agents could possibly be used to predict the distribution of adenovi-ruses in the brain and prevent undesired toxicity. We conclude that CED of Adenoviruses in the brain is feasible when regional anatomical differences in the brain are taken into account, and spread of adenoviruses may be predicted by MION infusion.



GENOMICS

GE-01. HFE GENETIC VARIANTS ARE A RISK FACTOR FOR BRAIN TUMORS AND INCREASE RESISTANCE TO THERAPYSang Lee, Becky Slagle-Webb, Elana Farace, Jonas Sheehan, and James Connor; Penn State College of Medicine, M.S. Hershey Medical Center, Hershey, PA, USA

HFE polymorphisms, the most common genetic variants in Caucasians, are associated with several cancers and neurodegenerative diseases. In pre-vious studies, we reported that HFE expression was observed in major-ity of high-grade astrocytoma tumors. In addition, brain tumor cells with the C282Y allele were resistant to Temodar and gamma radiation. In this study, migration ability and known resistance mechanisms were evaluated to begin to develop insights into how the C282Y allele could be influencing these cells. We also determined frequency of HFE polymorphisms in brain tumor patients. Two cell lines were studied, a human neuroblastoma cell line (SH-SY5Y) that we stably transfected and human astrocytoma cells representing the different HFE polymorphisms (WT, H63D and C282Y). C282Y stably transfected human neuroblastoma cells (SH-SY5Y) have the ability to migrate but wild type (WT) or H63D cells do not. The C282Y positive cells (both neuroblastoma and astrocytoma) are resistant to Temo-dar and gamma radiation. One of the Temodar resistance mechanisms is increased O6–methylguanine-DNA methyltransferase (MGMT) expression resulting from lack of MGMT promoter methylation. However, MGMT gene methylation was increased and protein expression was decreased in the cells carrying the C282Y allele suggesting this mechanism is not part of the resistance profile in C282Y cells. Poly (ADP-ribose) polymerase (PARP) fragmentation was less in the C282Y neuroblastoma cells following Temo-dar exposure. DNA mismatch repair protein 2 (MSH2) was also decreased in cells carrying the C282Y allele. The expression of p53 was increased in C282Y cells and the expression levels of p53 were cell type dependent rather than genotype dependent. Overall the expression of the resistance mechanisms was consistent with the phenotype of treatment resistance in the C282Y cells. Furthermore we determined the frequency of the genetic variants of HFE in our brain tumor population at the Hershey Medical Center. A total of 94 controls and 139 adult brain tumor patients were genotyped. The frequencies of H63D and C282Y polymorphisms in the brain tumors were 29% and 7%. This is similar to control population. However, homozygosity for the H63D allele of the HFE gene was increased over three-fold in individual with brain tumors compared to the control population. Among the female adult brain tumor patients (n 5 68) 12% were C282Y heterozygotes compared to only 7% in the control popula-tion. Glioblastoma multiform (GBM) tumor is associated with an increase in H63D allelic frequency (36.4 %) compared to the general population. Meningioma (13%) and metastasis (12%) have increased C282Y alleles fre-quency compare to total tumor patients. The data appear to suggest that there is a gender by tumor type by genotype effect. Taken together the data strongly suggest that HFE allelic variants should be part of the molecular stratification strategy for determining in evaluating treatment efficacies. (Temodar used in the study was a gift from Schering Plough).

GE-02. THE HEMOPOIETIC- AND NEUROLOGIC-EXPRESSED SEQUENCE 1 (HN1) GENE IS EXPRESSED IN HUMAN AND MURINE MALIGNANT GLIOMASKatharine Laughlin,1 Defang Luo,1 Che Liu,1 Gerry Shaw,2 Kenneth Warrington,3 Jingxin Qiu,4 Anthony Yachnis,4 and Jeffrey Harrison1; 1Pharmacology & Therapeutics, University of Florida, Gainesville, FL, USA; 2Neuroscience, University of Florida, Gainesville, FL, USA; 3Pediatrics, University of Florida, Gainesville, FL, USA; 4Pathology, University of Florida, Gainesville, FL, USA

The hemopoietic- and neurologic-expressed sequence 1 (Hn1) gene encodes a 154 amino acid protein. Its unique sequence is similar to Jupi-ter, a microtubule-associated protein found in Drosophila (Cell Motil. Cytoskeleton 63:301, 2006). Hn1 is highly expressed in a number of tis-sues in the developing mouse, including fetal brain, yolk sac and liver, as well as adult mouse tissues such as bone marrow, spleen and brain (Mamm. Genome, 8:695, 1997). We determined previously that Hn1 expression is increased in a robust manner within facial motoneurons that undergo suc-cessful regeneration following peripheral nerve injury (J. Neurosci. Res., 82:581, 2005). These data, coupled with recently published results that identify human HN1 as a potential marker for epithelial ovarian cancer (Clin. Cancer Res., 10:3291, 2004), suggest that this gene is involved in processes associated with cell growth and regeneration. The goals of this study were directed toward evaluating the expression of Hn1 in a murine model of malignant glioma as well as in tissue sections from a variety of human brain tumors. Northern blot analysis of the murine glioma cell line, GL261, identified two mRNA species (0.7 and 1.4 kb), which is consistent with previously published data. Western blot analysis using either mouse

monoclonal or rabbit polyclonal anti-Hn1 antibodies detected a prominent 25 kDa protein which co-migrated on SDS-PAGE with recombinant forms of Hn1. Cell fractionation and immunocytochemical analyses determined that the Hn1 protein resides within the cytoplasm. To evaluate the role of Hn1 in this and other murine cancer models, we developed a recombinant adeno-associated virus (serotype 6) engineered to express a siRNA target-ing murine Hn1. The in vitro growth rate of the Hn1-depleted cells was similar to both untreated cells and cells transduced with a control viral vec-tor. In situ hybridization analysis of GL261 tumors established in murine (C57BL/6) brains confirmed high levels of expression of Hn1 mRNA within the tumor. Mice implanted with Hn1-depleted cells had a tendency to form smaller tumors as compared to animals that received control virus-treated cells. The incidence of large tumors was greater in the animals implanted with control virus-treated cells, while the prevalence of animals display-ing no evidence of tumors was higher in the animals implanted with Hn1- depleted cells. Evaluation of a human brain tumor tissue microarray, using the mouse monoclonal anti-Hn1 antibody, identified strong immunoreac-tivity in human malignant gliomas including glioblastoma and anaplastic forms of astrocytoma and oligodendroglioma. These data identify Hn1 expression in human and murine gliomas, and suggest a role for this gene in the biology of malignant brain tumors.

GE-03. EXON-LEVEL EXPRESSION PROFILING IDENTIFIES EXON-SKIPPING MUTATIONS AND TRANSLOCATIONS IN GLIAL BRAIN TUMORSPim French,1 Linda Bralten,1 Mieke Schutte,2 Elza Duijm,1 Fons Elstrodt,2 Jord Nagel,2 Antoinette Hollestelle,2 Marijke Wasielewski,2 Justine Peeters,3 Martin Van Den Bent,1 Johan M. Kros,4 Peter Van Der Spek,3 and Peter Sillevis Smitt1; 1Neurology, Erasmus MC, Rotterdam, Netherlands; 2Medical Oncology, Erasmus MC, Rotterdam, Netherlands; 3Bioinformatics, Erasmus MC, Rotterdam, Netherlands; 4Pathology, Erasmus MC, Rotterdam, Netherlands

Human exon arrays have recently been developed to measure the expression-level of virtually all known and predicted exons present in the human genome (1.4 million). Apart from the identification of differentially regulated splice variants (PJF et al., Cancer Res., 67, in press), we hypoth-esized that exon-level expression profiling can also identify different types of genetic mutations in cancer. For example, exon-level expression profil-ing may identify genetic changes that result in the skipping of one or more exons in the encoded transcript. Such exon-skipping mutants represent an estimated 10%–20% of all cancer-related gene mutations and are caused by intragenic deletions or single nucleotide substitutions within the consensus splice site. We therefore developed a strategy, termed Outlier Exon Screening (OES), to identify candidate exon skipping mutations by screening for exons that are markedly lower expressed than predicted based on the expression level of their transcripts. As a proof-of-principle, we tested the OES strategy on human cancer samples of which the complete coding sequence of eight tumor suppressor genes and oncogenes had been screened for mutations. OES detected all seven exon-skipping mutants among 12 cancer cell lines and three of seven mutants among 14 surgically resected glioblastoma speci-mens. We are currently using the candidates identified by OES to screen for novel exon-skipping mutations. A second type of genetic aberration that may be identified by exon-level expression profiling are translocations that result in the expression of a fusion gene. Such genetic changes are identified by an aberrant ratio of start (5') and end (3') exons in the fusion partners. Screen-ing for transcripts with aberrant 5'/3' exon ratios identified at least nine strong candidate 3' fusion partners. In one of these candidates, RACE-PCR identified a 5' fusion partner of a gene located on chromosome 2. RT-PCR using a forward primer in the 5' fusion candidate and a reverse primer in the 3' fusion candidate confirmed expression of this fusion construct. Our current work is aimed at further characterizing this novel fusion gene and screening for translocations in other candidate genes.

GE-04. AN INK4 TUMOR SUPPRESSOR CIRCUIT CONSTRAINS GLIOBLASTOMA DEVELOPMENT AND PROMOTES COORDINATE SILENCING OF G1 CYCLIN-DEPENDENT KINASE INHIBITORS P18INK4C AND P16INK4ACameron Brennan,1 Ruprecht Wiedemeyer,2 Keith Ligon,3 P Andrew Futreal,4 and Lynda Chin2; 1Neurosurgery, Memorial Sloan-Kettering Cancer Center, New York, NY, USA; 2Dana Farber Cancer Institute, MA, USA; 3Dana-Farber/Brigham and Women’s Cancer Center, MA, USA; 4Wellcome Trust Sanger Institute, United Kingdom

RB pathway compromise is a common finding in GBM, either through deletion/mutation of the RB tumor suppressor itself, amplification of the G1 cyclin-dependent kinases 4 or 6 (CDK4/6), or deletion/silencing of the G1 CDK inhibitor (CKI), p16INK4A. In individual tumors, these principal



components of the pathway—RB, CDK4/6, and p16INK4A—are targeted in a mutually exclusive manner, consistent with their overlapping functions in regulating the G1–S transition of the cell cycle. Against this background, we report here an integrated analysis of both genome-wide transcriptional and chromosomal copy number profiles of primary GBM samples and cell lines, revealing numerous focal and recurrent amplifications and deletions targeting cancer-relevant genes with associated altered expression. This analysis has uncovered unexpected genomic co-deletion of two highly related G1 CKIs, p18INK4C, and p16INK4A. Gene resequencing confirms loss-of-function variants of p18INK4C in p16INK4A deficient tumor cells and we document frequent absence of both p18INK4C and p16INK4A pro-tein expression in human GBM samples. This strongly indicates that these highly related CKIs cooperate to suppress gliomagenesis. Correspondingly, reconstitution of p18INK4C in GBM cells null for both p16INK4A and p18INK4C impaired cell cycle progression and tumorigenic potential, while RNAi-mediated depletion of p18INK4C in p16INK4A deficient GBM cells had the opposite biological impact. Interestingly, RNAi-mediated deple-tion of p16INK4A caused an acute increase in p18INK4C levels in primary human and mouse astrocytes. These findings, together with the presence of E2F-binding sites in the p18INK4C promoter, indicate the existence of an INK4 feedback regulatory circuit in glioma. Thus, integrated high resolu-tion genomic analysis has uncovered a tumor suppressor role for p18INK4C in human GBM and an unexpected genetic interaction among members of the INK4 family of CKIs that functions to constrain inappropriate prolif-eration in astrocytes.

GE-05. A MOLECULAR-BASED SIGNATURE OF MENINGIOMASCarvalho Lucia,1 Ivan Smirnov,2 Baia Gilson,2 Zora Modrusan,2 Justin Smith,1 Peter Jun,2 Joseph Costello,2 Michael McDermott,1 Scott VandenBerg,1 and Anita Lal1; 1University of California, San Francisco, San Francisco, CA, USA; 2CA, USA

Meningiomas account for ~30% of all primary brain cancers and are classified into three World Health Organization (WHO) grades (benign, atypical and malignant) based on histopathological criteria. We have used a combination of microarray and array comparative genomic hybridiza-tion (arrayCGH) to profile the transcript levels and chromosomal aberra-tions of 23 meningiomas derived from all three histopathological grades. SAM analysis and unsupervised clustering of the expression data reveal that meningiomas of all grades fall into two major molecular subgroups designated the low-proliferative and the high-proliferative meningiomas. While all benign meningiomas fall into the low-proliferative group and all malignant meningiomas fall into the high-proliferative group, the atypi-cal meningiomas distribute into either group. The high-proliferative atypi-cal meningiomas had a higher median MIB-1 labeling index and a greater number of total copy number aberrations (CNAs) when compared to the low-proliferative atypical meningiomas. We have identified a set of genes that can classify benign and malignant meningiomas and have identified gain of cell proliferation markers and loss of transforming growth factor-beta signaling as the main functions distinguishing malignant from benign meningiomas. Additionally, we have identified CNAs that are exclusively found in the high-proliferative subgroup. Our data suggest that molecular signatures and CNA markers will potentially be more accurate predictors of atypical meningioma tumor growth than histopathological criteria.

GE-06. OVER-EXPRESSION OF SEC61 INCREASES GLIOBLASTOMA GROWTH AND SURVIVAL IN CULTUREAnjan Misra,1 Ivan Smirnov,2 M.S. Madhusudhan,1 and Burt Feuerstein1; 1Neurology, Barrow Neurological Institute, St. Joseph Hospital and Medical Center, Phoenix, AZ, USA; 2CA, USA

SEC61 is located on the short arm of chromosome 7, ~0.5 Mb from the epidermal growth factor receptor (EGFR). It is a subunit of the mammalian endoplasmic reticulum (ER) translocon, a highly conserved transmembrane protein that moves proteins across the ER. Crystal structures of the bacte-rial and yeast Sec61 translocon suggest that Sec61 a, b, and subunits form a complex that transports newly synthesized protein from the ribosome into golgi, plasma membrane or cytoplasm. Since EGFR is frequently amplified in GBM, and the EGFR amplicon is often large, encompassing the Sec61 region, it is likely that SEC61 is amplified and over-expressed in many GBM with EGFR amplification. The effect of SEC61 over-expression on GBM behavior is unknown. We obtained published Affymatrix GBM gene expression data from the University of California, San Francisco (Nigro et al., 2005), M.D. Anderson Cancer Center (Phillips et al., 2006) and the University of California, Los Angeles (Freije et al., 2004), and used an array of traditional and novel statistical methods, bioinformatics, structural mod-eling, and biological assays to investigate effects of SEC61 overexpression in GBM. There was extremely high correlation between Sec61 expression

and EGFR expression (p 5 2.2 e-16) in this set of 195 GBM. Almost all EGFR amplification cases had SEC61 amplification and overexpression. SEC61 was over-expressed in the poorly surviving mesenchymal and pro-liferative subgroups (Phillips et al., 2006), but not in the longer surviv-ing proneural subgroup. Furthermore, Sec61 was associated with poor survival (p 5 0.005), and was a strong negative predictor of . one year survival (p 5 7.6 e-6). Structural modeling of human SEC61 complex indi-cated its similarity to that of lower species. Human SEC61 is composed of two a helices of unequal length forming a motif we call “7” with a proline residue at the junction of the arms. This proline residue is strongly conserved through evoluation. Over-expression of Sec61 in U251NCI cells in culture increased growth rate (p , 0.05) and survival (p , 0.01) but had no discernable effect on morphology, adhesion or migration. Our analysis of gene expression profiles in 195 GBM indicated very strong cor-relations between Sec61 and cell cycle regulation genes, suggesting that SEC61 may increase cell growth via these genes. Similar analyses indicate that Sec61 may exert its prosurvival effects via key genes in the AKT and NFκb survival pathways. A protein-protein interaction database search sug-gested that Sec61 interacts with 107 known and hypothetical proteins. Pathway Enrichment analyses indicate associations between Sec61 and 26 biological pathways (e.g., calcium signaling, cell communication etc.). We are experimentally validating bioinformatics results to develop evidence whether and how Sec61 exerts neoplastic effects. This work was supported by NIH (NS42927 and CA85799), National Brain Tumor Foundation, and the Barrow Neurological Foundation.

GE-07. ISOLATION OF THE NORMAL CHROMOSOMES 1 AND 19 AND T(1;19)(Q10;P10) FROM OLIGODENDROGLIOMA CELLS USING CONVERSION TECHNOLOGYGobinda Sarkar,1 Heather Flynn,2 Hilary Blair,2 Kevin Meyer,2 Peggy Schneider,2 Victoria Marley,2 Karen Schowalter,2 Edward Highsmith,2 and Robert B Jenkins3; 1Mayo Foundation for Medical Education and Research, Rochester, MN, USA; 2Mayo Clinic and Foundation, Rochester, MN, USA; 3Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA

Oligodendrogliomas frequently show whole-arm deletions involv-ing chromosomes 1 and 19 along with presence of a t(1;19)(q10;p10) that involves the centromeres of the two chromosomes. Patients with such chro-mosomal abnormalities have better prognosis and response to radiation- and chemotherapy than patients with tumors without the translocation. However, it is difficult to culture oligodendrogliomas to generate sufficient quantities of the t(1;19) to study the genomic (and epigenomic) structure of the translocation. To facilitate such genomic studies, we have imple-mented “Conversion Technology” (a technique for the isolation of human chromosomes in mouse-human somatic cell hybrids) to isolate various chromosomes 1 and 19 from oligodendroglioma specimens. For conver-sion, msh2 deficient, embryonic mouse fibroblasts (E2) and human cells are fused and cultured with HAT selection. The hybrids are then genotyped for the chromosome(s) of interest to determine their haplotype. Individual cell lines are expanded once hybrids containing the desired chromosomes are identified. We report use of this technology for the isolation of chro-mosomes 1 and 19, and t(1;19)(q10;p10) from primary, overnight-cultured human oligodendrogliomas with and without combined 1p and 19q dele-tion. Genotypes were identified through mapping by 5 STR markers each for chromosomes 1 and 19. Based on the distribution of the STR markers, we were able to predict the genotypes of hybrids that contained the normal chromosome 1 and 19, and the derived t(1;19). We were able to isolate all three chromosomes from 3 gliomas with combined 1p and 19q deletion and all four chromosomes 1 and 19 from 4 gliomas without combined deletion. FISH analysis of cells derived from passages 2 to 4 of the expanded hybrids confirmed the STR genotyping results. This study demonstrates that Con-version Technology can be used to isolate and clone normal and derived chromosomes 1 and 19 from fresh, uncultured, primary glioma specimens. In addition, the successful isolation and expansion of these chromosomes will facilitate several future studies: For example, are the normal chromo-somes 1 and 19 in oligodendrogliomas structurally and/or functionally normal? Is the t(1;19) identical in every patient? Are epigenetic alterations associated with such translocations?



GE-08. BAC-ARRAY-BASED COMPARATIVE GENOMIC HYBRIDIZATION ANALYSIS OF ANAPLASTIC OLIGODENDROGLIAL TUMORS INCLUDED IN THE EORTC 26951 TRIALAhmed Idbaih,1 Catherine Carpentier,1 Mathilde Kouwenhoven,2 Emmanuelle Criniere,1 Judith Jeuken,3 Pim French,4 Jljm Teepen,5 Yannick Marie,1 Blandine Boisselier,1 Karima Mokhtari,1 Max Kros,6 Olivier Delattre,7 Marc Sanson,1 Jean-Yves Delattre,1 Thierry Gorlia,8 Martin Van Den Bent,9 and Khê Hoang-Xuan1; 1Groupe Hospitalier Pitié-Salpêtrière, Paris, France; 2Daniel den Hoed Oncology Center, Rotterdam, Netherlands; 3Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands; 4Cancer Genomics Center, Erasmus Medical Center, Rotterdam, Netherlands; 5St. Elisabeth Hospital, Tilburg, Netherlands; 6Erasmus Medical Center, Rotterdam, Netherlands; 7Institut Curie, Paris, France; 8EORTC DataCenter, Brussels, Belgium; 9Daniel den Hoed Oncology Center–Erasmus University Medical Center Rotterdam, Rotterdam, Rotterdam, Netherlands

Our objective was to characterize the genomic profile of anaplastic oligodendroglial tumors (AOT) using the BAC-array-based comparative genomic hybridization (CGHa) technique and to evaluated its prognostic value. The EORTC 26951 trial compared the efficacy of radiotherapy (RT) versus RT plus PCV adjuvant chemotherapy in AOT (van den Bent et al., J Clin Oncol., 2006). Tumor frozen samples from 60 patients of this trial were available for DNA extraction and CGHa analysis using 1 megabase CGHa. The population included 34 men and 26 women. The median age was 49 years old (range 20–69). Patients underwent a biopsy, partial and complete surgical resection in 2, 34, and 24 patients respectively. Twenty-nine patients were included in RT plus adjuvant chemotherapy arm and 31 in the RT arm. The most frequent chromosome arm imbalances were gain of chromosome 7p, 7q, 19p, 20p, and loss of chromosomes 1p, 9p, 10p, 10q, 13, 14, 19q, and 22. Five groups of tumors were identified: (1) 1p/19q codeleted tumors (n 5 10); (2) EGFR amplified tumors (n 5 17); (3) tumors harboring chromosome 7q gain and chromosome 10q loss (n 5 9); (4) tumors exhibiting chromosome 19q loss (n 5 8) and (5) tumors with other pattern (n 5 16). These genomic subgroups were associated with distinct outcome both in terms of progression free survival (p , 0.0001) and overall survival (p , 0.0001). Interestingly, the same results were obtained in the 45 tumors which were confirmed as AOT after centralized pathological review. Multivariate analysis including age, tumor localization, treatment, phenotype and genomic pattern revealed that age and genomic pattern were independent prognostic factors. CGHa analysis identifies in AOT several genomic patterns associated with distinct prognosis.

GE-09. PROTEIN BIOMARKER IDENTIFICATION IN THE CEREBROSPINAL FLUID IN PATIENTS WITH CENTRAL NERVOUS SYSTEM LYMPHOMAJames Rubenstein,1 Cigall Kadoch,1 Scott Josephson,2 Jane Fridlyand,3 Sandeep Kunwar,4 Juliana Karrim,1 Marc Shuman,1 Ted Jones,5 Chris Becker,5 Howard Schulman,6 and Sushmita Roy5; 1Medicine, University of California, San Francisco, San Francisco, CA, USA; 2Neurology, University of California, San Francisco, San Francisco, CA, USA; 3Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, CA, USA; 4Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA; 5PPD, Menlo Park, CA, USA; 6Stanford University, Mountain View, CA, USA

Characterization of the cerebrospinal fluid (CSF) proteome may provide insights into the pathogenesis of central nervous system malignancy. We tested the hypothesis that individual CSF proteins distinguish CNS lym-phoma and other brain tumors from benign focal brain lesions. We used a liquid chromatography-mass spectrometry-based method to differentially quantify and identify several hundred CSF proteins in control subjects and in patients with CNS lymphoma. We used immunologic techniques includ-ing ELISA to confirm results for two of these markers using an additional validation set of 106 cases. We identified approximately 80 CSF proteins which were found to be present at significantly different concentrations, both higher and lower, in training and test studies which were highly concordant. To further validate these observations, we defined in detail the expression of two of these candidate biomarkers; one, a serine protease inhibitor, and the other, a B-cell chemokine. RNA transcripts for each of these were identified within CNS lymphomas. Protein expression within tumors was confirmed by immunohistochemistry. Determination of the CSF concentration of one of these putative biomarkers by ELISA was more accurate than cytology in the identification of cancer (sensitivity 70%; specificity 98%) and was found to potentially enhance determination of prognosis as well as the monitor-ing of clinical response to therapy. We demonstrate for the first time that proteomic analysis of the CSF using two-dimensional liquid chromatogra-phy/mass spectrometry yields individual biomarkers with greater sensitivity than CSF cytology in the identification of CNS malignancy. The discovery of CSF protein biomarkers, used alone or in combinations, may facilitate

early and noninvasive diagnosis in patients with lesions not amenable to brain biopsy and may provide improved surrogates of prognosis and treat-ment response.

GE-10. GENOMIC EXPRESSION PATTERNS DISTINGUISH LONG-TERM FROM SHORT-TERM GLIOBLASTOMA SURVIVORSNicholas Marko, Steven Toms, Gene Barnett, and Robert Weil; Cleveland Clinic Foundation, Cleveland, OH, USA

We used microarray analysis to investigate associations between geno-typic expression profiles and survival phenotypes in glioblastoma patients. Tumor samples from 7 long-term glioblastoma survivors (. 24 months) and 13 short-term survivors (, 9 months) were analyzed to detect differential patterns of gene expression between these groups and to identify genotypic subclasses of glioblastomas that correlate with survival phenotypes. Five unsupervised and three supervised clustering algorithms consistently and accurately grouped the tumors into genotypic subgroups corresponding to the two clinical survival phenotypes. Three unique prospective math-ematical classification algorithms were subsequently trained to use expres-sion data to stratify unknown glioblastomas between survival groups and performed this task with 100% accuracy in validation studies. A set of 1478 genes with statistically significant differential expression (p , .01) between long-term and short-term survivors was identified, and additional mathematical filtering was used to isolate a 43 gene “fingerprint” that dis-tinguished survival phenotypes. Differential regulation of a subset of these genes was confirmed using RT-PCR. Gene ontology analysis of the finger-print demonstrated pathophysiologic functions for the gene products that are consistent with current models of tumor biology, suggesting that differ-ential expression of these genes may contribute etiologically to the observed differences in survival. These results demonstrate that unique expression profiles characterize genotypic subsets of glioblastomas associated with dif-ferential survival phenotypes, and these profiles can be used in a prospective fashion to assign unknown tumors to survival groups. Future efforts will focus on building more robust classifiers and identifying additional sub-classes of gliomas with phenotypic significance with the ultimate goal of developing a molecular classification system for gliomas.

GE-11. COMPARISON OF RNA AND PROTEIN EXPRESSION PROFILES IN GLIOBLASTOMA MULTIFORMEOscar Persson,1 Morten Krogh,2 Peter James,3 Lao Saal,4 Åke Borg,4 Bengt Widegren,5 and Leif Salford6; 1Neurosurgery, Clinical Sciences, Lund, Sweden; 2Theoretical Physics, Sweden; 3Protein Technology, Sweden; 4Oncology, Sweden; 5Tumor Biology, Sweden; 6Neurosurgery, Sweden

Gliomas are among the most aggressive malignant tumors and the most refractory to therapy, and despite resent therapeutic advances the prognosis still remains poor. Many studies have shown the ability of both gene and protein expression analysis as a diagnostic and prognostic tool in gliomas. We have used 27K cDNA microarrays to investigate global gene expression changes between normal brain and glioblastoma multiforme, as well as gliomas of lower grade. In parallel we have undertaken a proteomics-based approach to analyze the protein expression profile in the same set of tumor samples. We show that both the mRNA and the protein assay can efficiently discriminate between normal brain and tumors, and between tumors of dif-ferent grade. Both approaches further identify subsets of genes/proteins—both well-known and novel ones—that are significantly altered in the dif-ferent groups. The gene expression profiling shows strength in the large number of genes analyzed in each sample, which in turn makes it possible to apply different bioinformatic approaches and enrich for groups of genes of special interest (e.g., functional categories or sub-cellular location). In our study we were looking for genes upregulated in gliomas that could be poten-tial targets for different immunebased therapies. We therefore enriched for membrane associated or secreted proteins, and came up with a focused list of potential targets. By analyzing the data from a chromosomal posi-tional perspective, the mRNA assay further reveals large clusters of adjacent genes that are synchronically up- or downregulated. The protein approach however—while limited in the number of proteins possible to identify—has the advantage that it also takes into account post-translational alterations, and for some proteins multiple isoforms were identified. Approximately 100 overlapping genes were identified in both assays. The comparison of the mRNA and protein profiles for these genes is in process. Preliminary data suggest that there is a high concordance between mRNA and protein levels in gliomas, but a small number of genes show an inverse pattern. To our knowledge this is the first time a comparison of this kind has been per-formed in gliomas, and the results requires further investigation.



GE-12. THE EXPRESSION OF ASPP2, BCCIP, AND BIRC5 IN MODELING PROGNOSIS OF MALIGNANT ASTROCYTIC GLIOMA PATIENTSVinay Ravindrakumar,1 Eric Siegel,2 Kenneth Hess,3 Mark Linskey,4 Alfred Yung,3 Steven Jennings,5 and Yi-Hong Zhou4; 1Bioinformatics, University of Arkansas at Little Rock, Little Rock, AR, USA; 2University of Arkansas for Medical Sciences, AR, USA; 3UT M. D. Anderson Cancer Center, TX, USA; 4University of California, Irvine, CA, USA; 5University of Arkansas at Little Rock, AR, USA

Malignant astrocytic gliomas are aggressive, highly invasive and neu-rologically destructive brain tumors considered being among the deadliest of human cancers. A wide range of survival outcomes occur for patients afflicted by this disease that cannot be explained only by clinical variables like age, histology, recurrent status etc. Zhou et al. (2005) developed a prognostic model for patients with anaplastic astrocytoma (AA) and glio-blastoma multiforme (GBM) using clinical variables and gene expression variables responsible for suppressing/promoting tumor aggressiveness, which has shown great promise in predicting survival. The results from the modeling study elucidated important relationships among genes involved in tumor suppression, oncogenic activation, angiogenesis, cell proliferation, and cell invasion. The objective of the present study is to assess the improve-ment in the model developed in the prognosis modeling study, by adding three new gene expression variables and also increasing the sample size to 116 (AA and GBM) glioma samples. Using a bioinformatics approach, the gene expression variables of ASPP2, BCCIP, and BIRC5 were identi-fied from text mining frame works. The gene expressions of the variables were measured by real-time quantitative reverse transcription polymerase chain reaction (QRT-PCR). Wilcoxon rank sum tests show a significant differential expression for PAX6 (p , 0.0001), VEGF (p , 0.0001), RPS9 (p , 0.0001), EGFR (p , 0.01), IGFBP2 (p , 0.0001), BCCIP (p , 0.0001) and BIRC5 (p 5 0.03) between AA and GBM glioma samples. Spearman Rank correlation tests show a significant positive correlation between PAX6 and RPS9, PAX6 and ASPP2, PAX6 and BCCIP, PTEN and ASPP2, VEGF and EGFR, VEGF and IGFBP2, RPS9 and BCCIP, EGFR and MMP2, IGFBP2 and MMP2, and BCCIP and BIRC5. A significant negative cor-relation exists between PAX6 and IGFBP2, PTEN and MMP2, PTEN and BCCIP, PTEN and BIRC5, VEGF and RPS9, VEGF and BCCIP, IGFBP2 and ASPP2, and BIRC5 and ASPP2. The expression of the BCCIP gene is low in the GBM samples. Also it exhibits a significant positive correlation with other tumor suppressor genes like PAX6 and RPS9 and a negative correlation with the VEGF that is involved in vascular formation. These data suggest a tumor suppressor function for the BCCIP gene. Statistical analyses will be performed in the enhanced dataset to assess the predictive accuracy of the model. The improvement in the model will be measured by the improvement of the R2 of the model.

GE-13. EXPRESSION PROFILING IDENTIFIES CKS2 AS A GENE INVOLVED IN MENINGIOMA PROGRESSIONFrancesca Menghi, Marica Eoli, Donata Bianchessi, Lorella Valletta, Sergio Giombini, Bianca Pollo, and Gaetano Finocchiaro; Fondazione Istituto Neurologico Besta, Milano, Italy

Meningiomas are the most frequent of primary tumors affecting the brain. The large majority of meningiomas is biologically and clinically benign (grade I meningiomas) but in about 15% of the cases they become, or are since their first appearance, malignant (grade II or atypical men-ingiomas; grade III or malignant meningiomas). Understanding the biol-ogy behind the different form of meningiomas is intrinsically interesting and could also help to find markers alerting the clinician to the potentially malignant evolution of the tumor. To gain information on this, we have compared by DNA microarray using Affimetrix HG U133A chip hybrid-ization, expression profiles of 10 grade I, 10 grade II, and 3 grade III men-ingiomas. We also analyzed loss of heterozygosity (LOH) on 60 menin-giomas (37 grade I, 20 grade II and 3 grade III), investigating chromosomal regions on 1p, 9p, 10q, 14q, and 22q. Of 22,283 probe sets represented on the chip, 5,954 were selected by normalization and filtering and further selection of probe sets according to their p value identified 336 probe sets down-regulated and 344 up-regulated in grade II-III meningiomas. Genes down-regulated in high-grade meningiomas were over-represented in chro-mosomes 1, 6, 10, and 14. Fractional allelic loss, as defined by LOH, was higher in high grade meningiomas. EASE functional enrichment analysis helped to categorize genes according to their function. Expression ratio in high vs6.7) for CKS2, encoding cyclin-dependent kinase subunit 2. CKS2 expression is controlled by p53 and is high in metastasis of colorectal car-cinomas, in esophageal carcinomas and uterine cancers, and in progressing melanomas. A gene strongly down-regulated in high grade meningiomas is LEPR, encoding the leptin receptor. Interestingly, leptin interacts with the JAK-STAT pathway that appears to play a relevant role in meningioma biology. Expression trends of these genes were validated by real time PCR

on a wider number of meningiomas. These data may help the identification of new genetic markers for the clinical follow-up of meningiomas, as well as that of pathways of therapeutic relevance.

GE-14. GENETIC DETERMINANTS OF SUSCEPTIBILITY TO NERVOUS SYSTEM TUMORS IN A MOUSE MODEL OF NEUROFIBROMATOSIS TYPE 1Karlyne Reilly,1 Robert Tuskan,1 Kristi Fox,2 Erika Truffer,2 Karl Broman,3 Shirley Tsang,2 David Munroe,2 and Keiko Akagi1; 1National Cancer Institute-Frederick, Frederick, MD, USA; 2SAIC-Frederick, Frederick, MD, USA; 3Johns Hopkins University, Baltimore, MD, USA

Neurofibromatosis type 1 (NF1) is one of the most common genetic diseases affecting the nervous system and affects a wide variety of organs through alteration of the ras signaling pathway. One of the major clini-cal challenges of dealing with NF1 is the variable expressivity of the dis-ease: being diagnosed with NF1 does not give much information on what to expect from the disease because the phenotype is so variable. A study in monozygotic twins with NF1 has demonstrated that modifier genes unlinked to NF1 affect the variable expressivity of NF1 (Easton et al., 1993) and we have shown in mice that complex genetic and epigenetic interac-tions control susceptibility to different NF1-associated tumor types, spe-cifically astrocytoma/glioblastoma and malignant peripheral nerve sheath tumor (Reilly et al., 2004; Reilly et al., 2006). Our data demonstrate that knowing the genotype at a single modifier is of limited predictive value. Susceptibility to complex diseases such as cancer and NF1 can be thought of as a genetic barcode made up of the genotypes at multiple modifier loci, where a single bar in the code has no predictive value but the combination of bars produces a unique and highly predictive code. We are using the Nf1–/1; p53–/1 cis mouse model of the malignancies associated with NF1 to dissect the genetic determinants of cancer susceptibility and understand the mechanisms underlying tumorigenesis.

GE-15. A GENE EXPRESSION-BASED ASSAY FOR IDENTIFYING TISSUE OF ORIGIN: APPLICATION TO METASTATIC BRAIN LESIONSSamuel Cheshier,1 Ljubomir Buturovic,2 C. Theodore Rigl,2 W. David Henner,3 Glenda Anderson,3 and Steven Chang1; 1Stanford University, Stanford, CA, USA; 2Pathwork Diagnostics, Sunnyvale, CA, USA; 3Pathwork Diagnostics, CA, USA

Metastatic brain lesions can present difficult diagnostic and manage-ment problems, particularly when the primary is not apparent. A gene expression-based test to identify the tissue of origin (primary) for such metastatic lesions could be useful in targeting the diagnostic workup. The Pathwork Tissue of Origin Test (TOO) is a microarray-based test utiliz-ing hybridization of tumor derived RNA to identify the tissue of origin from a panel of 15 tissue types. The performance and reproducibility of this test for biopsy specimens other than brain have been reported previously. Eight tumor specimens from biopsy or resection of brain lesions where the primary tissue of origin was known were processed to RNA, labeled and hybridized to a Pathchip™ microarray. Resulting hybridization patterns were standardized and analyzed according to the Pathwork TOO algorithm to yield similarity scores (SS) for the specimen to each of the 15 tissues of origin on the Pathwork TOO panel. For each specimen, all SS total to 100, and an SS of [. . .] 30 (indicating a . 95% likelihood of a match) was a posi-tive call. The TOO Test was able to make a call with an SS [. . .] 30 for all 8 samples. Five of the TOO calls (2 lymphoma, 1 breast, 1 colorectal, 1 lung) were exact matches to the clinical impression for the primary. One of the TOO calls was a non-match in which the clinical impression was non-small cell lung cancer on the basis of a lung mass. However, the TOO call for the presumed lung tumor showed a very high SS score for ovarian carcinoma (SS 5 89.4). For two of the samples the clinical impression of the primary could not be matched to the TOO call because the primary was not one of the 15 tissue types on the panel. This study demonstrates the feasibility of using the Pathwork TOO Test to identify the primary tissue of origin for samples derived from biopsy or resection of metastatic brain tumors. Where the tissue of origin was one of the 15 tissue types on the TOO panel, the results of the TOO test matched the clinical impression in 5/6 (83.3%). For samples where the TOO Test provides a strong call but is discordant from the clinical impression, it may useful in targeting further histologic and imaging studies that might confirm either the initial clinical impression or the TOO result. In those cases for which the clinical impression was a pri-mary from a tissue site not included in the TOO panel, the TOO call may still provide some potentially useful indications regarding either the loca-tion (gastric/esophageal) or biology (squamous non-small cell lung cancer/squamous cell head and neck) of the primary.



GE-16. DIFFERENTIAL SERUM PROTEIN PROFILING OF GBM PATIENTS IMMUNIZED WITH AUTOLOGOUS GLIOMA CELLS TRANSFECTED WITH IFN-GAMMA GENE, AND THEIR CONTROLSLeif Salford,1 Oscar Persson,2 Peter Siesjö,3 Gunnar Skagerberg,4 Edward Visse,5 Bengt Widegren,6 Linda Geironson,5 Anders Carlsson,5 Johan Ingvarsson,5 Christer Wingren,5 and Carl Borrebaeck5; 1Lund University Hospital, Eblocket, Lund, Skåne, Sweden; 2Dept. of Neurosurgery, Lund, AE Europe, Sweden; 3AE (Europe); 4Skåne, Sweden; 5Sweden; 6Dept of Tumour Immunology, Lund University, Skåne, Sweden.

Malignant brain tumors are still among the most therapy resistant human malignancies. The malignant gliomas which infiltrate the brain are fatal in 99,8% and the mean survival time after diagnosis of a glioblastoma multiforme (GBM) is less than a year in spite of surgery and radiotherapy and chemotherapy administered separately. Based upon our results in ani-mal experiments, we have explored the possibilities of adding immune therapy utilizing the patients own tumor cells, transfected with the human IFN-gamma gene by the use of adenoviral vector, followed by 8–14 intra-dermal immunizations every 3rd week. Eight patients, all in the age group 50–69 years, who received this treatment after surgery and radiotherapy, had a significantly longer time to progression as well as survival time than a control group of nine patients. The treatment did not induce observ-able adverse effects and the treated patients reported improved quality of life during the immunization period. Sera from all patients were spared before and after initial surgery and before and after each immunization and now we have performed differential serum protein profiling of the non- immunized and the immunized GBM patients as well as of an age-matched group of healthy controls. We have developed a state-of-the art recombinant antibody microarray technology platform for high-throughput proteom-ics of non-fractionated biotinylated proteomes. The platform is based on human recombinant single-chain Fv (scFv) antibody fragments, microarray adapted by design, on black polymer Maxisorb slides. By the use of this platform we have found that GBM patients and healthy controls display more similar serum protein signatures prior to immunization, while distinct signatures were observed after immunization. Furthermore, we have identi-fied tentative serum protein biomarker signatures distinguishing between short-time (average 345 days) survivors and long-time survivors (average 694 days) among the immunized patients. In the long-term run, such serum protein signatures may be used, for example, disease diagnostics, monitor-ing of the effects of immunization, and prediction of survival, based on a simple non-invasive blood test.

IMMUNOLOGY AND IMMUNOTHERAPY

IM-01. CHARACTERIZATION OF BRAIN TUMOR EXOSOMES: BIOLOGY, BIOCHEMISTRY, IMMUMOLOGYMichael Graner, Anjelika Dechkovskaia, Ling Wang, Ian Cumming, and Darell Bigner; Duke University, Durham, NC, USA

Exosomes are 30–100 nm vesicles derived from the multivesicular bod-ies of late stage endosomes that cells release into the extracellular milieu. The biological relevance of exosomes is unclear, but they may be involved in the removal of extraneous proteins and distal signaling. While many dif-ferent cell types are documented to produce and release exosomes, there are few, if any, reports of exosomes from brain tumor cells. We have isolated these vesicles from the spent media of a number of different brain tumor cultured cell lines, as well as from CD1331 cells from tumor xenografts and from a patient tumor sample. Biochemical and proteomic analyses show that brain tumor exosomes contain proteins which are common to exosomes from many cell types, as well as proteins/potential antigens that are specific to brain tumors. Electron microscopic images of material from buoyant density gradient centrifugation reveal membrane vesicles of the appropriate size for exosomes, and FACS analyses demonstrate the outer membrane leaflet localization of certain proteins. As exosomes have been associated with immunostimulatory as well as immunosuppressive capaci-ties, we are currently examining that aspect of exosome biology in a synge-neic mouse model of anaplastic astrocytoma.

IM-02. ROLE OF STAT3 IN MICROGLIA INACTIVATION IN GLIOMASLeying Zhang,1 Michelle Vanhandel,1 Babak Kateb,2 and Behnam Badie3; 1Surgical Research, City of Hope, Duarte, CA, USA; 2Department of Neurosurgery, City of Hope, Duarte, CA, USA; 3City of Hope, Duarte, CA, USA

Microglia are considered to be the main immune effector-cell popula-tion of the central nervous system. During CNS inflammation, microglia secrete a number of immunoregulatory mediators that can lead to T cell activation. Our studies, however, have suggested that the immune effector function of microglia may be suppressed in brain tumor models. To study the mechanism of microglia inactivation in gliomas, N9 microglia cells were exposed to GL261 glioma conditioned-medium (GCM) in vitro. Real-time RT-PCR demonstrated upregulation of anti-inflammatory cytokines and downregulation of pro-inflammatory cytokines by N9 cells in response to GCM. Interestingly, this cytokine change was associated with increased Stat3 activity in N9 microglia as measured by EMSA. Inhibition of Stat3 by CPA7 or siRNA reversed GCM-induced cytokine expression profile in N9 cells. Furthermore, inactivation of Stat3 in intra cranial GL261 tumors by siRNA resulted in microglia activation and tumor growth inhibition in vivo. We conclude that glioma-induced microglia suppression may be mediated thorough Stat3. Inhibition of Stat3 function in microglia may result in their activation and can potentially be used as an immunotherapy approach for gliomas.

IM-03. SYSTEMIC INHIBITION OF STAT3 PROMOTES THE EFFICACY OF ADOPTIVE TRANSFER THERAPY USING TYPE-1 CTLS BY MODULATION OF THE IMMUNOLOGICAL MICROENVIRONMENT IN INTRACRANIAL GLIOMASMitsugu Fujita,1 Xinmei Zhu,1 Kotaro Sasaki,2 Ryo Ueda,1 Keri Low,1 Ian Pollack,1 and Hideho Okada1; 1Department of Neurosurgery, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA; 2Department of Dermatology and Immunology, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

A variety of cancers, including malignant gliomas, shows aberrant activation of signal transducers and activators of transcription 3 (STAT3), which plays a pivotal role in negative regulation of anti-tumor immunity. We hypothesized that inhibition of STAT3 signals would improve the efficacy of T-cell adoptive transfer therapy by reversal of STAT3-induced immunosup-pression in intracranial (i.c.) gliomas. In vitro treatment of mouse GL261 glioma cells with a STAT3 inhibitor JSI-124 reversed highly phosphorylated status of STAT3, and suppressed their growth at high concentrations. Sys-temic intraperitoneal (i.p.) administration of JSI-124 in syngeneic C57BL/6 mice bearing i.c. GL261 glioma resulted in prolonged survival, which was associated with enhanced tumor-infiltration of CD11c1 cells producing type-1 cytokine/chemokines, and reduction of CD11b1/Gr11 myeloid suppressor and CD41/CD251 regulatory T cells in the glioma. The same treatment in athymic mice bearing GL261 glioma did not prolong their survival, suggesting that T-cell immune response, but not the direct suppres-sion of tumor-growth, was significantly involved in the JSI-124–induced prolonged survival in C57BL/6 mice at the employed dose (1mg/kg/day) of JSI-124. When i.p. JSI-124 administration was combined with intravenous (i.v.) adoptive transfer with Pmel-1 mouse-derived type-1 CTLs (Tc1), mice bearing i.c. GL261 glioma exhibited prolonged survival compared to i.v. Tc1 therapy alone. Taken together, these data suggest that systemic inhibi-tion of STAT3 signaling can reverse the suppressive immunological envi-ronment of i.c. tumors and promote the efficacy of immunotherapy using anti-tumor CTLs, especially Tc1.

IM-04. CHEMOKINE RECEPTORS CX3CR1 AND CXCR3 AND THEIR ASSOCIATED LIGANDS IN THE GL261 MURINE MODEL OF MALIGNANT GLIOMADefang Luo,1 Wolfgang Streit,2 and Jeffrey Harrison1; 1Pharmacology & Therapeutics, University of Florida, Gainesville, FL, USA; 2Neuroscience, University of Florida, Gainesville, FL, USA

A characteristic of human high grade glioma is the marked presence of tumor infiltrated microglia and/or macrophages. This phenomenon is also readily observed in a variety of rodent models of this disease. Neither the specific function(s) of these immune competent cells within the tumor microenvironment nor the mechanism(s) by which they traffic into the tumor are clearly understood. Chemokine receptors present on microglial cells and macrophages are attractive candidates to mediate the directed migration of these cells into the tumor. The purpose of this study was to evaluate the expression of, and to begin to identify roles for, two chemokine receptor systems, namely CX3CL1 (fractalkine/FKN) and its receptor CX3CR1, as well as CXCL9 (MIG), CXCL10 (IP-10), and CXCL11 (I-TAC) and



their receptor, CXCR3, in the GL261 murine model of malignant glioma. C57BL/6 mice implanted intracranially with GL261 cells (2x105) develop large tumors within 3 weeks; at this time point a significant number of CD11b1 cells have infiltrated the tumor. In situ hybridization analysis of tumor sections indicated that a number of CX3CR11 cells are found within the tumor. When present, weakly expressing CX3CL11 cells were seen near the rim of the tumor. Neither CX3CL1 nor CX3CR1 were found to be expressed in the cultured GL261 glioma cells. Evaluation of microglial cell infiltration in heterozygous Cx3cr11/GFP (Cx3cr11/–) and homozygous knockout Cx3cr1GFP/GFP (Cx3cr1–/–) animals determined that a similar density of microglial cells were present inside the tumor. Immunostain-ing with anti-CD11b revealed that the majority of the tumor infiltrating mononuclear cells also expressed CX3CR1. The morphology of the intra-tumoral CX3CR1-expressing cells was similar when comparing tumors in the heterozygous and homozygous mice; these cells displayed morphologi-cal characteristics consistent with both activated and phagocytic microglial cell phenotypes. In situ hybridization analysis of GL261 tumor sections also indicated that CXCL9, CXCL10, and CXCL11, as well as the recep-tor for these chemokines, CXCR3, were all expressed within the tumor. CXCL9- and CXCL10-expressing cells were evident in far greater numbers within the tumor, as compared to CXCL11- and CXCR3-expressing cells; CXCL10 expression was also readily detected in the GL261 cell cultures. In addition to CXCL9- and CXCL10-expressing cells within the primary tumor, CXCL9 and CXCL10 expression were also notable in secondary tumor foci. In addition, CXCL9 and CXCL10 expression was found asso-ciated with blood vessels in peritumoral tissue. These data identify two chemokine/chemokine receptor systems present within the murine GL261 model of malignant glioma. Moreover, trafficking of CD11b1 microglia/macrophages into the tumor appears to be independent of CX3CR1.

IM-05. CHARACTERIZATION OF CARBON NANOTUBE INTERNALIZATION IN A MURINE GLIOMA MODELMichelle Vanhandel,1 Babak Kateb,1 Leying Zhang,1 Michael Bronikowski,2 Harish Monahara,2 and Behnam Badie1; 1City of Hope, Duarte, CA, USA; 2Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA, USA

There is a pressing need for new therapeutic, diagnostic and drug deliv-ery approaches for treating brain cancers. Nanotechnology offers a new method for targeted brain cancer therapy and could play a major role in gene and drug delivery. The goal of our study was to evaluate differen-tial uptake and toxicity of Multi-Walled Carbon Nanotubes (MWCNTs) in gliomas. GL261 glioma and BV2 microglia cells were incubated with MWCNTs that were synthesized through catalytic chemical vapor depo-sition technique. Real-time RT-PCR, cell proliferation analysis, confocal and electron microscopy, and flow cytometry were performed. MWCNTs were internalized more efficiently by BV2 microglia as compared to GL261 cells, and did not result in proliferative or cytokine changes. For in vivo experiments, PKH-labeled MWCNTs were injected directly into intrac-ranial GL261 gliomas. MWCNTs were ingested predominantly by tumor macrophages as compared to glioma or lymphocytes. Interestingly, PKH-positive cells were able to track away from the site of injection, demonstrat-ing macrophage migration. This study suggests that MWCNTs could be used as a novel, non-toxic, and biodegradable nano-vehicle for targeted therapy in malignant brain tumors.

IM-06. VACCINE COMBINED WITH POLY-ICLC TREATMENT PROMOTES THE TYPE-1 POLARIZATION OF THE CENTRAL NERVOUS SYSTEM GLIOMA ENVIRONMENTXinmei Zhu,1 Mitsugu Fujita,2 Ryo Ueda,2 Keri Low,2 Andres Salazar,3 and Hideho Okada2; 1Department of Neurological Surgery, Cancer Institute of Pittsburgh University, Pittsburgh, PA, USA; 2PA, USA; 3Washington, DC, USA

We have demonstrated that the administration of a TLR-3 ligand, poly-ICLC promotes the efficacy of peripheral vaccinations with tumor antigen-derived peptide epitopes in murine central nervous system (CNS) tumor models and identified some critical mechanisms related to the antigen specific T cells homing to brain tumor site. We next sought to determine whether poly-ICLC could promote type-1 polarization as type-1 status is critical for antitumor immune response. We investigated how poly-ICLC exerts its effect on brain antigen-presenting cells (APCs), including CD11c1 dendritic cells, and immunosuppressive macrophages, which comprise a sig-nificant proportion of tumor-infiltrating inflammatory cells in the regula-tion of the local anti-tumor immune response. To this end, C57BL6 mice bearing syngeneic CNS GL261 gliomas received subcutaneous vaccinations with synthetic peptides designed in GL261–derived CTL epitopes (TRP-2[180–188], hGP100[25–33] as well as mEphA2 [671–679]). The mice

also received intramuscular injections with poly-ICLC twice/week. We then evaluated the status of brain infiltrated APCs. Our data demonstrate that combination strategy of vaccine and poly-ICLC treatment modulates the microenvironment of CNS glioma by: (1) promoting mature and func-tional CD11c1 dendritic cells recruitment in brain tumor site; (2) enhanc-ing type-1 cytokine (TNF-a, IL-12, etc.) and chemomkine (e.g., CXCL-9) production whereas inhibiting type-2 chemokine (CCL-22) production; and (3) reducing the number of Tie2 and Fas-ligand (FasL) expressing CD11b1 macrophages which were reported to be associated with the tumor pro-gression . Taken together, these data suggest that vaccine in combination with poly-ICLC treatment may reverse the immunosuppressive status of CNS-tumor environment by reducing the immunosuppressive macrophages and promote the type-1 polarization of CNS-tumor environment which is thought to contribute to the efficacy of combination therapy strategy of vaccine and poly-ICLC.

IM-07. ENHANCED IMMUNITY TO INTRACEREBRAL BREAST CANCER IN MICE IMMUNIZED WITH A cDNA-BASED VACCINE ENRICHED FOR IMMUNOTHERAPEUTIC CELLSTerry Lichtor,1 Roberta Glick,2 Lisa Feldman,3 Goro Osawa,2 Julian Hardman,2 Insug O-Sullivan,2 and Edward Cohen2; 1Neurosurgery, Rush University Medical Center, Chicago, IL, USA; 2University of Illinois at Chicago, Chicago, IL, USA; 3Rush University, IL, USA

Antigenic differences between normal and malignant cells of the cancer patient form the rationale for clinical immunotherapeutic strategies. As the antigenic phenotype of neoplastic cells varies widely among different cells within the same malignant cell-population, immunization with a vaccine that stimulates immunity to the broad array of tumor antigens expressed by the cancer cells is likely to be more efficacious than immunization with a vaccine for a single antigen. Previously, we reported the efficacy of a multi-epitope vaccine prepared by transfer of genomic DNA from breast can-cer cells into a highly immunogenic fibroblast cell line (LM) where genes specifying breast cancer antigens were expressed. Here, we report a new cell-based vaccination strategy for the treatment of intracerebral breast cancer using a vaccine enriched for immunotherapeutic cells. In particular aliquots of the transfected fibroblast were divided into pools which were expanded and selected for increased numbers of immunogenic cells. In mice with an established intracerebral breast cancer, the strongest systemic anti-tumor immune response detected in either the spleen cells or cervical lymph nodes by an ELISPOT Interferon-gamma assay was found in those animals treated with the enriched vaccine. The immunity was mediated predomi-nantly by CD41, CD81, and NK/LAK cells. Furthermore a PCR and FACS analysis of the spleen, cervical lymph nodes and brain revealed a decreased expression of Foxp31 regulatory T cells, which are known to be involved in immune suppression, in mice injected intracerebrally with the enriched vaccine. Finally the enhanced immunity to the neoplasm generated in mice injected intracerebrally into the tumor bed with a vaccine enriched for immunotherapeutic cells translated into prolongation of survival. We con-clude that an array of undefined tumor-associated antigens was expressed in a highly immunogenic form by the cells transfected with tumor DNA, and the enrichment technique strategy enables the generation of highly immuno-genic pools of transfected cells with enhanced immunotherapeutic proper-ties. Immunization in conjunction with conventional treatment strategies may result in improved survival in patients with intracerebral tumors.

IM-08. INTRATUMORAL MANIPULATION OF PLASMACYTOID DENDRITIC CELLS BY FLT3L AND HSV1–TK TREATMENT IN A SYNGENEIC MODEL OF GLIOMA ELICITS TUMOR REGRESSION AND IMMUNOLOGICAL MEMORYGwendalyn King,1 James Curtin,1 Ghulam Muhammad,1 Daniel Larocque,1 Carlos Barcia,1 Marianela Candolfi,1 Bresee Catherine,2 Chunyan Liu,1 Sarah Honig,1 Nico Van Rooijen,3 Antoni Ribas,4 Robert Pechnick,2 Kurt Kroger,1 Pedro Lowenstein,1 and Maria Castro1; 1Gene Therapeutics Research Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA; 2Psychiatry Research, Cedars Sinai Medical Center, Los Angeles, CA, USA; 3VU MC FdG, Netherlands; 4Department of Medicine, Hematology and Oncology, UCLA, Los Angeles, CA, USA

Tumor recurrence post-resection of glioblastoma multiforme (GBM) hinders the long-term efficacy of current treatment modalities. We tested the hypothesis that in a large syngeneic rat model of glioma, intra-tumoral expression of the immunostimulant, FMS-like tyrosine kinase 3 ligand (Flt3L) and conditionally cytotoxic HSV1–TK followed by treatment with ganciclovir (GCV) would induce dendritic cell recruitment into the tumor mass with concomitant release of tumor antigen for uptake and antigen presentation. If anti-tumor immunity initiated by this combined treatment



initiates adaptive immune response, long-term protection from tumor recurrence would be observed. Upon analysis 15 and 22 days after CNS-1 tumor implantation followed by treatment with AdFlt3L and AdTK (1gan-ciclovir; GCV), we demonstrated infiltration of plasmacytoid dendritic cells (pDCs), B cells, macrophages, cDCs, and NK cells into the tumor mass. PDCs isolated from treated tumors were capable of in vitro antigen uptake and induced proliferation of T cells. These results suggest their capacity to act as antigen presenting cells. PDCs are known to influence adaptive immu-nity as the primary physiological source of IFN. To determine whether IFN secretion could mediate tumor regression in the syngeneic GBM model, we treated the animals 3 days post tumor implantation with AdINF. Our data shows tumor regression and long-term survival in 60% of the tumor bear-ing animals. We further wished to assess if INF could mimic the effects of Flt3L, when used in combination with AdTK in the large intracranial GBM model. When AdIFN and AdTK were used in combination, we observed tumor regression and prolongation of the survival time in the GBM bear-ing animals. Although the effects were not as potent when we compared them to AdFlt3L combined with AdTK in the same GBM model. To test whether long-term immunological memory was induced by Flt3L and HSV1–TK treatment, treated animals surviving to day 60 post implanta-tion, were re-implanted with CNS-1 cells into the contralateral hemisphere. While 80% of animals survived the initial tumor implantation with Flt3L and HSV1–TK treatment, all animals rechallenged survived long-term. Immunological memory responses to CNS-1 tumors were dependent on the presence of CD81 T cells. Animals implanted with CNS-1 tumors dis-played amphetamine induced rotational preference at the time of treatment, when the tumors were very large. Following tumor elimination, behavioral abnormalities resolved completely and fine motor skills were not disrupted. Neuropathological evaluation of long-term survivors after rechallenge showed low levels of immune infiltration restricted to the immediate tumor injection sites. In summary, treatment of GBM in rats with Flt3L in com-bination with TK (1GCV) induces infiltration of antigen presenting cells capable of inducing long-term immunological memory that protects against tumor recurrence. These data provide experimental evidence to support the further development and implementation of AdFlt3L combined with AdTK in a GBM clinical trial. Supported by NIH/NINDS Grant 1R01 NS44556.01, Minority Supplement NS445561; 1R21-NSO54143.01; 1UO1 NS052465.01to M.G.C.; NIH/NINDS Grants 1 RO1 NS 054193.01; RO1 NS 42893.01; U54 NS045309-01, and 1R21 NS047298-01 to P.R.L. GDK is supported by NINDS F32 NS0503034-01.

IM-09. HIGH-CAPACITY ADENOVIRAL VECTORS EXPRESSING HSV1-TK ELICIT TUMOR REGRESSION IN A SYNGENEIC INTRACRANIAL GLIOMA MODEL EVEN IN THE PRESENCE OF SYSTEMIC IMMUNITY AGAINST ADENOVIRUSES AS WOULD BE ENCOUNTERED IN CLINICAL TRIALSGwendalyn King,1 Akm Muhammad,1 Kurt Kroeger,1 Mariana Puntel,1 Daniel Larocque,1 Chunyan Liu,1 Donna Palmer,2 Phillip Ng,2 Pedro Lowenstein,1 and Maria Castro1; 1Gene Therapeutics Research Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA; 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA

Malignant glioma (GBM) is the most common subtype of primary brain tumor. In preclinical models, replication deficient adenoviruses (Ad) expressing herpes simplex virus type 1-hymidine kinase (Ad-TK) combined with prodrug ganciclovir (GCV) are highly efficient in causing tumor regression. Further, Ad-TK is currently been tested in phase III clinical tri-als and the results of a small phase II trial are encouraging (Ali S et al., Molecular Therapy, 2004 10:1071). The majority of humans have been infected with adenovirus over the course of their lives and develop long-term immunological memory that could eliminate therapeutic transgene expression when using first generation Ad vectors, therefore hampering efficacy in clinical applications (Thomas CE et al., PNAS, 1997 97:7482). To overcome this pitfall, we developed high-capacity replication deficient viral vectors expressing HSV1-TK (HC-AdTK) under the control of the murine CMV promoter. Because these vectors are devoid of all Ad coding regions, they become invisible to the host’s immune system and can sustain very long-term transgene expression even in the presence of anti-Ad circu-lating antibodies as could be encountered in human patients undergoing clinical trials for GBM. We assessed HC-Ad mediated HVS1-TK expression in the presence of a strong systemic immune response to Ads as could be encountered in the clinic. As expected, animals peripherally immunized with Ad vectors developed systemic immunity to adenovirus (as detected by neutralizing antibody and IFN elispot assays). Thirty days after HC-Ad injection in brain, comparable expression of TK was observed in all ani-mals regardless of systemic immunity. Meanwhile, TK expression from the first generation AdTK was completely abolished in the animals with pre-existing immunity to adenovirus. We next wished to determine whether treatment with HC-AdTK would elicit tumor regression and long-term survival even in the presence of a systemic immune response against Ads.

To do so, Lewis rats were peripherally immunized by injection of either Ad vector or saline (non-immunized controls). Fourteen days later only animals peripherally immunized with Ad vectors had developed systemic immunity to adenovirus (as detected by neutralizing antibody and IFN elispot assays). 5000 CNS-1 rat glioma cells were unilaterally implanted in the striatum and treatment was administered seven days later by intratumoral injection of HC-AdTK (with peripheral GCV). Regardless of adenoviral peripheral immune status, treatment with HC-AdTK resulted in tumor regression and long-term survival in ~60% of the tumor bearing animals. Utilizing an identical experimental paradigm, treatment with first generation AdTK, resulted in the death of Ad immunized animals. In conclusion, even in the presence of anti-adenoviral immunity, HC-Ad-TK vectors induced glioma regression and long-term survival indicating this treatment could be highly effective in human GBM patients regardless of previous exposure to ade-novirus. Supported by NIH/NINDS Grant 1R01 NS44556.01, Minor-ity Supplement NS445561; 1R21-NSO54143.01; 1UO1 NS052465.01 to M.G.C.; NIH/NINDS Grants 1 RO1 NS 054193.01; RO1 NS 42893.01; U54 NS045309-01, and 1R21 NS047298–01 to P.R.L. G.D.K. is supported by NINDS F32 NS0503034-01.

IM-10. ENDOGENOUS, TUMOR-DERIVED TLR2 LIGANDS MEDIATE T CELL–DEPENDENT BRAIN TUMOR REGRESSIONJames Curtin,1 Gwendalyn King,1 Kathrin Michelsen,1 Carlos Barcia,2 Tamer Fakhouri,1 Daniel Larocque,1 Marianela Candolfi,1 Begonya Comin-Anduix,3 Chunyan Liu,2 Kurt Kroeger,2 Daniel Resnick,1 Moshe Arditi,1 Antoni Ribas,4 Pedro Lowenstein,2 and Maria Castro1; 1Cedars-Sinai Medical Center, Los Angeles, CA, USA; 2Gene Therapeutics Research Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA; 3Department of Surgery, UCLA, CA, USA; 4Department of Medicine, Hematology and Oncology, UCLA, Los Angeles, CA, USA

Immune privilege, tolerance, immune-evasion and a lack of intra-tumoral dendritic cells (DC) all contribute to immunological ignorance against glioblastoma (GBM). To successfully induce T cell–dependent GBM regression, many or all of these limiting factors must be overcome. Herein we demonstrate that endogenous TLR2 signaling is necessary to overcome immunological ignorance against GBM antigen. We show that TLR2 sig-naling independently regulates two key stages in the initiation of adaptive immune responses against brain tumor antigen; infiltration of DC into the tumor in response to Flt3L and TK treatment, and DC activation. We found that TLR2 expression was required for the infiltration of bone marrow derived dendritic cells (BMDC) into syngeneic GL26 tumors in response to the intratumoral adenoviral-mediated expression of Flt3L and TK (1GCV). We also found that BMDC activation was induced by co-incubating wild-type but not TLR2–/– BMDC with necrotic and apoptotic GBM cells as assessed by the secretion of TNFa. Necrotic mouse and rat tumor cell lines stimulated NFκB activation in HEK-293 cells transfected with TLR2, but not TLR4. This confirms the presence of endogenous TLR2 ligands in dead tumor cells. Wild type BMDC loaded with necrotic GL26 cells were shown to stimulate tumor antigen specific T cell proliferation whereas TLR2–/– BMDC loaded with GL26 tumor extract fail to stimulate the proliferation of tumor antigen specific T cells. Moreover, wild type BMDC loaded with necrotic GL26 cell lysate induced allogeneic (Balb[c]) T cell proliferation. This was also significantly lower when allogeneic T cells were incubated with TLR2–/– BMDC, suggesting that activation of BMDC via TLR2 sig-naling is sufficient to stimulate allogeneic and syngeneic antigen dependent T cell proliferation. We next determined whether TLR2 signaling mediated clonal expansion of tumor antigen specific T cells. We observed that when T cells from wild type C57BL/6 mice were incubated with BMDC loaded with GL26 tumor extract IFN was produced. This did not occur using T cells isolated from TLR2–/– mice, suggesting that clonal expansion of tumor antigen specific T cells was ablated in TLR2–/– mice, which we sub-sequently confirmed using Trp2(180–188) loaded, H2–Kb MHC tetram-ers. We demonstrated that intratumoral expression of Flt3L and TK failed to improve survival in TLR2–/– mice bearing intracranial GL26 tumors, compared with long term survival observed in half of wild type C57BL/6 mice. Furthermore, TLR2 expression on bone-marrow derived APC, and not on resident microglial cells was sufficient and necessary to induce tumor regression and improve survival using Flt3L and TK. In conclusion, our data show that TLR2-dependent DC migration into an intracranial GBM mass and subsequent TLR2-dependent DC activation plays essential role in promoting a CD81 T cell dependent anti-GBM immune responses. Our data suggest that TLR2 signaling might be useful as a novel adjuvant therapy to overcome immunological privilege in the brain. Supported by NIH/NINDS Grant 1R01 NS44556.01, Minority Supplement NS445561; 1R21–NSO54143.01; 1UO1 NS052465.01 to M.G.C.; NIH/NINDS Grants 1 RO1 NS 054193.01; RO1 NS 42893.01; U54 NS045309–01, and 1R21 NS047298–01 to P.R.L.



IM-11. B CELLS ARE REQUIRED FOR PRESENTATION OF TUMOR ANTIGEN TO T CELLS AND FOR T CELL–DEPENDENT TUMOR REGRESSIONJames Curtin, Tamer Fakhouri, Chunyan Liu, Pedro Lowenstein, and Maria Castro; GTRI, Cedars-Sinai Medical Center, Los Angeles, CA, USA

B cells can present foreign antigen to naïve T cells and stimulate T cell proliferation and clonal expansion in response to infectious agents. How-ever, the role of B cells as brain tumor antigen presenting cells is not clear. Immunotherapy against brain tumors must first overcome immune igno-rance to tumor antigen. We have successfully induced T cell–dependent brain tumor regression by the intratumoral expression of Fms-like tyrosine kinase 3 ligand (Flt3 ligand) and Herpes Simplex Virus 1 Thymidine Kinase (TK) (p , 0.001, One Way ANOVA). In this study, we demonstrate that B cells are necessary to mount effective, tumor specific T cell immune responses in vivo. We found that bone-marrow-derived CD191 B cells infiltrated syngeneic GL26 brain tumors, in both saline and Flt3L and TK treated mice. We found that tumor infiltrating B cells engulfed CellTracker Green-labeled tumor antigen and confirmed that B cells containing Cell-Tracker Green labeled tumor antigen were present in the draining (cervical) lymph nodes. This suggested that B cells can endocytose tumor antigen in the brain tumor and traffic from the tumor to the draining lymph nodes (DLN). We found that intratumoral expression of Flt3L and HSV1–TK increased the total number of B cells in the DLN and we also found that B7–1 and B7–2 was upregulated on B cells in the DLN 7 days after treat-ment with Flt3L and TK compared with saline-treated control mice. We observed significant (p , 0.01, One Way ANOVA) increases in the total number of tumor-infiltrating T cells in wild type C57BL/6 mice 7 days after treatment with Flt3L and TK. Moreover, we used ELISPOT to determine that while IFN-producing tumor antigen–specific T cells were increased in wild type mice, no such increase was observed in B cell–deficient mice bearing GL26 tumors and treated with Flt3L and TK. We did not detect any increase in tumor specific antibodies in the sera of treated mice, nor did we observe any increase in the presence of immunoglobulins that opsonized GL26 cells that we had isolated from the brains of Flt3L and TK treated mice, compared with saline-treated control mice. Together, our data sug-gest that the primary role of B cells in brain tumors treated with Flt3L and TK is to initiate T cell–dependent tumor regression. They may do this either by directly presenting tumor antigen to T cells or by trafficking tumor antigen from the tumor to the DLN where DC can present the antigen to T cells. In conclusion, our data support the hypothesis that B cells are tumor antigen–presenting cells and are required for the clonal expansion of tumor antigen–specific T cells and brain tumor regression. Supported by NIH/NINDS Grant 1R01 NS44556.01, Minority Supplement NS445561; 1R21-NSO54143.01; 1UO1 NS052465.01to M.G.C.; NIH/NINDS Grants 1 RO1 NS 054193.01; RO1 NS 42893.01; U54 NS045309-01, and 1R21 NS047298-01 to P.R.L.

IM-12. SYSTEMIC IMMUNOLOGICAL MEMORY ELICITED BY AD.FLT3L AND AD.TK TREATMENT OF RAT INTRACRANIAL GLIOMA RESULTS IN REGRESSION OF A SECOND TUMOR IMPLANTED IN THE PERIPHERYA.K.M. Muhammad, Gwendalyn King, James Curtin, Marianela Candolfi, Chunyan Liu, Kurt Kroeger, Pedro Lowenstein, and Maria Castro; GTRI, Cedars-Sinai Medical Center, Los Angeles, CA, USA

Recurrence of human glioblastoma multiforme (GBM) is a common phenomenon that renders the outcome of currently available treatment modalities, including surgery, radiotherapy and chemotherapy margin-ally effective. We have developed a gene therapy strategy that combines conditional cytotoxicity (Thymidine kinase, TK) and immune stimulation utilizing Flt3 ligand (Flt3L) to treat very large syngeneic, intracranial GBM model (Ali et al., Cancer Res., 2005, 65:7194–7204). The combined gene therapy not only resulted in .80% survival of treated animals bearing a very large intracranial GBM, but also induced CD81 T cell dependent immunological memory that was capable of eliminating a second tumor implanted into the contra lateral hemisphere of the long-term (over 60 days) survivors. In the present study, we evaluated the efficacy of such treatment in preventing growth of a second tumor implanted outside the neuroaxis. Male Lewis rats (200–250 g; Harlan, Indianapolis, IN, USA) were injected with 4,500 CNS-I cells into the striatum (11 mm anterior from bregma, 13 mm lateral and –5 mm from dura). Nine days later, they were treated with intratumoral injection of Ad.TK and Ad.Flt3L (1 3 108 pfu each); the controls received saline. Twenty-four hours later, gancyclovir (25 mg/kg) was injected i.p. twice daily for 10 consecutive days. All saline treated control animals succumbed to tumor by day 20 and 80 % of the TK-Flt3L treated rats survived long term (p , 0.05). The long-term survivors were rechallenged with CNS-I cell (3 3 106) implanted in the flanks; naïve rats implanted with CNS- I cell in flanks served as controls. The size of the tumor was measured daily in both groups for 10 days, that is, until the

control tumors started regressing in size due to ischemic necrosis as revealed by H&E staining. The volume of the tumor mass (mm3; mean 6SD) was estimated to be 13.0 63.3, 13.7 62.8, 11.5 65.9, 1.7 60.7, and 0.3 60.2 in treated and 26.3 62.5, 52.1 64.2, 127.5 614.5, 426.1 636.3, and 203.8 634.3 in control groups at post implantation day 2, 4, 6, 8, and 10, respec-tively. The inter-group difference in tumor volume mass was significantly different. Employing irradiated CNS-I cells, delayed type hypersensitivity (DTH) reaction was seen to be present in long-term survivors whereas it was absent in saline treated controls. These findings suggest that the long-term immunological memory induced by our combined gene therapy is capable of causing regression of a second tumor implanted peripherally two months after treatment of the primary intracranial tumor mass and attest to the systemic nature of the anti-GBM immune response elicited by the combined Flt3L/TK (plus GCV) gene therapy. Brains of long-term survivors displayed no residual tumor. We detected CD681 macrophages at and around tumor regression scar but CD81 T cells were not seen, indicating that tumor regression results in limited damage to the brain parenchyma and low level, site-specific innate inflammation. In conclusion, gene therapy combining TK and Flt3L can elicit a powerful systemic anti-GBM immune response and appears to be a promising adjuvant for the treatment of GBM. Supported by NIH/NINDS Grant 1R01 NS44556.01, Minority Supplement NS445561; 1R21-NSO54143.01; 1UO1 NS052465.01 to M.G.C.; NIH/NINDS Grants 1 RO1 NS 054193.01; RO1 NS 42893.01; U54 NS045309–01, and 1R21 NS047298-01 to P.R.L.

IM-13. EFFICACY OF SYSTEMIC DELIVERY OF AD-FLT3L IN A SYNGENEIC RAT INTRACRANIAL GLIOBLASTOMA MODEL TREATED WITH AD-HSV1–TK DELIVERED INTO THE TUMOR MASSA.K.M. Muhammad, Gwendalyn King, Marianela Candolfi, James Curtin, Chunyan Liu, Kurt Kroeger, Pedro Lowenstein, and Maria Castro; Cedars-Sinai Medical Center, Los Angeles, CA, USA

Although the brain has been described as an “immune privileged” organ, the subsequent discovery of a large number of interactions at molec-ular and cellular levels between the brain and the immune system opened up avenues for immunotherapy targeting brain diseases. In our quest of developing novel treatment modalities for glioblastoma multiforme (GBM), we wished to test the hypothesis that by recruiting antigen presenting cells into the tumor mass following antigen release by local cytotoxic tumor destruction, could elicit an effective anti-GBM immune response. To this end, we developed an effective gene therapy strategy that combines condi-tional cytotoxicity (Herpes Simplex Type1 Thymidine Kinase, HSV1–TK) and immune stimulation utilizing FMS-like tyrosine kinase 3 ligand (Flt3L) to treat syngeniec, rat intracranial GBM model where the viral vectors are delivered directly into the tumor mass by intratumoral injection. In the present study we administered Flt3L by intravenous tail vein injection with the aim to render the treatment non invasive. Male Lewis rats (200–250 g; Harlan, Indianapolis, IN, USA) were injected with 4,500 CNS-1 cells into the striatum (11 mm anterior from bregma, 13 mm lateral and –5 mm from dura). Six days later, predosing with a nonreporter virus, Ad.0 (4 3 1010 vp), was performed by intravenous (tail vein) injection in order to saturate the liver reticulo-endothelium system/Kuffer cells. Within 4–6 hours of pre-dosing, Ad.Flt3L (5 3 109 pfu) was injected intravenously in the treatment group; the controls received saline, either intravenously or intratumorally. The treated group received an intratumoral injection of Ad.TK (1 3 108 pfu). Twenty-four hours later, gancyclovir (25 mg/kg) was injected i.p. twice daily for 10 consecutive days. All saline treated control animals succumbed to tumor by day 17, whereas the Flt3L-TK treated rats survived tumor free long term (p , 0.05). H&E staining on liver specimens did not show any toxicity. Brains of long-term survivors displayed no residual tumor. While ED11 macrophages were detected at and around tumor regression scar, CD81 T cells were not detected. These findings indicate that tumor regression results in limited damage to the brain parenchyma and low level, site-specific innate inflammation. In conclusion, gene therapy with Flt3L administered systemically combined with a local cytotoxic/oncolytic ther-apy (i.e., HSV1–TK) appears to have prospects as an adjuvant treatment of GBM. Supported by NIH/NINDS Grant 1R01 NS44556.01, Minor-ity Supplement NS445561; 1R21-NSO54143.01; 1UO1 NS052465.01 to M.G.C.; NIH/NINDS Grants 1 RO1 NS 054193.01; RO1 NS 42893.01; U54 NS045309-01, and 1R21 NS047298-01 to P.R.L.



IM-14. PREFERENTIAL MIGRATION OF REGULATORY T CELLS MEDIATED BY GLIOMA-SECRETED CHEMOKINES CAN BE BLOCKED WITH CHEMOTHERAPYWei Sun, Justin T Jordan, S. Farzana Hussain, Guillermo DeAngulo, Sujit S. Prabhu, and Amy B. Heimberger; University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Despite the immunogenicity of glioblastoma multiforme (GBM), immune-mediated eradication of these tumors remains deficient. Regula-tory T cells (Tregs) in the blood and within the tumor microenvironment of GBM patients are known to contribute to their dismal immune responses. Here, we determined which chemokine secreted by gliomas can prefer-entially induce Treg recruitment and migration. In the malignant human glioma cell lines D-54, U-87, U-251, and LN-229, the chemokines CCL22 and CCL2 were detected by intracellular cytokine analysis. Furthermore, tumor cells from 8 patients with GBM had a similar chemokine expression profile. However, only CCL2 was detected by enzyme-linked immunosor-bent assay, indicating that CCL2 may be the principal chemokine for Treg migration in GBM patients. Interestingly, the Tregs from GBM patients had significantly higher expression levels of the CCL2 receptor CCR4 than did Tregs from healthy controls. Glioma supernatants and the recombinant human chemokines CCL2 and CCL22 induced Treg migration and were blocked by antibodies to the chemokine receptors. Production of CCL2 by glioma cells could also be mitigated by the chemotherapeutic agents temozo-lomide and carmustine [3–bis (2–chloroethyl)-1–nitrosourea]. Our results indicate that gliomas augment immunosuppression by selective chemokine-mediated recruitment of Tregs into the tumor microenvironment and that modulating this interaction with chemotherapy could facilitate the develop-ment of novel immunotherapeutics to malignant gliomas.

IM-15. THERAPEUTIC EFFICACY OF COMBINED PRO-APOPTOTIC WITH IMMUNE STIMULATORY HIGH-CAPACITY GUTLESS ADENOVIRUS (HC-AD)–MEDIATED GENE THERAPY FOR GBM: FROM RODENT MODELS TO SPONTANEOUS GBM IN DOGSMarianela Candolfi,1 Akm Muhammad,1 Gwendalyn King,1 James Curtin,1 Weidong Xiong,1 Mariana Puntel,1 Chunyan Liu,1 W. Nichols,2 G. Pluhar,3 E. McNiel,3 John Ohlfest,4 Andrew Freese,4 Peter Moore,5 John Young,6 Phillip Ng,7 Donna Palmer,8 Pedro Lowenstein,1 and Maria Castro1; 1Gene Therapeutics Research Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA; 2Department of Pathology, Cedars Sinai Medical Center, Los Angeles, CA, USA; 3University of Minnesota, St. Paul, MN, USA; 4Neurosurgery, Pediatrics, University of Minnesota, Minneapolis, MN, USA; 5University of California, Davis, Davis, CA, USA; 6Cedars Sinai Medical Center, Los Angeles, CA, USA; 7Baylor College of Medicine, TX, USA; 8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA

Glioblastoma Multiforme (GBM) is the most common primary brain tumor in adults, and has a dismal prognosis. To identify the optimal pre-clinical model to test gene therapies for GBM, we characterized intracranial human xenografts in nude mice, syngeneic GL26 GBMs (C57BL/6 mice), CNS1 GBMs (Lewis rats) and spontaneous dog GBM, and compared them with human GBM. All GBMs exhibited necroses, neovascularization, pleo-morphism, glial markers, tumor infiltration into non-neoplastic brain, and inflammatory cell infiltration. Endothelial proliferation was only observed in dog GBM. We then tested transgene delivery using adenovirus serotype 5 (Ad5) in mouse, rat, dog and human glioma cells (cell lines and primary cultures intra-operative biopsies) and determined the expression of Ad receptors (CAR, integrin, MHCI). Although we found high variability in Ad receptor expression levels among the different GBM cells, Ad5 mediated therapeutic gene expression was very efficient in all of them. Further, we found no correlation between the levels of CAR, INT or MHCI molecules and levels of transgene expression, or the number of GBM cells transduced. We also administered Ad5 vectors in the brain of mice, rats, and Beagle dogs and found widespread distribution of transgene expression in astrocytes and neurons without clinical or neuropathological side effects, attesting to the suitability and efficacy of Ad5 to drive effective and safe therapeutic trans-gene expression for the treatment of GBM. To identify the most potent cyto-toxic gene therapy approach to kill GBM and release tumor antigens in situ to be engulfed by immune phagocytic/antigen presenting cells recruited into the tumor mass, we tested several Ad5 expressing pro-apoptotic transgenes, i.e., Herpes simplex type 1–thymidine kinase (Ad-TK), TNF-alpha (Ad-TNF-alpha), FasL (Ad-FasL) or TRAIL (Ad-TRAIL). HSV1–TK selectively kills dividing cells in combination with the prodrug ganciclovir (GCV), while TNF-alpha, FasL or TRAIL kill cells expressing the respective death receptor. We found that Ad-TK and Ad-FasL significantly improved the sur-vival of rats bearing established CNS-1 tumors (day 4 after implantation) when compared to saline, Ad-TNF-alpha and Ad-TRAIL. Then, we treated larger tumors (day 9 after implantation) with Ad-TK or Ad-FasL alone or combined with Ad-Flt3L, encoding fms-like tyrosine kinase ligand (Flt3L),

which recruits and activates dendritic cells into the tumor mass, improv-ing the presentation of tumor antigens released by proapoptotic Ads. The combination of Ad-TK with Ad-Flt3L induced the most significant GBM regression and long term survival. However, since most humans exhibit a pre-existing systemic anti-Ad immune response which could preclude both Ad infection/transduction and eliminate therapeutic transgene expression (Proc Natl Acad Sci U S A., 2000 97: 7482–7487; J Exp Med., 2006 203: 2095–2107), we developed non-immunogenic HC-Ad vectors expressing Flt3L and TK and tested their efficacy in rats bearing large intracranial GBMs. This treatment led to long term survival in 70% of the tumor bear-ing rats and immunological memory. Our results show that HC-Ad vectors encoding HSV1–TK and Flt3L constitute an attractive therapeutic approach for human GBM. Supported by National Institutes of Health/National Institute of Neurological Disorders & Stroke (NIH/NINDS) Grant 1R01 NS44556.01, Minority Supplement NS445561; 1R21-NSO54143.01; 1UO1 NS052465.01 to M.G.C.; NIH/NINDS Grants 1 RO1 NS 054193.01; RO1 NS 42893.01; U54 NS045309-01, and 1R21 NS047298-01 to P.R.L. M.C. is supported by NIH/NINDS 1F32 NS058156.01.

IM-16. CHEMO-IMMUNOTHERAPY: COMBINATION OF TEMOZOLOMIDE CHEMOTHERAPY AND DENDRITIC CELL VACCINATION IN MURINE GLIOMA MODELYong-Kil Hong1 and Tai-Gyu Kim2; 1Neurosurgery, Kangnam St. Mary’s Hospital, Seoul, South Korea; 2The Catholic University of Korea, Korea

Although chemotherapy remains among the best treatment options for most cancers, adjuvant therapies such as dendritic cell (DC)–based immu-notherapy have been added to treatment protocols to destroy residual tumor cells. IFN-secreting T cells specific for survivin was found after temozolo-mide (TMZ) treatment in C57BL/6 mice intracranial inoculated with GL26 cells. Furthermore, combination treatment with low-dose TMZ (2.5 mg/kg/day, i.p.) chemotherapy followed by vaccination with survivin RNA-transfected DCs (1 3 106 cells/mouse, s.c.) enhanced T cells responses spe-cific for survivin and improved survival rate compared with DC vaccination alone or TMZ treatment alone in tumor inoculated mice. However, these enhancements of T cells responses by TMZ treatment were not observed in mice without tumor inoculation. These results suggest that cross-priming by TMZ may enhance the antitumor effect of DC vaccination in malignant gliomas.

IM-17. INTRATUMORAL INJECTION OF DENDRITIC CELLS LOADED WITH LYSATE FROM GLIOMA CELLS PROLONGS SURVIVAL IN TUMOR-BEARING MICE: IMMUNOLOGICAL ASSESSMENT OF THE LOCAL ANTITUMOR EFFECTSerena Pellegatta,1 Pietro Luigi Poliani,2 Daniela Corno,1 Francesca Orzan,1 Elena Stucchi,1 Chiara Agnese Colombo,1 Valentina Caldera,1 Silvia Musio,1 and Gaetano Finocchiaro1; 1Istituto Neurologico C. Besta, Milan, Italy; 2University of Brescia, Italy

Recent work has suggested that the intra-tumoral (IT) and peripheral injection of dendritic cells (DC) may prolong survival of patients affected by malignant gliomas (Yamanaka et al., 2005). To evaluate the effect of it injections of DC on survival of mice bearing GL261 gliomas, immune-competent mice were injected with GL261 cells and treated with DC loaded with a GL261-lysate. All mice received three subcutaneous (SC) injections with 1 3 106 pulsed DC (pDC) spaced one week, one group also received one it injection of 2 3 105 pDC (it-sc). The combination of systemic and it vaccination cured 70% of the mice, whereas sc injection cured 50% of mice ( p ,0.0001 and p 5 0.002 vs. controls, respectively). To characterize the effect of the treatments on tumor microenviroment we used RT-PCR and found that TGF-beta expression is 1.4-fold higher in brains of mice treated by DCsc than by DCit-sc (p 5 0.04) and IFN-gamma expression 2-fold higher in brains of mice treated by DC it-sc than by DCsc (p 5 0.02). The analysis of regulatory T cell (Treg) by flow cytometry and RT-PCR on CD41/CD251 cells isolated from lymph nodes and spleen 15, 24, or 31 days after tumor implantation revealed increased expression of FoxP3 at each time-point in controls and a slight decrease in vaccinated mice. Tumor infiltrating lymphocytes (TIL) were characterized by histological analy-sis. In controls, a significant infiltration of CD41 cells and Foxp31 cells was observed in the tumor mass. In vaccinated mice CD41 and Treg cells appeared preferentially located in perivascular zones outside the tumors, while CD81 T cells mostly infiltrated the tumor mass. Histological evalua-tions revealed the persistence of viable pDC IT and the interaction with TIL, suggesting that DC can mediate biological activities in situ. Also of interest are in vitro observations that pDC produce higher level of IFN-gamma and TNF-alpha and lower level of IL-6 than unpulsed DC. Overall the results suggest that SC DC are effective in eliciting T cell responses and creating a convergence of immune response elements to tumor the site and and that



IT DC can modulate tumor microenvironment and potentiate anti-tumor immune responses, thus exerting a dual function on tumor biology and immune suppression.

IM-18. SELECTIVE MODIFICATION OF ANTIGEN-SPECIFIC T CELLS USING RNA ELECTROPORATIONDuane Mitchell, Isaac Karikari, Xiuyu Cui, Weihua Xie, Robert Schmittling, and John Sampson; Surgery, Duke University, Durham, NC, USA

We have investigated the use of RNA based gene transfer to modify the function of in vitro expanded T cells for use in adoptive immunotherapy. We and others have observed that the efficient transfection of T cells using RNA electroporation requires prior activation of T cells using mitogens or anti-CD3 antibody stimulation. We hypothesized that this requirement for T cell activation could be leveraged to express marker genes within activated T cells responding to antigen-pulsed dendritic cells and allow for the selective enrichment and modification of antigen-specific T cells. Uti-lizing electroporation of mRNA encoding for green fluorescent protein as a marker gene, we demonstrate that RNA electroporation can efficiently allow for the separation of human Cytomegalovirus (CMV)-specific CD81 and CD41 T cells from bulk culture responding to CMV pp65 antigen-pulsed DC. Furthermore, we demonstrate that CMV-specific T cells can be functionally modified using RNA transfection of the C-X-C chemokine receptor, CXCR2, to migrate efficiently toward a variety of CXCR2–specific chemokines in vitro and in vivo. These studies demonstrate the utility of RNA transfection as a simple method to purify and selectively modify the function of antigen-specific T cells for use in adoptive immu-notherapy, and importantly provide evidence that transient expression of proteins using RNA transfection is an efficient means of modulating the in vivo function of activated T cells. This work was supported by grants from the Howard Hughes Medical Student Fellowship Award (Karikari), NIH/NINDS SPORE in Brain Cancer 1P50 CA108786 01 (Sampson), NIH R01 CA-97222–01 (Sampson), The Brain Tumor Society Billy Grey Chair of Research Award (Mitchell), and Accelerate Brain Cancer Cure Foundation Young Investigator’s Award (Mitchell).

IM-19. INDUCTION OF ENHANCED IMMUNOLOGIC RESPONSES DURING HEMATOPOEITIC RECOVERY FROM TEMOZOLOMIDE-INDUCED LYMPHOPENIADuane Mitchell,1 Xiuyu Cui,1 Tracy Chewning,1 Henry Friedman,2 and John Sampson3; 1Surgery, Duke University, Durham, NC, USA; 2Medicine, Duke University, Durham, NC, USA; 3Durham, NC, USA

Homeostatic proliferation involves the expansion of residual or trans-ferred lymphocytes in hosts recovering from lymphopenia, and has been leveraged to enhance immune responses against tumors in experimental animals and human patients. We sought to determine whether temozolo-mide (TMZ), an alkylating agent given routinely to patients with malignant gliomas (MGs), could be utilized as an induction agent for enhancing sub-sequent adoptive immunotherapy and dendritic cell (DC) and/or peptide vaccination. Mice were lymphodepleted using TMZ or total body irridation (TBI), and subsequently infused with lymphocytes from syngeneic or T cell receptor transgenic mice (OT-I mice) labeled with an intracellular fluores-cent tracer (CFSE). Some animals received concurrent subcutaneous DC or peptide vaccination against the model antigen chicken ovalbumin (OVA). The proliferation of transferred T cells was monitored by following frac-tionation of CFSE with cellular division and the OVA-specific T cell precur-sor frequency in the peripheral blood was evaluated using tetramer analy-sis. Mice lymphodepleted with TMZ or TBI exhibited markedly elevated responses to vaccination with some mice achieving a precursor frequency of 70%–80% of their circulating CD81 T cells being specific for the OVA antigen. These responses remained elevated in TMZ pretreated animals over untreated mice for as long as 70 days post vaccination. These results demonstrate that TMZ induces homeostatic proliferation of transferred T cells in treated animals and enhances the response to antigen-specific vac-cination. These studies provide a rationale for evaluating TMZ, an agent with known efficacy against MG, as an induction agent for use in cellu-lar immunotherapy. This work was supported by funds from the NIH/NINDS SPORE in Brain Cancer 1P50 CA108786 01 (Sampson), NIH R01 CA-97222–01 (Sampson), The Brain Tumor Society Billy Grey Chair of Research Award (Mitchell), and Accelerate Brain Cancer Cure Foundation Young Investigator’s Award (Mitchell).

IM-20. IMMUNOSUPPRESSIVE MOLECULE EXPRESSION BY DIFFERENTIATED GLIOMA CELLS BUT NOT GLIOMA STEM CELLSIan Parney,1 Lei Zhang,2 John Kelly,2 and Samuel Weiss3; 1University of Calgary, Calgary, Alberta, Canada; 2Alberta, Canada; 3Calgary, Alberta, Canada

Glioma cells initiate a potent immunosuppressive cascade in malignant glioma patients. We hypothesized that glioma stem cells are particularly immunosuppressive in order to avoid eradication by immune surveillance. Human glioma stem cell cultures were established from fresh operative specimens by culture in stem cell media supplemented with EGF/FGF. These cells were either maintained in similar media or forced to differenti-ate by addition of 10% fetal bovine serum. Immunological surface marker expression (HLA-ABC, HLA-DR, B7–1, B7–2, B7–H1) was assessed (1/– interferon-gamma) by flow cytometry. Secreted factors (VEGF, IL-6, IL-10, TGF-B, PGE2) were assessed by ELISA in culture supernatants. Glioma stem cell cultures exposed to serum ceased to grow as neurospheres and adopted morphology similar to standard human glioma cell lines. Both glioma stem cells and differentiated glioma cells expressed HLA-ABC and VEGF. However, only differentiated glioma cells expressed immunosup-pressive TGF-B, PGE2, IL-6, and (constitutively) B7–H1. Neither stem cells nor differentiated cells expressed HLA-DR, B7–1, B7–2, or IL-10. Surprisingly, glioma stem cells express less immunosuppressive molecules than differentiated glioma cells. These findings suggest that they rely on neighboring differentiated glioma cells to protect them from immune sur-veillance through bystander mechanisms. Glioma stem cells themselves may be attractive targets for immunotherapy.

IM-21. INNATE AND ADAPTIVE IMMUNE RESPONSES BY GLIOMA-ASSOCIATED MICROGLIACourtney Crane, Tshilumna Kabongo, Joseph Murray, and Andrew Parsa; University of California, San Francisco, San Francisco, CA, USA

Glioma, the most common of central nervous system (CNS) cancers, is uniformly fatal and current treatments frequently extend a patient’s survival only at the expense of quality of life. As a result, immunotherapies are being applied with increasing frequency, the ultimate goal being to use a patient’s own immune system to prevent tumor recurrence while avoiding damage to surrounding tissue. Because inflammation is generally restricted in the CNS, an issue that is confounded by the immunosuppressive envi-ronment created by a tumor, successful immuotherapies will likely exploit the ability of local cells to initiate a specific anti-tumor response. Micro-glia are immune regulatory cells resident to the CNS, capable of evaluating the local environment and alerting cells outside of the CNS to insult or injury. In the case of many inflammatory diseases of the CNS, initiation of immune responses and local tissue damage are the work of locally activated microglia and the cells that they recruit. Microglia also retain some level of plasticity in the steady state and are sensitive to soluble factors present in the local milieu. As a result, these cells may be recruited to promote tumor invasiveness while skewing local immunity to prevent anti-tumor responses. Because of their role in innate and adaptive immune responses within the CNS as well as their sensitivity to the local environment, microglia are ideal targets for immunotherapy. We hypothesized that tumor cells influ-ence the functions of microglia, thereby affecting local immune responses that are essential for productive and specific anti-tumor responses. We find that microglia are sensitive to soluble factors secreted by tumor cells in the absence of direct interaction. Extended exposure to these secreted factors induces morphological, phenotypic and functional changes in microglia. Specifically, molecules involved in antigen presentation and phagocytosis are upregulated while expression of chemokine receptors are decreased. Following innate stimulation, pro- and anti-inflammatory cytokines are produced at significantly greater levels than by cells that are freshly isolated. Coculture of glioma conditioned microglia with stimulated CD4 T cells sig-nificantly decreases IL-17 production and increases FoxP3 positive regula-tory T cells. To determine the role of constitutively active Ras expression by tumor cells on microglia, we treated tumor cells with simvastatin or shRNA specific for H-Ras. Carryover of simvastatin to microglial cultures shows that Ras inhibition in microglia affects their morphology and function more than inhibiting Ras in tumor cells. Ras activators are present in the tumor cell supernatant and their cognate receptors are expressed on the surface of microglia. As a result, Ras is activated in microglia in response to tumor supernatant, suggesting that Ras activation in microglia plays a significant role in the morphological, phenotypic and functional changes observed in vitro. Together, these data suggest that the ability of microglia to act as mediators of immunity, with the potential to influence the magnitude and specificity of local responses, can be useful in developing an effective immu-notherapy for intracranial tumors.



IM-22. PROPHYLAXIS AGAINST RECURRENCE OF AGGRESSIVE F98 RAT GLIOMAS AFTER RADIOSURGERY: TREG DEPLETION AND INJECTIONS OF GMCSF-TRANSFECTED IRRADIATED F98 CELLS WITH IMMUNOMODULATION USING MONOSODIUM URATE CRYSTALSHenry M. Smilowitz,1 Timothy Graham,1 George Tellides,2 Martin Oaks,3 James F. Hainfeld,4 and Daniel N. Slatkin4; 1University of Connecticut Health Center, Farmington, CT, USA; 2Yale University, New Haven, CT, USA; 3St. Lukes Medical Center, Milwaukee, WI, USA; 4Nanoprobes Inc., Yaphank, NY, USA

Our objective was to test the enhancement of LINAC-radiosurgery for weakly immunogenic, aggressive, advanced F98 malignant rat gliomas by Treg depletion followed by weekly injections of GMCSF-transfected, then irradiated F98 cells, alone (GMI*) or with monosodium urate (MSU) crystals (GMI**). Fischer 344 rats (149) were implanted (I) ic with 10,000 F98 cells d0. Each of the 39 untreated rats (controls) died from a large ic tumor before d30. On d14, the tumor-bearing brains of the remaining 110 rats were treated with a single radiosurgical 12.5–25 Gy dose of electron- equilibrated 6 MV photons. Of those, 45 received no further treatment (LINAC alone), 38 received LINAC 1 GMI* and 26 received LINAC 1 GMI** All untreated rats (n 5 39) died (median, 24.5d post-I). The median survivals of rats that received LINAC alone (n 5 45) or LINAC 1 GMI* (n 5 38) were the same (41 days post-I). However, LINAC 1 GMI* prolonged the lives of about 1/3 of the rats compared to those that received LINAC alone. The addition of monosodium urate crystals to LINAC 1 GMI** caused a marked rightward shift of the survival curve, prolonging post-radiosurgery survival (median 56d post-I, n 5 26) by ~50% over LINAC alone (median 41d post-I, n 5 45); ~10% of the former survived ~3–4 months. The immunomodulator monosodium urate crystals significantly increased the effectiveness of immunotherapy against the post-radiosurgical recurrence of an aggressive malignant glioma model. This information also appeared in abstract #6591 of the annual AACR meeting, 2007.

IM-23. A NOVEL SMALL MOLECULE INHIBITOR OF STAT3 REVERSES IMMUNE TOLERANCE IN MALIGNANT GLIOMA PATIENTSLing-Yuan Kong, S. Farzana Hussain, Justin Jordan, Charles Conrad, Timothy Madden, Isabella Fokt, Waldemar Priebe, and Amy B. Heimberger; University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Several immunotherapeutic clinical trials in glioma patients have shown promise, especially in patients with minimal microscopic residual disease which are minimally immunosuppressed. However, in patients with advanced cancers and grossly evident disease, the objective immunotherapy response rates have remained low. Overcoming the profound immunosup-pression in glioma patients has impeded efficacious immunotherapy. Potent immune activators are necessary to counteract the immunosuppressive fac-tors that overwhelm an induced immune response. Recently signal trans-ducers and activators of transcription (STATs), which are over-expressed in gliomas, have emerged as a potential target for effective immunotherapy. We have pharmacologically created a novel small molecular inhibitor of STAT3. This compound can penetrate the central nervous system (CNS) in physiologically relevant doses and has marked in vivo efficacy against neo-plasms including gliomas. Furthermore, we can reverse immune tolerance of immune cells isolated from glioblastoma multiforme (GBM) patients. Specif-ically, the STAT3 inhibitor induced the expression of the co-stimulatory mol-ecules CD80 and CD86 on peripheral macrophages and tumor-infiltrating microglia, which was in marked contrast to known antigen presenting cell toll-like receptor agonists, which are unable to do so. Additionally, the STAT3 inhibitor induced the production of the immune-stimulatory cytokines IL-2, IL-4, IL-12, and IL-15. Of significance, was that the STAT3 inhibitor could induce the proliferation and activation of effector T cells from GBM patients that are refractory to T cell receptor agonist (CD3) stimulation. To determine how this compound could induce cytotoxicity in neoplasms but in contrast would induce immune activation and prolif-eration, using Western blots, we show that the functional enhancement of immune responses following STAT3 inhibition is accompanied by up regu-lation of several key intracellular signaling molecules that critically regulate T cell and monocyte activation. Specifically, the phosphorylation of Syk (Tyr352) in monocytes and ZAP-70 (Tyr319) in T cells are enhanced by STAT3 inhibition. This novel small molecule STAT3 inhibitor has tremen-dous potential for clinical applications with its penetration into the CNS, easy parental administration, direct tumor cytotoxicity and potent immune adjuvant responses in immunosuppressed cancer patients. This study is sup-ported by the SPORE in Pancreatic Cancer P20 CA101936 and the Morton and Angela Topfer Pancreatic Cancer Research Fund awarded to W.P. and The University of Texas M.D. Anderson Cancer Center Department of Neu-rosurgery Recruitment start-up funds and the National Institute of Health RO1 CA120813 awarded to A.B.H.

IM-24. DEFECTIVE DENDRITIC CELL MATURATION IN MALIGNANT GLIOMA PATIENTSIan Parney,1 Jennifer Rodrigues,2 Lei Zhang,2 and Guido Gonzalez2; 1University of Calgary, Calgary, Alberta, Canada; 2Alberta, Canada

Glioma patients are systemically immunosuppressed. We hypothesized that dendritic cell (DC) maturation defects may be partly responsible for this and that generating DC in vitro from malignant glioma patients’ peripheral blood mononuclear cells (PBMC) would be more difficult than doing so from normal donor PBMC. PBMC from malignant glioma patients and normal donors (n 5 10, each) were analyzed by flow cytometry for myeloid (CD11c1/HLA-DR1/Lin–) and plasmacytoid (CD1231/HLA-DR1/Lin–) DC precursors (Lin 5 CD3/CD14/CD19/CD56). DC were cultured from adherent PBMC in the presence of GM-CSF, IL-4, and TNF-a. At day 8, they were harvested and stained for class I and II MHC molecules (HLA-ABC, HLA-DR), the monocyte marker CD14, T cell costimulatory mol-ecules (B7-1, B7-2), and the DC maturation marker CD83. Expressed as a percentage of Lin– cells, myeloid DC precursors were decreased in glioma patients compared with normal donors (mean 6sem 5 6.6 62.5% vs. 48.8 64.2%, p , 0.001). Plasmacytoid DC precursors were similarly decreased (4.7 61.9% vs. 22.2 63.7%, p , 0.01). Cultured DCs were HLA-ABC1/HLA-DR1/CD14–/B7-11/B7-2–. This pattern did not differ significantly between normal donors and glioma patients. Glioma patients’ DC showed a trend towards decreased CD83 expression. Glioma patients have markedly reduced circulating DC precursors. This may contribute to immunosuppres-sion in these patients. Despite this, DC can be cultured from PBMC using standard culture techniques equally well for glioma patients and normal donors. This has important implications for glioma dendritic cell vaccine strategies utilizing autologous blood-derived DC.

IM-25. IMMUNE MODULATION BY COMBINED RADIATION AND TEMOZOLOMIDE IN GLIOBLASTOMA MULTIFORME PATIENTSCamilo Fadul,1 Jan Fisher,1 Eugene Demidenko,2 Mary Jo Turk,3 Edward Usherwood,3 and Marc Ernstoff1; 1Dartmouth-Hitchcock Medical Center, Lebanon, NH, USA; 2Dartmouth College, Hanover, NH, USA; 3Dartmouth College, Lebanon, NH, USA

Although glioblastoma multiforme (GBM) patients exhibit a CD41T cell lymphocytopenia, an increase in the percentage of regulatory (CD41CD251) T cells (Tregs) may render ineffective otherwise promis-ing immune therapy approaches. Temozolomide (TMZ) decreases Tregs and when given after immune therapy seems to improve survival of GBM patients. We studied the treatment effects of TMZ and RT on peripheral blood mononuclear cell subpopulations to determine if there may be impor-tant interactions with a vaccine intervention against GBM. Blood samples from 11 newly diagnosed GBM patients were obtained before (PRE), the last day (END) and 4 weeks (POST) after RT/TMZ. All patients had surgi-cal resection of the tumor. Six of the patients at the PRE time point and none at the END and POST time points were on steroids. Using multi-color flowcytometry, CD3, CD4, CD8, CD45RA, CD45RO, CD25, FOXP3, valpha24, vbeta11, CD11b, CD56, CCR6, and CCR7 antibodies identified T cells subpopulations, while Lineage specific markers (LIN), 4–1–BBL , HLA-DR, CD123 , and CD11c antibodies identified dendritic cells (DC). A significant increase in the percentage of CD41CD251hi Treg occurred POST treatment as compared to PRE. Our percentage of CD41CD251hi Treg at the PRE time point was lower than that reported in the literature for Treg fraction present in peripheral blood of GBM patients before surgical resection, and comparable to our healthy volunteers. Intracellular FOXP3 staining was present in 82% of the PRE and POST CD41CD251hi T cells and in 52% of the PRE and 53% of the POST CD41CD251lower T cells. When combined CD41CD251 subpopulations were used the percent-age of CD41 T cells was 3.6% at PRE and 8% POST. The CD8 effector memory T cells (CD81CD45RO1CCR7–), and effector myeloid DC (Lin-DR1CD11c1) increased from PRE to POST, while a significant decrease in naïve CD8 T cells (CD81CD45RA1CCR7–) and activated CD81NKT cells (CD81CD561) was observed. These results suggest that 4 weeks after RT/TMZ in patients with GBM there is an increase in Tregs that could provide an inauspicious milieu for the addition of immune therapy, but the increase in memory effector CD8 T cells suggests that treatment-related tumor killing might expand circulating effector cells that have seen anti-gen. RT/TMZ combined with an intervention that selectively depletes Tregs could offer optimal conditions for future DC vaccination trials in patients with GBM. Updated information on the shift of mononuclear subpopula-tions when DC vaccination follows RT/TMZ and results of Treg suppressor assays will also be presented.



IM-26. GLIOBLASTOMA MULTIFORME: A 20-YEAR EXPERIENCE USING RADIOIMMUNOTHERAPY AND TEMOZOLOMIDELinna Li,1 Tony S Quang,2 Edward Gracely,3 Jacqueline G. Emrich,4 Ji Kim,4 Theodore E. Yaeger,5 Joseph M. Jenrette,6 and Luther W. Brady4; 1Radiation Oncology, Drexel University, Philadelphia, PA, USA; 2Radiation Oncology, U.C. Davis Medical Center, CA, USA; 3Department of Epidemiology and Biostatistics, Drexel University College of Medicine, Philadelphia, PA, USA; 4Radiation Oncology, Drexel University College of Medicine, PA, USA; 5Radiation Oncology, Wake Forest University School of Medicine, NC, USA; 6Radiation Oncology, Medical University of South Carolina, SC, USA

The standard treatment of patients (pts) with glioblastoma multiforme (GBM) is surgery and radiation therapy (XRT) 6chemotherapy. Recently, temozolomide (TMZ) has been shown to offer a survival benefit. Adjuvant radioimmunotherapy with 125I-labeled anti-epidermal growth factor recep-tor monoclonal antibody 425 (125I-EGFR MAb 425) may also increase survival. In a phase II clinical study, we compared the survival of 3 treat-ment groups: surgery1XRT (CTL), 125I-EGFR MAb 4251surgery1XRT (RIT), and TMZ1125I-EGFR MAb 4251surgery1XRT (TMZ1RIT). Pts with newly diagnosed and histologically proven GBM were eligible. Post-operative XRT was give to a median dose of 60 Gy following debulking surgery. 125I-EGFR MAb 425 was given by 3 weekly intravenous injections of 1.8 GBq of 125I-EGFR MAb 425 following surgery and XRT. TMZ was given concomitantly (75 mg/ m2/d, 35–42d) with XRT followed by 6 cycles of adjuvant TMZ (150–200 mg/ m2/d 3 5d, q28d). Median survival was the primary endpoint. From 1988 to 2007, 273 GBM pts were treated at Hahnemann University Hospital: CTL (n 5 81), RIT (n 5 132), and TMZ1RIT (n 5 60). Median age in years for CTL, RIT, and TMZ1RIT group was 66.0, 52.5, and 53.5, respectively (p , 0.001). The percentage of pts who received debulking surgery for CTL, RIT, and TMZ1RIT group was 70.4, 74.2, and 86.7, respectively (p 5 NS). The percentage of pts who received XRT in CTL, RIT, and TMZ1RIT group was 67.9, 98.5, and 100, respectively (p , 0.001). Pts who did not receive both surgery1XRT were excluded from median survival calculations. Median survival in months overall (n 5 187), CTL (n 5 39), RIT (n 5 97), and TMZ1RIT (n 5 51) group was 14.0, 10.2, 14.5, and 20.4, respectively. Log rank analy-sis resulted in RIT vs. CTL, p , 0.001, TMZ1RIT vs. CTL, p , 0.001, and TMZ1RIT vs. RIT, p 5 0.011. On multivariate analysis (proportional hazards) using all 273 pts controlling for age, surgery, and XRT, the hazard ratio for RIT vs. CTL, TMZ1RIT vs. CTL, and TMZ1RIT vs. RIT was 0.49 (p , 0.001), 0.30 (p , 0.001), and 0.62 (p 5 0.008), respectively. Historical control pts differ from both treatment groups being older in age and less likely to receive or complete XRT. However, after controlling for these factors, the differences in median survival between each of the treat-ment groups and control remain statistically significant. Treatment with 125I-EGFR MAb 425 confers survival benefits over controls. Combination 125I-EGFR MAb 4251TMZ provides the greatest survival benefit. This study represents the largest reported series using radioimmunotherapy in the treatment of GBM.

IM-27. OPTIMIZING DENDRITIC CELL PRODUCTION AND CHARACTERIZATION OF IMMUNE PHENOTYPE IN PATIENTS WITH NEWLY DIAGNOSED OR RECURRENT GLIOBLASTOMAKent New,1 Michael Gustafson,2 Peggy Bulur,2 Susan Steinmetz,2 Debra Sprau,2 Brian O’Neill,2 and Allen Dietz2; 1Mayo Clinic, Jacksonville, FL, USA; 2Mayo Clinic, Rochester, MN, USA

No effective therapy exists for patients with glioblastoma (GBM). Animal models and some human clinical trials indicate that GBM may be susceptible to directed stimulation of the immune system. Specifically, autologous antigen presenting cells known as dendritic cells (DC) have been combined with tumor derived antigens and used as a vaccine in patients with recurrent GBM. Cumulatively, these data suggest that GBM may be amenable to immunotherapy; however, additional work is needed to improve the efficacy of DC vaccines. A particularly acute area of deficiency in DC based immunotherapy is the uniformity and quality control of cul-tured cells used for vaccination. In addition, DC vaccination regimens will be enhanced if used at times of maximum potency; that is at times when patients have the best ability to respond to immune stimulation. To better understand these two influences we have begun to characterize the immune competence of patients with GBM by peripheral blood immune phenotyp-ing. Firstly, we evaluated four culture conditions for generation of DC in these patients. Using CD141 cells from GBM patients, we have been able to produce similar numbers of high viability DC as those seen in normal volun-teers. However, in all four culture conditions tested, the DC generated from the blood of GBM patients were of lower purity and potency than cells from normal volunteers (65.5% 66.1 CD831 in the best condition for GBM patients versus 95.2 6CD831 in normal volunteers; p , 0.001, n 5 13).

Secondly, the same patients were immune phenotyped to look for abnor-malities in immune suppression. GBM patients had more granulocytes as a percent of white blood cells (76.5 63.0% vs. 58.4 63.0%; p 5 0.0005) and more regulatory T cells (CD251CD127– as a percent of CD4; 7.8 62.2% vs. 3.0 60.7%; p 5 0.046). GBM patients also showed reduced expression of HLA-DR on circulating lineage positive cells (47.7 65.7% vs. 65 62.5%; p 5 0.005). We found that the ability to recover CD141 cells during our isolation procedure was inversely proportional to our ability to culture pure DC (p 5 0.014; n 5 9), suggesting a pre-existing deficiency of monocytes in their ability to differentiate into DC. We believe that this deficiency may represent a novel mechanism of immune suppression in patients with GBM. This work was supported through a Developmental Research grant from the Mayo Clinic Brain Tumor SPORE CA108961-03.

IM-28. VACCINATION WITH TYPE-1 DENDRITIC CELLS PULSED WITH MULTIPLE GLIOMA-ASSOCIATED ANTIGEN PEPTIDES IN COMBINATION WITH POLY-ICLC IN PATIENTS WITH RECURRENT MALIGNANT GLIOMAHideho Okada,1 Frank Lieberman,1 Pawel Kalinski,1 Xinmei Zhu,1 L. Dade Lunsford,1 Amin Kassam,1 Arlan Mintz,2 David Bartlett,1 Charles Brown,3 Herbert Zeh,1 Theresa Whiteside,1 Lisa Butterfield,1 Ronald Hamilton,1 Andres Salazar,4 and Ian Pollack1; 1University of Pittsburgh, Pittsburgh, PA, USA; 2Pittsburgh, PA, USA; 3PA, USA; 4Oncovir, Inc., Washington, DC, USA

We implemented two early phase trials in patients with recurrent malig-nant gliomas (UPCI 04–136 and 05–115) to evaluate vaccine safety as the primary objective, and secondarily, to assess induction of immune response against glioma-associated antigen (GAA) peptides and clinical response in an exploratory manner. These trials were designed based on our previous studies/observations as follows: (1) isolation and characterization of human leukocyte antigen (HLA)-A2–restricted cytotoxic T-lymphocytes (CTL) epitopes derived from human GAAs, such as IL-13Ralpha2 (Okano et al., 2002; Eguchi et al., 2006) and EphA2 (Hatano et al., 2005); (2) develop-ment of a novel culture method to produce alpha-type-1–polarized den-dritic cells (alphaDC1), which are superior to conventional DCs in inducing antigen-specific type-1 CTL (Mailliard et al., 2004); and (3) intramuscular (i.m.) administration of toll-like receptor-3 ligand poly-ICLC remarkably enhance the effect of peptide-based vaccines in mice bearing brain tumors (Zhu et al., 2007). In a pilot trial UPCI 04–136, HLA-A21 patients with recurrent glioblastoma multiforme (GBM) or anaplastic astrocytoma (AA) were vaccinated with alphaDC1s (1 3 107/injection) loaded with 4 synthetic GAA epitopes every two weeks (minimum 4 vaccinations) by ultrasound-guided intranodal (i.n.) injections. We treated three patients; two with recurrent AA and one with recurrent GBM, all of whom were surgically resected before initiation of the vaccination. All three patients successfully received 4 i.n. injections of GAA peptide-loaded alphaDC1 vaccines with no evidence of progression and no adverse events nor need for administration of corticosteroids during the course of therapy. Positive tetramer-response against IL-13Ralpha2 and EphA2-derived peptides was observed in one of three patients. All three patients remained progression-free at least for 16 weeks following the initial vaccine. Tumor tissues obtained from one patient with radiological progression demonstrated mostly inflammatory response, but not many tumor cells. Real-time PCR and immunohistochemical analy-ses demonstrated expression of vaccine-targeted GAAs in pre-vaccine tumor tissues, but not in the post-vaccine tumor tissues from this patient. Based on the data from UPCI 04–136 regarding the safety and feasibility of type-1 alphaDC1-based vaccines in 3 participants, we implemented a novel trial UPCI 05–115, in which i.m. injections of poly-ICLC (20 micrograms/kg, twice/week) are added to the regimen employed in UPCI-04–136. The first patient in UPCI 05–115 with recurrent GBM received 4 alphaDC1 vaccina-tion and a total 16 i.m. poly-ICLC treatment with no adverse events, and exhibited partial response based on over 30% reduction of the diameter of gadolinium enhancing mass in post-vaccine MRI (Week 9) compared to the baseline (Week 0) scan. As we perform immunological assays for all samples obtained from at least one patient to avoid inter-assay variability, evalua-tion of immunological response awaits the withdrawal of this patient, who currently receives additional booster vaccines. In 05–115, dose escalation of alphaDC1 is also planned to find the maximal tolerated dose and most immunologically efficient dose. While still preliminary, these data support safety and feasibility of poly-ICLC assisted alphaDC1-based vaccines.



IM-29. TEMOZOLOMIDE THERAPY INDUCES AN INCREASE IN REGULATORY T-CELLS WITHOUT ABBROGATING A SPECIFIC TUMOR ANTIGEN IMMUNE RESPONSE IN PATIENTS WITH GBMGary Archer,1 Duane Mitchell,1 Amy Heimberger,2 Henry Friedman,1 David Reardon,3 James Vredenburgh,4 Mark Gilbert,5 Allan Friedman,6 Raymond Sawaya,2 Kenneth Aldape,2 Darell Bigner,1 and John Sampson3; 1Duke University, Durham, NC, USA; 2University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3Durham, NC, USA; 4Duke University Medical Center, Durham, NC, USA; 5Houston, TX, USA; 6Surgery, Duke University, Durham, NC, USA

Traditional dogma would suggest that the combination of immuno-therapy with cytotoxic chemotherapy would be domed to failure due to the induced lymphopenia accompanying chemotherapy. However new para-digms are emerging that suggest that immunotherapy may benefit from this induced lymphopenia if prior thought is given to the timing of the two agents. We have previously carried out a Phase II clinical trial for patients with newly diagnosed GBM that are EGFRvIII positive at Duke and M.D. Anderson. In this trial patients with newly diagnosed, completely resected GBM were vaccinated with an EGFRvIII-specific peptide q2 weeks 3 3 after radiation (XRT) (~60Gy) and Temozolomide (TMZ) (75mg/m2/d) and then monthly until progression. The initial finding of this trial was a median time to progression of 64.5 weeks (p , 0.0001, compared with a matched control group) and a median survival of 126.1 weeks. Immunomonitoring showed the induction of peptide specific humoral immune response. The recurrent tumors in the vaccinated patients with shown to be EGFRvIII negative by immunohistochemistry. In a second Phase II trial, the same patient population was vaccinated as before, q2 weeks 3 3 after radiation (XRT) (~60Gy) and TMZ (75mg/m2/d), and then monthly with the addition of 5 day TMZ cycles (200mg/m2/d). In these patients the TMZ induces a transient, Grade 3 lymphopenia (, 500 cells/mL) in 70% of patients after the first TMZ cycle with nadirs occurring 14–21 days after treatment (n 5 10). Regulatory T cell (TReg) (CD41CD2511CD45RO1FOXP31) levels increased from 5.2 1 1.5% (3.3–7.5) to 11.8 1 2.6% (6.9–13.8) (p 5 0.0004; paired t-test) with TMZ and XRT and averaged 12.2 1 4.0% (6.4–18.1) after the second TMZ cycle (p 5 0.007) (n 5 6). Despite these findings, in patients assayed, both humoral and cellular EGFRvIII-specific immune responses appear to be enhanced with TMZ. Median survival and TTP after vaccination is 22.1 weeks with no patients progressing (n 5 8). These initial results demonstrate that both chemotherapy and immunotherapy can be delivered concurrently without negating the effects of immunotherapy. TMZ-induced lymphopenia in patients with GBM may prove to be synergistic with a tumor specific peptide vaccine.

IM-30. A PHASE II CLINICAL TRIAL TO TEST THE EFFICACY OF DCVax-BRAIN, AUTOLOGOUS DENDRITIC CELLS PULSED WITH AUTOLOGOUS TUMOR LYSATE, FOR THE TREATMENT OF PATIENTS WITH GLIOBLASTOMA MULTIFORMEMarnix Bosch,1 Alton Boynton,1 Robert Prins,2 and Linda Liau3; 1Northwest Biotherapeutics, Inc., Bothell, WA, USA; 2University of California, Los Angeles, CA, USA; 3CA, USA

Northwest Biotherapeutics Inc. has initiated a Phase II clinical trial to test the activity and efficacy of DCVax-Brain, autologous dendritic cells pulsed with autologous tumor cell lysate, for the treatment of patients with newly diagnosed glioblastoma multiforme (GBM). GBM is the most aggres-sive form of brain cancer, with median time to tumor progression (TTP) of approximately 8 months and median survival time of 15–18 months. Patients treated with DCVax-Brain in Phase I trials at UCLA have median TTP of 18 months (p , 0.00001, log-rank) and median survival times of 33.8 months (p 5 0.0015, log-rank). Twenty-five percent of patients treated in these trials are alive for more than 42 months without progression. Evi-dence for immune responses induced by DCVax-Brain included detection of CTL responses and tetramer analysis of CD81 T cells. The trial will enroll 141 patients at 15 institutions in the United States over approximately 9 months. Patients are screened for eligibility during standard primary treat-ment consisting of surgery, external beam radiation therapy and concur-rent temozolomide, and eligible patients are randomized to the treatment arm or the control arm at a 2:1 ratio. Patients in the treatment group will receive immunizations at frequent intervals, as well as continued adjuvant temozolomide. The primary endpoint is progression-free survival (PFS), and the first secondary endpoint is overall survival (OS). Other secondary endpoints include safety, as well as the monitoring of immune responses induced by DCVax-Brain. This is the first large, multicenter, randomized trial to test the efficacy of dendritic cell-based immunotherapy for primary brain cancer.

IM-31. RNA-LOADED DENDRITIC CELLS TARGETING CYTOMEGALOVIRUS IN PATIENTS WITH MALIGNANT GLIOMADuane Mitchell,1 Gary Archer,1 Darell Bigner,2 Allan Friedman,1 Henry Friedman,3 David Reardon,3 James Vredenburgh,3 James Herndon,4 Roger McLendon,2 and John Sampson1; 1Surgery, Duke University, Durham, NC, USA; 2Pathology, Duke University, NC, USA; 3Medicine, Duke University, Durham, NC, USA; 4Biostatistics and Bioinformatics, Duke University, Durham, NC, USA

We recently confirmed the detection of CMV antigens in the majority of newly diagnosed GBM specimens (Mitchell et al., NeuroOncology, in press). A Phase I/II clinical trial targeting the CMV integument protein pp65 in patients with newly-diagnosed GBM using pp65–LAMP RNA-transfected DC has been completed. The clinical trial was opened in Febru-ary 2006 and has now accrued 6 patients that are CMV seropositive and 7 patients that are seronegative. Patients were treated with standard doses of temozolomide (TMZ) throughout vaccination and were randomized to receive an autologous lymphocyte transfusion (ALT) prior to vaccination. No vaccine-induced toxicities were observed. Standard TMZ therapy, how-ever, induced transient, Grade 3 lymphopenia (, 500 cells/mL) in 70% of patients after the first TMZ cycle with nadirs occurring 14–21 days after treatment. Regulatory T cell (TReg) (CD41CD2511CD45RO1FOXP31) levels increased from 5.2 1 1.5% (3.3–7.5) to 11.8 1 2.6% (6.9–13.8) (p 5 0.0004; paired t-test) with TMZ and XRT and averaged 12.2 1 4.0% (6.4–18.1) after the second TMZ cycle (p 5 0.007). Nearly complete radio-graphic responses were observed. Median TTP is 60.5 weeks and median survival has not been reached. There are no statistically significant differ-ences based on serologic status (p 5 0.3633) or ALT (p 5 0.0447). Our preclinical data in TMZ treated mice, demonstrates significant increases in immunologic responses in mice vaccinated during hematopoietic recovery from TMZ-induced lymphopenia. Cellular and humoral immune responses in vaccinated patients are under current evaluation. These results indicate that chemotherapy and immunotherapy can be delivered concurrently with-out negating the effects of immunotherapy, and TMZ-induced lymphopenia may prove to be synergistic with tumor-specific immunotherapy. This work was supported by funds from the NIH/NINDS SPORE in Brain Cancer 1P50 CA108786 01 (Sampson), NIH R01 CA-97222–01 (Sampson), The Brain Tumor Society Billy Grey Chair of Research Award (Mitchell), and Accelerate Brain Cancer Cure Foundation Young Investigator’s Award (Mitchell).

IM-32. SAFETY OF AN AUTOLOGOUS GLIAL TUMOR LYSATE-PULSED, DENDRITIC CELL-BASED VACCINE IN COMBINATION WITH THALIDOMIDE FOR PATIENTS WITH RELAPSED MALIGNANT GLIOMASAlbert Ty,1 Who Whong Wang,2 Han Chong Toh,3 Cong Ju Zhu,4 Khong Bee Kang,4 and Meng Cheong Wong5; 1Singapore General Hospital, Singapore, Singapore; 2Cell Therapy and Cancer Vaccine Laboratory, National Cancer Centre, Singapore, Singapore; 3Medical Oncology, National Cancer Centre, Singapore, Singapore; 4Brain Tumour Research Laboratory, National Cancer Centre, Singapore, Singapore; 5Neurology, Singapore General Hospital, Singapore, Singapore

Survival among patients with malignant gliomas who relapse follow-ing conventional treatment (surgery, concurrent radiotherapy and chemo-therapy) remains poor and range from 3–6 months. Poor survival outcome for patients who relapse is partly due to the lack of effective second-line or third-line treatment. Immunotherapy represents a theoretically viable alternative as it offers the potential for high tumor-specific cytotoxicity with minimal adverse events. An increasing number of reports have dem-onstrated systemic immunotherapy using dendritic cells (DCs) as capable of inducing a glioma anti-tumor response within the brain in both murine and human models. The possible synergistic effect between immunotherapy and chemotherapy has been documented in a number of small studies suggesting that such combinations are not entirely ineffective. In this study, our objec-tive was to investigate the safety of an autologous glial tumor lysate-pulsed, dendritic cell-based vaccine in combination with thalidomide for patients with relapsed malignant gliomas. From January to June 2006, 2 patients (1 male, aged 51 and 1 female, aged 36) with histologically proven malignant glioma (1 glioblastoma multiforme and 1 pleomorphic xanthoastrocytoma with anaplastic features) received autologous glioma lysate-pulsed, den-dritic cell-based vaccines concurrently with daily oral thalidomide. Vac-cines were prepared using the patients’ own dendritic cells derived from peripheral blood monocytes and pulsed with a lysate of autologous glial cells obtained during surgical resection. Each patient received intradermal injections of the prepared vaccine every 2 weeks until tumor progression or if patient declines further treatment. Incremental doses of daily thalidomide (maximum of 300 mg BID) were given concurrently. A delayed hypersensi-tivity reaction (DTH) test was obtained after the 4th injection to determine immune response. MRI scans were done after the 5th and 10th injection. Adverse events were recorded using the NCI Common Toxicity Criteria,



version 3. Both patients were heavily pre-treated prior to immunotherapy. Patient 1 (glioblastoma multiforme) received standard concurrent radio-therapy (RT) and Temozolomide (TMZ) followed by 19 monthly cycles of single agent TMZ (75 mg/ m2 3 14 days) (Progression-free interval 5 20.3 months). He underwent a second surgical resection followed by 2 cycles of Bevazucimab (5 mg/kg) 1 Cyclosphosphamide (800 mg/m2) and 1 cycle of single agent Bevazucimab with salvage radiotherapy (Progression-free interval 5 5 months). Patient 2 (Pleomorphic xanthoastrocytoma with ana-plastic features) underwent 3 previous surgical resections, standard concur-rent RT and TMZ followed by 7 monthly cycles of single agent TMZ (75 mg/m2) before progressive disease was detected (Progression-free interval 5 8.6 months). Patient 1 received 12 injections while Patient 2 received 7 injections. Doses of thalidomide were escalated to 400 mg/day. Patient 2 received symptomatic doses of Dexamethasone (8 to 16 mg OD). DTH test done on the post-4th injection yielded a .2 mm skin induration in both patients. No CTC grade 3 to 4 adverse events were reported. Transient grade 1 somnolence was observed in Patient 2. Post-5th injection MRI showed stable disease (Patient 1) and progressive disease (Patient 2). Post-10th injec-tion showed stable disease (Patient 1). Survival from the start of vaccination 1 thalidomide was 8.1 months for patient 1 and 5 months for patient 2. In conclusion, the combination of autologous glial tumor lysate-pulsed, den-dritic cell-based vaccination and thalidomide is safe in heavily pre-treated patients with relapsed malignant glioma. More data is needed to assess the frequency and the risk of serious adverse events in patients receiving this combination.

IM-33. COMPLEMENT ACTIVATION AND RESISTANCE WITHIN THE CSF FOLLOWING INTRAVENTRICULAR ADMINISTRATION OF ANTI-CD20 IN THE TREATMENT OF CENTRAL NERVOUS SYSTEM LYMPHOMACigall Kadoch, Juliana Karrim, Marc Shuman, and James Rubenstein; Medicine, University of California, San Francisco, San Francisco, CA, USA

We recently conducted the first phase I dose escalation study of intraven-tricular rituximab (anti-CD20 antibody) in immunocompetent patients with recurrent central nervous system (CNS) non-Hodgkin’s lymphoma (NHL). The goals of this study have been to define the safety and pharmacokinetics of intraventricular rituximab administration and to gain insight into the molecular and cellular basis of rituximab efficacy and resistance in the treat-ment of high-grade lymphomas. Maximum tolerated dose was determined to be 25 mg rituximab. Responses were observed within leptomeninges, brain and intraocular compartments. We observed a rapid lymphocytotoxic effect of intraventricular rituximab administration in six out of ten subjects treated, resulting in four complete responses at the completion of five weeks of therapy. Cytologic responses were evident within one hour of rituximab treatment and were not likely related to the action of corticosteroids as these were held stable, tapered, or withheld in the week before intrathecal therapy. Three potential mechanisms have been proposed to explain the basis for rituximab efficacy in the treatment of NHL: (1) direct induction of apoptosis; (2) complement-dependent cytotoxicity (CDC); (3) antibody dependent cellular cytotoxicity (ADCC). Given the rapid rate of response detected in the leptomeningeal compartment and the relative paucity of Fc-receptor bearing natural killer cells in the CSF, we tested the hypothesis that complement activation is responsible for the rapid rituximab-mediated tumor cell lysis which we observed. We focused on the potential generation of C3a anaphylatoxin in the CSF after rituximab administration as C3 is the central component in the classical and alternative complement path-ways. Quantitative measurement of C3a concentration in CSF by ELISA demonstrated rapid activation of the complement cascade within one hour of intrathecal anti-CD20 antibody administration. C3a concentrations increased between 3–8-fold to peak concentrations of greater than 10 ng/ml. These findings were confirmed by immunoblot analysis of C3a levels in CSF which also suggested a trend toward a decreasing concentration of total C3 in the CSF over the five week course of therapy. In addition, we identified a soluble complement inhibitory protein within the CSF which we observed to increase in concentration beginning at one hour after the first intrathecal rituximab administration. This complement inhibitor has not previously been associated with rituximab resistance. Increasing levels of this comple-ment inhibitory protein were detected in the CSF with repeated dosing in each of the patients and correlated with rituximab-resistance.

INVASION

IN-01. ELMO1 AND DOCK180, A BIPARTITE RAC1 GUANINE NUCLEOTIDE EXCHANGE FACTOR, PROMOTE HUMAN GLIOMA CELL INVASIONMichael Jarzynka,1 Bo Hu,1 Kwok-Min Hui,1 Ifat Bar-Joseph,1 Weisong Gu,2 Takanori Hirose,3 Lisa Haney,4 Kodi Ravichandran,4 Ryo Nishikawa,5 and Shi-Yuan Cheng1; 1University of Pittsburgh, Pittsburgh, PA, USA; 2Ohio Supercomputer Center, OH, USA; 3Saitama Medical University, Oak Ridge, TN, USA; 4University of Virginia, Charlottesville, VA, USA; 5Saitama Medical University, Japan

Malignant gliomas have an intrinsic ability to disperse throughout the brain, contributing to their high frequency of recurrence and progression in patients afflicted with these deadly cancers. In spite of the utilization of multi-modal therapies including surgery, radiation and chemotherapy, the mean survival time in patients with high grade gliomas is less than 1 year. Regrettably, the mechanisms underlying the inherent propensity of glioma cells to invade are poorly understood. Therefore, the identification of novel molecules that are crucial for glioma cell invasion is paramount. Recent reports demonstrate that the bipartite guanine nucleotide exchange factor (GEF), engulfment and cell motility 1 (ELMO1) and dedicator of cytokinesis 1 (Dock180) cooperatively function to activate Rac1 and induce cell migration in C. elegans, Drosophila, and normal mammalian cells. However, the role of these proteins in human cancer progression has not been investigated. Here, we report that ELMO1 and Dock180 markedly contribute to the invasive phenotype of malignant human gliomas. Immu-nohistochemical analyses of primary human glioma specimens containing a center area and invasive region reveal a co-upregulation of ELMO1 and Dock180 in the glioma cells infiltrating the brain parenchyma. Consistent with these findings, elevated expression of ELMO1 and Dock180 is detected in human glioma cell lines compared to normal human astrocytes. Inhibi-tion of endogenous ELMO1 and Dock180 expression in LN229, U373MG and SNB19 glioma cells using small interfering RNA significantly decreases glioma cell migration and invasion in vitro and in an ex vivo murine brain slice invasion assay with a concomitant reduction in Rac1 activation. Con-versely, exogenous expression of ELMO1 and Dock180 in U87MG and U251MG glioma cells that have low endogenous expression of these two proteins increases their migratory and invasive capacity in vitro and ex vivo. Taken together, these data show for the first time that the bipartite Rac1 GEF, ELMO1, and Dock180 play a critical role in promoting cancer cell invasion thus establishing these molecules as potential therapeutic targets for the treatment of malignant gliomas.

IN-02. HYPOXIA-INDUCIBLE CARBONIC ANHYDRASE IX ENHANCES INVASION OF GLIOMA CELLS IN VITROMartin Proescholdt,1 Christina Mayer,1 Eva-Maria Störr,1 Alexander Brawanski,1 and Marsha Merrill2; 1Neurosurgery, University Regensburg Medical Center, Regensburg, Germany; 2Surgical Neurology Branch, National Institutes of Health, Bethesda, MD, USA

Malignant gliomas display a distinct metabolic pattern with high glu-cose utilization rates and increased lactate levels. Despite that, the intracel-lular pH has been shown to be rather alkaline, whereas the extracellular pH is highly acidic, however the molecular mechanisms for this effect are unknown. The cell-membrane associated, hypoxia-inducible carbonic anhy-drase (CA) IX may be involved in the maintenance of this intra-extracellular pH gradient. We hypothesized that CA IX may acidify the immediate extra-cellular milieu and create a microenvironment conductive to tumor invasion. We therefore induced CA IX expression by hypoxia in vitro and analyzed the invasive potential of the cells in the presence of a specific carbonic anhy-drase inhibitor. Method: U251 glioblastoma cells were cultured in a Biocoat Matrigel invasion chamber purchased from Becton Dickinson. A matrigel-coated polycarbonate filter was situated between upper and the lower well plates The chambers were incubated in a humified 5% CO2 modular with either 0% or 21% oxygen for 6 and 12 hours. Expression of CA IX was dem-onstrated with real-time PCR and Western blotting using antibodies spe-cific for CA IX. Subsequently, cells were treated either with carrier or with acetazolamide at a concentration range between 40 nm and 40 mM. At the end of the incubation time, the cells that had penetrated the matrigel-coated filter were stained and counted visually. Percent invasion was corrected for proliferation and calculated accordingly. Real-time PCR and Western blot analysis confirmed a strong and long lasting induction of the CA IX mRNA and protein expression by hypoxia in U251 glioblastoma cells. In addition, the invasive behavior of the glioma cells was significantly enhanced after hypoxic treatment (p 5 0.01). This invasive behavior was again strongly inhibited by the treatment with acetazolamide in a dose dependent fashion (p 5 0.002). Our data suggest that hypoxic overexpression of CA IX may be functionally involved in glioma invasion by the acidification of the extracel-lular milieu and subsequent activation of proteases such as cathepsin B.



IN-03. TWIST1 ACTIVATES A PRO-INVASIVE MOLECULAR AND CELLULAR PHENOTYPE IN GLIOMA CELLS CONSISTENT WITH EPITHELIAL MESENCHYMAL TRANSITION (EMT)Svetlana Mikheeva,1 Andrei Mikheev,1 Leila Khorasani,1 Rob Oxford,1 Isidro Sanchez-Garcia,2 and Robert Rostomily1; 1Department of Neurological Surgery, University of Washington, Seattle, WA, USA; 2Institute of Molecular and Cellular Biology of Cancer, Salamanca, Spain

Epithelial mesenchymal transition (EMT) occurs normally during mesodermal tissue morphogenesis and orchestrates intravasation of epithe-lial cancer cells required for metastasis by converting stationary epithelial cells to an invasive mesenchymal phenotype. We previously demonstrated that expression of TWIST1, a master regulator of mesodermal morphogen-esis and EMT driven metastasis, correlated with human glioma malignancy and promoted invasion. Of interest, a great deal of overlap exists between the molecular determinants and cellular features of EMT and those associ-ated with glioma cell invasiveness. These observations and the recent rec-ognition of clinically relevant mesenchymal phenotypes in gliomas suggest the possibility that recapitulation of a process similar to epithelial cancer EMT may be fundamental to the malignant phenotype of human gliomas. To address this hypothesis, we analyzed the EMT-related phenotype of TWIST1 over-expression in SNB19 (SNB19 TW), a glioma cell line with low endogeneous levels of TWIST1. In microarray analysis TWIST1 over-expression was associated with up-regulation of functional groups linked to cell motility (cytoskeleton organization, regulation of cell motility and loco-motion, enzyme linked receptor signaling pathway) and target genes previ-ously associated with cancer cell invasion or EMT, including SNAI2(SLUG). SNB19 TW cells demonstrated increased invasion through matrigel filters (~70%), increased motility (~40%), greater adhesion to fibronectin (~70%), and markedly decreased cell aggregation. In accord with an invasive phe-notype, SNB19 TW cells had more pronounced lamellipodia formation, altered F-actin cytoskeletal organization and increased focal adhesion kinase (FAK) phosphorylation. The TWIST1 target gene SNAI2, identi-fied in microarray studies, functions to trigger avian EMT, contributes to maintenance of mesenchymal phenotype in mammalian EMT and is highly correlated with invasive and metastatic epithelial cancers. Accordingly, SNB19 cells over-expressing SNAI2 demonstrated an ~80% increase in invasion through matrigel filters compared with vector controls. Transient transfection of SNB19 TWIST1 cells with a SNAI2 promoter reporter led to a 1.8-fold increase in luciferase activity suggesting that TWIST acti-vates SNAI2 either through direct or indirect transcriptional regulation. The clinical relevance of pro-invasive functions of TWIST1 and SNAI2 was demonstrated by the strong correlation between their message levels determined by quantitative RT-PCR in 39 human glioma tissue samples of varying types and grades (r 5 0.72; p 5 0.001). In addition, relative to normal brain, each gene was significantly upregulated in grade IV compared with grade II and III gliomas, and notably, the highest expression levels for each gene were detected in samples of gliosarcoma, a grade IV glioma variant with highest degree of mesenchymal differentiation. These results demonstrate that TWIST1, a known regulator of EMT in epithelial cancers, activates EMT related molecular and cellular changes in glioma cells and strongly supports the hypothesis that recapitulation of an EMT-like process in neural tissue, or neural EMT, contributes to the genesis, evolution and progression of human gliomas. Given that both TWIST1 and SNAI2 acti-vate pro-invasive phenotypes in glioma cells and promote cell survival and resistance to chemotherapy in other cancers, they warrant consideration as novel targets for glioma therapy.

IN-04. IL-8 MEDIATES NF-KAPPAB–DRIVEN GLIOMA CELL INVASIONBaisakhi Raychaudhuri and Michael Vogelbaum; Cleveland Clinic Foundation, Cleveland, OH, USA

We have shown recently that aberrant constitutive activation of nuclear factor kappa B (NF-kB) is present in large number of malignant glioma cell lines as well as in primary cultures derived from tumor tissue and that one consequence of its superactivation is an enhancement of the invasive pheno-type (Raychaudhuri et al., Journal of Neurooncology, 2007). We hypoth-esize that IL-8, a pleotropic chemokine which is transcriptionally regulated by NFkB, mediates the pro-invasive phenotype resulting from NF-kB superactivation. We used flow cytometry to evaluate glioma cell lines (U87, LN229, D54, U251) for the presence of CXCR1 and 2, the receptors for IL-8. We then evaluated the effects of interruption of IL-8 autocrine signal-ing (by use of IL-8 or CXCR blocking antibodies) on glioma cell invasion, as measured using a matrigel invasion assay. Finally, we examined the effects of interruption of IL-8 signaling on activity of MMP2 and 9 by zymogra-phy. We found that GBM cells express CXCR-1 but not CXCR-2. Treatment of LN229 cells with an IL-8 neutralizing antibody markedly decreased the number of cells that invaded through matrigel (651.7 6441.2) compared to those treated with control IgG (1615 6324.7) (Student’s t-test, p , 0.001). Treatment of U87 cells with a blocking antibody against CXCR-1 reduced

the number of U87 cells that invaded through matrigel (159.0 639.7) com-pared to IgG treated controls (64.3 631.1) (p , 0.02). IL-8 treatment of GBM cells had no effect in itself on MMP-2 or 9 activities, indicating that IL-8 is not likely to modulate NF-kB dependent regulation of MMPs in gliomas. Our data are the first to indicate that IL-8 is involved in glioma cell invasion and suggest an autocrine mechanism of action.

IN-05. ROLE OF NEUROPILIN1-SEMAPHORIN3A–MEDIATED ACTIVATION OF PLEXINA1 IN REGULATING GBM MIGRATION AND INVASIONJoydeep Mukherjee,1 Amparo Wolf,2 and Abhijit Guha3; 1Cell Biology, The Arthur and Sonia Labatt Brain Tumor Research Centre, The Hospital for Sick Children Research Institute., Toronto, Ontario, Canada; 2University of Toronto, Ontario, Canada; 3Neurosurgery and Cell Biology, Toronto Western Hospital & Hosp. for Sick Children Res. Ins., Toronto, Ontario, Canada

Neuropilin1 (NRP1) can act as a co-receptor regulating both tumor angiogenesis and invasion, two cardinal features underlying the pathologi-cal heterogeneity between the “center-pseudopalisade” and “periphery-infiltrating” regions of human GBMs. NRP1 acts as a co-receptor for VEGF165 to facilitate activation of VEGFR2 and thereby angiogenesis, an important mechanism of the aberrant hypervascularity in central regions of GBMs. NRP1 can also be a co-receptor of Semaphorin3A (Sema3A) to acti-vate PlexinA1 in regulating cell guidance and tumor invasion. Our interest in NRP1–Sema3A mediated activation of PlexinA1 in GBMs stemmed from the observation that levels of all three of these transcripts were increased by Real-Time qRT-PCR in the RNA isolated from Laser Capture Micro-dissected (LCM) “periphery-infiltrating” GBM cells. NRP1 expression was elevated in GBM endothelial cells, compared to those from normal brain, though there was no difference between endothelium from the two GBM regions. These RNA expression results were validated by immuno-histochemistry (IHC) on corresponding paraffin sections. To determine the functional role of NRP1–Sema3A mediated activation of PlexinA1, in-vitro studies on U87GBMs and HUVECs were undertaken with NRP1 siRNA and a PlexinA1 neutralizing antibody. NRP1 knock down in both U87 and HUVECs resulted in decreased cell migration & invasion, without any alteration in proliferation. Exogenous administration of PlexinA1 neutral-izing antibody also decreased migration and invasion, plus augmented the results of the NRP1 knock down experiments. We postulated that promo-tion of GBM and HUVEC migration and invasion by NRP1–Sema3A acti-vation of PlexinA1 may be mediated by integrins and expression of MMPs. Initial results showed activation of avb3 integrins and secretion of MMP2 but not MMP9, by activation of PlexinA1 in GBM cells. Therefore, our results demonstrate that NRP1–Sema3A mediated activation of PlexinA1 may promote the migration and invasion phenotype of GBM tumor and endothelial cells in the periphery.

IN-06. THE INTERMEDIATE FILAMENT PROTEIN SYNEMIN REGULATES ASTROCYTOMA CELL MOTILITY AND PROLIFERATION BY AFFECTING THE DYNAMICS OF THE ACTIN CYTOSKELETONYihang Pan,1 Runfeng Jing,1 and Omar Skalli2; 1Cellular Biology and Anatomy, Louisiana State University, Shreveport, LA, USA; 2Louisiana State University Medical Center at Shreveport, Shreveport, LA, USA

Intermediate filaments (IFs) are a prominent component of the cytoskel-eton, which role in impacting the malignant properties of astrocytoma cells is relatively uncharted. In previous studies, we have shown that the IF pro-tein synemin is expressed in grade I-IV astrocytomas, but not in normal human astrocytes. These studies have also shown that synemin is present in the leading edge of astrocytoma cells and that it forms a complex with alpha-actinin, a protein important for cell motility. To determine the func-tion of synemin in astrocytoma cells, we have now used a lentivirus-based RNAi system. Six days after lentivirus infection and puromycin selection, Western blot analysis of U-373 MG, U-118 MG, and A172 astrocytoma cells revealed a ~20 fold decrease in synemin protein level when compared to cells infected with lentivirus containing a non-target shRNA. Concomitant with this decrease in synemin protein level was a 3- to 10-fold reduction in the motility of astrocytoma cells, as determined with a Boyden chamber assay. To rule out off-target effects, we have infected prostate carcinoma cells not expressing synemin with control or synemin shRNA-containing lentivirus and found that cells in these two groups had similar motility. In addition to alteration in cell motility, astrocytoma cells treated with synemin shRNAs took longer to spread out after trypsinization and were smaller than their control counterparts. Furthermore, the rate of prolifera-tion of astrocytoma cells treated with synemin shRNA was decreased and cell cycle analysis showed that this involved the accumulation of cells in G2/M phase. Synemin-silenced astrocytoma cells did not show a reduc-



tion in total actin, alpha-actinin, or vinculin protein levels when compared to controls. However, in synemin-silenced cells, there was a decrease in filamentous actin and in the amount of alpha-actinin, but not of vinculin, associated with filamentous actin. Altogether, these results demonstrate that synemin expression contributes to several malignant properties of astrocy-toma cells by a mechanism involving modifications in the dynamics of the actin cytoskeleton.

IN-07. SN38 (ACTIVE METABOLITE OF IRINOTECAN) INHIBITS INVASION UNDER HYPOXIC ENVIRONMENT IN MALIGNANT GLIOMASYasutomo Momii, Tatsuya Abe, Yukihiro Wakabayashi, Masaki Morishige, Minoru Fujiki, and Hidenori Kobayashi; Department of Neurosurgery, Oita University, Yufu, Oita, Japan

Hypoxia has long been known as a major stimulator of angiogenesis in human tumors including malignant gliomas. Recently, hypoxia was shown to also stimulate tumor cell migration, invasion and metastasis. Hypoxic tumors are more resistant to chemotherapy and radiotherapy. These effects are mediated, at least in part, by targets of the hypoxia-inducible factors (HIFs). HIF regulates the expression of at least 150 genes involved in the metabolism, cell survival, vascular remodeling and invasion. In this study, we investigate whether SN38 inhibit hypoxia-induced invasion. SN38 sig-nificantly decreased the HIF and other related gene’s expressions of glioma cells under normoxic and hypoxic conditions, in addition to suppression of hypoxia-induced invasion activity. SN38 could be attractive agent as an invasion inhibitor in glioma cells.

IN-08. MECHANISMS OF AMPA RECEPTOR–MEDIATED INVASIONJohn De Groot, Li Lu, and W.K. Alfred Yung; Neuro-Oncology, U.T. M.D. Anderson Cancer Center, Houston, TX, USA

High-grade gliomas release excitotoxic concentrations of glutamate which has been shown to enhance tumor proliferation and migration. Alpha- amino-3–hydroxy-5–methylisoxazole-4–propionic acid (AMPA) receptors are abundantly expressed at the invading edge of glioblastoma specimens suggesting they may play an important biologic role in tumor invasion. Although therapeutic trials aimed at targeting glutamate receptors for the treatment of glioma are ongoing, there remains an incomplete understand-ing of the mechanisms by which glutamate enhances tumor invasion. In this study, we examined some of the potential mechanisms by which AMPA receptor expression promotes glioma migration and invasion. Over expres-sion of GluR1, the most abundant AMPA receptor subunit in gliomas, posi-tively correlated with glioma cell adhesion to collagen and fibronectin (FN), but not vitronectin (VN) suggesting that AMPAR expression modulates glioma adhesion to the extracellular matrix. This adhesion was blocked with antibodies to b1 integrin but not a5 or a8 integrins. Knockdown of GluR1 using shRNA also abrogated glioma cell adhesion to the extracellu-lar matrix, similar to that observed with anti- â1 integrin antibodies. Using immunoprecipitation techniques, we show that GluR1 associates with the actin cytoskeleton-linked protein Band 4.1B which may serve as a link between GluR1 and the cytoskeleton via the spectrin-actin binding (SAB) domain of Band 4.1B. In the U87 cell line, over expression of GluR1 cor-related with increased cell surface expression of b1 integrin and resulted in increased phosphorylation of focal adhesion kinase (FAK-Y397). Immuno-fluorescence staining demonstrated there was a direct correlation between the level of GluR1 expression and the number of focal adhesion complexes. Furthermore, cells with over expressed GluR1 had more organized actin filaments and increased co-localization of actin and paxillin at focal adhe-sions. GluR1 over expression in U251 and U87 glioma cells significantly increased invasion in an in vitro matrigel transwell assay. Likewise, in an intracranial xenograft model, over expression of GluR1 led to perivascular and subependymal glioma cell invasion similar to patterns of tumor dissemi-nation described in human glioblastoma. Together, these results suggest that AMPA receptors may integrate signals from glutamate and from binding to the extracellular matrix to promote tumor invasion.

IN-09. MicroRNA ALTERATIONS IN GLIOBLASTOMA INVASIONJakub Godlewski, Agnieszka Bronisz, Michal Nowicki, Herbert Newton, E. Antonio Chiocca, and Sean Lawler; Department of Neurological Surgery, Ohio State University Medical Center, Columbus, OH, USA

Gliomas are therapeutically challenging because tumor cells infiltrate normal brain tissue, thereby evading surgical resection. There are currently no therapies available to block glioma invasion, although a large number of

genes are known to play a role in the process. MicroRNAs are a recently identified group of non-protein coding genes that modulate expression of protein coding genes. Aberrant expression of microRNAs plays an important role in tumorigenesis by affecting the expression of oncogenes and tumor suppressor genes. However, little is known about the role of microRNAs in gliomas. We therefore used a custom microarray to examine the global expression of microRNAs in patient tumor samples, and also in glioma cells from an in vitro invasion assay. Several major changes in microRNA expression were observed in this study, and validated by RT-PCR. These changes included substantial alterations in mir-21, mir-451 and mir-128 levels. The functional significance of these alterations is currently being explored by overexpression of these microRNAs, and identification of bio-logically relevant target genes. This will provide the basis for future stud-ies in which microRNAs may be used as diagnostic or therapeutic agents, and also provides a novel approach to invasion that may provide significant mechanistic insights.

IN-10. SYNAPTOJANIN 2 STIMULATES GLIOMA INVASION BY STABILIZING THE SRC FAMILY KINASE LYNXiaonan Xu, Ya-Yu Chuang, and Marc Symons; The Feinstein Institute for Medical Research, Manhasset, NY, USA

We previously identified a critical role for the Rac1 effector synaptojanin 2 in controlling the migration and invasion of glioblastoma cells (Chuang et al., (04) Cancer Res. 64, 8271). We also demonstrated that synaptojanin 2 is essential for the formation of invadopodia, specialized membrane structures that coordinate the action of metalloproteinases. Synaptojanin 2 has five proline rich domains (PRDs) that contain consensus sites for binding to SH3 domains. To identify novel binding partners of synaptojanin 2 that could mediate its role in invasion, we performed a screen of SH3 domain arrays (Panomics). Using this methodology, we identified a number of candidate proteins that potentially interact with synaptojanin 2, including several Src family members. The Lyn and Fyn kinases are thought to be the major Src family kinase members that are activated in glioblastoma tumors and we therefore focused on these protein kinases. We confirmed the interaction between synaptojanin 2 and Lyn/Fyn by co-immunoprecipitation of these proteins in 293T cells. We showed that small interfering RNA (siRNA)-mediated depletion of Lyn, but not Fyn, strongly inhibits invasion of SNB19 and U87MG glioblastoma cells through Matrigel and thus identified Lyn as a novel glioma invasion gene. We also found that siRNA-mediated knock down of synaptojanin 2 in SNB19 cells significantly decreases the protein level of endogenous Lyn, but not Fyn, suggesting that synaptojanin 2 regu-lates the protein stability of Lyn. A potential mechanism for the latter find-ing is that synaptojanin competes with Cbl binding to the Lyn-SH3 domain. We are currently testing this hypothesis.

IN-11. COMPUTATIONAL MODELING IDENTIFIES MORPHOLOGIC PREDICTORS OF TUMOR INVASIONVittorio Cristini1 and Elaine Bearer2; 1University of Texas Health Science Center at Houston, Houston, TX, USA; 2Providence, RI, USA

Mathematical modeling based on first principles quantifies tumor growth’s dependence on interactions between a set of variables—including genomic instability producing variations in sub-tumor clonal expansion and generating nutrient diffusion gradients—and demonstrates that these determinants of heterogeneity, and not angiogenesis per se, conspire to produce the typical morphologic patterns of infiltrative tumor boundaries in histopathology. We demonstrate that heterogeneity in sub-tumor clonal expansion and nutrient consumption drives migration and proliferation of the emerging more aggressive clones up a nutrient concentration gradient within and beyond the central tumor mass. This heterogeneity and loss of cell adhesion trigger a gross morphologic instability that leads to replace-ment of less aggressive clones and separation of tumor cell strands or clus-ters infiltrating into adjacent tissue. This model allows all variables that characterize the biophysics of tumor growth to be considered and could be applied to determine the probabilistic behavior of tumors given their pathologic appearance.

IN-12. PTEN REGULATION OF SPARC-MEDIATED GLIOMA INVASION AND SURVIVALStacey Thomas and Sandra Rempel; Henry Ford Hospital, Detroit, MI, USA

The malignant brain tumor glioblastoma (GBM) is difficult to treat due to the highly infiltrative growth into adjacent brain tissue and the enhanced survival of the cells. Our laboratory has found that SPARC is expressed by all grades of astrocytomas, including GBM. SPARC promotes



both glioma cell invasion and survival. In contrast, the tumor suppressor PTEN inhibits tumor cell invasion, proliferation, and survival; and its loss often contributes to de novo GBM development. Since SPARC and PTEN inversely regulate signaling pathways involving Akt and ILK, we hypoth-esize that reconstitution of PTEN will suppress SPARC-mediated signal transduction, invasiveness, and survival. In this study, we have transfected wild-type PTEN or empty vector (control) into GBM cells that have mutant PTEN and express high levels of SPARC. We have found that PTEN resto-ration alters cell morphology and induces strong peripheral focal adhesions in SPARC-expressing GBM cells. Using a wound healing migration assay, we found that PTEN significantly reduces migration in SPARC-expressing cells by 38% (p , 0.001), whereas PTEN expression does not change migra-tion in control GBM cells. In addition, PTEN reduces proliferation in both SPARC-expressing (60%, p , 0.001) and control (36%, p , 0.05) GBM cells, with a greater suppression of proliferation in SPARC-expressing cells. These changes in cell behavior were accompanied by a suppression of ERK phosphorylation in SPARC-expressing but not control GBM cells. Further studies will determine if PTEN reconstitution can reduce activation of the SPARC-induced signaling pathways ILK-Akt and p38MAPK-HSP27 and if these changes in signaling will lead to a suppression of cell invasion and survival both in vitro and in vivo. The results of this research will provide us with a better understanding of signaling pathways that can be used to regulate SPARC-induced invasiveness and cell survival in GBM.

IN-13. CHARACTERIZATION OF SPARC-INDUCED UPREGULATION OF ILK, AKT, ERK1/2, P38MAPK, AND HSP27 IN GLIOMA MIGRATIONSandra Rempel, Chad Schultz, William Golembieski, Christopher Yunker, and Stacey Thomas; Neurosurgery, Henry Ford Hospital, Detroit, MI, USA

Secreted Protein Acidic and Rich in Cysteine (SPARC) is a matricellular protein implicated in cell-extracellular matrix interactions that regulate cell adhesion, migration, and survival. We have previously demonstrated that SPARC is highly expressed in human gliomas, and that it promotes brain tumor invasion in vitro and in vivo. To further our understanding regard-ing SPARC function in glioma migration, SPARC-green fluorescent pro-tein (GFP) and control GFP vectors were transfected into U87MG glioma cells. We confirmed that the level of SPARC-GFP expression was similar to that observed in other established glioma cell lines and in primary human GBM tissues. The effects of SPARC-GFP expression on downstream sig-naling, proliferation, cytoskeletal structure, and migration on fibronectin were assessed in DMEM plus serum (non-stress) versus Opti-MEM (stress). In DMEM plus serum for 24 hr, SPARC expression was associated with increases in total protein and/or phosphorylation of proteins that participate in the regulation of actin reorganization (HSP27, p38MAPK, and AKT) as well as in the regulation of survival and proliferation (ILK, AKT, and ERK1/2). In Opti-MEM for 24 hr, SPARC-induced signaling was primarily associated with actin reorganization (p38MAPK, ILK, Akt, HSP27) and decreased proliferation (p , 0.05). SPARC siRNA treatment over 3 days reduced total and pAkt, total and pERK1/2, and total and pHSP27. Under stress conditions for 24 hr, the SPARC-induced changes included a two-fold increase in elongated morphology as assessed by length/width ratio (con-focal microscopy; p , 0.05), coincident localization of SPARC with ILK and SPARC with HSP27 (confocal microscopy), co-immunoprecipitation of SPARC with ILK and HSP27 (Western blot analysis), and increased migra-tion (spheroids and wound assay [p , 0.05]). Since HSP27 has been impli-cated in the regulation of actin microfilaments and migration, the relation-ship between SPARC and HSP27 was further investigated. Increasing SPARC expression was associated with increasing HSP27 in different cell lines. Immunohistochemistry of orthotopically implanted SPARC-transfected U87 cells and primary human GBM spheroids into nude rat brains showed that SPARC and HSP27 are colocalized in invading tumor cells in vivo. Treatment with HSP27 siRNA in vitro was sufficient to abrogate the three-fold SPARC-induced increase in tumor cell elongation as assessed by length/width ratio (p , 0.05). These data provide provisional mechanisms whereby SPARC regulates cell motility as an inducer and participant of the ILK/Akt and p38MAPK/Akt/Hsp27 signaling pathways that regulate stress response, proliferation, actin polymerization, and migration. Target-ing both SPARC-ILK and SPARC-HSP27 interactions may be necessary to inhibit SPARC-induced migration and invasion.

IN-14. BREVICAN, A CNS-SPECIFIC PROTEOGLYCAN, PROMOTES FIBRONECTIN SYNTHESIS AND SUBSTRATE-SPECIFIC MOTILITY IN CULTURED GLIOMA CELLSBin Hu1 and Mariano Viapiano2; 1Ohio State University, Columbus, OH, USA; 2Dept. of Neurological Surgery, Ohio State University, Columbus, OH, USA

A fundamental challenge in treating malignant gliomas is their insidi-ous invasion of adjacent normal nervous tissue. Their individual, highly motile cells evade immune detection, resist cytotoxic therapies, and ulti-mately cause all therapy to fail. The adult neural parenchyma is a notori-ously inhibitory terrain for cell movement, with a distinctive extracellular matrix (ECM) that effectively prevents the dispersion of most tumors that metastasize to the central nervous system (CNS). However, gliomas can uniquely infiltrate, disrupt, and replace this matrix with their own abnor-mal ECM. A major component of the neural ECM is a family of secreted chondroitin sulfate proteoglycans (CSPGs) that strongly inhibit cellular movement. Brevican, a CNS-specific CSPG, is the most abundant member of this family in the adult brain. We have shown it to be highly upregulated in gliomas and to promote glioma invasion in what seems a surprising sub-version of its perceived structural role in the CNS. Based on our descrip-tion of novel brevican isoforms in human gliomas (Viapiano et al., Cancer Research 2005), we have hypothesized that specific processing of this pro-tein may lead to disruption of the ECM in a manner conducive to glioma cell infiltration. Nevertheless, almost nothing is known about the molecular and cellular changes caused by the overexpression and processing of brevi-can in glioma cells. Here, we show that brevican overexpression in several glioma cell lines has a pro-adhesive effect that stimulates cell motility on specific substrates. Brevican strongly increased cell adhesion and motility on fibronectin, a major component of the basal lamina in brain blood ves-sels, and more modestly on hyaluronic acid, the main scaffolding molecule of the neural matrix. This motogenic effect, however, was not detected with cells plated on poly-D-lysine, type-I laminin, or uncoated surfaces. Con-cordantly, brevican-overexpressing cells showed an increase in the levels of beta3 integrin, which binds to fibronectin, but not in beta1 integrin, which promotes adhesion to laminin. In addition, brevican overexpres-sion caused a significant increase in fibronectin secretion by glioma cells. This effect was mimicked by an ADAMTS-produced fragment of brevican found to be upregulated in human gliomas, but not by an “uncleavable” form of brevican that cannot be processed by the metalloproteases of the ADAMTS family. Taken together, our results provide first evidence of the cellular mechanisms that support brevican’s pro-invasive role in primary brain tumors and suggest that the regulated processing of this ECM mol-ecule has important functional implications in glioma progression. In addi-tion, these findings underscore the relevance of specific ECM-processing metalloproteases, such as the members of the ADAMTS family, as novel targets in glioma therapy.

IN-15. YKL-40 INDUCES LUMICAN EXPRESSION AND PROMOTES INVASION IN HUMAN GLIOBLASTOMAChristopher Pelloski,1 Lixin Zhang,1 Krishna Bhat,2 and Kenneth Aldape2; 1Radiation Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Pathology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

YKL-40 (CHI3L1) is one of the most over-expressed gene in glioblas-toma (GBM) when compared to other brain tumors and normal brain. High expression of YKL-40 is correlated with decreased overall survival and resistance to treatment. We have shown that YKL-40 induces invasion; a hallmark of this aggressive disease. To elucidate the mechanisms of invasion induction mediated by YKL-40, we performed gene expression profiling of 2 sets of human GBM tumor specimens as well as modified, isogenic cell lines (via forced expression or knockdown of YKL-40). Expression of lumi-can (LUM) correlated with the expression of YKL-40 and had, on average, a 21-fold increased expression in the 4 comparison sets. We validated this finding using RT-PCR. To explore this further, we added soluble YKL-40 protein to the media of YKL-40–deficient cells and found the expression of LUM to increase by 20–3000-fold. Because lumican is a protein involved with extacellular matrix remodeling, we hypothesized that it was involved in YKL-40–induced invasion. Further, since YKL-40 is known to simu-late the Ras/PI3K pathways, we hypothesized that the induction of LUM and invasion occurred via Ras/PI3K stimulation. We performed transient siRNA knockdown of LUM in cells that produce YKL-40 and are highly invasive. Knockdown of LUM resulted in reduced invasion rates that were 1/4 of those observed in controls. This reduction in invasive potential, resulting from LUM knockdown, could not be rescued by addition of sol-uble YKL-40 protein. Pharmacologic inhibition of Ras and PI3K (but not of MAPK, mTOR, p38, and JNK) suppressed the induction of LUM and invasion rates in a dose dependent manner. Collectively, these data suggest that YKL-40 induces invasion, at least in part, through stimulation of the Ras/PI3K pathways and subsequent upregulation of lumican.



IN-16. SPHINGOSINE-1–PHOSPHATE REGULATES GLIOBLASTOMA CELL INVASION THROUGH CCN1/CYR61 AND THE UROKINASE PLASMINOGEN ACTIVATOR SYSTEMJames Van Brocklyn and Nicholas Young; Pathology, Ohio State University, Columbus, OH, USA

Sphingosine-1–phosphate (S1P) is a bioactive lipid that signals through a group of five G protein-coupled receptors, three of which, S1P1, S1P2, and S1P3, are commonly expressed in glioblastoma (GBM) cell lines and tissue. S1P signaling stimulates proliferation and invasiveness of GBM cells. In addition, high expression levels of the enzyme that forms S1P, sphingosine kinase, correlate with short survival time of GBM patients. In this study we investigated the mechanism by which S1P regulates glioma cell invasiveness. cDNA microarray data of GBM cells treated with or without S1P indicate that S1P induces expression of several genes, including CCN1/Cyr61 and urokinase plasminogen activator (uPA), both of which are known to be related to glioma invasion. CCN1 is a matricellular protein that increases cellular motility and invasiveness. In addition, overexpression of CCN1 in GBM correlates with short survival time of GBM patients. uPA binds to its receptor uPAR on the surface of glioma cells leading to plasmin activation and enhanced invasiveness. In this study, S1P1, S1P2, or S1P3 was overex-pressed in U-118 MG glioma cells, which normally express low levels of S1P receptors, in order to examine the contributions of individual receptor subtypes to the effects of S1P on glioma invasion and expression of pro-invasion genes. S1P1 or S1P2 overexpression enhanced S1P-induced CCN1 expression. Blocking CCN1 with a neutralizing antibody decreased S1P2-stimulated invasion. S1P1 or S1P2 overexpression also enhanced expression of both uPA and its receptor uPAR. uPA activity in conditioned medium was increased most potently by S1P1 overexpression, as determined by fibrin zymography. S1P3 overexpression enhances U-118 motility and invasiveness but had no effect on CCN1 expression and minimal effect on the uPA/uPAR system. In summary, S1P1 and S1P2 both contribute to enhanced invasive-ness of glioma cells through induction of the uPA system and CCN1. S1P3 may further contribute to glioma invasion by enhancing cell motility. Thus, S1P-stimulation of glioma invasion involves multiple pathways mediated by the various S1P receptors expressed in these tumors. Although some func-tional overlap exists, each receptor contributes uniquely and cooperatively to this process.

IN-17. THE RELATIONSHIP OF E-CADHERIN EXPRESSION AND SRC-DEPENDENT PHOSPHORYLATION OF P120 CATENIN TO GLIOMAGENESIS AND INVASIONLaura Lewis-Tuffin,1 Jann Sarkaria,2 Caterina Giannini,3 Nhan Tran,4 Michael Berens,4 and Panos Anastasiadis1; 1Cancer Cell Biology, Mayo Clinic, Jacksonville, FL, USA; 2Radiation Oncology, Mayo Clinic, Rochester, MN, USA; 3Pathology, Mayo Clinic, Rochester, MN, USA; 4Translational Genomics Research Institute, Phoenix, AZ, USA

p120 catenin is a member of the Armadillo-domain family of proteins which play a variety of roles in modulating cell-cell adhesion and cell motil-ity. p120 interacts with the cytoplasmic tail of classical cadherins such as E-cadherin, N-cadherin, and cadherin-11, thereby regulating cadherin clus-tering and the stabilization of intercellular junctions. p120 also regulates the activity of the small GTPases RhoA, Rac, and Cdc42, which organize the cytoskeleton so as to regulate cell adhesion and cell motility. p120 was also originally identified as a substrate for the Src tyrosine kinase whose tyrosine phosphorylation correlated with cellular transformation. p120 is increasingly recognized as an important protein in the processes of epithelial tumor cell invasion and metastasis. In contrast, relatively little is known about the importance of p120 and cadherins to gliomagenesis or glioma invasiveness. Our previous data indicate that p120 exerts its pro-invasive actions, in part, through its interaction with mesenchymal cadherins that are not normally expressed in epithelial tissues. However, mesenchymal cad-herin expression is the norm in most of the central nervous system, while E-cadherin expression is limited to neuroepithelial tissues. Our initial char-acterization of cadherin expression in a panel of human glioma cell lines grown conventionally on plastic or propagated as xenografts in nude mice indicated that E-cadherin expression was rare. However, a small number of xenograft lines do express E-cadherin; this expression correlates with increased invasiveness relative to E-cadherin-absent lines when the cells are implanted orthotopically in mouse brain. In addition, Src activity and Src-dependent phosphorylation of p120 tyrosine 228 is also elevated in sev-eral of the xenograft cell lines; again these results correlate with increased invasiveness in the orthotopic mouse model. We are currently confirming the relationship between E-cadherin expression, p120 Y228 phosphoryla-tion, and cell growth and motility using conventional glioma cell lines. We hypothesize that abnormal E-cadherin expression coupled with increased Src signaling and Src-dependent phosphorylation of p120 may contribute to the highly invasive nature of gliomas. We anticipate that an understanding of E-cadherin-, Src-, and p120-dependent signaling will lead to novel targets for glioma therapy.

IN-18. ROLE OF THE SMALL RHO GTPASE RND3/RHOE IN BRAIN CANCER, GLIOMA MORPHOLOGY AND INVASIONAbdelhamid Liacini,1 Angela Johnston,2 Stephen M. Robbins,3 Donna L. Senger,3 and Peter A. Forsyth3; 1University of Calgary, Calgary, Alberta, Canada; 2Calgary, Alberta, Canada; 3Biochemistry and Mol. Biology, University of Calgary, Calgary, Alberta, Canada

Malignant astrocytomas are highly invasive neoplasms infiltrating dif-fusely into regions of normal brain. Migration or invasion of tumor cells into normal tissue is a principal cause of mortality and is thought to be a multi-factorial process, consisting of cell interaction with ECM and adjacent cells, as well as accompanying biochemical processes supportive of active cell movement. Whereas the molecular and cellular mechanisms governing astrocytoma invasion remain poorly understood, evidence in other cell sys-tems have implicated Rho-GTPases in cell motility and invasion. To identify candidate glioma invasion genes, we used a process of serial in vivo selection where we selected highly invasive tumor cells from a non invasive malignant glioma cell line U87. We found that these two populations of cells had mark-edly different morphologies and different rates of migration. Subsequent cDNA microarray experiments identified several candidate genes including the Ras homolog gene family member E (RhoE). We selected RhoE as a promising candidate to study further. We first confirmed the differential expression of RhoE at the RNA and protein level in the tumor and invasive cells. Next, we constructed a mammalian expression plasmid containing human RhoE and ectopically expressed it in U87 (U87-RhoE). We estab-lished several clones and selected two for further studies. Ectopic expression of RhoE was found to inhibit cell growth, lead to an accumulation of cells at the G1/S transition of the cell cycle and induced membrane ruffle forma-tion. In addition, expression of RhoE increased migration and invasion both in vitro and in vivo and altered the relative levels of GTP-bound Rac1 and RhoA. Finally and most importantly, expression of RhoE was significantly higher in patients with highly invasive gliomas.

IN-19. THE NEUROFIBROMATOSIS 2 PROTEIN, MERLIN, REGULATES GLIAL CELL GROWTH AND MOTILITY IN A SRC-DEPENDENT MANNERS. Sean Houshmandi, Ryan J. Emnett, and David H. Gutmann; Neurology, Washington University in St. Louis, St. Louis, MO, USA

Individuals with the inherited tumor predisposition syndrome, neurofi-bromatosis type 2 (NF2), develop ependymomas and spinal astrocytomas. Both ependymomas and astrocytomas are glial cell malignancies (gliomas) that arise from neoplastic glial cells. Studies from several laboratories, including our own, have shown that NF2 gene inactivation and loss of expression of the NF2 gene product, merlin or schwannomin, represents the most common genetic change in ependymoma, both in individuals with NF2 as well as in individuals with sporadic ependymomas. To define the mechanism underlying merlin growth regulation in the nervous system, we have used Nf2-deficient glial cells, to demonstrate that merlin regulates growth and motility in astrocytes. Nf2-deficient astrocytes show increased activation of the Src oncoprotein and its downstream effectors. In addition, we show that merlin regulation of growth and motility in Nf2–deficient glial cells is Src-dependent. We have also determined that merlin regulates Src in a receptor tyrosine kinase-dependent manner and that this regulation controls glial cell growth and motility. Our studies, elucidate one of the key growth control pathways de-regulated in gial cell tumors. These studies will be beneficial for the identification of potential targets for therapeutic drug design aimed at ameliorating one of the growth control defects in brain tumors, which may lead to improved treatments for these human cancers.

MEDICAL THERAPIES, ADULT

MA-01. BEVACIZUMAB, A MONOCLONAL ANTIBODY TO THE VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF), AND IRINOTECAN FOR TREATMENT OF RECURRENT PRIMARY MALIGNANT BRAIN TUMORS IN ADULTSBenedikte Hasselbalch,1 Ulrik Lassen,2 Kirsten Grunnet,1 Michael Kosteljanetz,3 Henning Laursen,4 and Hans Skovgaard Poulsen1; 1Radiation Biology, Rigshospitalet, Copenhagen, Denmark; 2Department of Oncology, Rigshospitalet, Copenhagen, Denmark; 3Department of Neurosurgery, Rigshospitalet, Copenhagen, Denmark; 4Department of Neuropathology, Rigshospitalet, Copenhagen, Denmark

The prognosis of recurrent malignant brain tumors is poor, and no effi-cacious therapy exists in patients previously treated with radiotherapy and standard chemotherapy. Bevacizumab (B) binds to VEGF and inhibits tumor angiogenesis, and treatment with this drug might induce tumor regression and prolongation of life. Irinotecan (I) is a topoisomerase 1 inhibitor with



modest effect on recurrent primary brain tumors. The combination of B and I in recurrent malignant gliomas was presented at ASCO 2006 and published in January 2007, showing very encouraging responses. We report confirmatory results of the combination of B and I in a consecutive series of patients with primary malignant brain tumors recurring after standard primary and secondary treatment (surgery, radiotherapy and standard and/or secondline chemotherapy).With standard inclusion criteria, including PS 0–2, patients received B as 10 mg/kg, and I 125 mg/m2 in patients not treated with enzyme inducing antiepileptic drugs (EIAED) or 340 mg/m2 in patients treated with EIAED every other week until progression or non-manageable toxicity. Response evaluation was performed by MacDonald criteria and MRI scans. The results of 31 patients is presented, 15 with grade IV tumors (glioblastoma multiforme), 7 with grade III anaplastic astrocytomas, 5 with anaplastic oligodendrogliomas, 1 with anaplastic ependymoma, 1 with hemangiopericytoma, 1 with prolactinoma, and 1 with medulloblastoma. Four patients had complete response, 3 grade IV tumors and 1 anaplastic oligodendroglioma. One patient had partial response (. 50 % tumor reduc-tion), 12 had stable disease (3 had tumor reduction between 31%–45%). 14 patients progressed. No grade 4 toxicity was observed and most patients experienced grade 1–2 toxicity. Two tromboembolic events and 1 intestinal perforation were observed. The combination of B and I is safe, induces tumor regression in a substantial number of patients, and can be used as treatment to patients recurring after standard treatment. An update will be presented at the meeting.

MA-02. PHASE I/PHARMACOKINETIC (PK) STUDY OF ENZASTAURIN PLUS TEMOZOLOMIDE DURING AND FOLLOWING RADIATION THERAPY IN PATIENTS (PTS) WITH NEWLY DIAGNOSED GLIOBLASTOMA MULTIFORME (GBM) OR GLIOSARCOMANicholas Butowski,1 Kathleen Lamborn,1 Susan Chang,1 Rebecca DeBoer,1 Margaretta Page,2 Jane Rabbitt,2 Anne Fedoroff,2 Rupa Parvataneni,1 Valerie Kivett,1 Steven Nicol,3 Luna Musib,4 Astra Liepa,5 Donald E. Thornton,3 and Michael Prados1; 1Neurosurgery, University of California, San Francisco, San Francisco, CA, USA; 2Neuro-Oncology, University of California, San Francisco, San Francisco, CA, USA; 3Oncology, Eli Lilly and Company, Indianapolis, IN, USA; 4Biopharm, Eli Lilly and Company, Indianapolis, IN, USA; 5Global Health Outcomes, Eli Lilly and Company, Indianapolis, IN, USA

Enzastaurin is a potent, selective, oral serine/threonine kinase which suppresses signaling through PKCb and the PI3K/AKT pathway to induce apoptosis, reduce cell proliferation and suppress angiogenesis. PKCb is activated by vascular endothelial growth factor (VEGF), one of the most potent angiogenic factors which is upregulated in most cases of GBM. The goal of this study was to assess the tolerance of two doses of enzastaurin given concurrently with radiation therapy (RT) and temozolomide (TMZ) and adjuvant with TMZ in newly diagnosed GBM to define the phase II dose for further study. Secondary objectives were to characterize the PK parameters of enzastaurin and explore the utility of perfusion imaging in the evaluation of this agent. Patients .18 years with histologically con-firmed, newly diagnosed GBM with a KPS of at least 60, were eligible. Pts must have had discontinued enzyme inducing antiepileptic drugs. Treatment was started within 5 weeks of diagnosis. RT consisted of 60 Gy over 6 weeks and TMZ was given at 75 mg/m2 daily, during XRT and at 200mg/m2 daily, 5/28 days. One adjuvant cycles was 5 28 days. Enzastaurin was given once daily, with a starting dose of 250 mg/day. Dose was escalated to 500 mg/day if no more than 1/6 patients experienced a dose limiting toxic-ity (DLT) during XRT and the first adjuvant cycle of TMZ. The primary objective of phase II is to determine the efficacy of enzastaurin as measured by overall survival. Multiple PK and pharmacodynamic (PD) endpoints will be evaluated using perfusion imaging and plasma biomarkers. Patients will continue treatment for one year unless disease progression or unacceptable toxicity occurs. From Sep. 2006, 12 pts have enrolled. There was no DLT in the first 6 pts treated at 250 mg enzastaurin, and the second cohort of 6 pts at the 500 level, continue to be assessed for toxicity. One pt experienced a DLT of grade IV thrombocytopenia during radiation, which has been previously observed with TMZ. If no further DLTs are seen, the phase II dose will be defined at 500 mg/day enzastaurin. Enzastaurin with concur-rent XRT and TMZ appears to be well-tolerated at a dose of 250 mg daily. Cohort 2 (enzastaurin 500 mg daily) is being evaluated. Final phase I safety results, recommended phase II dose of enzastaurin, and preliminary PK, PD and symptom data, as well as results using perfusion imaging, will be presented.

MA-03. COMPLETE BILATERAL THIRD NERVE PALSIES AS THE INITIAL MANIFESTATION OF ANTI-HU PARANEOPLASTIC ENCEPHALOMYELITISLynne Taylor; Neurology, Virginia Mason Medical Center, Seattle, WA, USA

A previously healthy 44-year-old woman presented with the acute onset of diplopia, right-sided hearing loss and gait ataxia. On examination she had normal cognition, mildly dysarthric speech, diminished hearing in the right ear, dramatic, complete 3rd nerve palsies with 8mm fixed pupils, sig-nificant ptosis, and a wide-based, ataxic gait. CSF initially showed 16 white cells and an elevated protein of 76, cytology was negative. One year later, her neurologic clinical syndrome remained and her CSF had normalized. MRI of the brain and orbits with and without contrast was normal. CT of the chest showed a left anterior mediastinal mass, which was small cell lung cancer by needle biopsy. Staging studies at presentation were unremarkable and she had a CR to cis-platin and VP-16. One year later she is without active disease. Evaluation at Rockefeller University revealed 1anti-HU by Western blot. Cranial nerve involvement in paraneoplastic syndromes has been reported, usually involving 2, 3, 4, 6, and 8th nerve palsies. Crino et al. (1996) reported ophthalmoparesis as an early manifestation of para-neoplastic brainstem encephalitis in three patients. At autopsy, gliosis and neuronal cell loss with an inflammatory component was found within the ocular motor nuclei and brainstem tegmentum. Dalmau termed these “oto-neuro-ophthalmologic syndromes” and stressed the importance of quick recognition that these syndromes are paraneoplastic. Literature review reveals very few well-documented cases similar to our patient. Since 1980 there have been 6 patients with cranial nerve palsies and paraneoplastic syndromes reported. One patient was 1anti-HU and had bilateral 6th nerve palsies associated with breast cancer, 5 were small-cell patients with some degree of 3rd nerve involvement. One had an INO, one had multiple cranial nerve palsies 5–10 and two were reported as having “ophthalmoparesis”, one with involvement of both 3rd and 4th nerves at autopsy. Detailed exam-ination by a neuro-ophthalmogist will be reviewed as well as a videotape of the index patient. Her ptosis was so profound that it worsened her mild gait ataxia and rendered her functionally blind. Rehabilitative measures such as the use of “ptosis crutches” will be addressed.

MA-04. RETROSPECTIVE STUDY OF BEVACIZUMAB FOR RECURRENT MALIGNANT GLIOMAS AND ANALYSIS OF THE PATTERNS OF RECURRENCEAndrew D. Norden,1 Geoffrey Young,2 Kian Setayesh,2 Alona Muzikansky,3 Roman Klufas,2 Gina Ross,2 Jan Drappatz,1 Santosh Kesari,1 Abigail Slate Ciampa,1 Laura G. Ebbeling,1 Brenda Levy,1 and Patrick Wen1; 1Dana-Farber/Brigham and Women’s Cancer Center and Harvard Medical School, Boston, MA, USA; 2Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; 3Massachusetts General Hospital, Boston, MA, USA

There is increasing evidence that bevacizumab, a humanized mono-clonal antibody against VEGF, may have activity in recurrent malignant gliomas. At recurrence some patients appear to develop non-enhancing diffuse infiltrating disease rather than enhancing tumor. This may be explained in part by preclinical studies suggesting that angiogenesis block-ade by anti-VEGF agents facilitates co-option of the normal vasculature and tumor invasion (Kunkel et al., Cancer Res. 2001;61:6624). We retro-spectively reviewed our experience with 47 consecutive, heavily pre-treated patients with recurrent malignant gliomas who received bevacizumab and chemotherapy to determine efficacy, toxicity, and patterns of recurrence. Thirty-seven patients had adequate MRI data which was blindly reviewed by three neuroradiologists, except in a small number of cases in which only one review was possible. Responses were classified according to conven-tional Macdonald Criteria, as well as by quantitative volumetric analysis. The recurrence patterns of patients treated with bevacizumab were com-pared to recurrence patterns in 19 malignant glioma patients treated with conventional cytotoxic chemotherapy on clinical trials. There were 29 GBM and 18 anaplastic glioma (AG) patients (27 men; 20 women). Median age was 50 years (range 23–71). Median number of prior therapies was 2 (range 0–6). Thirty-nine patients received CPT-11 in conjunction with bevacizumab. Other patients received carboplatin (6), BCNU (1), and temo-zolomide (1). Treatment was generally well tolerated. Adverse effects were mostly attributable to the concurrently administered chemotherapy agent rather than to bevacizumab. Eight patients experienced Grade 1–2 hemor-rhages, primarily epistaxis. Two patients developed asymptomatic brain hemorrhages that were detected on routine neuroimaging. One patient experienced spontaneous Grade 4 colon perforation. Of the 7 patients who received anticoagulation with low molecular weight heparin, the only bleed-ing complication observed was epistaxis in one patient. Five Grade 4 throm-boembolic complications occurred. There were no complete responses. 32.4% of patients had a partial response, 29.7% minimal response, and 24.3% had stable disease. Median time to radiographic progression (MTP) was 17.7 weeks. Overall 6-month progression-free survival (6M-PFS) was



34%, and 6-month overall survival 64%. 6M-PFS for GBM patients was 41%. In 21 patients who progressed on their initial therapy, bevacizumab was continued and the concurrent chemotherapy agent changed, mainly to carboplatin or a nitrosourea. In no case did the change from one chemo-therapy agent to another produce a radiographic response, but 2 patients had prolonged PFS of 20 and 31 weeks. Overall MTP was 7 weeks. We did not detect a difference in recurrence pattern between patients treated with bevacizumab and conventional cytotoxic chemotherapy. Most recur-rences in both groups were classified as “local recurrence.” Combination therapy with bevacizumab and chemotherapy appears to be active against recurrent malignant gliomas, even in the setting of extensive pre-treatment. Therapy is generally well-tolerated. At recurrence, continuing bevacizumab and changing the chemotherapy agent provided long-term disease control only in a small subset of patients. This small, retrospective study was unable to confirm that bevacizumab promotes tumor infiltration. However, a larger prospective study evaluating this phenomenon is warranted.

MA-05. ASSESSMENT OF TREATMENT RESPONSE TO BEVACIZUMAB (BEV) WITH MRI IN RECURRENT MALIGNANT GLIOMA (MG)Nimish Mohile and Lauren Abrey; Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

Preliminary reports of BEV response in the treatment of recurrent MG are promising. Response assessment based on Gadolinium enhanced magnetic resonance imaging (Gd-MRI) is complicated by effects of BEV on vasculature; standard response criteria may be inadequate to measure treatment effect. The objective of this study is to describe findings of non-contrast MRI sequences and to determine how they may aid in the inter-pretation of BEV response. MRI scans from all recurrent MG patients treated with BEV on clinical trials at our institution were evaluated in an IRB approved study. Macdonald criteria were used to describe Gd enhanced sequences. FLAIR changes were defined as increased (. 25% increase in bi-dimensional area), decreased (. 25% decrease) or stable. Diffusion weighted imaging (DWI) was evaluated for changes from baseline abnor-malities or for new foci of restricted diffusion. T1 scans were reviewed to identify new hyperintense lesions. 129 MRI scans, representing 176 cycles of treatment, were reviewed in 28 patients who received BEV alone, BEV 1 CPT-11 or BEV 1 re-irradiation. 28 scans were baseline and 101 were done for response assessment. 56% of scans (57/101) had CR or PR. FLAIR abnormality was stable in 60 scans and decreased in 22, all of which were concordant with Gd-MRI. In 19 scans, FLAIR abnormality increased, cor-relating with PD in 10. Increased FLAIR in 9 scans was associated with SD in 2 and objective responses in 7; in 3 of these, this finding pre-dated PD by 1 to 3 cycles. Changes in restricted diffusion were noted in 30 scans. 10 scans demonstrated resolution of baseline restricted diffusion and correlated with Gd-MRI response. In 20 scans, the extent of restricted diffusion increased, corresponding with PD in 11; 9 scans were associated with objective responses and in 3, anticipated PD by 1 cycle. New T1 hyperintense foci suggestive of hemorrhage were noted in 5 scans from 5 patients with no clinical correlate. Increase in FLAIR abnormality, consistent with growth of non-enhancing tumor, correlated with or predicted PD in most patients. DWI abnormalities were also associated with progression and suggest that restricted diffusion is secondary to tumor growth and hypoxia rather than a positive response to therapy. FLAIR and DWI findings discordant with Gd-MRI may be clarified with longer follow up. T1 abnormalities in 18% of patients likely represent clinically asymptomatic hemorrhage related to treatment.

MA-06. PHASE II TRIAL OF BEVACIZUMAB AND ERLOTINIB IN RECURRENT GLIOBLASTOMA MULTIFORME (GBM)Sith Sathornsumetee,1 James Vredenburgh,2 Jeremy Rich,3 Annick Desjardins,1 Jennifer Quinn,1 Daniela Bota,4 Krishna Goli,5 Alyssa Mathe,5 Sridharan Gururangan,3 Allan Friedman,5 Henry Friedman,3 and David Reardon3; 1Medicine, Duke University, Durham, NC, USA; 2Medicine, Surgery, Duke University, Durham, NC, USA; 3Duke University, Durham, NC, USA; 4Neuro-Oncology, Duke University Medical Center, Durham, NC, USA; 5Surgery, Duke University, Durham, NC, USA

Bevacizumab (Avastin), a neutralizing monoclonal antibody to vas-cular endothelial growth factor (VEGF), has demonstrated remarkable radiographic response and promising survival benefit in combination with irinotecan in patients with recurrent malignant glioma. Erlotinib, an EGFR tyrosine kinase inhibitor, has shown anti-tumor activity in some glioma patients. In this study, we evaluate the combinatorial efficacy of bevaci-zumab and erlotinib in patients with recurrent GBM. Twenty-four patients with recurrent GBM have enrolled in this Phase II study to evaluate a 6 month progression-free survival as a primary outcome measure. Radio-

graphic response, pharmacokinetics and correlative biomarkers are outcome measures. Eligibility criteria include: histologically confirmed GBM; age at least 18 years; KPS of at least 60%; no radiographic evidence of intracra-nial hemorrhage; adequate hepatic, renal, and bone marrow function and lack of prior failure or significant toxicity following treatment with either bevacizumab or erlotinib. Patients are stratified based on concurrent use of enzyme-inducing anticonvulsants (EIAC; phenytoin, carbamazepine, oxcar-bazepine and phenobarbital). Bevacizumab is dosed at 10 mg/kg intrave-nously every two weeks. Erlotinib is orally administered daily with 200 mg/day for patients not on EIAC and 450 mg/day for patients on EIAC. At present, enrollment is complete. Median age is 51.2 years (range 24 to 70); 52% are male and 35% are on EIAC. Seven patients have completed their first cycle with only one patient experienced progressive disease. There are no grade 3–4 toxicities. Twenty three patients continue on treatment. Pharmacokinetic sampling has been performed in 78% of patients. Archival tumor samples are undergoing analysis for identifying correlative biomark-ers of response and survival. A companion study with this regimen for recur-rent grade 3 malignant glioma patients continues to accrue. Combination of bevacizumab and erlotinib appears to be safe. Further clinical follow-up of this completely enrolled study is ongoing.

MA-07. A PHASE II TRIAL OF LAPATINIB AND PAZOPANIB FOR PATIENTS WITH RECURRENT GLIOBLASTOMA MULTIFORME (GBM)David Reardon,1 Morris Groves,2 Patrick Wen,3 Louis Nabors,4 Tom Mikkelsen,5 Steve Rosenfeld,6 Dean Harris,7 Douglas DeMarini,8 Ben Suttle,8 Thangam Arumugham,8 Jeffrey Hodge,8 and Joanne Lager8; 1Duke University, Durham, NC, USA; 2University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3Dana Farber Cancer Institute, Boston, MA, USA; 4University of Alabama at Birmingham, AL, USA; 5Henry Ford Health Systems, Detroit, MI, USA; 6Columbia University, New York, NY, USA; 7The Royal Marsden Hospital, Surrey, United Kingdom; 8GlaxoSmithKline, Durham, NC, USA.

The ErbB1/ErbB2, VEGF, and PDGF receptors activate critical growth, survival, and angiogenic pathways in malignant glioma, making them attrac-tive therapeutic targets. Preclinical studies confirm synergistic anti-glioma activity with combinatorial regimens against these targets. Pazopanib and lapatinib are potent, orally active, tyrosine kinase inhibitors of VEGFRs, PDGFRs and c-kit, and ErbB-1/ErbB-2, respectively. This study includes a multi-center, single-arm, open-label phase 2 design to determine the 6-month progression-free survival (PFS) of patients with recurrent GBM treated with daily lapatinib and pazopanib. Secondary endpoints include safety and tolerability, pharmacokinetics (PK), and radiographic response. Eligibility is limited to adult patients with GBM who have received no more than 2 prior cytotoxic treatment regimens, ECOG 0–1, adequate organ function and no prior therapy against VEGFR, ErbB-1, or ErbB-2. At present, patients can not be receiving cytochrome P450 enzyme inducing anti-epileptics (EIAEDs: phenytoin, carbamazepine, phenobarbitol, oxcar-bazepine, primidone). Patients are enrolled into 2 strata: those with archival tumors positive for either PTEN or EGFRvIII (stratum 1; n 5 35) and those with archival tumors that do not express these gene products (stratum 2; n 5 40). Each stratum incorporates a 2-stage design with a stopping rule which leads to termination of the stratum at the end of the first stage if PFS results at 2 months provide evidence that the regimen is not effective. After 20 patients have been enrolled in a given stratum, PFS rate at 2 months will be analyzed. If fewer than 10 patients in stratum 1 or fewer than 12 patients in stratum 2 have been observed to have died or experienced disease progression at 2 months, further enrollment into that stratum will continue. The daily doses of pazopanib and lapatinib are 400 mg and 1000 mg once daily, respectively. Treatment is continued until disease progression, with-drawal for toxicity or withdrawal of consent. Each treatment cycle is 28 days. Response is evaluated using MacDonald criteria after cycle 1, cycle 2, and every second cycle thereafter. Pharmacokinetic (PK) studies are per-formed on day 1 of cycle 2. Thirty-two patients have enrolled including 16 on stratum 1 and 16 on stratum 2. The median age is 55 years (range: 20 to 76) and 66% are male. Serious adverse events reported to date include seizure (n 5 3), altered mental status (n 5 2) and the following in one sub-ject each: abdominal pain, pneumonitis, pneumonia, CNS hemorrhage, and fatigue. PK analyses are ongoing. The following investigator-assessed results have been seen at 2 months: in stratum 1, 3 patients with stable disease, 2 with progression, and 11 that are too early to evaluate, and in stratum 2, 5 patients with stable disease, 2 with progression, and 9 that are too early to evaluate. Further accrual is ongoing. An update of outcome, toxicity, and PK analyses will be presented.



MA-08. AN UPDATE OF THE PHASE IIA TRIAL OF CILENGITIDE (EMD121974) SINGLE-AGENT THERAPY IN PATIENTS WITH RECURRENT GLIOBLASTOMA: EMD 121974–009David Reardon,1 Karen Fink,2 Louis Nabors,3 Timothy Cloughesy,4 Alison O’Neill,5 David Schiff,6 Jeffrey Raizer,7 Stefan Krueger,8 Martin Picard,8 and Tom Mikkelsen9; 1Duke University Medical Center, Durham, NC, USA; 2Baylor University Medical Center at Dallas, Dallas, TX, USA; 3Neurology, University of Alabama at Birmingham, Birmingham, AL, USA; 4University of California, Los Angeles, Los Angeles, CA, USA; 5Neurology, Massachusetts General Hospital, Boston, MA, USA; 6Neurology, University of Virginia Health Science Center, Charlottesville, VA, USA; 7Feinberg School of Medicine, Northwestern University, Chicago, IL, USA; 8Merck KGaA, Darmstadt, Germany; 9Hermelin Brain Tumor Center/Neurology, Henry Ford Hospital, Detroit, MI, USA

We evaluated the safety, toxicity, and clinical activity of the cyclic RGD pentapeptide cilengitide (EMD121974), an inhibitor of integrins avß3 and avß5, as a single agent at doses of 500 and 2000 mg in patients (pts) with recurrent glioblastoma (GBM). In this phase IIa study multicenter, open-label, randomized, uncontrolled study, pts with GBM and measurable dis-ease that was progressive after prior temozolomide and radiotherapy were randomized to receive cilengitide at either 500 mg or 2000 mg i.v., 2×/week, until progression. Neurologic exams were performed after every cycle (4 weeks) while MRIs were evaluated every other cycle. The study included central, blinded pathology and independent radiology review. The primary endpoint was Progression Free Survival (PFS) at 6 months (6-mth PFS). Secondary endpoints included radiographic response, overall survival (OS), time to disease progression, safety, tolerability and pharmacokinetics (PK). Accrual included 81 patients (median Karnofsky Performance Status 80%; median age 57 yrs) at 15 sites with 41 at the 500 mg and 40 at the 2000 mg dose levels. Demographic and pretreatment variables were comparable between dose level cohorts. The median number of infusions was16 [range, 4–189]. PK studies revealed significantly greater exposures among the 2000 mg cohort. Treatment related NCI CTC grade 3 adverse events (AEs) were limited to elevated transaminases (at 500 mg), arthralgia/ myalgia (at 500 mg), and weight increase/ edema (at 2000 mg) in 1 patient, respectively. No grade 4 therapy related AEs occurred. One CTC grade 2 cerebral hemor-rhage was reported in a pt at progression. Although not statistically signifi-cant, there was a trend towards better outcome among pts treated at the 2000 mg dose level. Cilengitide was well tolerated and demonstrated single agent activity in recurrent GBM, with long-term disease stabilization in a subset of pts.

MA-09. DIC IN A PATIENT WITH GBM MANIFESTING BOTH HEMORRHAGIC AND EMBOLIC PHENOMENASarah Taylor,1 Allan Fleming,2 and Ania Pollack3; 1University of Kansas School of Medicine, Kansas City, KS, USA; 2KS, USA; 3Kansas City, KS, USA

Cancer patients(pts) frequently present with a hypercoagulable syn-drome. In pts with high-grade glioma, this usually presents as thromboem-bolic phenomena. DIC has also been reported in high-grade gliomas. It has been considered to have little clinical consequence because of the usual lack of hemorrhagic clinical manifestations due to compensated cellular and humoral clotting factors. Physicians may also fail to consider the pos-sible etiologic relationship between the thromboembolic complications of high grade gliomas and DIC. We present a patient with chronic DIC, with marked hypofibrinogenemia, diagnosed during evaluation of protracted thrombocytopenia, initially attributed to bone marrow suppression by temozolomide(T). CASE: 43 yo WM presented with acute mental status changes. PMH : 2 elective surgeries without abnormal bleeding. PE: fluc-tuating mental status changes with GCS of 14. Imaging revealed a large butterfly mass with associated hemorrhage. Intraventricular bleeding neces-sitated repeated bilateral ventricular drainage. Biopsy revealed GBM. XRT and temodar (T) per Stupp were started. (T) was held on day 27 due to thrombocytopenia. Day 49, fibrinogen 77, FDP . 20, plts 43,000. Rx’d with cryoglobulin to maintain fibrinogen . 100. Day 69, plts recovered and maintenance (T) begun. Day 88, DVT, Rx’d wtih IVC filter and ASA due to prior IHC. Day 98, fibrinogen normal. Day 124, PE, Rx’d with LMWH. Day 135, recurrent hypofibrinogenemia with abdominal wall bleeding. Pt continues now on maintenance (T) with improving MRI and neurologic status(PS 5 2) but chronically low fibrinogen. Grade 3 thrombocytopenia is uncommon with (T) but may be prolonged and result in cessation of Rx. Bone marrow exam in some pts has documented reduced or absent mega-karyocytes. In this pt, myelosuppression from (T) was initially considered the cause of his thrombocytopenia, but (T) may have only impaired the mar-row’s ability to compensate for the consumption of platelets by DIC. The clinical relevance of DIC may be under appreciated in high grade gliomas, particularly in reference to thromboembolic events. The relationship between the hypercoagulable state and angiogenesis and its contribution to

the aggressiveness of high grade gliomas, as appreciated in other malignan-cies, deserves further consideration.

MA-10. MATURE RESULTS OF A PHASE I/IIA TRIAL OF THE INTEGRIN INHIBITOR CILENGITIDE (EMD121974) ADDED TO STANDARD CONCOMITANT AND ADJUVANT TEMOZOLOMIDE AND RADIOTHERAPY (TMZ/RT) FOR NEWLY DIAGNOSED GLIOBLASTOMA (GBM)Roger Stupp,1 Roland Goldbrunner,2 Bart Neyns,3 Uwe Schlegel,4 Paul Clement,5 Gerhard Grabenbauer,6 Monika Hegi,7 Johannes Nippgen,8 Martin Picard,8 and Michael Weller9; 1Multidisciplinary Oncology Center, University of Lausanne, Lausanne, Switzerland; 2Department of Neurosurgery, University of Munich, Grosshadern, Munich, Germany; 3Department of Medical Oncology, AZ-VUB University Hospital, Brussels, Belgium; 4Department of Neurology, Ruhr University Hospital, Bochum, Germany; 5Academisch Ziekenhuis Leuven, Leuven, Belgium; 6Department of Radiation Oncology, University Hospitals, Erlangen, Germany; 7Laboratory of Tumor Biology and Genetics, Department of Neurosurgery, University of Lausanne, Lausanne, Switzerland; 8Merck KGaA, Darmstadt, Germany; 9Department of General Neurology, University of Tuebingen, Tübingen, Germany

GBM is a highly vascularized tumor with tumor cells and tumor vas-culature expressing v3 and/or v5 integrins. Based on preclinical and early clinical data, inhibition of the integrins with the specific inhibitor cilen-gitide, a cyclic RGD pentapeptide targeting integrins v3 and v5 may be a promising strategy against GBM. 52 pts (PS 0–1: 92%, 2: 8%; median age 57 yrs) after tumor resection (n 5 43, 83%) or biopsy (n 5 9, 17%) were treated with standard TMZ/RT (Stupp et al., NEJM 2005). In addition cilengitide (500 mg i.v., 2×/week) was started one week before TMZ/RT and given throughout for the duration of chemotherapy or until progres-sion. The primary endpoint was progression free survival rate at 6 months. Pts were followed with MRI every 2 months. Histopathologic diagnosis and MRI imaging were independently reviewed, O6–methylguanine-DNA methyltransferase (MGMT) promoter methylation status was assessed in 45 (86.5%) pts. 52 pts were treated; 50 pts (96%) completed RT; 23 (44%) pts completed 6 cycles of maintenance TMZ and cilengitide; and thereafter 7 (13%) pts optionally continued monotherapy with cilengitide. Observed hematological grade 3 and 4 toxicities were: lymphopenia (29/52, 55.8%), thrombocytopenia (7/52, 13.5%), neutropenia (5/52, 9.6%). As severe treat-ment related non-hematologic toxicities were reported: constitutional symp-toms (asthenia n 5 1, fatigue and anorexia n 5 1), elevated liver function test (n 5 1), deep vein thrombosis and pulmonary embolism (n 5 1). Overall the median PFS was 7.9 mo (95% CI, 6.0, 10.7), and 36/52 pts (69.2% [95% CI, 0.55, 0.81]) and 18/52 pts (34.6% [95% CI, 0.22, 0.49]) were progres-sion free and alive after 6 and 12 months, respectively. The median OS was not reached, and the OS rate at 6 and 12 months were 90.4% (47/52, [95% CI, 0.79, 0.97]) and 63.5% (33/52, [95% CI, 0.49, 0.76]). The subgroup of pts. with a methylated MGMT gene promoter (n 5 23) had a median PFS of 13.4 mo (95% CI, 8.6, NA) with a 1 year PFS rate of 56.5% (95% CI, 0.34, 0.77). The median OS was not reached, and the 1yr OS rate was 91.3% (95% CI, 0.72, 0.99). The study strongly suggests activity of the specific integrin inhibitor cilengitide in combination with TMZ/RT with regard to PFS and survival, most prominent in the pts subgroup with methy-lated MGMT promoter. The treatment was well tolerated. These results are encouraging and warrant further investigation.

MA-11. EARLY TREATMENT WITH BEVACIZUMAB IN PATIENTS WITH GLIOBLASTOMA MULTIFORME AND GLIOSARCOMA FACILITATES ADMINISTRATION OF RADIATION THERAPY BY RAPIDLY ALLEVIATING CEREBRAL EDEMA AND IMPROVING NEUROLOGICAL FUNCTIONRichard Green,1 Emily Woyshner,1 George Carvajal,1 Phioanh Nghiemphu,2 Albert Lai,2 and Timothy Cloughesy2; 1Neurology, Kaiser Permanente-Los Angeles Medical Center, Los Angeles, CA, USA; 2University of California, Los Angeles, Los Angeles, CA, USA

Bevacizumab is an inhibitor of the vascular endothelial growth factor receptor. A recent Phase II study of bevacizumab and irinotecan in recur-rent malignant glioma showed promising activity. Three patients with glio-blastoma multiforme and one with gliosarcoma who developed worsening neurological function from early postoperative mass effect and cerebral edema, despite high dose corticosteroids, were given bevacizumab 5 mg/kg biweekly. All patients were receiving temozolomide; two patients started bevacizumab prior to regional radiation therapy (RT) and two during RT. All patients showed rapid improvement in neurological function and neuroimaging and were rapidly weaned from corticosteroids. Complica-tions included pulmonary embolism in two patients. No patient developed intracranial hemorrhage. Treatment benefits appear to be durable in patients



treated for up to 11 months; no patients have developed recurrent edema or tumor progression to date. The early addition of bevacizumab before or during RT rapidly alleviated corticosteroid-refractory cerebral edema, improved neurological function, and provided an opportunity for standard RT and concurrent temozolomide to be administered.

MA-12. TEMOZOLOMIDE IN ADDITION TO GLIADEL WAFER AND RADIOTHERAPY IN ADULTS WITH NEWLY DIAGNOSED GLIOBLASTOMA MULTIFORME: THE IMPACT OF MGMT EXPRESSIONRichard Rovin,1 Robert Winn,2 and Nicholas Brown2; 1Upper Michigan Brain Tumor Center, Marquette, MI, USA; 2Biology, Northern Michigan University, Marquette, MI, USA

The purpose of this study was twofold. First, to determine if the addi-tion of temozolomide to our standard management protocol of maximal surgical resection, Gliadel wafer implant and radiation therapy had an impact on time to progression in patients with glioblastoma multiforme. Second, to correlate time to progression in the two cohorts with MGMT expression status. Two cohorts of patients with glioblastoma multiforme were reviewed. The first group consisted of 10 patients treated with our standard management protocol (TMZ–). The second group included 9 patients who were additionally given temozolomide (TMZ1). The only difference in management between the two groups, then, was the use of temozolomide. Time to progression was defined as the interval between surgery and the first imaging study that was compelling for tumor pro-gression and lead to a change in management. MGMT expression was evaluated using a RT-PCR protocol. The median time to progression for the temozolomide group (TMZ1) was 5.8 months (range 1.6 to 9.4). The median time to progression for the group without temozolomide (TMZ–) was 5.2 months (range 2.4 to 16.7). This was not significantly different. In the TMZ– group, 50% of tumors expressed MGMT. In the TMZ1 group, 89% of tumors were MGMT positive. The addition of temozolomide to our aggressive management protocol did not extend the time to progression in this group of patients with glioblastoma multiforme. This result may be related to the preponderance of MGMT expressing tumors in the TMZ1 group, or to the effect of other DNA repair mechanisms or to the time course of BCNU release.

MA-13. PHASE I STUDY OF VORINOSTAT (SUBEROYLANILIDE HYDROXAMIC ACID [SAHA]) IN COMBINATION WITH TEMOZOLOMIDE IN PATIENTS WITH MALIGNANT GLIOMAS (NABTC 04–03)Patrick Wen,1 Vinay Puduvalli,2 John Kuhn,3 Joel Reid,4 Timothy Cloughesy,5 Wka Yung,2 Jan Drappatz,1 Kathleen Lamborn,6 Mark Gilbert,2 Nicholas Butowski,6 Victor Levin,2 Susan Chang,6 Santosh Kesari,1 Hi Robbins,7 Renee McGovern,4 Victoria Richon,8 Stanley Frankel,9 Matthew Ames,4 James Zweibel,10 and Michael Prados6; 1Dana-Farber/Brigham and Women’s Cancer Center, Boston, MA, USA; 2M.D. Anderson Cancer Center, Houston, TX, USA; 3UT San Antonio, San Antonio, TX, USA; 4Mayo Clinic, Rochester, MN, USA; 5University of California, Los Angeles, Los Angeles, CA, USA; 6University of California, San Francisco, San Francisco, CA, USA; 7University of Wisconsin, WI, USA; 8Merck, MA, USA; 9Merck, PA, USA; 10National Cancer Institute, Washington, DC, USA

Vorinostat is an oral inhibitor of histone deacetylase. In preclinical studies it inhibits growth of glioblastoma (GBM) cell lines and has supra-additive activity when combined with temozolomide (TMZ). The North American Brain Tumor Consortium (NABTC) is conducting a phase I study of vorinostat in combination with TMZ in patients with malignant gliomas (MG). Eligibility criteria are histologically proven GBM and anaplastic gliomas (AG) who have received radiotherapy and have not progressed on temozolomide, . 18 yrs old, life expectancy . 8 weeks, KPS . 60, ade-quate bone marrow reserve and organ function. There was no limitation to the type of antiepileptic drugs that could be used. All patients received TMZ at a dose of 150 mg/ m2/day on days 1–5 days every 28 days. Variable doses of vorinostat were administered with food on days 1–14 every 28 days. Dose-limiting toxicities (DLT), determined during the first 4 weeks of therapy, were defined as any grade 4 hematologic toxicity or any grade 3 non-hematologic toxicity or thrombocytopenia. Escalation was performed in standard groups of 3. The maximum tolerated dose (MTD) was defined as the dose at which DLTs occurred in no more than 1/6 patients. PKs were determined on cycle 1 for TMZ, vorinostat and its metabolites. To date, 30 eligible patients have been enrolled (21 GBM; 2 gliosarcoma, 7 AG). Patients characteristics are 17 male, 13 female; median age 58 yrs (24–78); median KPS 90 (70–100). DLTs were encountered at 300 mg bid of vorinostat (1 grade 3 thrombocytopenia; 1 grade 3 fatigue in 3 patients), 200 mg tid (1 grade 3 nausea, 1 grade 4 thrombocytopenia in 3 patients) and 200 mg

twice daily (grade 3 fatigue in 2/6 patients). No DLTs were encountered in 16 patients receiving 300 mg daily. TMZ PKs included [n 5 16: Cmax 5.2 (61.55) mcg/ml; AUC 20.1 (64.66) mcg 3 hr/ml; t1/2 2.0 (60.32)/hr]. Vorinostat PKs (300 mg dose level) were [Cmax 267 (6174) ng/ml; AUC0–8 603 (6197) ng 3 hr/ml]. The t1/2 for vorinostat and its glucuronide metabo-lite were identical (1.6 hrs) vs. 5.6 hrs for the acid metabolite. The MTD of vorinostat in combination with TMZ is 300 mg daily on days 1–14 every 28 days. Although this was well-tolerated, the exposure of vorinostat, based on plasma AUC, achieved at this dose was low. To increase the exposure of vorinostat in combination with temozolomide, a schedule of 400 mg of vor-inostat administered on days 1–7 and 15–21 of every 28-day cycle, together with TMZ at a dose of 150 mg/ m2/day on days 1–5 days every 28 days is being examined. The tolerability of this revised regimen, final PK results and toxicities will be presented.

MA-14. PHASE I STUDY OF TEMSIROLIMUS (CCI-779) AND SORAFENIB IN RECURRENT GLIOBLASTOMA: NORTH CENTRAL CANCER TREATMENT GROUP (NCCTG) N0572David Schiff,1 Jann Sarkaria,2 Paul Decker,2 Jan Buckner,2 Evanthia Galanis,2 Janet Dancey,3 Caterina Giannini,2 Paul Brown,2 Martin Wiesenfeld,4 and Kurt Jaeckle5; 1University of Virginia Health Science Center, Charlottesville, VA, USA; 2Mayo Clinic, Rochester, MN, USA; 3National Cancer Institute, Bethesda, MD, USA; 4Cedar Rapids Oncology Project CCOP, Cedar Rapids, IA, USA; 5Mayo Clinic, Jacksonville, FL, USA

The ras/raf/MEK/ERK (MAPK) and PI3K/Akt/mTOR pathways are thought to be key overactive pathways in glioblastoma. Temsirolimus, a prodrug of sirolimus (Rapamycin), is an intravenously administered mTOR inhibitor. Sorafenib is an inhibitor of multiple kinases including raf, PDGFR, and VEGFR 2 and 3. Sorafenib has shown modest single-agent activity in phase I glioblastoma trials; temsirolimus has shown limited single-agent activity in prior NCCTG and NABTC studies in recurrent GBM. Hori-zontal inhibition of these critical pathways is a logical strategy. Standard single-agent dose of sorafenib is 400 mg bid; the MTD of temsirolimus is 225–250 mg. NCCTG began a phase I study of temsirolimus with sorafenib in June 2006. Eligibility was restricted to patients with progressive glio-blastoma, not taking enzyme-inducing anticonvulsants, with [. . .] 2 prior chemotherapy regimens. Sorafenib was administered orally on a continu-ous daily schedule and temsirolimus given intravenously every week of each four-week cycle. To date, 11 patients have been enrolled. At dose level 0 (sorafenib 200 mg bid and temsirolimus 25 mg), 0/3 patients experienced DLT. Two additional patients at dose level 0 required replacement for inad-equate dosing, including one patient with grade 2 mucositis. At dose level 1 (sorafenib 400 mg bid and temsirolimus 25 mg), 2/3 patients had DLT: one patient had grade 4 gastrointestinal perforation possibly related to treat-ment, and another patient experienced grade 3 anorexia, vomiting, and fatigue. Per protocol, 3 additional patients were enrolled at dose level 0 and are currently being observed for toxicities. This is one of the first studies of horizontal inhibition of multiple signal transduction pathways in glioblas-toma. In this combination, the dose of temsirolimus tolerated by recurrent GBM patients is approximately 10-fold less than that observed with single agent temsirolimus. Provided that no DLTs are observed in the additional patients enrolled at Dose level 0, this will represent the Phase II dose for non-EIAC patients. Updated results of this phase I study will be presented.

MA-15. CLINICAL AND MOLECULAR-METABOLIC PHASE II TRIAL OF PERIFOSINE FOR RECURRENT/PROGRESSIVE MALIGNANT GLIOMAAndrew Lassman, Eric Holland, Lisa DeAngelis, Katherine Panageas, and Lauren Abrey; Memorial Sloan-Kettering Cancer Center, New York, NY, USA

Recent efforts to treat recurrent malignant gliomas have focused on small molecule inhibitors targeting single signaling molecules. However, drugs that inhibit only one target are relatively ineffective, in part because of the redundancy of signal transduction cascades. Perifosine is an alky-lphospholipid that inhibits membrane localization of AKT and also inhibits RAS signaling in glia. Blocking both the AKT and RAS cascades may be critical because they are co-activated in most human glioblastomas (GBMs), and forced co-activation induces GBMs in mice. Therefore, we initiated a phase II study of perifosine in patients (KPS at least 50, age at least 18 years, not taking EIAEDs, unlimited prior therapies) with recurrent GBM or other malignant gliomas with a primary goal of determining efficacy. Secondary goals include determination of molecular and metabolic effects of perifosine by tumor tissue analysis and brain PET imaging. Perifosine was administered as an oral loading dose of 600 mg (150 mg 3 4) on day 1 followed by 100 mg nightly thereafter. Patients were treated until disease progression or unacceptable toxicity and monitored by brain MRI every 2 months. Eighteen patients (9 men, 9 women; median age 54 years, range



34–78) have been treated to date: 11 GBM, 4 anaplastic astrocytoma, and 3 anaplastic oligodendroglioma. Two (both GBM) enrolled on a surgical sub-study, receiving perifosine for 1 week before resection of recurrent disease and re-initiating therapy following recovery from surgery. Best radiographic response (Macdonald criteria) among 13 evaluable patients was 2 PR, 3 SD, and 8 PD. Three patients remain on study which continues to accrue (maxi-mum n 5 49). Updated response data will be presented at the meeting as will progression-free, 6-month progression free, and overall survival. Pretreat-ment tissue will be analyzed to identify molecular predictors of response, and tissue resected through the surgical substudy will be analyzed to deter-mine effects of perifosine on oncogenic signaling in vivo.

MA-16. UPDATE ON PHASE II TRIAL OF IMATINIB MESYLATE (GLEEVEC) AND HYDROXYUREA FOR ADULTS WITH RECURRENT/PROGRESSIVE LOW-GRADE GLIOMADaniela Bota,1 Annick Desjardins,1 Jeremy Rich,1 James Vredenburgh,1 Jennifer Quinn,1 Sith Sathornsumetee,1 Kristna Goli,1 August Salvado,2 Henry Friedman,1 and David Reardon1; 1Duke University Medical Center, Durham, NC, USA; 2Novartis Pharmaceutical Corporation, East Hanover, NJ, USA

We evaluated the combination of imatinib mesylate and hydroxyurea in recurrent/progressive low-grade gliomas (LGG) following the encouraging response of this combination demonstrated among adults with recurrent malignant glioma. Key eligibility criteria: age over 18 yrs; histologically confirmed grade II LGG that is recurrent/progressive following at least prior surgical intervention; Karnofsky > 60% and adequate organ func-tion. Imatinib plus hydroxyurea were given on a continuous oral daily schedule for 28-day cycles; Imatinib was administered at 400 mg daily to patients not on CYP3A-enzyme inducing antiepileptic drugs (EIAED) while those on EIAED received 500 mg twice a day. All patients received 500 mg twice a day of hydroxyurea. Patients were evaluated every other month with physical and MRI examinations. Forty-three patients with recurrent or progressive LGG have enrolled including 26 with astrocytoma and 17 with oligodendroglioma. Seventeen (39%) are on EIAED. Median age was 41 years (range 20–74 years). 33% of patients had received prior therapy other than surgery (XRT, n 5 10; chemotherapy, n 5 14). The most fre-quent toxicities at least possibly related to the study regimen were fatigue (grade 2, n 5 5; grade 3, n 5 5), neutropenia (grade 2, n 5 2; grade 3, n 5 2), anemia (grade 2, n 5 2), rash (grade 2, n 5 2), nausea (grade 2, n 5 4; grade 3, n 5 1), anorexia (grade 2, n 5 1), and AST elevation (grade 2, n 5 1). There were no grade 4 or 5 treatment-related toxicities. Among patients evaluable for response, 39 (91%) achieved stable disease and 3 (7%) had progression. Twenty-six patients continue on study having received between 2 and 20 months of therapy. Additional accrual and follow-up is ongoing. Continuous daily treatment of imatinib plus hydroxyurea is well tolerated and associated with encouraging preliminary activity among adults with recurrent/progressive LGG.

MA-17. IMATINIB PLUS HYDROXYUREA VERSUS HYDROXYUREA MONOTHERAPY IN PROGRESSIVE GLIOBLASTOMA (GBM)—AN INTERNATIONAL OPEN LABEL RANDOMISED PHASE III STUDY (AMBROSIA-STUDY)Gregor Dresemann,1 Mark Rosenthal,2 Klaus Höffken,3 Wolfgang Wagner,4 E. Engel,5 B. Heinrich,6 E. Kettner,7 H. Döhner,8 A. Karup-Hansen,9 Anna Nowak,10 M Mehdorn,11 Eberhard Schleyer,12 Zariana Nikolova,13 Christian Hosius,14 Kea Franz,15 D. Krex,16 S. Green,13 S. Selgav-Nayagam,17 R. Parker,13 and Michael Weller18; 1German Cancer Research Center, Duelmen, Germany; 2Royal Melbourne Hospital, Parkville, Victoria, Australia; 3University of Jena, Jena, Germany; 4Osnabrück, Germany; 5Onkologische Praxis Altona, Hamburg, Germany; 6Klinikum Augsburg, Augsburg, Germany; 7Klinikum Magdeburg, Magdeburg, Germany; 8Klinikum Ulm, Ulm, Germany; 9Clinic Herlev, Denmark; 10University of Western Australia, Nedlands, Western Australia, Australia; 11University of Schleswig Holstein, Germany; 12Merseburg, Germany; 13Novartis Pharma, Basel, Switzerland; 14Novartis Pharma, Nürnberg, Germany; 15University of Frankfurt, Frankfurt, Germany; 16University of Dresden, Dresden, Germany; 17Adelaide, Australia; 18Neurology, University of Tuebingen, Germany

GBM is a highly malignant brain tumor with a median survival of about 15.6 months. Dysregulated signaling of platelet derived growth factor recep-tors (PDGF-Rs) is implicated in pathogenesis. Imatinib (I) plus Hydroxyu-rea (HU) is effective in patients (pts) with recurrent progressing GBM. In independent publications the 6 and 24 month progression free survival estimates were about 30 % and 15 % respectively. More than 30 % of pts achieved disease stabilization (SD). As HU could not enhance I uptake into the brain the role of HU in this concept remains unclear. Therefore a multi-

center open label randomized phase III trial comparing the combination of I plus HU versus HU alone was initiated for progressive GBM pts. Between October 2004 and July 2006 240 pts with progressing recurrent GBM were randomized. Pts were stratified according to enzyme inducing antiepilep-tics (EIADS). Patients were randomized to receive either 600 mg Glivec 1 1000 mg HU per day, or 1500 mg HU per day. Patients were followed up for response every 6 weeks using magnetic resonance imaging (MRI) in conjunction with neurological and steroid data. Additionally, patients were followed for safety (which included weekly blood tests). Patients who pro-gressed when receiving I 1 HU could be dose escalated to 800mg Glivec 1 1000mg HU per day. Patients who progressed when receiving HU mono-therapy were allowed to cross over and start Glivec 600mg 1 HU 1000mg per day. The primary endpoint was to measure 6 month progression free survival (PFS). Secondary assessments included response rate, clinical ben-efit, PFS at 12 months, overall survival and safety. 240 pts were randomized, of which 152 were male and 88 were female. The median age was 51 years (range: 19–73). All pts had prior irradiation and chemotherapy with Temo-zolomide. 138 pts had received one prior chemotherapy, 77 had received two. Following local site histology, 27, 201, and 12 patients were initially diagnosed with AA, GBM, or Gliosarcoma respectively. In the combination therapy arm there were two confirmed responders out of 120 patients (1.7%) compared with one confirmed responder in the mono-therapy arm out of 120 patients (0.8%). The percentage of patients with a best overall response of SD or better (CR1PR1SD) was very similar for each treatment group at approximately 25%. Additionally, 67.5% of patients recorded progressive disease or death in both arms, a further 7% being not assessable. There were no significant differences between the treatment groups in any of the end-points. Main reason for discontinuation was suspected progression and/or ECOG grade 3–4 in 30.8 % of the pts (36.7% combination arm vs. 25.0% mono arm) followed by an unsatisfactory therapeutic effect (21.3% in total, 20.8% in the combination, and 21.7% in mono therapy). Furthermore 15.8% of pts discontinued due to adverse events (15.0% in combination vs. 16.7% in mono therapy). Safety and additionally efficacy data regarding cross over will be available and presented by November 2007. Efficacy and safety data of this trial following evaluation of 240 patients following one, two or more relapses for patients suffering from glioblastoma will determine if there is superiority of I plus HU compared to HU monotherapy in pro-gressive Temozolomide-resistant GBM. The influence of EIADS on efficacy profile will be analyzed.

MA-18. A PHASE II TRIAL OF IMATINIB MESYLATE (GLEEVEC) AND HYDROXYUREA FOR ADULTS WITH RECURRENT/PROGRESSIVE MENINGIOMAKrishna Goli,1 Daniela Bota,1 Annick Desjardins,1 Jeremy Rich,1 James Vredenburgh,1 Jennifer Quinn,1 Sith Sathornsumetee,1 August Salvado,2 Henry Friedman,1 and David Reardon1; 1Duke University Medical Center, Durham, NC, USA; 2Novartis Pharmaceutical Corporation, East Hanover, NJ, USA

Limited therapeutic options exist for patients with recurrent menin-gioma. Monotherapy with either imatinib mesylate or hydroxyurea is active in preclinical meningioma models, while each of these agents has clinical benefit in small series of meningioma patients. We are therefore conducting the current study to evaluate the combination of imatinib mesylate plus hydroxyurea in patients with recurrent/progressive meningioma. Key eligi-bility criteria include age over 18 yrs, histologically confirmed meningioma that is recurrent/progressive following at least prior surgical intervention, Karnofsky > 60% and adequate organ function. Imatinib plus hydroxyurea are given on a continuous oral daily schedule for 28-day cycles that includes imatinib administered at 400 mg daily to patients not on CYP3A-enzyme inducing antiepileptic drugs (EIAED) and 500 mg twice a day to those on EIAED. Hydroxyurea dosing is 500 mg twice a day for all patients. Physical examinations and MRI scans are performed every other month. To date, six patients have enrolled including one on EIAED and 5 who are not on EIAED. The median age is 45 years (range, 43–69 years) and all patients have a KPS of 80%–90%. The median number of prior treatment regimens is 2 (range, 1–5). Grade 2 or greater toxicities at least possibly related to the study regimen include: neutropenia (grade 2, n 5 1; grade 4, n 5 1); anemia (grade 3, n 5 1); constipation (grade 2, n 5 1); and nausea (grade 2, n 5 1). All patients achieved a best radiographic response of stable disease. Three patients have discontinued therapy including one with stable disease after 17 cycles and two with progressive disease after 8 and 10 cycles, respectively. Three patients continue on study with stable disease after 2 (n 5 2) and 10 cycles, respectively. Continuous daily treatment of imatinib plus hydroxyu-rea is well tolerated and associated with encouraging preliminary activity among adults with recurrent/progressive meningioma. Additional accrual and follow-up is ongoing.



MA-19. GLIOBLASTOMA MULTIFORME IN THE ERA OF TEMOZOLOMIDERamin Altaha, Amir Anis, and Mohammed Almubarak; West Virginia University, Morgantown, WV, USA

The FDA has approved temozolomide in conjunction with radiation and also as single agent for treatment of Glioblastoma Multiforme (GBM), we conduct a retrospective study to look at our patient population and their outcome that were treated with chemoradiation, followed by chemotherapy with temozolomide. Thirty eight patients with GBM diagnosed between February 2002 and September 2006 were identified. Their medical charts and brain-MRIs were reviewed. Median age of the diagnosis in this cohort of 38 patients (pts) with GBM was 60 (range 41–81). MRI of the brain showed that 13 pts had the typical butterfly lesions, 10 pts had left hemispheric lesions, 8 pts had right hemispheric lesions, 3 pts had multifocal lesions, and 2 pts had brainstem lesions. The average tumor size of the lesions based on the MRI scan was 4.4 cm. Most common presenting signs and symptoms at the time of diagnosis were headaches and change in mental status predomi-nantly speech and memory deficits. Seizure was only reported in 4 pts (11%), and focal motor weaknesses were reported only in 6 pts (16%). Gliadel wafer was placed in 10 pts (26%). Twenty nine patients (76%) were treated with concomitant radiation and daily temozolomide 75 mg/m2. Eighteen patients (47%) received monthly temozolomide (200 mg/m2 day 1–5). Progression of the disease was noted in 19 pts (65%). Disease progression in average was noted after 6–8 cycles of monthly temozolomide. Two patients (7%) are still in complete remission (CR) after 24 months of temozolamide. This small retrospective study shows that GBM has still extremely poor prognosis, current treatment with temozolomide in conjunction with brain radiation, followed by monthly temozolomide has modest activity. Long-term survival has been reported in few patients. Different treatment strategies needed to be developed to improve the outcome of GBM patients.

MA-20. PATTERNS OF INITIAL TREATMENT, SURVIVAL, AND COST FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME (GBM): AN ANALYSIS OF SEER-MEDICARE DATALisa McGarry,1 Nathalie Morissette,1 Craig Earle,2 Astra Liepa,3 and David Thompson1; 1i3 Innovus, Medford, MA, USA; 2Dana-Farber Cancer Institute, Boston, MA, USA; 3Eli Lilly and Company, Indianapolis, IN, USA

Published data on the economic burden of glioblastoma multiforme (GBM) are extremely limited. In this study, we used data from the Sur-veillance Epidemiology and End Results (SEER) registry linked with Medi-care claims data to assess patterns of initial treatment, survival and costs among patients with this disease. GBM patients (ICD-O-3 histology codes 9440–9442) were identified in the SEER-Medicare database from 1993 to 2002 and followed through 2004 or until death. Included in the analysis were patients aged 651 years at diagnosis with no prior cancer diagnosis, enrolled in Medicare Parts A and B, and not enrolled in a Medicare health maintenance organization (HMO). Initial treatment was defined based on patterns of care observed during the period 1 month prior to through 3 months after diagnosis. Costs of hospitalization, outpatient clinic visits, physician visits, home health care, skilled nursing facilities, and hospice were summed from 30 days prior to diagnosis until death or end of fol-low-up. All costs were adjusted to 2006 values. Lifetime costs and costs per month of follow-up were calculated. Kaplan-Meier methods were used to assess survival by initial treatment. 3,407 patients met all eligibility criteria. Median age at GBM diagnosis was 75 years; 51.3% of patients were male and 91.9% were white. Mean length of follow-up was 5.9 months. Survival and costs by initial treatment are summarized in the table. Overall, 39% of costs were for hospitalization, 34% for outpatient visits, and 12% for physi-cian visits. Despite a mean survival of , 6 months, the economic burden of treating GBM patients is high. More aggressive initial therapy is associated with longer survival and higher lifetime costs, but cost per month is similar to less aggressive therapies.

MA-21. CHANGING ISSUES IN LEPTOMENINGEAL CARCINOMATOSIS: ISOLATED OCCURRENCES, SYSTEMIC TREATMENTPamela New,1 Yee-Lu Tham,2 Richard Elledge,2 C. Kent Osborne,2 Amy Curtis,3 and Bin Teh3; 1Neurosurgery, The Methodist Hospital Neurological Institute, Houston, TX, USA; 2Internal Medicine, Baylor College of Medicine, Houston, TX, USA; 3Radiation Oncology, Baylor College of Medicine, Houston, TX, USA

Four cases of isolated leptomeningeal disease (LMD)/brain metastases are presented. Courses and responses to treatment will be discussed. LMD usually develops in the setting of progressive systemic cancer and is not commonly described in patients who are free of tumor. Survival is usually

3 to 6 months post diagnosis. Specific receptor status in breast cancer and treatment regimens have been examined for a possible correlation, but no statistically significant data have been found. Reports of treatment with Capecitabine have been encouraging. Four cases are presented here which prompt further questions concerning occurrence and treatment of this dis-ease. Three patients with history of infiltrating ductal breast carcinoma and one with prior prostate carcinoma were diagnosed with isolated central nervous system (CNS) cancer. Case #1 was estrogen(ER)/progesterone(PR) receptor and HER-2 negative, treated for isolated LMD for 2 years with capecitabine. Case #2 was ER, PR, and HER-2 negative. Three months after completion of therapy, she developed brain metastases and LMD, treated successfully with intrathecal (IT) and systemic chemotherapy. Case #3 was ER, PR, and HER-2 positive. LMD was diagnosed by MRI, cytologies nega-tive, 3 years after diagnosis, and was treated with radiation and IT chemo-therapy with improvement. Case #4 is a 63-year-old male diagnosed with prostatic adenocarcinoma, stage T3b, treated with a radiation-gene therapy protocol, and hormonal therapy. Three years later he developed LMD, treated successfully with radiation and hormonal therapy. Disease recurred one year later and he was treated with IT and systemic chemotherapy. All four patients presented with LMD 1/– brain metastases, in the absence of systemic disease. Case #1 had an excellent clinical response to systemic chemotherapy but with persistently positive cytology. Case #2 responded promptly to IT and systemic chemotherapy, and case #3 responded to radio-therapy and IT chemotherapy. Case #4 responded to hormonal therapy, IT chemotherapy and systemic chemotherapy. The occurrence of LMD in patients with successfully treated systemic cancer may be increasing. Iso-lated LMD/brain metastases seem to occur regardless of receptor status in breast cancer patients and approaches incorporating systemic treatment require further investigation. Survival rather than cytology should be the major endpoint in these trials.

MA-22. TARGETED THERAPY WITH AP 12009 FOR RECURRENT OR REFRACTORY HIGH-GRADE GLIOMA: RESULTS OF A PHASE IIB STUDYUlrich Bogdahn,1 Z. H. Rappaport,2 Guenther Stockhammer,3 Victor Oliushine,4 Valery Parfenov,5 Ashok Mahapatra,6 Chandra Mouli,7 K. Neelam Venkataramana,8 Hubert Heinrichs,9 and Karl-Herrmann Schlingensiepen9; 1Department of Neurology, University of Regensburg, Regensburg, Germany; 2Neurosurgery Department, Rabin Medical Center (Beilinson), Petach-Tikva, Israel; 3Department of Neurology, University Hospital of Innsbruck, Innsbruck, Austria; 4Polenov Neurosurgery Research Institute, St. Petersburg, Russia; 5Neurosurgery Department, Military Medical Academy, St. Petersburg, Russia; 6Department of Neurosurgery, All India Institute of Medical Sciences, New Delhi, India; 7Department of Neurosurgery, National Institute of Mental Health and Neurosciences, Bangalore, India; 8Manipal Institute for Neurological Disorders, Manipal Hospital, Bangalore, India; 9Antisense Pharma, Regensburg, Germany

High-grade gliomas (HGG) are very aggressive tumors characterized by transforming growth factor-beta2 (TGF-beta2) overexpression. Above all TGF-beta2 is the most potent immunosuppressor but it also plays a key role in tumor progression by regulating key mechanisms including prolifera-tion, metastasis, and angiogenesis. The TGF-beta2 specific phosphorothio-ate antisense oligodeoxynucleotide AP 12009 was developed for targeted suppression of this protein. In three Phase I/II dose escalation trials in HGG patients, AP 12009 proved to be well tolerated and revealed a very good safety profile. Moreover, complete and long lasting tumor responses were observed. The Phase IIb clinical trial G004 is an international, open-label, randomized and actively controlled dose finding study. Main trial objective is a comparison of two doses of AP 12009 (10 mM or 80 mM) and stan-dard chemotherapy (temozolomide [TMZ] or PCV [procarbazine / CCNU (5 lomustine) / vincristine]) with regard to response rate, survival and safety. 145 patients with either recurrent or refractory anaplastic astrocy-toma (AA, WHO grade III) or glioblastoma (GBM, WHO grade IV) were randomized into three arms. AP 12009 was administered into the tumor via convection-enhanced delivery (CED) during a 6-month active treatment period with 7-day treatment cycles at bi-weekly intervals. 134 patients including 96 GBM and 38 AA patients have received study medication. 89 patients received AP 12009 (40 pts. 10 mM, 49 pts. 80 mM). In both AP 12009 groups, long-lasting tumor responses were observed in AA as well as in GBM patients. The duration of long-term tumor responses until now exceeds the active treatment period by far. In AA patients, the response data (CR 1 PR at 12 and 14 months) and the survival data of the 10 mM dose group after 24 months are superior to the 80 mM dose group, and either AP 12009 dose is superior to the chemotherapy control. Median overall survival has not yet been reached in the AP 12009 dose groups in contrast to 91 weeks in the chemotherapy treatment group. Overall survival at pres-ent is 30.6 months for the AP-10 mM group, 27.4 months for the AP-80 mM group, and 21.7 months for the control group. Dose finding was achieved, as efficacy and safety results for the AP-10 mM group are superior to those of the AP-80 mM group in both AA and GBM patients. Due to the very encouraging results the follow-up period for the Phase II study has been



prolonged to collect further data regarding survival, response, and safety. In addition, preparations for a Phase III study in recurrent or refractory HGG patients are ongoing.

MA-23. POST-TRANSPLANT LYMPHOPROLIFERATIVE DISORDER (PTLD) PATIENTS TREATED WITH HIGH-DOSE METHOTREXATELouis Nabors,1 Bruce Julian,2 Clifton Kew,2 and Cheryl Palmer3; 1Neurology, University of Alabama at Birmingham, Birmingham, AL, USA; 2Medicine, University of Alabama at Birmingham, Birmingham, AL, USA; 3University of Alabama at Birmingham, Birmingham, AL, USA

Post-transplant lymphoproliferative disorder (PTLD) is a relatively uncommon neoplastic complication of solid organ, as well as bone marrow transplantation. Generally associated with Epstein-Barr virus (EBV) infec-tion of B-lineage lymphocytes, there is little clinical experience with treat-ment of the condition other than reduction or withdrawal of immunosup-pression due to the relative rarity of the disease. We describe seven patients, 5 with renal transplants, 1 with heart transplant, and 1 with combined kidney/pancreas transplants, with histologically proven central nervous system PTLD, who were treated with high-dose intravenous methotrexate therapy. Tissue analysis from the patient’s PTLD biopsy specimens for EBV-genomic material was positive in 6 of the 6 patients tested. Dosages of 8 gm/ m2 methotrexate were employed, with adjustments for creatinine clearance. With this therapy, five patients have had a sustained complete response, and two had progressive disease and were referred for radiation therapy. No unexpected toxicities were encountered over 50 courses of treatment. High-dose intravenous methotrexate chemotherapy should be considered as an alternative for the treatment of CNS PTLD.

MA-24. REVERSIBLE POSTERIOR LEUKOENCEPHALOPATHY SYNDROME IN A PATIENT TREATED WITH BEVACIZUMAB AND IRINOTECANAllison Weathers and Nina Paleologos; Neurology, Evanston Northwestern Healthcare, Evanston, IL, USA

To our knowledge this is the first reported case of reversible posterior leukoencephalopathy syndrome (RPLS) in a patient treated with bevaci-zumab and irinotecan for progressive glioblastoma multiforme (GBM). A 61-year-old woman with a left posterior temporal GBM and well-controlled hypertension progressed after treatment with subtotal resection, radiother-apy with concomitant daily temozolomide, and 11 cycles of maintenance temozolomide. She was neurologically stable with mild aphasia, minimal right hemiparesis, and rare aphasic seizures well controlled with levetirac-etam. At radiographic progression she was treated with bevacizumab (10mg/kg) and irinotecan (125mg/m2) at two week intervals on the Genentech investigational protocol AVF3708g. Blood pressures (BP) during infusions were 110–160 mmHg systolic and 58–84 mmHg diastolic. Approximately 62 hours after completion of the 11th infusion she had 2 episodes of emesis, followed by a focal seizure with secondary generalization. She had two more generalized seizures in the local emergency room, where maximum systolic BP was 215 mmHg and maximum diastolic BP was 111 mmHg. Examina-tion revealed somnolence, dense aphasia, left gaze preference, no response to visual threat, and a dense right sided hemiparesis. There were no signs of infection. She had proteinuria, but no other significant laboratory abnormal-ities. MRI, compared to 2 weeks prior, revealed new bilateral hemispheral (primarily posterior) and cerebellar diffuse increased signal abnormalities on T2/ FLAIR images and new gadolinium enhancement in the cerebellum. She was treated with IV steroids, medical management, and an increased dose of levetiracetam. She improved rapidly and was discharged 3 days later. Examination 6 days after discharge revealed full visual fields, improvement in the aphasia and right hemiparesis, and mild left sided neglect compared to baseline. MRI showed a marked decrease in the T2/FLAIR signal abnor-malities in both hemispheres and cerebellum and near resolution of the cer-ebellar enhancement. There was new enhancement in the left hemisphere with associated restricted diffusion consistent with ischemia. Three weeks after discharge, she was close to her previous neurologic baseline. A repeat MRI showed further improvement. RPLS is the syndrome of headache, altered mental status, seizures, and visual disturbances in association with diffuse vasogenic edema, predominantly in the parieto-occipital lobes, with involvement of the cerebellum, brain stem, basal ganglia, and frontal lobes also seen. It is seen in patients with eclampsia, hypertensive encephalopathy, renal failure, and is associated with a number of immunosuppressive and cytotoxic drugs. Bevacizumab is a monoclonal antibody against vascular endothelial growth factor. Cases of RPLS have been reported in patients receiving bevacizumab in clinical studies of other malignancies and post-marketing experience at a rate of ,0.1%. One hundred and sixty patients have been enrolled on Genentech protocol AVF3708g. This regimen is also being used off protocol and in other protocols. We conclude that our patient had RPLS as a direct complication of treatment with bevacizumab. RPLS is

not a known complication of irinotecan. As of 4/26/07 there were no other reported cases of RPLS in trial subjects. To our knowledge no other cases of RPLS in patients with GBM treated with bevacizumab have been reported.

MA-25. TREATMENT-INDUCED IMMUNOSUPPRESSION IN NEWLY DIAGNOSED HIGH-GRADE GLIOMAS: A NABTT CNS CONSORTIUM STUDYStuart Grossman,1 Serena Desideri,1 Steven Piantadosi,1 Glenn Lesser,2 Marc Chamberlain,3 and Xiaobu Ye1; 1The Johns Hopkins University, Baltimore, MD, USA; 2Wake Forest University, Winston-Salem, NC, USA; 3Moffitt Cancer Center, Tampa, FL, USA

Vaccines and other immunologic interventions hold promise for patients with high grade gliomas (HGG). A prior study noted a dramatic fall in CD4 counts in newly diagnosed patients with HGG during their 6 weeks of radiation (RT). These patients received dexamethasone (DEX) but no temozolomide (TMZ). We now report preliminary results from the first prospective study to follow serial CD4 counts in newly diagnosed HGG treated with RT, DEX, and TMZ. The accrual goal is 100 newly diagnosed HGG. CD4 counts are obtained before RT and TMZ and monthly thereaf-ter for one year. Information on hospitalizations, infections, steroid doses, and survival is also collected. Demographic data are currently available on 75 pts: 55% are female, 97% are Caucasian, median age is 57 (range 31–77), median KPS is 90 (70–100), and 86% have glioblastoma. The median CD4 count before RT was 640 (range 145–1445) which fell to 312 (32–1055), 263 (8–558), and 250 (44–845) in 1, 2, and 3 months respectively. Median CD4 counts during months 4, 6, 8, 10, and 12 were 301, 319, 328, 300, and 269. At 2 months, a CD4 count of , 300 was noted in 58% of patients (95% CI 5 43%–72%) and , 200 in 35% (95% CI 5 22%–50%). We focused on CD4 count at 2 months, shortly after completing concomitant RT 1 TMZ, for which 48 patients had values for this analysis. Overall survival for all pts was 441 days (95% CI 5 351–579). In pts with CD4 counts , 200 at 2 months survival was 341 days (95% CI: 169–475) while it was 579 days (95% CI 5 579–**) in pts with CD4 counts > 200. This corresponds to a hazard ratio of 2.6 (log rank p value 0.02). Multivariable analysis using the proportional hazards model adjusted for age, histology, KPS, steroid use, and sex. Only steriod use and KPS were significant predictors of death. Their analysis yielded an adjusted hazard ratio of 2.7 (95% CI: 1.1 to 6.8; p 5 0.04) attributable to CD4 count , 200 as compared with CD4 count > 200. Analysis of hospitalizations, infections, and DEX are in progress. This study demonstrates that patients with newly diagnosed HGG receiving DEX, RT, and TMZ commonly develop severe immunosuppression from this therapy. CD4 counts are relatively normal prior to RT and TMZ, fall to a median of 250 during the first three months of therapy, and remain depressed during the first year of follow-up. Furthermore, CD4 counts , 200 after 2 months of therapy are associated with significantly shorter survival that appears to be independent of age, KPS, tumor grade, or DEX administration. These findings raise questions regarding the recommended duration of PCP prophylaxis, the role of routine CD4 count monitoring, and whether low CD4 counts should be considered in tailoring the duration or intensity of therapy for individual patients. These data should also be considered when planning therapeutic interventions that require an intact immune system (i.e., vaccines), or clinical trials seeking to increase the inten-sity or duration of TMZ.

MA-26. NEUROLEUKEMIOSIS: CASE REPORT OF LEUKEMIC NERVE INFILTRATION IN ACUTE LYMPHOBLASTIC LEUKEMIADawit Aregawi, Michael Douvas, Ted Burns, and David Schiff; University of Virginia, Charlottesville, VA, USA

Leukemic infiltration of leptomeninges leading to cranial and spinal nerve root involvement is a common complication of acute leukemia. In contrast, leukemic involvement of the peripheral nervous system outside of the subarachnoid space is extremely rare. Objective: To present a case of acute lymphoblastic leukemia with peripheral nerve infiltration. Case: A 21-year-old male presented for evaluation of foot drop in 2/06. He was diagnosed ten years earlier with acute lymphoblastic leukemia, achieved remission, but five years later had marrow relapse treated with matched sibling allogeneic bone marrow transplant with total body irradiation and cyclophosphamide. Subsequent right testicular relapse was treated with radiotherapy four years prior to referral. When seen, he noted painless left ankle weakness associated with numbness of lateral side of the lower leg that came on imperceptibly five months earlier. He had weakness of ankle dorsiflexion and eversion as well as dorsiflexion of all toes. Pinrick and light touch were decreased over the lateral lower leg and dorsal foot. He denied weight loss. Lumbar puncture was normal, including negative cytol-ogy. Electromyography (EMG) in 5/06 revealed active and chronic left com-mon peroneal neuropathy with no evidence of lumbosacral plexopathy or radiculopathy. The patient declined further investigations or follow-up. He



returned in 12/06 with a new complaint of painless numbness in ulnar nerve distributions bilaterally with stable left ankle weakness. Lower extremity MR scan revealed multiple bony lesions bilaterally in addition to linear enhancement, thickening and swelling of the left common peroneal nerve. Total body PET revealed a 16 cm segment of abnormal fluoro-deoxyglucose (FDG) uptake tracking along the neurovascular bundle in the left thigh, as well as multiple areas of abnormal FDG uptake in bones, pancreas, kidney, and pleural space. CT-guided fine needle aspiration and core biopsy of left proximal tibia was positive for acute lymphoblastic leukemia. Staging CSF examination showed an elevated protein, cell count and positive cytology. Bone marrow biopsy showed no evidence of recurrent ALL. The patient has received 5 cycles of high-dose intravenous methotrexate and cytarabine, hyperfractionated cyclophosphamide, and intrathecal liposomal cytara-bine and remains clinically stable. PET scan following the second cycle of chemotherapy showed complete resolution of neurovascular bundle uptake. Discussion: Although neurologic involvement in ALL is almost always attributable to leptomeningeal seeding, our patient had an unequivocal peripheral peroneal neuropathy (and possibly bilateral ulnar neuropathies) as the initial clinical manifestation of extramedullary ALL relapse. Periph-eral nerve invasion is a well-recognized albeit rare complication of systemic lymphoma (“neurolymphomatosis”); this case demonstrates that a similar phenomenon (“neuroleukemiosis”) may occur in ALL. MRI and PET were extremely useful in suggesting the diagnosis. As in neurolymphomatosis, the use of chemotherapies such as high-dose methotrexate and cytarabine that penetrate the blood-nerve barrier is likely key to successful management of this unusual complication.

MA-27. RANDOMIZED PHASE II TRIAL OF ERLOTINIB (E) VERSUS TEMOZOLOMIDE (TMZ) OR BCNU IN RECURRENT GLIOBLASTOMA MULTIFORME (GBM): EORTC 26034Martin Van Den Bent,1 Alba Brandes,2 Roy Rampling,3 Mathilde Kouwenhoven,4 Johan M. Kros,5 Antoine Carpentier,6 Paul Clement,7 Barbara Klughammer,8 Brenda Bakker,9 Denis Lacombe,10 and Thierry Gorlia10; 1Daniel den Hoed Cancer Center, Rotterdam, Netherlands; 2Bologna, Padova, Italy; 3Beatson Oncology Center, Glasgow, United Kingdom; 4EEA, Rotterdam, Zuid-Holland, Netherlands; 5Pathology, Erasmus MC, Rotterdam, Netherlands; 6Department of Neurology, CHU Pitié-Salpétrière, Paris, France; 7Academisch Ziekenhuis Leuven, Belgium; 8F Hoffmann-La Roche, Switzerland; 9Erasmus University Hospital, Netherlands; 10EORTC DataCenter, Brussels, Belgium

In 40%–50% of GBM epidermal growth factor receptor (EGFR) is amplified, and often constitutively activated (EGFRvIII mutant). EGFR is therefore a potential therapeutic target. Previous studies suggested activity of EGFR tyrosine-kinase inhibitors in recurrent GBM, particularly in spe-cific molecular subsets. This study explored erlotinib (E, Tarceva) activity in recurrent GBM. Randomized phase II trial. Eligibility criteria: histologically proven GBM, recurrent . 3 months after radiotherapy, Karnofsky perfor-mance status (KPS) [. . .] 70, no prior chemotherapy for recurrent disease, tis-sue sample for EGFR studies. Patients (pts) received E 150 mg/day (300 mg/day if on enzyme inducing anti-epileptic drugs [EIAEDs]), or control (TMZ 150–200 mg/m2 day 1–5 q4wk or BCNU 60–80 mg/ m2 i.v. day 1–3 q8wk). If no significant toxicity, E was escalated to 200 mg (500 mg in patients on EIAEDs). The primary endpoint was 6 months’ PFS in [?] 10/50 pts on E (20%); P0 was set at 15%, and P1 to 30%. Response was assessed with Macdonalds criteria. EGFR amplification (FISH) and expression of EGFR, EGFRvIII, and PTEN (immunohistochemistry [IHC]) were assessed. 110 patients were randomized (54 E, 56 control: 27 TMZ, 29 BCNU). Median age 55 years, median KPS 90. All but 2 patient started treatment; median number of cycles was 2 for E, 4 for TMZ and 1 for BCNU. Grade 3/4 toxicities likely related to E: dermatological (6) and bilirubin (2). Grade 3/4 toxicities for control were mainly hematological (4 TMZ, 13 BCNU). Three pts discontinued E due to toxicity. Six-month PFS was 11% for E, 24% for control. Six- and 12-month survival were 58% and 22% for E, and 59% and 27% for control. Five responses were seen on control; and 1 on E. Patients with EGFRvIII mutations (13 in E arm, 8 in control arm) had shorter PFS (p 5 0.007) and OS (p 5 0.004) regardless of treatment . Response to E was not correlated with EGFR expression, EGFR amplification, EGFRvIII muta-tion or PTEN. None of the 8 patients with combined EGFRvIII mutation and PTEN expression treated with E had 6-month PFS. PFS was associated with rash grade 2 or higher. This randomized, controlled phase II study did not find sufficient activity for erlotinib in the general population of recurrent GBM. The presence of EGFRvIII mutations with or without PTEN expres-sion was not predictive for PFS.

MA-28. PHASE II STUDY OF PROLONGED DAILY TEMOZOLOMIDE FOR LOW-GRADE GLIOMAS IN ADULTSSantosh Kesari,1 David Schiff,2 Jan Drappatz,3 Debra Gigas,3 Lisa Doherty,3 Keith Ligon,3 Alona Muzikansky,4 Abgail Ciampa,3 Joanna Bradshaw,3 Brenda Levy,3 Gospova Radakovic,2 Naren Ramakrishna,3 Peter Black,3 and Patrick Wen3; 1Society for Neuro-Oncology, Boston, MA, USA; 2University of Virginia, Charlottesville, VA, USA; 3Dana-Farber/Brigham and Women’s Cancer Center, Boston, MA, USA; 4Massachusetts General Hospital, Boston, MA, USA

Resistance to temozolomide chemotherapy may be mediated in part by the presence of O6–methylguanine-DNA methlytransferase (MGMT). Pro-longed treatment with temozolomide potentially overcomes MGMT resis-tance and improves outcome. We conducted a phase II study of prolonged daily temozolomide (TMZ) in adults with low-grade gliomas. Patients with newly-diagnosed oligodendroglioma with a MIB-1 index of .5%, or recur-rent low-grade gliomas (oligodendrogliomas, astrocytomas and oligoastro-cytomas) were given TMZ at a dose of 75 mg/ m2/day in 11-week cycles (7 weeks on/ 4 weeks off). Patients were treated until tumor progression, development of unacceptable toxicity, or for a total of 6 cycles. The primary endpoint was overall response rate; with progression-free survival (PFS), overall survival (OS), and toxicity being the secondary endpoints. We sought to correlate response with chromosome 1p or 19q deletion status, MGMT protein levels by immunohistochemistry, and MGMT promoter methyla-tion status by methylation specific PCR. Forty-five patients were enrolled (14 female, 31 male). Median age was 43 (range 20–68), and median KPS was 90 (range 70–100). One patient withdrew consent after registration and was never treated and another patient was discontinued because of non-compliance. The regimen was generally well tolerated with a median number of cycles of 5.6 (range 0–6 cycles). Toxicities included neutropenia (3 grade 3, 1 grade 4), thrombocytopenia (3 grade 3, 2 grade 4), leukopenia (5 grade 3), lymphopenia (6 grade 3), thromboembolic event (1 grade 4), nausea (1 grade 3), and elevated SGPT (1 grade 3). Three patients came off treatment during the first cycle for unacceptable toxicities; one for persis-tent grade 3 thrombocytopenia, 1 for grade 2 nausea, and 1 for worsening of preexisting depression and agitation which was felt to be unrelated to temozolomide. There were no treatment-related deaths. Of the 36 patients evaluable for best response, no patients had a complete response (0%), 8 had partial response (22%), 26 had stable disease (72%), and 2 had progressive disease (6%). Two patients progressed rapidly on cycle 1 of therapy; one in retrospect had gliomatosis cerebri. To date, a total of 16 patients have pro-gressed and the median progression-free survival was excess of 47.5 months. In a limited subset of patients deletions in 1p or 19q, and MGMT status did not predict response to treatment or PFS at the time of analysis. However, the number of events was small. Protracted course of daily temozolomide is a well-tolerated regimen that appears to produce effective tumor control; at least comparable to those obtained with the standard 5-day every 28-day regimen of temozolomide. Updated results and correlation of response with 1p, 19q, MGMT promoter methylation status, and other variables will be reported.

MA-29. LONG-TERM THERAPY OF BRAIN TUMORS WITH TEMOZOLOMIDE: REVIEW OF TOLERABILITY AND EFFICACY IN 47 PATIENTSHerbert Newton,1 Jennifer Dalton,2 Greg Figg,2 Carol Volpi,2 and Dennis Pearl3; 1Neurology & Neuro-Oncology, Dardinger Neuro-Oncology Center, Ohio State University, Columbus, OH, USA; 2Neurology & Neuro-Oncology, Ohio State University Medical Center, Columbus, OH, USA; 3Statistics, Ohio State University Medical Center, Columbus, OH, USA

TZM is a 2nd generation alkylating agent with significant efficacy for low-grade and malignant brain tumors. The drug is administered orally for 5 days every month (150–200 mg/m2/day), using a conventional schedule. Due to TZM’s excellent tolerability and lack of cumulative systemic toxic-ity, some patients are receiving treatment for 12 to 24 months. The effi-cacy, safety, and tolerability of this longterm therapeutic approach remains unclear. We performed a retrospective chart review of all Neuro-Oncology Center patients who had undergone temozolomide chemotherapy for 12 months or longer. A total of 47 patients (median age 46 years) met the cri-teria; tumor types included GBM (14), oligodendroglioma (10), anaplastic glioma (10), astrocytoma (7), other glioma (5), and primary CNS lymphoma (1). Thirty-seven patients had received irradiation; 9 had prior chemother-apy. The mean number of monthly TZM cycles was 18 (range 12–28), with a median TZM dose of 400 mg/day. Median time to progression was 221 months (range 3 to 601 months; 181 months in GBM cohort), with 10 objective responses by MRI (21.3%). Toxicity included mild to moderate fatigue (98%), mild nausea (85%), constipation (70%), and grade I/II leuko-penia (66%) and thrombocytopenia (47%). Of 852 total cycles of TZM, 14 (1.6%) were delayed at least one week by treatment-related side effects. No lymphoproliferative disorders have been documented. Longterm treatment



with TZM is feasible, and demonstrates durable activity and acceptable toxicity in patients with gliomas, including GBM.

MA-30. FOLLOW-UP STUDY OF ADULTS WITH LOW-GRADE GLIOMAS TREATED WITH TEMOZOLOMIDEThomas Kaley,1 Jonathan Finlay,2 Lauren Abrey,3 Patrick Kelly,4 Steven Pacia,1 Ravinder Tikoo,1 Dennis Kuo,5 and John Golfinos4; 1Neurology, New York University, New York, NY, USA; 2Pediatrics/Neurosurgery, New York University, New York, NY, USA; 3Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA; 4Neurosurgery, New York University, New York, NY, USA; 5New York University, New York, NY, USA

The management of patients with low-grade glioma is controversial. In particular, the efficacy of chemotherapy in this patient population is poorly defined. We previously reported radiographic response following temozolo-mide therapy in 20 patients with unresectable LGG (5 patients had a PR or CR, 4 minor response, 4 stable disease and 7 progressed). The objective of this IRB approved study was to report the long term follow up and outcome of these patients. From 1999 to 2003, 20 patients aged 22 to 68 years old were treated with temozolomide for biopsy-proven unresectable LGGs (1 juvenile pilocytic astrocytoma, 2 low-grade astrocytomas, 2 oligodendro-gliomas, and 15 low-grade mixed gliomas diagnosed as ganglioglioneuro-cytomas or glioneurocytomas on the basis of immunohistochemical stain-ing). This included both newly diagnosed (11) and recurrent (9) patients. 16 patients received a 42-day regimen (TM 75 mg/ m2/day 42 days every 56 days) and 4 patients were treated with the standard 5-day regimen (TM 150–200 mg/m2/day, 5 days every 28 days). We performed a retrospective chart review to document time to tumor progression and survival; phone calls were placed to those patients lost to follow up at our institution. The time from diagnosis to treatment obviously varied considerably between the groups (median of 1.6 months for newly diagnosed, 54.9 months for recur-rent patient). Median follow-up of surviving patients is 65 months (32–83) and the median overall survival was 71.4 months for entire group (71.4 months for newly diagnosed patients and not reached for recurrent patients). Median time to tumor progression from the start of TMZ treatment was 38.4 months in newly diagnosed patients as compared with 26.8 months for the recurrent patients (regardless of prior therapy). 88% of recurrent LGG patients (8/9) eventually progressed after TMZ but only 54% of newly diag-nosed patients (6/11) have progressed at last follow up. Adults with LGGs derived a benefit in terms of disease control and overall survival following treatment with temozolomide whether they are treated at diagnosis or recur-rence. It is likely that our patients treated at recurrence represent a selected group with better than usual prognosis. Prospective studies are needed to define the optimal dose and timing of TMZ or other chemotherapeutics.

MA-31. TEMOZOLOMIDE PLUS 13–CIS-RETINOIC ACID (TMZ/CRA) AS FIRST-LINE CHEMOTHERAPY FOR NEWLY DIAGNOSED GLIOBLASTOMA MULTIFORME (GBM): A RETROSPECTIVE STUDYMarta Penas-Prado, Vinay Puduvalli, Morris Groves, Charles Conrad, Mark Gilbert, Victor A. Levin, and W.K. Alfred Yung; Neuro-Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Conventional therapy has demonstrated only modest survival benefit in GBM patients. Prognosis is poor with a median survival of 12–14 months after standard therapy at diagnosis. 13–Cis-retinoic acid has demonstrated activity in recurrent malignant gliomas in Phase II trials, both as a single agent and in combination with TMZ. Our objective was to review our experience with TMZ/cRA in the post radiation setting of GBM. Adults with GBM treated with TMZ/cRA as first line chemotherapy after radiation (RT) from 01/1999 to 04/2004 were retrieved from the Neuro-Oncology departmental longitudinal database. Available medical, surgery, radiology and pathology reports were reviewed. Treatment response in patients with measurable disease, median time to progression (TTP), overall survival (OS), progression free survival (PFS) and OS at 6, 12, and 24 months were determined. PFS and OS were estimated by Kaplan-Meier method. Seventy patients fulfilling inclusion criteria were identified. Median age was 54 years (19–77). Two subgroups were defined based on radiological response in the first scan after RT (Group 1, stable/response, n 5 27; Group 2, progression, n 5 43). In Group 2, 8 patients underwent surgery after RT confirming pro-gression (6 were symptomatic and 2 asymptomatic). In Group 1, extent of definitive surgery at diagnosis was gross total (GTR) in 59%, subtotal (ST) in 29%, and biopsy (BX) in 11%. In Group 2, GTR in 35%, STR in 56%, BX in 7%, and unknown in 2%. Reasons for discontinuation of TMZ/cRA were progression or death in 50 patients (71%), toxicity in 10 (14%), completion of planned treatment in 4 (6%), intercurrent illness in 3 (4%) and unknown in 3 (4%). Fifty-four patients had measurable disease after surgery and RT; best response to treatment is summarized below. Median

duration of SD in Group 1 patients with measurable disease was 14 weeks (6–118) and 16 weeks in Group 2 (4–96). In Group 2 patients with surgi-cally confirmed progression after RT (n 5 8), there was 1 MR, 1 PR, and 3 SD. Outcome measures are summarized below. Though retrospective, out-come in our series compares favorably to recent prospective trials for newly diagnosed GBM. In contrast with other series, we included patients with imaging progression in the first scan after RT. Although some of them possi-bly had pseudoprogression, surgical biopsy confirmed recurrence in almost 20%. Treatment response and PFS are encouraging, even in the subgroup with post-RT progression; however, OS is similar to other series, reflecting variable effectiveness of salvage therapies. We believe that post-RT adjuvant chemotherapy with TMZ/cRA is more effective than TMZ alone and ongo-ing randomized trials should validate or refute this believe in the future.

MA-32. PHASE II TRIAL OF PATUPILONE IN PATIENTS WITH BREAST CANCER BRAIN METASTASESDavid Peereboom,1 Cathy Brewer,1 and Andrew Seidman2; 1Brain Tumor and Neuro-Oncology Center, Cleveland Clinic Foundation, Cleveland, OH, USA; 2Memorial Sloan-Kettering Cancer Center, New York, NY, USA

CNS metastases from breast cancer (CM) are becoming more prevalent with improved systemic therapies for this malignancy. As a result, recurrent brain metastases after whole brain radiation therapy (WBRT) are becom-ing more common as well. In addition, no standard treatment exists for patients with patients with leptomeningeal disease. Novel therapies for these patients are needed. Patupilone is an epothilone which crosses the blood brain barrier and is many-fold more potent than paclitaxel or docetaxel. This agent has activity against systemic breast cancer as well as several other histologies. Patupilone is therefore a logical drug to test for patients with CM. Eligibility includes recurrent or progressive CM after WBRT; asymp-tomatic untreated CM; or leptomeningeal disease. Patients may continue trastuzumab and hormonal therapy but no other chemotherapy during this trial. Patients must be at least 4 weeks past WBRT, 3 weeks past cytotoxic therapy (6 weeks for nitrosoureas), 2 weeks past noncytotoxic agents, and on a stable dose of steroids. Exclusions are prior treatment with epothilones and grade . 1 diarrhea or neuropathy. Patients receive patupilone 10 mg/m2 IV over 20 minutes every 3 weeks with MRI brain and CT chest/abdomen every 6 weeks. The trial is powered to assess the 3-month CNS progres-sion free survival in parenchymal metastases but also allows entry of two other subsets of patients in an exploratory, hypothesis-generating fashion: patients with asymptomatic, unirradiated metastases and those with lep-tomeningeal disease. Four patients have been enrolled, all of whom had recurrent or progressive parenchymal brain metastases. Two progressed in the CNS after 2 cycles, one of whom had a partial response in liver dis-ease, the other of whom had stable systemic disease but died rapidly of progressive CNS disease. One patient is stable after 2 cycles and the fourth has had a minor CNS response after 2 cycles. Grade 3–5 toxicities have included 1 pt with G3 diarrhea. G 1–2 toxicities have included diarrhea, dysgeusia, and fatigue. Preliminary results indicate that patupilone is active against systemic breast cancer. At this early follow-up, CNS activity is not yet assessable. Toxicities are tolerable. Accrual continues. As more patients with CNS metastases receive chemotherapy for treatment of CNS metasta-ses the dilemma of systemic response in the setting of CNS progression will become more common. Since most of the drugs for systemic breast cancer, including patupilone, are cytotoxic, it is necessary to develop combinations that will allow full dosing of both agents for optimal concurrent treatment of CNS and systemic disease.

MA-33. RESULTS OF A PHASE I TRIAL WITH THE NOVEL ANTHRACYCLINE DERIVATIVE RTA 744 IN PATIENTS WITH RECURRENT PRIMARY BRAIN TUMORSCharles Conrad,1 Elizabeth Maher,2 Timothy Cloughesy,3 Karen Fink,4 Waldemar Priebe,1 Timothy Madden,1 Colin Meyer,5 Paul Foster,5 Morris Groves,1 Mark Gilbert,1 Victor Levin,1 Vinay Puduvalli,1 Howard Colman,1 and W.K. Alfred Yung1; 1University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2The University of Texas Southwestern Medical Center, Dallas, TX, USA; 3University of California, Los Angeles, Los Angeles, CA, USA; 4Baylor University Medical Center at Dallas, Dallas, TX, USA; 5Reata Pharmaceuticals, Inc., Irving, TX, USA

RTA 744 is an anthracycline derivative that was shown preclinically to cross the blood-brain barrier, not be a substrate for P-gp or MRP, and improve survival in a rigorous model of glioblastoma (GBM). A trial of RTA 744 was conducted in patients with recurrent, high-grade gliomas. RTA 744 was administered as a 2-hour i.v. infusion either on the first 3 days of a 21-day cycle (qdx3) or once weekly (qwk) for four weeks of a five week schedule, with an MTD determined for each schedule. Pharmacokinetic



samples were taken at the beginning and end of Cycle 1. Tumor activity was assessed by the Macdonald criteria. As of May 2007, 28 patients have been enrolled, all at the qdx3 schedule. 21 patients (75%) were diagnosed with GBM, with AO (n 5 4), AA (n 5 2), and GS (n 5 1) representing the balance. Median age was 50 (range 29–70), and 62% were male. All patients were pre-treated with temozolomide and radiation therapy, and most had failed other chemotherapies (range of previous treatments 2–16). Plasma pharmacokinetics indicate dose proportionality with a mean half-life of 34.9 hours. In the qdx3 cohort, there was some accumulation by day 3. The MTD of the qdx3 schedule was found to be 7.5 mg/m2/day (22.5 mg/m2/cycle). MTD for the qwk cohort has not yet been determined, and results will be updated at the meeting. The dose-limiting toxicity in the qdx3 cohort was myelosuppression, specifically neutropenia. Toxicities observed in more than 10% of patients and at least possibly attributed to study drug include lymphopenia, leukopenia, fatigue, neutropenia, hypophosphatemia, elevated ALT, weight loss/anorexia, and headache. No cardiotoxicity, and no significant neurotoxicity, alopecia, or GI toxicity was reported. Evi-dence of anti-tumor activity has been seen in multiple patients. A Complete Response was observed in a GBM patient at 2.4 mg/m2/day and has been maintained for . 9 months (patient is currently tumor free). Additionally, a Partial Response (81% tumor reduction) was observed in a patient with AO at 7.5 mg/m2/day. A Minor Response was observed in a GBM patient at 2.4 mg/m2/day, and several patients have experienced Stable Disease of several months duration. Multiple patients have shown evidence of necrosis based on imaging and/or biopsy. Median time to progression was 7 weeks (range 2 to . 68). Median number of cycles was 2 (range 1 to 7). RTA 744 is well tolerated when administered at doses up to and including 7.5 mg/m2/day for the first three days of a 21-day cycle. Based on evidence of drug activ-ity, advanced clinical trials of RTA 744 in patients with both primary and metastatic brain tumors are planned to begin by the end of 2007.

MA-34. A PHASE I/II STUDY OF TEMOZOLOMIDE (TMZ) AND THE FARNESYLTRANSFERASE INHIBITOR (FTI), TIPIFARNIB (ZARNESTRA, ZAR) IN RECURRENT GLIOBLASTOMA (GBM)Mark Gilbert,1 Patricia Gaupp,1 Sandra Ictech,1 Ken Hess,2 Morris Groves,3 Victor Levin,1 Charles Conrad,1 Vinay Puduvalli,1 Howard Colman,1 John De Groot,1 Sigmond Hsu,1 Peter De Porre,4 and W.K. Alfred Yung1; 1Neuro-Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Biostatistics, M.D. Anderson Cancer Center, TX, USA; 3University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 4Johnson & Johnson, Belgium.

Farnesylation is an essential step in the post-translational modification of several proteins that play a role in cell proliferation and growth, including Ras, RhoB, centromere binding proteins (CENP-E/CENP-F), lamin B, and protein tyrosine phosphatase (PTP). Inhibition of farnesylation may inhibit tumor cell proliferation. Preclinical testing demonstrates antiproliferative effects of FTIs in a variety of tumor cell lines and xenograft models. Addi-tional laboratory data from animal models demonstrated enhanced efficacy of combining an FTI with TMZ compared with TMZ alone. Objectives: (1) Determine optimal dose combination of ZAR and TMZ using an alternating week dosing schedule. (2) Evaluate the efficacy as measured by response rate and 6-month progression-free survival rate (PFS6) of the combination. Eligi-bility Criteria: Histologically proven GBM; unequivocal evidence of tumor relapse or progression after radiation therapy; up to 2 prior chemotherapy regimens for recurrent disease but no prior TMZ or FTI; ability to provide informed consent; KPS 60 or better. Patients may not be on CYP450-induc-ing anticonvulsants. PHASE I: Using a 3 1 3 design, alternating dose escala-tions determined the maximal tolerated dosing schedule: TMZ 150 mg/m2 days 1–7, 15–21; ZAR 600 mg BID orally days 8–14, 22–28. Response was evaluated every 2 cycles. Standard response criteria established by MacDon-ald were used. Overall 55 patients were accrued (phase I 5 15, phase II 5 40). Average age was 51 years old (range 21 to 84), and median KPS was 90. Male to female ratio was 3:1. Five patients were not evaluated for response because of early death (n 5 1), refused treatment or withdrew consent (n 5 3) or discontinued for intercurrent illness (pneumonia, n 5 1). These patients were included in survival and progression free survival analysis. Nine patients have either an ongoing response or remain progression-free (381, 371, 271, 271, 261, 211, 151, 121, and 101 mos). The PFS6 rate was 31%, median PFS was 20 weeks, and overall survival from study entry was 44 weeks. Treatment was associated with a high percent of patients developing either grade 3 (n 5 14; 25%) or grade 4 (n 5 27; 49%) lymphope-nia, grade 3–4 leukopenia (n 5 20; 36%), neutropenia (n 5 15; 27%), or thrombocytopenia (n 5 12; 22%), however without opportunistic infections or major bleeding events. Five patients developed severe fatigue. There were no treatment-related deaths. The combination of TMZ with ZAR using a dose-dense TMZ dosing schedule demonstrated durable objective responses and was associated with modest toxicity. These data support further inves-tigation of using a dose-dense schedule of TMZ in combination with signal transduction modulators. Future studies should determine the contribution

of the FTI to the efficacy of this regimen as recent reports (W. Wick et al., Neurology 2004) suggest that an enhanced response rate and tumor control may be achieved with dose-dense schedules of TMZ.

MA-35. A PHASE I TRIAL OF THE ADDITION OF THE FARNESYL TRANSFERASE INHIBITOR SCH 66336 TO TEMOZOLOMIDE FOR PATIENTS WITH GRADE 3 OR 4 MALIGNANT GLIOMASAnnick Desjardins, Sith Sathornsumetee, David Reardon, Krishna Goli, Daniela Bota, Jennifer Quinn, Jeremy Rich, Sridharan Gururangan, Stevie Threatt, Allan Friedman, Henry Friedman, and James Vredenburgh; Duke University, Durham, NC, USA

Concurrent temozolomide (TMZ) and radiation therapy followed by six monthly cycles of TMZ is now recognized as the standard-of-care treatment for malignant glioma (MG) patients. The H-, K- and N-ras proto-oncogenes encode 21 kDa guanine nucleotide binding proteins (H-Ras, K-Ras, and N-Ras), which play a crucial role in growth factor signal transduction and in the control of cellular proliferation and differentiation. An essential step in the post-translational processing of Ras is farnesylation, the addition of a farnesyl or C15 isoprenoid moiety from farnesyl diphosphate to the cysteine residue at the C-terminal side of Ras. SCH 66336, a tricyclic farnesyl protein transferase inhibitor, inhibits farnesyl transferase, the crucial enzyme in this process. Preclinical studies have shown that the antitumor activity of SCH 66336 is enhanced upon combination with standard cytotoxic agents. We report a phase I trial of TMZ and SCH 66336 for the treatment of stable or recurrent MG. Patient eligibility included: adult patients with stable or recurrent/progressive MG (glioblastoma multiforme [GBM], anaplastic astrocytoma [AA], anaplastic oligodendroglioma [AO]) who were treated previously with conventional external beam radiation and with or without chemotherapy; an interval of at least three weeks between prior surgical resection, two weeks between prior radiotherapy, or four weeks between prior chemotherapy; Karnofsky of at least 60%; and adequate hematologic, renal, and liver function. Patients were divided in two strata: those receiv-ing enzyme-inducing anticonvulsant (EIAC) and those not receiving EIAC. Each patient were treated with TMZ for five days every 28 days. The first cycle of TMZ was dosed at 150 mg/m² and subsequent cycles at 200 mg/m². SCH 66336 was dosed orally daily and was dose escalated. Responses were assessed by functional and imaging criteria after two cycles (8 weeks). The primary endpoints of this study were to determine the maximum tolerated dose (MTD) of SCH 66336 when administered with TMZ, and to deter-mine the activity and toxicity of this combination. Twenty-four patients have been treated thus far, 17 with GBM, five with AA, and two with AO. Nine patients have been enrolled to the EIAC stratum at SCH 66336 doses of 125, and then to the present dose of 175 mg daily. Fifteen patients have been accrued to the non-EIAC stratum at SCH 66336 doses of 75, 100, and then to the present dose of 150 mg daily. Dose limiting toxicities observed thus far have been limited to non-hematologic toxicities, including deep venous thrombosis (1 grade 3), and elevated ALT (1 grade 3). Twenty-four patients are assessable for response. One patient showed a partial response, nine showed a stable disease for at least 4 cycles, and nine patients pro-gressed after either the 1st or 2nd cycle. Ten patients have completed at least six cycles. In conclusion, the MTD of this drug combination for the EIAC and the non-EIAC strata have yet to be identified. Partial response has been observed in one patient. Forty-two percent of the patients have completed at least six cycles.

MA-36. A PHASE II TRIAL OF TEMOZOLOMIDE AND VINORELBINE FOR PATIENTS WITH RECURRENT BRAIN METASTASESFabio Iwamoto,1 Antonio Omuro,2 Jeffrey Raizer,3 Craig Nolan,4 Adilia Hormigo,4 Igor Gavrilovic,4 and Lauren Abrey1; 1Memorial Sloan-Kettering Cancer Center, New York, NY, USA; 2Service de Neurologie Mazarin, Hôpital de la Salpêtrière, Paris, France; 3Northwestern University, Chicago, IL, USA; 4NY, USA

Temozolomide has shown modest efficacy in the treatment of recur-rent brain metastases. We designed a regimen combining temozolomide with vinorelbine, a lipophilic agent that crosses the blood-brain barrier, trying to improve efficacy. This is a phase II trial with 28-day cycles using temozolomide (150 mg/m2, days 1–7 and 15–21) and vinorelbine on days 1 and 8. We previously reported a phase I trial that established an MTD of 30 mg/m2 of vinorelbine in this combination, but dose was decreased to 25 mg/m2 in the phase II trial. The phase II component was planned as a two-stage clinical trial. Since two or more responses were observed after the 20 initial patients, 15 more assessable patients were required. This design had a 91% probability to detect a true response rate of 20% or more. The primary endpoint was objective radiographic response. Secondary end-points include OS, PFS and toxicity. Patients 18 years or older with KPS of 60 or higher, adequate organ function and progressive or recurrent brain



metastases were eligible. Thirty-eight patients (15 men, 23 women) with a median age of 57 years (range, 39–75) and median KPS of 80 were enrolled. The primary tumor sites were lung (n 5 20), breast (n 5 11), colorectal (n 5 2), kidney (n 5 2), bladder (n 5 1), endometrium (n 5 1), head/neck (n 5 1). Prior therapies included whole-brain radiation therapy (79%), che-motherapy (97%), radiosurgery (47%), and brain metastasis resection (53%). Objective radiographic response was 5% (1 CR and 1 minor response); 5 patients had SD and 29 PD. Two patients did not receive the planned treat-ment. The median follow-up was 12 weeks and 72% of patients have died. Median PFS and OS were 8 and 22 weeks, respectively. Grade 3/4 toxicities were mainly hematological and 3 patients were removed from the study due to myelosuppression. In this heavily pretreated population of patients with brain metastases, adding vinorelbine to temozolomide does not seem to improve response rates as compared to temozolomide alone. Single-agent temozolomide also has a more favorable toxicity profile.

MA-37. NABTT 0502: PHASE 2 TRIAL OF ERLOTINIB AND SORAFENIB FOR PATIENTS WITH PROGRESSIVE OR RECURRENT GLIOBLASTOMA MULTIFORMEDavid Peereboom,1 Jeffrey Olson,2 Burt Nabors,3 Myrna Rosenfeld,4 Tom Mikkelsen,5 Xiaobu Ye,6 and Stuart Grossman6; 1Cleveland Clinic Foundation, Cleveland, OH, USA; 2Emory University, Atlanta, GA, USA; 3University of Alabama, AL, USA; 4Philadelphia, PA, USA; 5Detroit, MI, USA; 6Johns Hopkins University, Baltimore, MD, USA

Patients with progressive or recurrent GBM have a poor prognosis with a 6-month progression-free survival of 15%–20%. Erlotinib inhib-its the epidermal growth factor receptor tyrosine which is over expressed in 40%–60% of patients with GBM. Erlotinib has single agent activity against recurrent high-grade gliomas. Activation of the ras signaling path-way promotes glioma proliferation and tumor associated angiognensis. Raf kinase is a critical enzyme in the ras signaling cascade. Thus, inhibition of raf kinase has the potential to inhibit the ras pathway and thereby inhibit glioma growth. Sorafenib is a raf kinase inhibitor with oral bioavailability and penetration of the CNS. Sorafenib has shown promise as a single agent against high grade gliomas. The combined inhibition of both EGFR and ras pathways is a logical combination for patients with recurrent GBM. Eligible patients must have contrast-enhancing measurable progressive or recurrent glioblastoma multiforme after radiation therapy (RT) and up to 2 chemo-therapy regimens. Patients must not be on hepatic enzyme inducing antiepi-leptic drugs. Patients must have tissue specimens available and agree to have their blood and tissue blocks (or slides) submitted to the NIH tissue bank for correlative studies. The minimum interval from prior therapy is 3 months for RT, 3 weeks for cytotoxic chemotherapy (6 weeks for nitrosoureas), and 2 weeks for non-cytotoxic agents. Patients must have KPS > 60 and Mini Mental status exam score > 15. Patients receive erlotinib 150 mg once daily and sorafenib 400 mg twice daily. Each treatment cycle is 28 days. MRI or CT is performed every 2 cycles. The primary endpoint for the trial is overall survival. Eighteen patients have been enrolled, 9 of whom are on study but too early to evaluate. Three patients have died of progressive disease; 3 have progressed but remain alive, and 3 have chosen to go off study with non-dose limiting toxicities (abdominal pain, diarrhea and fatigue). Overall 2 of 9 evaluable patients reached 2 cycles of therapy while 7 reached less than 2 cycles before going off study. The combination of erlotinib and sorafenib is tolerable in patients with recurrent or progressive GBM. To date response and survival data are immature. Accrual continues.

MA-38. PROPHYLACTIC PERI-OPERATIVE ANTI-EPILEPTIC MEDICATIONS IN MALIGNANT GLIOMA PATIENTSIan Parney,1 Shelly Lwu,2 Annabelle Deguzman,2 J. Gregory Cairncross,2 and Mark Hamilton3; 1University of Calgary, Calgary, Alberta, Canada; 2Alberta, Canada; 3Calgary, Alberta, Canada

Published clinical practice guidelines based on multiple randomized controlled trials suggest that prophylactic anti-epileptic drugs (AED) should be avoided in patients with newly diagnosed brain tumors as they provide no benefit and have significant side effects. However, a recent prospective multi-center North American outcome study in patient with newly diag-nosed gliomas found that 89% of patient received AED even though only 32% presented with seizures. We wished to review our own practice pat-terns to see if we conformed to the published guidelines. We performed a retrospective chart review on all patients in southern Alberta with newly diagnosed malignant gliomas between January 2003 and December 2005. Only adult patients with pathological confirmation of malignant (WHO grade 3 or 4) glioma were included. Presentation with seizures, use of AED, peri-operative seizures, and complications related to either AED or peri-operative seizures were noted. To date, 127 cases have been reviewed. A minority (28%) presented with seizures, all of whom received AED. Most patients did not have seizures initially (72%). Of these, 39% received pro-phylactic AED. No patients receiving prophylactic AED experienced peri-

operative seizures while 3.8% of patients without prophylactic AED seized (p 5 NS). Complications from either seizures or AED were rare in the first post-operative week (1 patient each). Most patients receiving prophylactic peri-operative AED remained on AED more than one week after surgery (65%). Despite clinical practice guidelines to the contrary, many malignant glioma patients receive prophylactic AED without any particular benefit. Complications from AED were limited in the peri-operative period but, notably, most patients receiving prophylactic AED continued to get these medications beyond the peri-operative period.

MA-39. TUMOR TISSUE DELIVERY OF CILENGITIDE AFTER INTRAVENOUS ADMINISTRATION TO PATIENTS WITH RECURRENT GLIOBLASTOMA (GBM): PRELIMINARY DATA FROM NABTC PROTOCOL 03–02Mark Gilbert,1 Kathleen Lamborn,2 Andrew Lassman,3 Timothy Cloughesy,4 Susan Chang,2 Frank Lieberman,5 Patrick Wen,6 Sven Potzsch,7 Martin Picard,8 Michael Prados,2 and John Kuhn9; 1Society for Neuro-Oncology, Houston, TX, USA; 2University of California, San Francisco, San Francisco, CA, USA; 3Memorial Sloan-Kettering Cancer Center, New York, NY, USA; 4University of California, Los Angeles, Los Angeles, CA, USA; 5University of Pittsburgh, Pittsburgh, PA, USA; 6Boston, MA, USA; 7Merck KGaA, Darmstadt, Germany; 8AE (Europe) 9UT San Antonio, San Antonio, TX, USA

Invasion of endothelial cells into the tumor is an important component of the angiogenic process. In addition to growth factors such as VEGF, integrins are an essential component of both angiogenesis and tumor cell migration. The ab integrin family, particularly avb3 and avb5 are thought to be critical components of angiogenesis, being present on endothelial cells. Studies have confirmed the presence of the avb3 integrin on small blood vessels in glioblastoma, but not on normal brain vessels. Celengitide is a cyclic pentapeptide that targets the RGD sequence on vitronectin and as a consequence is a specific inhibitor of both the avb3 and avb5 integrins. A recent phase I study (Nabors et al., JCO 2007) treated patients with malig-nant glioma and demonstrated an excellent toxicity profile and anti-tumor activity. The current study is a phase II trial for patients with recurrent GBM who require tumor resection. The main objectives are to evaluate efficacy as measured by 6-month progression free survival rate and examine drug delivery into tumor. Patients were randomized to receive Celengitide at either 500 mg or 2000 mg on days –8, –4, and –1 with tumor resec-tion on day 0. Plasma was obtained at the end-of-infusion of the 3rd dose (day –1) then at the time of tumor resection. All patients were then treated with the 2000 mg dose twice weekly, beginning 2 weeks after tumor resec-tion. To date, 30 patients have been accrued. Treatment has been well toler-ated with 8 patients developing grade 3 or 4 lymphopenia. Grade 3 fatigue (n 5 1), thrombocytopenia (n 5 1), myalgias (n 51), and non-cardiogenic pulmonary edema (n 51) were also seen. There were no episodes of post-operative hemorrhage. The preliminary results of plasma and tumor con-centrations demonstrate that at the end of the 3rd Celengitide infusion, the concentration averaged 26.5 mg/ml in plasma after the 500 mg infusion and 130.4 mg/ml after the 2000 mg infusion. At the time of tumor resection, plasma concentrations were at 371 ng/ml and 373 ng/ml for the 500 mg and 2000 mg dose, respectively. Tumor concentrations were 400 and 1190 ng/gram of tissue for the 500 and 2000 mg dosing, yielding a tissue to plasma ratio of 1.83 for the 500 mg dose and 4.17 for the 2000 mg dose. Treatment with Celengitide has been well tolerated. Preliminary pharmacokinetic stud-ies demonstrate good delivery of drug into the tumor that is dose-related. Analyses of additional samples are underway and will be incorporated into the presentation.

MA-40. LEVETIRACETAM AND SEIZURE FREEDOM IN PRIMARY AND METASTATIC BRAIN TUMORS: A RETROSPECTIVE STUDYJennifer Connelly, Romila Mushtaq, Hendrikus Krouwer, and Mark Malkin; Department of Neurology, Medical College of Wisconsin, Milwaukee, WI, USA

Levetiracetam (LEV) is an FDA approved anti-epileptic medication for adjunctive therapy of partial complex seizures and primary generalized epi-lepsy. Many large retrospective case series report efficacy and safety using LEV monotherapy for the treatment of both partial complex and generalized seizures. Several small retrospective studies of LEV demonstrated efficacy at seizure management in patients with brain tumors. We proposed that LEV provided significant seizure freedom among patients with brain tumors in a large retrospective study. This was an IRB-approved retrospective chart review of all patients referred to the Neuro-oncology Service at an academic medical center for management of primary brain tumors or brain metastases from January 2000 to December 2006. Inclusion criteria were: (1) diagnosis of a primary or metastatic brain tumor, (2) documented history of seizures, (3) age greater than 18 years, and (4) patients receiving LEV treatment as



either monotherapy or polytherapy. Exclusion criteria were: (1) documented history of non-compliance, (2) history of pseudoseizures, and (3) lack of complete documentation of seizure history. Additional information was obtained on tumor type, date of seizure onset, seizure frequency, dose of levetiracetam, and reasons for discontinuation of LEV therapy. Change in seizure frequency from baseline was calculated as 0%, ,25%, 26%–50%, 51%–75%, 76%–99%, and 100%. Note that data collection is ongoing at the time of abstract submission; it is anticipated that ultimately data on 200 patients will be reported. A total of 154 patients out of 214 charts screened met all the inclusion criteria. Patients were excluded due to brain biopsy negative for tumor and for incomplete documentation. Tumors sub-types included: 48 glioblastomas (31%), 19 anaplastic astrocytomas (12%), 19 WHO grade II astrocytomas (12%), 22 oligodendrogliomas (14%), 17 oligoastrocytomas (11%), 9 meningiomas (6%), 8 metastases (5%), and 12 other primary CNS tumors (8%). 124 out of 154 (80.5%) patients had complete seizure freedom on an average daily dose of 2100 mg of LEV. There was no apparent statistically significant difference in rates of seizure control between various tumor types. Discontinuation rates were higher among patients with seizure freedom than those without seizure freedom, 14.5% vs. 10% respectively. Seizures are not uncommon in patients with primary brain and metastatic tumors. Management of these seizures can be complicated due to concomitant therapy of the tumor. The majority of anti-epileptic medications are metabolized by the hepatic cytochrome P450 sys-tem. Unfortunately, many of the therapeutic interventions for brain tumors also affect this system, particularly chemotherapeutic agents and dexam-ethasone. This is the largest retrospective study to date that demonstrates excellent seizure freedom rates using LEV for patients with primary brain tumors or metastases. Interestingly, patients were more likely to discontinue LEV due to adverse events when they were seizure free. Larger prospective studies are needed to validate the efficacy and tolerability of LEV as mono-therapy among patients with brain tumors.

MA-41. RECURRENT MENINGIOMA: SALVAGE THERAPY WITH LONG-ACTING SOMATOSTATIN ANALOGUEMarc Chamberlain,1 Camilo Fadul,2 and Michael Glantz3; 1H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, USA; 2Department of Medicine, Division of Hematology/Oncology, Dartmouth Medical School Lebanon, New Hampshire, USA, NH, USA; 3Florida State University, Tallahassee, FL, USA

Somatostatin receptors, especially the sst2A subtype, are present on most meningiomas. The addition of somatostatin (SS) inhibits meningioma growth in vitro in some studies. There have been anecdotal reports of oct-reotide inhibiting growth in meningiomas. Objectives: A prospective pilot trial of sustained-release somatostatin (Sandostatin LAR) in 16 patients with recurrent meningiomas was conducted with a primary study objective of progression free survival at 6 months. 16 patients (11 women; 5 men: median age 58 years) with recurrent meningioma were treated prospectively with long-acting somatostatin. Patients had progressed radiographically after prior therapy with surgery (14/16; complete resection in 5; subtotal in 7; biopsy only in 2), radiotherapy (13/16), and chemotherapy (12/16). All patients had confirmation of the presence of somatostatin receptors in their tumor using indium 111-octreotide, a long-acting somatostatin agonist, SPECT scanning. Patients received 2–15 cycles (median 4.5) of somatostatin with minimal toxicity. 4 partial responses, 5 stable disease, and 7 progres-sive disease patterns were seen. Duration of response ranged from 2–201 months (median 5.0 months). Median survival was 7.5 months (range 3–201). The overall progression free survival was 44% (7 patients) at 6 months. In this small trial of patients with recurrent meningiomas shown to overexpress somatostatin receptors by octreotide scintigraphy, long-acting somatostatin (Sandostatin LAR) was administered on a monthly schedule. 31% of patients demonstrated a partial radiographic response and 44% achieved progression free survival at 6 months. Toxicity was minimal sug-gesting somatostatin analogues may offer a novel, relatively nontoxic alter-native treatment for recurrent meningiomas.

MA-42. SALVAGE CHEMOTHERAPY WITH TEMOZOLOMIDE AND CPT-11 FOR RECURRENT OR PROGRESSIVE HIGH-GRADE GLIOMAMizuhiko Terasaki, Shintaro Fukushima, and Minoru Shigemori; Neurosurgery, Kurume University, Kurume, Fukuoka, Japan

The best treatment for recurrent or progressive high-grade gliomas has yet to be determined. Recently, interest in the use of temozolomide has increased, but only a limited number of patients responded to therapy and responses are often short-lived. We therefore undertook a trial of temozolo-mide in combination with CPT-11 for patients with recurrent or progressive high-grade glioma. Patients were eligible for the study if they had recurrence or progressive of a glioma (glioblastoma, anaplastic glioma), and had not received prior therapy with CPT-11. All patients received temozolomide at

150 mg/m2/d on days 1–5 and CPT-11 at 325 mg/m2/d on day 6 in non-CPY3A4–enzyme-inducing anticonvulsants (EIAC) condition. Cycles were repeated every 28 days for up to 12 cycles. Sixteen patients were enrolled–8 with glioblastoma and 8 with anaplastic gliomas. Their median age was 55.5 years (16–74), and 69% were male. Eight were enrolled at first and 8 at second and more recurrence. All had had prior radiation and chemotherapy. Of the 8 subjects with glioblastoma, 1 had a PR (12.5%), 2 (25%) SD, and 5 (62.5%) PD. The median PFS was 1.5 mo. (1–251 mo), and the 6-month progression-free survival (PFS) was 37.5%. Of the 8 patients with anaplastic gliomas, 1 had a PR (12.5%), 2 SD (25%), and 5 PD (62.5%). Their median PFS and 6-month PFS were 3.5 mo and 37.5%, respectively. Of the entire cohort, during a total of 60 treatment courses, six had grade 3/4 neutro-penia; one patients developed fever and neutropenia and died of a Pneumo-cystis Carnii pneumonia. Remaining five patients had grade 3 neutropenia toxicity. Another two patients were prematurely withdrawn from the study due to toxicity (two for grade 2 allergy and two for grade 3 diarrhea). The histological subtype of the tumor, its location, and its maximum response to chemotherapy did not have an impact on the duration of disease control. The combination of CPT-11 and temozolomide has only modest efficacy and significant toxicity. The results of the study suggest the combination offers no advantage over either agent used alone.

MA-43. SALVAGE CHEMOTHERAPY WITH CPT-11 FOR RECURRENT TEMOZOLOMIDE-REFRACTORY ANAPLASTIC OLIGODENDROGLIOMAMarc Chamberlain,1 Denise Wei-Tsao,2 Deborah Blumenthal,3 and Michael Glantz4; 1H. Lee Moffitt Cancer Center, Tampa, FL, USA; 2Department of Preventative Medicine, CA, USA; 3University of Utah, Salt Lake City, UT, USA; 4Florida State University, Tallahassee, FL, USA

A prospective Phase II study of CPT-11 in adult patients with recurrent Temozolomide (TMZ)-refractory anaplastic oligodendroglioma (AO) with a primary objective of determining 6-month progression free survival (PFS). There is no standard therapy for alkylator-resistant AO and hence a need exists for new therapies. Twenty patients (10 men; 10 women) ages 27–47 (median 38), with radiographically recurrent AO were treated. All patients had previously been treated with surgery, involved-field radiotherapy, and adjuvant chemotherapy (TMZ in 14; BCNU in 6). 13 patients were treated at first recurrence with an alternative chemotherapy (TMZ in 6 all previously treated with BCNU; 5 with PCV and 1 each with carboplatin, sorafenib, or cyclophosphamide). 11 patients underwent repeat surgery. All patients were treated at either first or second recurrence with CPT-11 administered intra-venously once every 3 weeks (350mg/m2 in patients on non-enzyme induc-ing anticonvulsants or no anticonvulsant; 600mg/m2 in patients on enzyme inducing anticonvulsants), operationally defined as a single cycle. Neuro-logical and neuroradiographic evaluations were performed every 8–9 weeks. All patients were evaluable for toxicity and response. A total of 129 cycles of CPT-11 (median 4.5 cycles; range 3–18) was administered. CPT-11 related toxicity included diarrhea (4 patients; 2 [10%] grade 3), neutropenia (3; 3 [15%] grade 3), fatigue (3; 3 [15%] grade 3), and delayed nausea/vomiting (1; 1 [5%] grade 3). 5 patients (25%) demonstrated a partial radiographic response, 7 (35%) demonstrated stable disease, and 8 (40%) had progres-sive disease following three cycles of CPT-11. Time to tumor progression ranged from 2–13.5 months (median: 4.5 months). Survival ranged from 3–21 months (median: 6 months). 6-month and 12-month PFS were 30% and 25% respectively. CPT-11 demonstrated modest efficacy with accept-able toxicity in this cohort of adult patients with recurrent AO all of whom had failed prior TMZ chemotherapy.

MA-44. COMBINATION OF HIGH-DOSE SYSTEMIC METHOTREXATE AND INTRATHECAL LIPOSOMAL CYTARABINE IS WELL TOLERATED IN PATIENTS WITH CNS METASTASES FROM BREAST CANCERMaciej M. Mrugala,1 Jason Rockhill,2 Alexander M. Spence,1 and Julie Gralow3; 1Neurology and Neurosurgery, University of Washington/Fred Hutchinson Cancer Research Center, Seattle, WA, USA; 2Radiation-Oncology, University of Washington/Fred Hutchinson Cancer Research Center, Seattle, WA, USA; 3Medical Oncology, University of Washington/Fred Hutchinson Cancer Research Center, Seattle, WA, USA

Treatment options for patients with CNS metastases from breast cancer are limited. Surgical intervention can be helpful in patients with solitary lesions. Focal and whole brain radiation therapy is also frequently applied. For patients with leptomeningeal spread of the disease prognosis remains poor and therapy is predominantly palliative. Intrathecal treatment with methotrexate has been most commonly used, followed by whole brain or cranio-spinal radiation therapy. Even this aggressive approach is not asso-ciated with significant improvement in patient survival and the quality of life can be heavily impaired. Recent report showed that systemic high-dose methotrexate at 3.5 gm/m2 can result in disease control (partial response



or stable disease) in 56% of patients with CNS metastases within the study cohort. It was also shown that cytarabine liposome injection (DepoCyt®) demonstrates activity in leptomeningial breast cancer. Combining systemic treatment using agents penetrating CNS (methotrexate, thiotepa, cytara-bine) with intrathecal chemotherapy may be beneficial but can be associ-ated with increased neurotoxicity. We treated two patients, ages 35 and 40, both with parenchymal and leptomeningeal disease using a combination of HD-MTX at 8 gm/m2 and IT DepoCyt® at 50 mg. Total of 11 cycles of HD-MTX and 7 cycles of IT DepoCyt® were provided to date. MTX was given intravenously every two weeks alternating with IT DepoCyt®. First dose of DepoCyt® in both patients was administered via lumbar puncture. All subsequent doses were administered through the Ommaya reservoir. Both patients had normal CSF flow studies at the beginning of therapy. Pre-medication with Dexamethasone at 4 mg twice a day was given at the start of IT therapy and was continued for the total of five days. Steroid pre-medication for HD-MTX infusion was not routinely used. Both patients tolerated treatments well. Transient, reversible elevations of transaminases were noted following HD-MTX. Mild, occasional headaches were observed in one patient following administration of IT therapy but pharmacological intervention was not necessary. Cognitive changes were not observed as documented by periodic MMS assessments. No signs typical of chemical arachnoiditis were identified. Patient one suffered from severe headache and developed VI nerve palsy after having received 4 cycles of HD-MTX and 3 doses of IT DepoCyt®, however these symptoms were identified at the time of disease progression and improved with steroid therapy. Patient two showed complete resolution of parenchymal lesions after 6 cycles of HD-MTX and continued clearing of CSF after 3 doses of DepoCyt®. Based on this limited experience, we conclude that the combination of HD-MTX at 8gm/m2 with IT DepoCyt® at 50 mg is associated with minimal short term neurotoxicity and can be safely administered. In addition it may be associated with positive tumor response. This combination should be fur-ther studied in a clinical trial to examine long-term safety and efficacy.

MA-45. INTRAVITREAL METHOTREXATE WITH NO RADIATION THERAPY FOR TREATMENT OF INTRAOCULAR LYMPHOMA: TEN YEARS OF EXPERIENCEShahar Frenkel,1 Karen Hendler,1 Edna Shalom,2 Jacob Pe’er,1 and Tali Siegal2; 1Ophthalmology, Hadassah Hebrew University Medical Center, Jerusalem, Israel; 2Gaffin Center for Neuro-Oncology, Hadassah Hebrew University Medical Center, Jerusalem, Israel

Intraocular lymphoma (IOL) involves the retina, vitreous or optic nerve head, and appears in the context of either primary CNS lymphoma or as a relapse of systemic disease. It carries a high risk of ocular and CNS relapse. The optimal management is unknown. Objective: to describe our experience in treating IOL by intravitreal injections of methotrexate (MTX). Patients with suspected IOL underwent a diagnostic vitrectomy which allowed eval-uation of cytology, gene rearrangement and interleukins level. Treatment protocol includes intravitreal injections of 400 mg MTX twice weekly for 4 weeks, once weekly for 8 weeks and then monthly for 9 months, for a total of 25 injections. Radiation therapy was not given. In the past 10 years we have treated 44 eyes of 26 patients; 7 patients had monocular involvement, and 19 binocular. IOL was the primary manifestation of lymphoma in 6 patients (23%) and the mean interval between ocular and CNS involvement was 14 months (range 1–54 months). CNS involvement preceded IOL in 13 patients with a mean interval of 36 months (range 2.5–132 months). B-cell lymphoma predominated with T-cell lymphoma found in 3 patients (11.5%). Both the vitreal and serum samples showed a high IL-10 to IL-6 ratio, compatible with the diagnosis of lymphoma. Remission was reached after a mean of 6.1 63.5 injections, with 86% of the eyes needing 12 injec-tions or less to be cleared of malignant cells. None of the patients had an intraocular recurrence with a median follow-up of 21 months (range 3–120 months) with 8 (31%) patients followed for a median of 63 months after completing treatment protocol. The most common side effect was corneal epitheliopathy, usually appearing after the third injection and subsiding with the increase in injections interval. IOL can be controlled effectively and without serious adverse reactions by intravitreal MTX injections. The treatment protocol described herein has resulted in no intraocular recur-rence so far, and has had favorable profile of side effects.

MA-46. MACULOPATHY IN PATIENTS WITH PRIMARY CNS LYMPHOMA (PCNSL) TREATED WITH OSMOTIC BLOOD-BRAIN BARRIER DISRUPTION (BBBD) IN CONJUNCTION WITH CHEMOTHERAPYJoaquin Vicuna-Kojchen,1 Shahar Frenkel,1 Edna Shalom,2 Itay Chowers,1 Jacob Pe’Er,1 and Tali Siegal2; 1Ophthalmology, Hadassah Hebrew University Medical Center, Jerusalem, Israel; 2Gaffin Center for Neuro-Oncology, Hadassah Hebrew University Medical Center, Jerusalem, Israel

Maculopathy associated with osmotic BBBD was described before in 11 patients (Millay et al., Ophthalmology 1986 102:626). The observed abnormalities were of minimal functional significance. It was assumed that it probably represents direct toxicity of the chemotherapy, particu-larly the intraarterially administered methotrextae (MTX).Toxicity from multiple mannitol disruptions alone could not be ruled out. Retinopathy in survivors of PCNSL treated with high dose MTX (HD-MTX) and cra-nial irradiation occurred in 18.5% of 5-year survivors and was associated with reduced visual acuity in 80%. Although radiation therapy can induce delayed rethinopathy it is unclear whether prior HD-MTX plays a role in this delayed toxicity. Objective: To evaluate the presence of toxic maculopa-thy in patients with PCNSL treated by either intravenous (i.v.) HD-MTX-based protocol, BBBD (MTX-based or Carboplatin-based protocol) or by intravitreal MTX injections alone in patients with intra ocular lymphoma (IOL). 49 patients with PCNSL or IOL who had documented detailed ocu-lar examination were included. Those alive at time of data collection were invited for ophthalmologic follow up and fundus photography. Patients are divided into treatment groups as follows: group A—i.v. MTX-based pro-tocol (n 5 26) of whom 18 (69%) received in addition intravitreal MTX (400 mg/injection 3 25 doses) to treat IOL; group B—BBBD protocol (n 5 23) of whom 6 (26%) received in addition intravitreal MTX. None of the group A patients developed maculopathy while 16/23 (69.5%) of group B patients had maculopathy on ophthalmologic examination. The maculopa-thy was bilateral in 87.5% with a median severity graded as moderate. It did not affect visual acuity in any of the patients and only 2 patients had visual complains that improved over time. There was no statistical differ-ence between the total number of BBBD procedures and the total number of carotid procedures between patients who developed maculopathy and those who did not have maculopathy. However, patients with advanced macul-opathy had significantly higher number of carotid procedures as compared to patients with mild-moderate maculopathy (p 5 0.025). No association was found between the degree of BBBD (good-excellent vs. poor-moderate) and the severity of the maculopathy (advanced vs. mild-moderate.). BBBD often causes an asymptomatic maculopathy that does not affect the visual acuity. The severity of the maculopathy is related to the number of carotid procedures but not to the degree of BBBD. Intravitreal injections of MTX are not associated with toxic maculopathy and therefore it seems that the maculopathy is not related to the toxic effect of chemotherapy alone but rather to barrier disruption. Long-term consequences are unknown.

MA-47. UP-FRONT TEMOZOLOMIDE IN ELDERLY PATIENTS WITH GRADE III AND IV ASTROCYTOMAS ELIGIBLE FOR RADIOTHERAPY VS SUPPORTIVE CARE TRIAL (NCT00430911)Florence Laigle-Donadey,1 Gentian Kaloshi,2 Khe Hoang-Xuan,2 Marc Sanson,2 and Jean-Yves Delattre2; 1Service de Neurologie Mazarin, Hôpital Pitié-Salpêtrière, Paris, France; 2Service de Neurologie Mazarin, Hôpital de la Salpêtrière, Paris, France

Background Upfront temozolomide (TMZ) is often proposed to elderly patients as an alternative to radiotherapy (RT). However RT, as recently shown by a phase III trial, provides a survival benefit in elderly glioblastoma patients (>70 years) with good performance status (KPS > 70), as com-pared to supportive care alone (MS 5 29.1 weeks vs. 16.9 weeks), without reducing the quality of life or cognition (Keime-Guibert et al., NEJM 2007 356(15):1527–35). In order to better evaluate the benefit of TMZ in the elderly, we analyzed here a cohort of eligible patients, who were treated with TMZ alone because they refused to participate to this trial. Methods We retrospectively reviewed all patients from our institution, who met the inclu-sion criteria (patients > 70 years, KPS > 70, WHO grade III astrocytoma or glioblastoma), and were proposed to participate to the study but refused and received upfront chemotherapy by TMZ (150 to 200 mg/m2, 5 days a month, every 28 days). Seventeen eligible patients (mean age: 76 years, [range 71–85], mean KPS: 70 [range 70–90], 3 grade III astrocytomas and 14 glioblastomas) declined to participate to the radiotherapy versus sup-portive care randomized study and were treated up-front with oral TMZ for 1 to 10 cycles (mean 5 4). Four partial responses were observed. Overall median survival (MS) and median progression-free survival (PFS) were 28 weeks and 16 weeks, respectively. Survival was related to KPS (MS 5 27 weeks for KPS 5 70 and 60 weeks for KPS . 70; p 5 0.007), but not to age (, 76 years vs. . 76 years) histology (grade III vs. IV), or response (responders vs. non-responders). Two grade III/IV haematological toxicity, one grade II cutaneous toxicity and one grade IV gastro-intestinal toxicity



were observed. No treatment related death was observed. Conclusion: The survival of TMZ-treated patients seems comparable to the RT arm of the trial. These preliminary results support further randomized studies compar-ing TMZ to RT. In addition they show the major prognostic impact of KPS in this population.

MA-48. TEMOZOLOMIDE AS FIRST-LINE TREATMENT FOR ELDERLY PATIENTS WITH ANAPLASTIC OLIGODENDROGLIOMA OR OLIGOASTROCYTOMAFrançois Ducray,1 Wafa Iraqi,1 Karima Mokhtari,2 Soledad Navarro,3 Sophie Taillibert,1 Florence Laigle-Donadey,1 Alexandra Amiel-Benouaich,1 Antonio Omuro,1 Antoine Carpentier,1 Marc Sanson,1 Jean-Yves Delattre,1 and Khe Hoang-Xuan1; 1Service de Neurologie Mazarin, Hôpital de la Salpêtrière, Paris, France; 2Neurolopathology, Hôpital de la Salpêtrière, Paris, France; 3Neurosurgery, Hôpital de la Salpêtrière, Paris, France

There are no specific data concerning the optimal management of oli-godendroglial gliomas in elderly patients. Objective: To assess the efficacy and safety of first-line chemotherapy with temozolomide in elderly patients with a diagnosis of anaplastic oligodendroglioma (AO) or oligoastrocytoma (AOA). Between March 2000 and November 2005, 44 consecutive patients aged 70 years or over with a diagnosis of AO or AOA (WHO grade III) were treated with first-line temozolomide. Temozolomide was given at a dose of 150–200 mg/m² per day for 5 consecutive days every 28 days until progression; Macdonald criteria were used to evaluate the tumor response. Patients and tumor characteristics were as followed: median age was 74 years (range, 70–90 years), median KPS was 70 (range, 30–100). Contrast enhancement was present on preoperative CT scan or MRI in 43 patients (98%) with 32 patients (73%) having an annular ring enhancement. Surgery consisted in gross total surgical resection in 2 patients, in partial resection in 9 patients and in biopsy in 33 patients. AO was the pathological diagnosis in 30 patients and AOA in 14 patients. Median OS from the time of diagnosis was 12.9 months (range, 2–38 months) and median PFS was 6.9 months (range 1–25 months). A partial response (PR) was observed in 13 patients (30%), 19 patients (44%) had stable disease (SD), and 11 (26%) patients had progressive disease (PD). Median OS was 18 months in patients with PR (range, 5–37.9), 14.1 months in patients with SD (range, 6.7–29.4), and 5.4 for patients with PD (range, 1.8–16) (p 5 0.0001). Clinical improvement was observed in thirteen patients (30%). Grade 3 or 4 hematologic toxicities occured in 9 patients (20%). This study suggests that first-line temozolo-mide is an interesting therapeutic option in elderly patients with anaplastic oligodendroglial tumors.

MA-49. IMPACT OF CONCURRENT AND ADJUVANT TEMOZOLOMIDE ON THE PATTERN OF RECURRENCE IN NEWLY DIAGNOSED GLIOBLASTOMAErin Dunbar, Lawrence Kleinberg, Jaishri Blakeley, and Stuart Grossman; Johns Hopkins University, Baltimore, MD, USA

In the past, approximately 90% of newly diagnosed glioblastoma (GBM) failed locally despite aggressive surgery, radiation, and chemother-apy. Since 2005, standard therapy has changed to include temozolomide given during and after radiation therapy (RT 1 TMZ) which prolongs both the median survival and the number of survivors at two years. This retro-spective study was conducted to determine if RT 1 TMZ alters the pattern of recurrence or merely delays the time to local recurrence. An IRB approved review of patients with newly diagnosed GBM treated with concurrent and adjuvant temozolomide from 12/01/2004 through 12/31/2006. Allowing for a thorough review of radiographic patterns of recurrence, patients stud-ied were limited to only those who received their radiation, chemotherapy, and follow-up scans at Johns Hopkins. In addition, recurrences occurring in less than 6 months were not studied to minimize confusion caused by radiation effect. Patients reviewed were required to have: age . 18, KPS . 60, and received .5 of 6 weeks of chemo-radiation. Of the 157 potential patients reviewed, 42 met the above criteria. Twenty-four were 24 males (57%). Their median age was 54 years, and the estimated median survival and 1 and 2 year survivals were 603 days, 60% and 40%. At this time, 26 of the 42 (59%) patients have recurred (median 494 days, range 219 to 967). A total of 22/26 (85%) recurred within the radiation field, with 18 (69%) recurring only within and 4 (15%) recurring within and outside. Only 4 patients (15%) recurred exclusively outside the radiation field and no patients recurred exclusively at the radiation margin (within 1 cm of the 95% dose line). Eighty-five percent of the recurrences to date have occurred within the radiation field. This is remarkably similar to that reported by Hochberg in 1980 and suggests that the vast majority of recurrence in newly diagnosed glioblastoma treated with RT1TMZ will continue to be within the original radiation field. Follow-up studies of the long-term survivors of RT1TMZ will be critical in answering this important question and setting further research priorities in patients with glioblastoma.

MA-50. PHASE II STUDY OF TARCEVA PLUS TEMODAR DURING AND FOLLOWING RADIOTHERAPY IN PATIENTS NEWLY DIAGNOSED WITH GLIOBLASTOMA OR GLIOSARCOMAMichael Prados, Rebecca DeBoer, Susan Chang, Nicholas Butowski, Eric Burton, Jane Rabbitt, Margaretta Page, Anne Fedoroff, Hannah Carliner, Rupa Parvataneni, Valerie Kivett, Penny Sneed, Paul Kabuubi, David Larson, Mitchel Berger, Michael McDermott, Kathleen Lamborn, David Stokoe, and Daphne Haas-Kogan; University of California, San Francisco, San Francisco, CA, USA.

Tarceva is an orally active selective inhibitor of EGFR tyrosine kinase. GBM tumors frequently display amplification and/or mutation of the EGFR gene, causing over-expression of EGFR protein or constitutively activated EGFR signaling. This open label, prospective single arm phase II study combined Tarceva with radiation therapy (XRT) and Temodar (TMZ). The study objectives were to determine the efficacy of this treatment combina-tion as measured by overall survival, to evaluate toxicity, and to explore the relationship between molecular markers and treatment response. 65 adults with newly diagnosed GBM or GS with a KPS of > 60 were enrolled. Patients were stratified based on their use of enzyme-inducing antiepilep-tic drugs (EIAEDs). Patients not on EIAEDs (Group A) received Tarceva 100 mg/day during XRT and 150 mg/day after XRT. Patients on EIAEDs (Group B) received Tarceva 200 mg/day during XRT and 300 mg/day after XRT. Following XRT the Tarceva dose was escalated until the develop-ment of tolerable grade 2 rash or the maximum allowed dose (200 mg/day for Group A; 500 mg/day for Group B). All patients received Temodar 75 mg/m2/day during XRT and 200 mg/m2/day for 5 days every 28-day cycle after XRT. XRT was administered in 1.8–2.0 Gy fractions 5 days/week for 6 weeks to a total dose of 59.4–61.0 Gy. Treatment continued for at least 12 months unless tumor progression or unacceptable toxicity occurred. Molecular markers of EGFR (IHC and FISH), EGFRvIII (IHC), PTEN (IHC), and AKT phosphorylation (IHC) were analyzed from paraffin embedded tumor tissue from each patient. Survival results were compared to 2 previously completed phase II studies of thalidomide plus TMZ and cis-retinoic acid plus TMZ during and following XRT in newly diagnosed GBM and GS. Patient characteristics and outcome are shown in the table. Median survival was 84 weeks for the current study compared to 61 weeks in the two combined historical control studies, with a hazard ratio for sur-vival (treated/control) of 0.65 (c.i. 0.45, 0.95), adjusted for age, extent of resection and KPS (p 5 0.025). Treatment was well tolerated. Correlation with molecular markers will be presented. The combination of Tarceva plus Temodar during and following radiotherapy is an active regimen and the results of this phase II study were superior to previous studies done at UCSF. Updated analysis and additional clinical follow up will be presented. Further investigation is warranted.

MA-51. PHASE II TRIAL OF CONCOMITANT LOW-DOSE TEMOZOLOMIDE WITH RADIATION THERAPY (RT) FOLLOWED BY 12 MONTHS OF TEMOZOLOMIDE AND IRINOTECAN FOR NEWLY DIAGNOSED GLIOBLASTOMA (GBM): UPDATED RESULTS OF RTOG 04–20Frank Lieberman,1 Jennifer Moughan,2 Christina Tsein,3 Walter Curran,4 Maria Werner-Wasik,4 Ryan Smith,1 Linda Grossheim,5 Eugen Hug,6 Ian Robins,7 and Minesh Mehta7; 1University of Pittsburgh, Pittsburgh, PA, USA; 2American College of Radiology, Philadelphia, PA, USA; 3University of Michigan, Ann Arbor, MI, USA; 4Thomas Jefferson University, Philadelphia, PA, USA; 5Medical College of Wisconsin, Milwaukee, WI, USA; 6Radiation Oncology, University of Dartmouth, NH, USA; 7University of Wisconsin-Madison, Madison, WI, USA

.Irinotecan plus temozolomide produced improved 6-month progres-sion-free survival (PFS) in a recurrent GBM NABTC trial. RTOG 04–20 tested this as an adjuvant regimen, instead of temozolomide alone. Adults with newly diagnosed supratentorial GBM received temozolomide 75mg/m2 daily with RT for 6 weeks. Pneumocystis prophylaxis was required. Within 6 weeks after RT, subjects with stable or improved MRI received temozolomide 150mg/m2 on days 1–5, and irinotecan 200mg/m2 on days 1 and 15 of 28 day cycles 3 12. Clinical assessments and MRI were required prior to and after RT, and after every 2 treatment cycles. Only non-enzyme inducing anticonvulsants were allowed. 170 patients were accrued by Sep-tember 2005 with 154 evaluable patients. As of 5/9/07, 90/154 have died, and median f/u is 13.2 months (m). Median age is 57 years, 81% are RPA class III or IV. Median survival (MS) is 16.6 m for the entire cohort. MS is not reached for RPA class III, and is 16.9 and 9.2 m for RPA classes IV and V, respectively. PFS is 6.5 m, with 120/154 progressing. PFS is not reached for RPA class III and is 6.3 and 3.9m for classes IV and V. In spite of the use of a doublet, and treating for up to 12, instead of 6 cycles, MS for the entire cohort is not statistically significantly superior to the EORTC trial of temozolomide for adjuvant therapy of GBM (MS 5 14.6); the MS for RPA class III has not been reached.



MA-52. PSEUDOPROGRESSION AFTER CONCOMITANT RADIO-CHEMOTHERAPY TREATMENT IN NEWLY DIAGNOSED GLIOBLASTOMA PATIENTS AND POTENTIAL CORRELATION WITH MGMT METHYLATION STATUSAlba Brandes,1 Alicia Tosoni,1 Enrico Franceschi,1 Valeria Blatt,2 Stefania Bartolini,1 Daniela Grosso,2 Nicola Galtarossa,2 Cinzia Susini,1 Angela Tozzoli,1 Rina Renzi,1 and Mario Ermani3; 1Medical Oncology, Bellaria-Maggiore Hospital, Azienda USL di Bologna, Bologna, Italy; 2Medical Oncology, Istituto Oncologico Veneto-IRCCS, Padova, Italy; 3Neurosciences Department, Statistic and Informatic Unit, Padova University, Padova, Italy

In the treatment of glioblastoma (GBM) standard approach is temozo-lomide administration, initially concurrent with radiotherapy, and subse-quently as maintenance therapy. In this setting, the first post-radiotherapy radiological assessment is made earlier than before, when radiotherapy was given alone. It can be difficult to interpret the radiological picture obtained, as any changes observed may be due to treatment related pseudo progres-sion (psPD) rather than true disease progression. A prospective analysis was made of newly diagnosed GBM patients treated according to the EORTC 22981/26981/NCIC trial. The first MRI scan was performed 1 month after completion of combined chemo radiotherapy treatment. During follow-up patients underwent MRI every 2 months and treatment was suspended after 12 cycles if MRI was negative or if MRI showed enhancement suggesting tumor. In doubtful cases, MRS or PET 18FDG was performed whenever feasible. 103 patients were analyzed (m/f, 68/35; median age 53 years [range 20–73]); total/subtotal resection/biopsy: 51/52/1 (MGMT1/MGMT– 36/67). After a median follow-up of 16 months (range 4–62), psPD was detected in 32/103 patients (31.1%). Median time to psPD following chemo-radiation was 1.9 (range 0.1–10) months. psPD was recorded in 21/36 (58%) MGMT1 and 11/67 (16%) MGMT– cases (p , 0.0001). Clinical deteriora-tion at the onset of psPD was registered in 11/32 pts (34%), being detected in 5/21 (23.8%) MGMT1 vs. 6/11 (55%) MGMT– cases (p 5 0.09). OS was significantly influenced by MGMT methylation status (38 months in MGMT1 vs. 17 months in MGMT– cases) (p , 0.0001), and by the regis-tration of psPD (62 months vs. 18; p , 0.0001). psPD has a clinical impact on GBM treated with chemo radiotherapy and may express the glioma kill-ing effects of treatment; it is moreover significantly correlated with MGMT status. The early detection of psPD patterns and a better knowledge of the biological mechanisms underlying this phenomenon are crucial to obviating biases in evaluating the results of clinical trials, and to preventing patients from being denied effective treatment.

MA-53. NATURAL HISTORY AND MANAGEMENT OF BRAINSTEM GLIOMAS IN ADULTS: A RETROSPECTIVE ITALIAN STUDYRiccardo Soffietti,1 Laura Fariselli,2 Ida Milanesi,2 Emanuela Lamperti,2 Antonio Silvani,2 Antonio Bizzi,2 Elio Maccagnano,2 Elisa Trevisan,1

Elena Laguzzi,1 Roberta Rudà,1 Amerigo Boiardi,2 and Andrea Salmaggi2; 1Neuro-Oncology, University of Turin, Turin, Italy; 2Neuro-Oncology, Istituto Nazionale Neurologico C. Besta, Milan, Italy

Brain stem gliomas in adults are rare tumors with heterogeneous clinical course, and few studies only are available in the MRI era. In this retro-spective study, we report the clinical characteristics, neuroimaging findings and outcome after treatments of 32 adult patients with either biopsy-proven or radiologically suspected brain stem gliomas from 2 Centers for Neuro- Oncology in Italy. There were 18 males and 14 female with a median age of 31. Twenty-one patients had an histological diagnosis (2 pilocytic astro-cytoma, 9 astrocytoma grade II, 8 astrocytoma grade III, 1 glioblastoma, 1 inconclusive sample). Contrast enhancement on MRI was present in 14 patients; H-MR spectroscopy was performed in 9 patients, with high degree of concordance with the histology; PET with FDG was performed in 8 patients, with some discrepancies with the histology. In 8 patients an initial “watch and wait” policy was adopted, due to the paucity of symptoms and the low grade appearance on MRI, whereas 24 patients underwent an early antineoplastic treatment with either radiotherapy alone (4) or radiotherapy combined with chemotherapy (20) (mostly with temozolomide). We did not observe any complete or partial response on MRI, whereas a minor response or a stable disease was reported in 15/31 pts (48%) after radiochemotherapy and 10/31 pts (32%) after radiotherapy alone. Overall, a significant neuro-logical improvement was observed in 15/31 patients (48%), but in 6 only some degree of radiological improvement was associated. Median TTP was 10 months (35 months in pts with initial watch and wait and 6 months in pts with early treatment), whereas median survival was 59 months (95 months in pts with initial watch and wait and 52 months in pts with early treat-ment). No significant differences in survival emerged between patients with or without an histological diagnosis. After multivariate analysis the time between onset and diagnosis was the sole statistically significant prognostic factor, and a trend for a negative prognostic impact of age . 40 years, a high-grade histology and the presence of necrosis on MRI was evident. when a biopsy cannot be performed in suspected brainstem gliomas, MRS can be

useful to improve the diagnostic accuracy; radiation therapy remains the mainstay of treatment, whereas the efficacy and timing of chemotherapy are to be clarified in prospective multicenter trials.

MA-54. IMPACT OF PHASE II TRIALS WITH PROGRESSION-FREE SURVIVAL AS END-POINT ON SURVIVAL-BASED PHASE III STUDIES IN PATIENTS WITH ANAPLASTIC GLIOMASVictor A. Levin,1 Sandra Ictech,1 and Kenneth R. Hess2; 1Neuro-Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Biostatistics, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Since patients with anaplastic gliomas (AG) have median survival times of 4 to 6 years, Phase III trials can, at times, take a decade to conduct. In order to design and justify shorter more informative trials, we sought to define the potential impact of phase II AG studies on survival-based phase III studies and to probe the utility of progression-free survival (PFS) as an alternative endpoint. Data were compiled from the computerized database of the Department of Neuro-Oncology at The University of Texas M. D. Anderson Cancer Center between 1995 and 2004. The 16 studies inter-rogated represented published and a few unpublished studies (n 5 529) that shared common response criteria. The datasets had similar fields and entries. These data were analyzed to determine PFS at 6, 9, and 12 months and the impact of age, Karnofsky performance score (KPS), number of prior che-motherapies, and response to treatment on PFS. We found that the specific chemotherapy used was the major effecter of PFS at 6, 9, and 12 months. Age, KPS, treatment response rate, and number of prior chemotherapies did not affect PFS to the same extent. Hierarchical cluster analyses and linear least squares fitting of PFS9 versus PFS12 demonstrated the existence of three therapeutic efficacy groups with PFS rates at 6, 9, and, 12 months ranging from lowest (A) to highest (C). The PFS6 was 15% in group A and 41% in group C (p , .0001); the PFS12 was 9% in group A, and 33% in group C (p , .0001). Furthermore, we determined that 80% of patients at recurrence had a 23% likelihood that each chemotherapy regimen would provide greater than 1 year of additional life. Based on PFS rates at 6, 9, and 12 months for AG patients, a differential of 1.5 to 2 years is the norm and could invalidate overall survival as an end-point for phase III studies in patients with AG. Even if a statistical work around could be invoked, a survival-based phase III study of AGs would be prohibitive in length of time to conduct and, therefore, not likely to be supported by pharmaceutical companies. It is concluded that PFS is a reliable end-point that reflects the true antitumor benefit of the chemotherapy being investigated and, there-fore, is more appropriate for phase II and phase III trials in AG patients than survival.

MA-55. PRIMARY CEREBRAL GLIOSARCOMA: THREE CASES FROM A SINGLE INSTITUTIONAngel F. Campos-Gines, Juan Herrada, and Anuradha Gupta; Texas Tech University Health Sciences Center, El Paso, TX, USA

Gliosarcoma is a rare malignant neoplasm of the central nervous system characterized by a biphasic histological pattern, with areas of gliomatous and sarcomatous differentiation. Accounting for about 2%–8% of the glioblas-tomas, gliosarcomas have been classified as a separate clinico-pathological entity (Kleihues et al., J Neuropathol Exp Neurol 61:215225–225, 2002), even though they remain closely related to glioblastomas in terms of clinical features and therapeutic approach. Retrospective review. Medical records, diagnostic imaging procedures, and pathology reports (including GFAP, vimentin, EMA, CD34 and factor VIII immunostains) were reviewed. Three patients (pts) were identified. Pt #1 was a 73-year-old female with a tempo-ral location. Pt #2 was a 44-year-old male with also a temporal location. Both were treated with surgical debulking, postoperative combined temo-zolomide with radiation, followed by six months of temozolomide. Both pts experienced local relapse at 10 and 24 months, respectively, and no addi-tional treatment was given. Both patients died of progressive disease 11 and 28 months after initial diagnosis, respectively. Pt #3 was a 38-year-old male with an occipital location. His treatment consisted of surgical debulking, postoperative radiation, followed by twelve months of procarbazine, lomus-tine, and vincristine. Ten years after diagnosis the pt remains disease-free. Gliosarcoma is a highly aggressive disease. As in glioblastomas, younger age appears to be associated with slightly better prognosis.



MA-56. GEMCITABINE AS RADIOSENSITIZER FOR NEWLY DIAGNOSED GLIOBLASTOMA MULTIFORME (GBM): PRELIMINARY RESULTS OF A PHASE II STUDYAlessandra Fabi,1 Alessandra Mirri,2 Andrea Pace,3 Alessandra Felici,1 Antonello Vidiri,4 Giulio Metro,1 Francesco Cognetti,1 Emanuele Occhipinti,5 Giorgio Arcangeli,2 and Carmine Carapella5; 1Medical Oncology, Regina Elena Nat Cancer Institute, Roma, Italy; 2Radiotherapy, Regina Elena Nat Cancer Institute, Roma, Italy; 3Neurology, Regina Elena Nat Cancer Institute, Roma, Italy; 4Diagnostic Imaging, Regina Elena Nat Cancer Institute, Roma, Italy; 5Neurosurgery, Regina Elena Nat Cancer Institute, Roma, Italy

Gemcitabine is a deoxycytidine analogue with a wide range of antitu-mor activity, presenting powerful radiosensitizing activity at non-cytotoxic concentrations. In malignant glioma few data are presently available on the effects of gemcitabine, with unsatisfactory results as a single antiblastic agent. In a previous phase I study, conducted in our Institution, where fixed dose rate (FDR) gemcitabine at 10/mg/m²/min was tested in association with radiotherapy (RT) for the treatment of newly diagnosed GBM, a maximum tolerated dose of 175 mg/m2/wk was identified. Observed activity has been considered interesting enough to support a phase II study. After surgery for GBM, patients presenting measurable residual tumor were treated with fractionated focal RT at a daily dose of 2.0 Gy per fraction, five days per week for six weeks (total dose of 60 Gys). FDR gemcitabine at 175 mg/m2/wk was given concomitantly starting 24–72 hours prior to RT, and then for the whole duration of RT. MRI evaluation was performed at 7 and 40 days from the end of chemo-radiotherapy for early therapeutic assessment. Standard oral temozolomide 150–200 mg/m² was administrated follow-ing the combined experimental treatment, at least until tumor progression or relevant side effects. Tumor response rate, progression free survival, and overall survival time have been considered as main objectives. From 07/2004 19 patients (11 male, 8 female) have been enrolled. Characteristics of patients were: median age 56 years (42–72), median KPS at baseline 90 (70–100), surgery/biopsy 16/3. Median time from diagnosis to the initia-tion of gemcitabine was 45 days (28–54). Among the 14 valuable patients 3 (21.4%) partial responses, 7 (50%) stable disease, and 4 (28.5%) progres-sive diseases were recorded. At a median follow-up of 18 months (2–33) time to tumor progression was 6 months (1.5–24). Toxicity was manageable with only one G3 neutropenia and hypertransaminasemia in two patients respectively. Grade 1 hypertransaminasemia was registered in 6 patients (43%). These preliminary results show that in patients with newly diagnosed GBM, radiosensitizing FDR gemcitabine at 175 mg/m2/wk is a well tolerated regimen with an interesting activity. Accrual is ongoing and more extensive results will be presented.

MA-57. A RANDOMIZED PHASE II TRIAL OF CONCURRENT TEMOZOLOMIDE (TMZ) AND RADIOTHERAPY (RT) FOLLOWED BY DOSE-DENSE COMPARED TO METRONOMIC TMZ AND MAINTENANCE CIS-RETINOIC ACID FOR PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA MULTIFORME (GBM)Jennifer Clarke,1 Joohee Sul,1 Lisa DeAngelis,1 Andrew Lassman,1 Adilia Hormigo,1 Craig Nolan,1 Igor Gavrilovic,1 Bin Gu,2 Kathy Panageas,2 and Lauren Abrey1; 1Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA; 2Memorial Sloan-Kettering Cancer Center, New York, NY, USA

Metronomic and dose-dense scheduling are alternatives to conventional TMZ regimens designed to overcome drug resistance in part by depleting O6-methylguanine-DNA methyltransferase (MGMT). Metronomic TMZ may also inhibit endothelial recovery and act as an anti-angiogenic therapy; dose-dense TMZ increases the intensity of drug delivery. This phase II trial evaluates two alternative TMZ dosing schedules for newly diagnosed, histologically confirmed GBM. All patients receive standard radiotherapy with concurrent daily TMZ (75 mg/m²). For adjuvant therapy, patients are randomized to dose-dense TMZ, at 150 mg/m² days 1–7 and 15–21 out of a 28-day cycle, or metronomic TMZ with 50 mg/m² daily in a continuous fashion. Upon completion of six months of adjuvant TMZ, patients are prescribed maintenance oral cis-retinoic acid. Each arm of the study was designed as a two-stage Simon minimax study. If either arm shows 70% survival probability at one year, further evaluation in a phase III trial will be recommended. Correlative tissue analysis of MGMT status is planned from prospectively collected samples. Primary outcome is overall survival (OS); secondary objectives include progression-free survival (PFS), the prognostic impact of methylated MGMT status, and preliminary data on the efficacy of this regimen and impact of MGMT status in other malignant glioma sub-types. Of a planned 78 GBM patients, 67 have started treatment to date. 32 of the patients with GBM have been randomized to metronomic and 35 to dose-dense TMZ. Median age is 54, and median KPS is 90. Median PFS for the entire GBM cohort is 5.8 months. Median PFS for the metronomic and dose-dense cohorts are 4.4 months and 6.6 months, respectively. Median OS has not yet been reached; 19% (13 of 67 patients) have died, and median follow-up of survivors is 7.5 months. 14 of a planned 20 patients with grade

III tumors have begun treatment. Of these, 11 are anaplastic astrocytoma and 3 are anaplastic oligodendroglioma. 7 have been randomized to dose-dense treatment and 7 to metronomic treatment. Median OS and PFS have not yet been reached; 14% (2 of 14 patients) have died with a median fol-low-up of survivors of 6.3 months to date. The most common grade 3 or 4 toxicities among the GBM patients were hematologic (leukopenia, 13%; thrombocytopenia 6%). The most significant grade 3 nonhematologic toxic-ity was hepatic (13%); 7% had thrombotic events, 3% had infections, and 6% had grade 3 or higher fatigue. Of the entire 81 patient cohort, 8 patients have received treatment with cis-retinoic acid; 4 were discontinued for pro-gression of disease, 2 were discontinued due to side effects, and 2 remain on therapy. One additional patient was eligible for cis-retinoic acid but refused. Our patient population is comparable to that of other upfront GBM treat-ment trials. Both metronomic and dose-dense TMZ regimens appear to be well tolerated with modest toxicities, though hematologic toxicity is higher than with the Stupp regimen. In early analysis, both arms appear to be equally effective.

MA-58. A PHASE II SAFETY STUDY OF BEVACIZUMAB IN PATIENTS WITH MULTIPLE RECURRENT OR PROGRESSIVE MALIGNANT GLIOMASJeffrey Raizer,1 Lilia Gallot,2 Robert Levy,3 Christopher Getch,3 Ann Mellot,3 Steven Newman,4 Claudia Tellez,4 Hunt Batjer,3 Maryanne Marymont,5 and James Chandler3; 1Neurology, Northwestern University, Chicago, IL, USA; 2Northwestern University, IL, USA; 3Northwestern University, Chicago, IL, USA; 4Chicago, IL, USA; 5Radiation Oncology, Northwestern University, Chicago, IL, USA

Limited effective therapies are available for patients (pts) with malig-nant gliomas at relapse. Target specific agents maybe more effective with less systemic toxicity but none are effective to date. A single agent trial was designed to determine the safety and efficacy of Bevacizumab in pts with recurrent MG after 2 relapses. The study eligibility was modified after the first 16 pts to allow first relapsed patients to enroll. All pts had to sign an IRB informed consent. The first 16 pts had to have at least two relapses; there after only 1 relapse was required. Patients had to be . 18 year of age with Karnofsky Performance Status of . 60. Adequate bone marrow, liver, and renal function was required, as well as normal urine protein to creatinine ratio. Although an interaction with enzyme inducing anti-convulsants was not anticipated, all patients were required to be on a non-enzyme inducing anti-convulsants. An MRI with perfusion was done at baseline (if patient consented) and then every 6 weeks. Patients received Bevacizumab 15 mg/kg every 3 weeks as a 60–90 minute infusion. Patients continued on trial until tumor progression assessed by imaging or clinical deterioration. To date, 26 pts with recurrent MG have been treated. 23 pts had a glioblastoma and 3 had anaplastic gliomas (2, oligodendroglioma and 1 astrocytoma). Median number of prior chemotherapies was 2 (range 1–5); 8 pts had 3 or more chemotherapies. A total number of 111 doses have been given. The median number of doses was 2 (range 1–18). No patient had an intracranial hemor-rhage and the only significant toxicities were a DVT in a patient with prior DVT and an intestinal perforation. Best Responses per McDonald Criteria were: PR in 2 pts, SD in 5 pts, PD in 8 pts, and non-evaluable in 11 pts: 6 pts are too early to evaluate, 2 declined before first follow up MRI, 1 each was removed from trial after 2, 2, and 3 doses with stable MRIs for non-compliance, clinical decline and patient wishes. Only 2 out of 26 pts treated with single agent Bevacizumab had a significant toxicity. Partial responses and stable disease were seen in about 30% of patients but many patients are not evaluable yet. Our response rates to date are lower then previous reports of patients treated with CPT-11 and Bevacizumab. One reason for this dis-crepancy may be how heavily pre-treated out initial 16 pts were. We may see improved activity in patients recruited after first relapse. Alternatively, the schedule we used and the absence of combined chemotherapy may have also lead to a lower response rate. Matured data including PFS at 6 months and overall survival will be presented and will also compare outcomes of less heavily pre-treated patients.

MA-59. DELAYED LEUKOENCEPHALOPATHY WITH STROKE-LIKE PRESENTATION IN CHEMOTHERAPY RECIPIENTSJoachim Baehring1 and Robert Fulbright2; 1Neurology and Neurosurgery, Yale University, New Haven, CT, USA; 2Diagnostic Radiology, Yale University, New Haven, CT, USA

A transient leukoencephalopathy (LEP) mimicking cerebrovascular acci-dent has been described as a complication of chemotherapy, most commonly in recipients of intrathecal methotrexate (MTX) for childhood leukemia. Recently published neuroimaging data suggest a common pathophysiology associated with a variety of chemotherapy agents and modes of admin-istration. We will refer to the syndrome as delayed LEP with stroke-like presentation (DLEPS). We reviewed the PubMed database and the regis-



try of the Yale Brain Tumor Center using the following search terms in various combinations: diffusion-weighted MRI, DWI, apparent diffusion coefficient map, ADC, leukoencephalopathy, methotrexate, capecitabine, 5-fluorouracil, carmofur, and tegafur. In order to be included, patients had to have a clinical syndrome characterized by acute onset and focal or diffuse central neurological deficits in combination with new onset of leukoenceph-alopathy displaying decreased proton diffusion on diffusion weighted MRI (DWI and ADC maps). The vast majority of patients presented with focal neurological signs and symptoms (hemiparesis [15], dysphasia [9], hemidy-saesthesia [3], cerebellar dysfunction [4], subcortical and brainstem signs [dysarthria (5), dysphagia (1), dysphonia (1), and pseudobulbar affect (1)]). We identified reports of DLEPS in recipients of intrathecal methotrexate (n 5 18), intravenous methotrexate (1), 5-fluorouracil (5-FU; 2), carmo-fur (1), and capecitabine (5). Among patients treated with MTX, median age was 14 years (range 6 to 20) and female to male ratio was 1:1. DLEPS occurred between six hours and 11 days after chemotherapy administration. All patients recovered within hours to one month. DLEPS on capecitabine chemotherapy occurred between 3 to 7 days of treatment initiation (age range 40–74). All patients were female, suffered from breast (4) or pancre-atic cancer, and recovered within days of discontinuation of capecitabine. Abnormalities of proton diffusion on DWI and ADC maps were consis-tent with reduced water diffusion rates, as the lesions were hyperintense on DWI and hypointense on ADC maps (21/27 patients). ADC maps were not reported in six patients. Diffusion abnormalities almost invariably involved the white matter of the cerebral hemispheres and were reversible in all cases in which follow-up imaging data were available except for the patient with carmofur toxicity. MRI abnormalities were unilateral in 5 and bilateral in 21 (one unknown). MRI scans obtained between 2 weeks and 6 months in six patients after DLEPS revealed residual FLAIR/T2 signal abnormalities in five and normalization in one. DLEPS has been described after chemo-therapy with methotrexate, 5-FU, capecitabine, and carmofur. It is charac-terized by an acute stage defined by decreased proton diffusion on MRI. It remains unknown if there is a unifying neuropathological correlate. Such has been described in animal models of 5-FU neurotoxicity and consists of intramyelinic sheath edema and formation of vacuoles. Reversibility of markedly decreased ADC values without clinically apparent permanent defi-cit is in striking contrast with findings in acute ischemia. Recognition of the clinical syndrome supported by a comprehensive neuroimaging evaluation can spare the patient from unnecessary invasive procedures or therapies. Interruption of exposure to the suspected toxin may be crucial in order to prevent permanent neurological injury.

MA-60. CYTOREDUCTIVE CHEMOTHERAPY PRIOR TO DEFINITIVE RESECTION IN PATIENTS WITH PINEAL PARENCHYMAL TUMORSMilan G. Chheda,1 Frederick G. Barker,2 David H. Ebb,3 David N. Louis,4 and Tracy T. Batchelor5; 1Department of Neurology, Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, MA, USA; 2Department of Neurosurgery, Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, MA, USA; 3Department of Pediatrics, Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, MA, USA; 4Department of Pathology, Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, MA, USA; 5Departments of Neurology and Radiation Oncology, Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, MA, USA

While chemosensitive solid tumors of the body may be treated with chemotherapy prior to definitive resection, chemosensitive brain tumors are usually resected prior to chemotherapy. However, based on tumor location and size, cytoreduction with chemotherapy may allow more definitive resec-tion in some cases. Prospective case series. Three patients with potentially chemosensitive pineal tumors received chemoreduction prior to resection. Case 1 was a 49-year-old woman who presented with a 4.6 3 3.7 3 3.9 cm3 pineoblastoma; after two cycles of cisplatin, cyclophosphamide, and vincristine the tumor decreased in size to 4 3 3 3 3.3 cm3 and she under-went gross total resection. The pathology specimen revealed densely packed small blue cells with mitoses; while there were apoptic bodies and foci of necrosis, much of the tumor consisted of viable-appearing tumor. Case 2 was a 34-year-old woman who presented with a 3.2 3 2.8 3 3.0 cm3 pineal parenchymal tumor of intermediate differentiation; after several cycles of chemotherapy, its volume decreased to 2 3 2 3 2.5 cm3 and she underwent a gross total resection. The pathology specimen revealed one small focus of viable tumor with the vast majority of the tumor composed of collagen and scattered atypical cells. Case 3 is a 21-year-old who presented with a 2.5 3 2.3 3 4.1 cm3 pineal parenchymal tumor of intermediate differentia-tion who is currently receiving cisplatin, cyclophosphamide and vincristine chemotherapy. Cases 1 and 2 are alive and without disease progression at 9 months and 8.1 years, respectively, from the time of their resections. Follow-up data on Case 3 will be available at the time of presentation. For some chemosensitive pineal tumors, cytoreduction with pre-surgical chemo-therapy may allow safer, more definitive surgical resections.

MA-61. RESULTS FROM PRECISE: A RANDOMIZED PHASE 3 STUDY IN PATIENTS WITH FIRST RECURRENT GLIOBLASTOMA MULITFORME (GBM) COMPARING CINTREDEKIN BESUDOTOX (CB) ADMINISTERED VIA CONVECTION-ENHANCED DELIVERY (CED) WITH GLIADEL WAFERS (GW)Sandeep Kunwar,1 Manfred Westphal,2 Maximillian Medhorn,3 Zvi Ram,4 John Sampson,5 Richard Byrne,6 Frederick Lang,7 Stephen Tatter,8 Joseph Piepmeier,9 Gene Barnett,10 Abhijit Guha,11 Jan Mooij,3 Michael Vogelbaum,12 Susan Chang,1 David Croteau,13 Jeffrey Sherman,14 and Raj Puri15; 1University of California, San Francisco, San Francisco, CA, USA; 2Universitatsklinikum Hamburg-Eppendorf, Germany; 3Germany; 4Tel-Aviv Medical Center, Tel-Aviv, Israel; 5Duke University, Durham, NC, USA; 6University of Massachusetts at Boston, Boston, MA, USA; 7University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 8Wake Forest University, Winston-Salem, NC, USA; 9Yale University, New Haven, CT, USA; 10Cleveland Clinic Foundation, OH, USA; 11Neurosurgery and Cell Biology, Toronto Western Hospital & Hosp. for Sick Children Res. Ins., Toronto, Ontario, Canada; 12Brain Tumor and Neuro-Oncology Center, Cleveland Clinic Foundation, Cleveland, OH, USA; 13IL, USA; 14Waukegan, IL, USA; 15Bethesda, MD, USA

CB is a recombinant protein consisting of interleukin-13 (IL13) and truncated Pseudomonas exotoxin. CB binds selectively to its molecular tar-get, the IL13Ra2 receptor, which is over-expressed on malignant glioma cells. CED uses positive pressure infusion to achieve loco-regional delivery of therapeutic agents via intracerebral catheters. Despite aggressive mul-timodal treatment, patients with recurrent GBM have a median survival of approximately 6 months. Adults with GBM at first recurrence who met eligibility criteria were enrolled; additional criteria included tumor size [?] 1.0 cm, planned gross total resection > 95%, KPS > 70, and adequate hematologic status. Patients were randomized 2:1 to receive CB or GW. CB (0.5 mcg/mL; total flow rate 0.75 mL/hr) was administered over 96 hours via 2–4 intraparenchymal catheters placed 2–7 days after tumor resection. GW (3.85%/7.7 mg carmustine per wafer; up to 8 wafers per patient) was placed immediately after tumor resection. The primary endpoint was over-all survival from time of randomization. Secondary and tertiary endpoints were safety and health-related quality of life assessments. 276 intent-to-treat (ITT) patients were enrolled at 52 centers in North America, Europe, and Israel. Demographic and baseline characteristics were balanced between the treatment arms. Median survival was 36.4 weeks for CB and 35.3 weeks for GW (p 5 0.476) with a hazard ratio (HR) of 0.89 (95% CI: 0.67 to 1.18) in favor of CB (p 5 0.416). For the efficacy evaluable population which included patients in the ITT group who had histopathologic confirmation of viable glioma consistent with recurrent/progressive GBM from the central pathology review, met all eligibility criteria, underwent gross total resec-tion of the tumor, and received at least 90% of the planned dose of study drug, the median survival was 45.3 weeks for CB and 39.8 weeks for GW (p 5 0.310) with a hazard ratio (HR) of 0.81 (95% CI: 0.67 to 1.18) in favor of CB (p 5 0.234). The difference in median survival further increased when sites with > 6 ITT patients were considered, with 46.8 weeks for CB vs. 41.6 weeks for GW (p 5 0.288), and a HR of 0.77 in favor of CB (p 5 0.163). Median progression-free survival (PFS) for the ITT population was 17.7 vs. 11.4 weeks in favor of CB (p 5 0.008). Adverse events profile was similar in both arms, except pulmonary embolism was higher in the CB arm (8% vs. 1%, p 5 0.014) which can be addressed with preventive measures. CB appears to be at least comparable to GW in terms of effi-cacy. Neurosurgeon experience may enhance the degree of CB benefit in the future. Overall, CB is an active therapeutic agent with an acceptable safety profile and consequently a favorable benefit-risk ratio.

MA-62. INTRACRANIAL ANAPLASTIC EPENDYMOMAS IN ADULTS: A RETROSPECTIVE REVIEWPatricia S. Ritch, Michael Edgeworth, and Paul Moots, Vanderbilt University Medical Center, Nashville, TN, USA

The majority of adult ependymomas are well circumscribed masses aris-ing in the spinal cord. These tumors can sometimes be completely resected effecting a cure, albeit with residual deficits resulting from the surgical inter-vention. Intracranial ependymomas in adults are less common, therefore there is less clinical experience and evidence to help guide the patient and their physician in choosing appropriate treatment options once the diagnosis has been established. Most physicians rely on the results from additional studies such as MRI, CSF cytology, and tissue pathology prior to making their recommendations. The focus of this study was to review our experi-ence with the most aggressive form of intracranial ependymoma, anaplas-tic ependymoma (grade III, WHO classification) with a goal of providing clinical support for the recommendation of adjunctive radiation and chemo-therapy following tumor resection. Our analysis revealed only eight cases of anaplastic ependymoma over the past 15 years. In all patients, surgical resection was followed by radiation and chemotherapy.



MODELS, IN VITRO AND IN VIVO

MO-01. ROLE OF ACTIVATED STAT3 IN THE PROGRESSION OF GBM GROWTH IN MICEAtreyi Dasgupta,1 Baisakhi Raychaudhuri,2 Talat Haqqi,2 Richard Prayson,3 Erwin Van Meir,4 Michael Vogelbaum,2 and S. Jaharul Haque1; 1Cancer Biology, Cleveland Clinic Foundation, Cleveland, OH, USA; 2Cleveland Clinic Foundation, Cleveland, OH, USA; 3Neuropathology, Cleveland Clinic Foundation, Cleveland, OH, USA; 4Emory University, Atlanta, GA, USA

We have previously found that activated Stat3 plays roles in the pro-liferation and survival of GBM cells in vitro. To understand the role of activated Stat3 in the growth of GBM in vivo, we have generated U87-derived stable cell lines expressing varying levels of a dominant negative mutant (DN)-Stat3 in a hypoxia-inducible fashion. Hypoxia, necrosis and micro-vascular hyperplasia are successive events in the progression of GBM growth. We found that palpable tumors were formed by DN-Stat3 express-ing U87 cell lines within two weeks but growth of the tumors was halted once they reached ~2 mm in thickness, whereas the parental and vector control U87 cell line-derived tumors maintained rapid growth rates. The survival of mice bearing DN-Stat3–expressing U87-derived orthotopic tumors was significantly longer than the control groups, as evident from Kaplan-Meier analysis. Limited numbers of proliferating cells and signifi-cant numbers of apoptotic cells were found in flank tumors derived from DN-Stat3 expressing U87 cells, compared with control groups. In addition, new vessel formation was almost completely compromised in the DN-Stat3 expressing U87 tumors, which is in agreement with the observed inhibition of VEGF promoter activity by DN-Stat3 expression in hypoxic U87 cells, in vitro. These data suggest that activated Stat3 plays an essential role in the progression of GBM growth, by suppressing apoptosis and inducing angio-genesis. Supported by NIH R01 grants CA095006 to S.J.H. and CA87830 and CA86335 to E.G.V.M.

MO-02. PDGFB CONTRIBUTES TO THE INITIATION OF GLIOMAYasuyuki Hitoshi,1 April Kemper,2 Brian Popko,3 Brent Harris,1 and Mark Israel1; 1Norris Cotton Cancer Center, Lebanon, NH, USA; 2Pathology, Wake Forest University, Winston-Salem, NC, USA; 3Neurology, The Jack Miller Center for Peripheral Neuropathy, Chicago, IL, USA

Human platelet-derived growth factor B (hPDGF-B) has been exten-sively characterized in culture-based assays, where it has been shown to mediate numerous cellular responses including proliferation, survival, migration and the differentiation of glial, meningeal, and vascular cells. Overexpression of hPDGF and hPDGF receptors is detectable in many glial tumor cell lines and pathological surgical samples from human glioma patients. Also, extensive data point to the autocrine growth stimulation of glioma cells by PDGF. Expression of hPDGFB following the inoculation of recombinant retroviruses into the brains of normal mice is associated with the development of brain tumors that resemble either human glioblastoma multiforme or oligodendroglioma. Retroviral insertional mutagenesis has evaluated by others in these tumors utilizing retroviral tagging, and several common insertion sites were determined to mark genes implicated in known cancer- associated pathways. To examine whether PDGF was sufficient to initiate tumorigenesis, we created conventional transgenic mice that over-expressed hPDGFB in GFAP expressing cells. Two lines of the transgenic mice were created. One in which the human GFAP 2.2kb promoter was used to drive human hPDGFB production (GFAP-PDGFB) and another in which a tetracycline responsive promoter drives hPDGFB expression (TRE-hPDGFB). TRE-hPDGFB mice were mated with GFAP-tTA mice so that hPDGFB would be expressed in GFAP expressing glia in a manner respon-sive to doxycycline administration. We found that GFAP-hPDGFB mice developed malignant neoplasms resembling mixed oligoastrocytoma mainly in their spinal cord and occasionally in their brain, while GFAP-tTA:TRE-hPDGFB double transgenic mice developed oligodendroglioma in their spi-nal cords and brain. Tumor development in GFAP-tTA:TRE-hPDGFB mice was suppressed by doxycycline provided in the drinking water. Currently we are trying to identify if any reversal effect to the tumor growth could be induced by doxycycline as an evidence of ability to sustain tumor growth by PDGFB. This research was funded in part by the Theodora B. Betz Founda-tion (MAI) and NS034939 (BP).

MO-03. DE NOVO INDUCTION OF CENTRAL NERVOUS SYSTEM TUMORS USING THE SLEEPING BEAUTY TRANSPOSON FOR SOMATIC CELL GENE TRANSFERStephen Wiesner,1 Zachary Demorest,2 Walter Low,3 David Largaespada,4 and John Ohlfest5; 1Center for Allied Health Professions, University of Minnesota, Minneapolis, MN, USA; 2University of Minnesota, Minneapolis, MN, USA; 3Neurosurgery, University of Minnesota, Minneapolis, MN, USA; 4Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA; 5Neurosurgery, Pediatrics, University of Minnesota, Minneapolis, MN, USA

Animal models are powerful tools for translational research. Currently, there are basic two approaches to the study of central nervous system tumors in mice; implantation of tumor cells or in situ development of tumors in genetically modified strains of mice. Each has advantages and disadvan-tages. Human xenografts in immune deficient animals can facilitate the study of tumor biology and preclinical trials of drugs that target human signal transduction pathways. However, these models cannot account for the interaction of tumor cells with the host immune system. Even in synge-neic models, surgical tumor implantation causes disruption of normal host tissue, potentially confounding the study of tumor immune infiltrates. The development of genetically modified strains (GEMs) of mice in which the tumor develops in situ is an alternative model to study tumor biology. How-ever, combination of genetic elements from various GEMs is costly and time consuming. Not all of the offspring in any given cross may be useful, partic-ularly when more than two genetic elements are required to test the hypoth-esis. The tumors often develop with mixed penetrance and high throughput quantitation of tumor burden can be challenging. In addition, the tumors that develop may not express human-specific mutations that are targeted by drug and immunotherapy. In order to accelerate the study of tumor biol-ogy and high throughput preclinical trials, we have developed a technique allowing rapid induction of tumors in wild type mice using simple nonviral gene transfer. Polyethylenimine (PEI)/DNA complexes preferentially trans-fect progenitor cells in the mouse subventricular zone (SVZ). We enhanced the ability of these complexes to transfect cells in the SVZ and deeper into the parenchyma by pretreatment with mannitol (10 ml 25% mannitol/g) prior to injection of PEI/DNA complexes (1 mg total DNA/mouse) into the lateral ventricle of postnatal day one neonatal mice. Long-term expression of the transgene was achieved by using the Sleeping Beauty (SB) transposable element, which mediates integration of a transgene from plasmid DNA into the host cell genome by a “cut and paste” mechanism. We co-transfected the neonatal SVZ with three plasmids: a transposon encoding a mutant NRAS oncoprotein (G12V; provides continual pro-growth signal) and SV40LgT antigen (inhibits the Rb and p53 tumor suppressor function), along with a third vector encoding both firefly luciferase within a transposon (for biolu-minescence) and SB transposase (mediates integration). Tumors graded as malignant glioma developed within three weeks and progressed rapidly, as indicated by in vivo bioluminescence imaging. The location and intensity of bioluminescence was predictive of the location and size of the tumor. Tumor burden was detectable by imaging before overt symptoms of disease were present in most animals. The technique is highly penetrant, with all of the animals treated with both LgT and NRAS12 developing tumors. None of the control animals receiving either LgT or NRAS12 alone developed tumors. This technique enables de novo development of genetically relevant central nervous system tumors in immune competent mice. The approach is rapid, simple, and scalable for high throughput screening of genetic interactions, pharmacologic targets and therapeutically relevant molecules expressed in the tumor. Experiments may be performed in any mouse strain, including wild type animals and GEMs. Addition of newly defined genetic elements is rapid and simple by virtue of plasmid DNA preparation, without the labor-intensive development of new GEMs or viral vectors. These features make non-viral somatic cell gene transfer for tumor initiation a powerful tool for the study of central nervous system tumors. In combination with existing GEMs, this technique can rapidly advance our understanding of the genetic mechanisms of gliomagenesis and increase the capacity to study genetically distinct tumors in the mouse.

MO-04. AN IN VIVO MODEL OF CNS LYMPHOMAGeorgia Panagiotakos, Rory Abrams, Jayanthi Menon, George Alshamy, and Viviane Tabar; Memorial Sloan-Kettering Cancer Center, New York, NY, USA

Central Nervous System Lymphoma (CNSL) is a highly aggressive malignancy typically originating from B cells. This tumor exists in two forms within the CNS, either as a primary tumor originating within the CNS itself or as a systemic tumor resulting from metastasis in patients with uncontrolled systemic disease. To date, there exists no appropriate preclini-cal models of CNSL. This is largely due to the difficulty in obtaining enough primary patient tissue from these tumors, in addition to the inability to maintain lymphoma cells ex vivo for extended time periods. Current models make use of immortalized cell lines that can acquire numerous mutations in culture. Here we propose a long-term in vivo xenograft model for CNSL



that accurately recapitulates the infiltration pattern and phenotypic charac-teristics of human CNSL. Human primary and systemic B cell CNS lympho-mas were obtained intraoperatively according to institutional guidelines. Tumors were carefully dissected into explants measuring an average of 0.8 mm3 and surgically implanted into the striatum of immunodeficient mice (n 5 10). Mice were sacrificed at 8 weeks following transplantation and brains processed for histological analyses. Similar to the in vivo behavior of these tumors in patients, transplanted lymphoma cells formed a large tumor core with significant infiltration into the meninges and choroid plexus. Individual tumor cells were observed migrating across the corpus callosum and could be found at distances far from the injection site. Transplanted tumors also exhibited perivascular cuffing, a clustering of cells around blood vessels (stained for von Willebrand Factor) common to CNS lymphomas. As in the parent tumors, transplanted lymphoma cells expressed Bcl-6 (a marker for the B cell germinal center) as well as CD20 (B cell marker) widely. We also detected the presence of proliferating cells, as indicated by immunostain-ing for Ki67 (MIB-1), and observed significant expression of MMP-2, a potential indicator for enhanced tumor infiltration in lymphomas. Cytoge-netic studies of chromosomal rearrangements and abnormalities are ongo-ing. Future studies will also explore the feasibility of using this model for therapeutic targeting and a better understanding of key pathways critical for tumor maintenance and propagation. Our preliminary data suggest this to be a relevant preclinical model of CNS lymphomas with apparent repro-duction of main tumor characteristics and behavior. This is particularly relevant in the absence of appropriate genetic models and the difficulties in maintaining unaltered lymphoma cells in vitro.

MO-05. BIOLUMINESCENCE IMAGING IN BRAIN TUMOR—A POWERFUL TOOL IN DRUG DISCOVERYPatrizia Tunici,1 Cinzia Giordano,2 Massimiliano Salerno,1 Laura Calderan,3 Marco Rossi,1 Pasquina Marzola,3 Elena Nicolato,3 Federico Boschi,3 Andrea Sbarbati,3 Giovanni Gaviraghi,2 and Annette Bakker4; 1Siena Biotech, Siena, Italy; 2Siena, Italy; 3Dep Morphol & Biomed Sciences, University of Verona, Italy; 4Cancer Biology, Siena Biotech, Siena, Italy

Primary brain tumors, including gliomas, often represent the most dev-astating and difficult to treat tumors. Although enormous advantages in treating other solid tumors highlighted the last decade, the median survival of glioblastoma (GBM) remained the same over the last decades, averaging approximately 1 year. Our paramount inability to successfully treat brain cancer mostly stems from the lack of understanding of the underlying brain tumor biology. However, recent advances in laboratory techniques and the successful development of disease relevant brain tumor models may help the identification of relevant therapeutic targets and may provide an amenable field for testing and kinetic follow up of novel therapeutic agents. In the cur-rent study we successfully developed a GBM model in vivo which mimics its human counterpart. To visualize the tumors, we made use of a highly valu-able, recently developed technology to non-invasively image brain tumor development in living animals, known as in vivo bioluminescence imag-ing. This imaging method is based on light-emitting enzymes, luciferases, which require specific substrates for light production. When linked to a specific biological process/pathway in an animal model of human disease, the enzyme-substrate interactions become biological indicators that can be studied. Aiming at maximal exploitation of different imaging modalities (MRI, bioluminescence imaging and histology) we have validated the use of bioluminescence imaging to monitor glioblastoma progression in vivo. Typically, we have used glioblastoma cells derived from invasive recur-rent tumors. In these cells pathway RE-luciferase constructs were stably expressed. After their orthotopic implantation, the use of bioluminescence technology to kinetically follow tumor growth has been counter-validated using the classical Magnetic resonance imaging on the same animals. Con-trast enhanced MRI allowed us to show the existence of heterogeneous areas in the tumors. Subsequently complete molecular characterization of this new GBM model has been performed.

MO-06. CHARACTERIZING THE EFFECTS OF PDGF ON GLIAL PROGENITOR MIGRATION AND PROLIFERATION IN SLICE CULTUREMarcela Assanah,1 Satoshi Suzuki,2 Amy Chen,1 Jeffrey Bruce,1 James Goldman,3 and Peter Canoll3; 1Columbia University, New York, NY, USA; 2Naka-gun, Japan; 3New York, NY, USA

Previous studies have shown that PDGF can induce glial progenitors to form tumors that closely resemble human gliomas. In this study we used time-lapse microscopy to monitor the effects of PDGF on the migration and proliferation of glial progenitors in slice cultures generated from retrovirus-infected brains. This analysis provided the first dynamic view of how PDGF drives normal glial progenitors to form diffusely infiltrating gliomas. To characterize the short-term effects of PDGF stimulation, a retrovirus that

expresses GFP (pNIT-GFP) was injected into the subventricular zone of neonatal rat pups (P3). The rats were sacrificed at 3 days post-injection (dpi) and 300 um thick slice cultures were generated in the coronal plane at the level of injection. Time-lapse analysis was performed for up to 13.5 hours, then PDGF-B (100 ng/ml) was added to the slice culture media, the slices were allowed to equilibrate and the slices were again filmed for up to 13.5 hour. To characterize the long-term effects of PDGF stimulation, rat pups injected with retroviruses that express PDGF-IRES-DsRed and pNIT-GFP (alone or in combination). At 10 days post-injection the animals were sacrificed, sliced cultures were prepared and time-lapse was performed. The migration of individual cells was tracked and speed and directionality were measured using the DIAS dynamic image analysis system (Soll Technolo-gies, Inc.) The time-lapse analysis also allowed us to directly monitor cell division and the number of cells that underwent mitosis was counted for each condition. Several hundred cells from three or more slices (generated from separate experiments) were analyzed for each condition. Statistical analysis (ANOVA) was performed using Instat and SPSS software. Results At three days post-injection with pNIT-GFP many GFP1 glial progenitors had migrated into the overlying white matter and cortex and started to dif-ferentiate into astrocytes and oligodendrocytes. Time-lapse microscopy showed that only a small subset of GFP1 cells in the white matter were migrating. Mitotic activity was rare. Treating the slices with PDGF-B (100 ng/ml) induced a marked increase in the percentage of cells migrating in the white matter. The PDGF treated cells also migrated significantly faster with significant increases in both the mean and maximum speeds. PDGF also caused a marked increase in proliferation of GFP1 cells, with many migrating cells proliferating en route. By 10 dpi with pNIT-GFP alone the majority of GFP1 cells had differentiated into mature glia and time-lapse microscopy showed that these cells were no longer migrating or proliferat-ing. In contrast, brains injected with PDGF-IRES-DsRed and pNIT-GFP had formed large tumors composed of DsRed1 and GFP1 cells. Time-lapse analysis showed that both populations were highly migratory and prolifera-tive and migrated with similar speeds and directionality. Conclusions These results show that PDGF is a potent mitogen and motogen for glial progeni-tors. Furthermore, constitutive expression of PDGF keeps glial progenitors migrating and proliferating for an extended time and this leads to the rapid formation of infiltrating gliomas.

MO-07. STROMAL INFLUENCES ON LOW-GRADE GLIOMA GROWTHGirish C. Daginakatte and David H. Gutmann; Neurology, Washington University, St. Louis, MO, USA

Recent studies in other cancers have implicated the tumor microenviron-ment in modulating tumor formation and progression. In this regard, cells in the local tumor environment (stroma) provide both negative and positive sig-nals that influence tumor cell growth. We have developed a model system in which to examine the interaction between stroma-derived signals and low-grade glioma growth using mice genetically-engineered with neurofibroma-tosis-1 (Nf1) inactivation in glial cells. Nf1 loss in glial cells is insufficient for glioma formation; however, as in children with NF1, optic gliomas form in mice when Nf1 loss in glia is coupled with a microenvironment composed of cells heterozygous for a targeted Nf1 mutation (Nf11/– cells). Using NF1 as a model system, we show that Nf11/– microglia are stromal cell types that promote the proliferation of Nf1-deficient astrocytes. We further identify one soluble factor (MGEA5; hyaluronidase) produced by Nf11/– micro-glia that promotes Nf1–/– astrocyte growth in MAPK-dependent manner in vitro. Lastly, we demonstrate that inhibition of microglia activation in genetically-engineered Nf1 mice significantly reduces mouse optic glioma proliferation in vivo. Collectively, these studies identify Nf11/– microglia as an important stromal cell type that promotes Nf1–/– astrocyte and optic glioma growth relevant to the pathogenesis of NF1-associated brain tumors, and suggest that future brain therapies might be directed against paracrine factors produced by cells in the tumor microenvironment.

MO-08. PDGF DRIVES NEONATAL GLIAL PROGENITORS TO FORM MALIGNANT GLIOMAS VIA AUTOCRINE AND PARACRINE SIGNALINGMarcela Assanah,1 James Goldman,2 Jeffrey Bruce,1 and Peter Canoll2; 1Columbia University, New York, NY, USA; 2New York, NY, USA

In a previous study we showed that infecting glial progenitors in the adult white matter with retroviruses that express high levels of PDGF induced the rapid and consistent formation of tumors that closely resemble human glioblastomas. The tumors were composed of a mixture of retro-virus-infected cells that expressed PDGF and uninfected glial progenitors that were recruited to proliferate via paracrine growth factor signaling. In this study, we tested the effects of the same retroviruses when injected into the neonatal SVZ. As with adults, malignant gliomas formed that were composed of a mixture of infected and uninfected (recruited) progenitors.



To test the tumorigenic potential of the recruited progenitors we separated these 2 populations via FACS and reinjected them into naïve rats. DsRed1 cells formed new tumors whereas the GFP1 cells did not form tumors unless they were co-injected with DsRed cells. Retroviruses that express PDGF-IRES-GFP or PDGF-IRES-DsRed and pNIT-GFP were stereotacti-cally injected into the SVZ of neonatal rats. Animals were sacrificed and analyzed by immunohistochemistry at several time-points post injection. Some animals co-injected with PDGF-IRES-DsRed and pNIT-GFP were allowed to survive until they showed signs of tumor induced morbidity and these were used for tumor cell isolation. Isolated cells were sorted using GFP (for control) and DsRed (for PDGF) and the cells were reinjected into neonatal rats. Animals were sacrificed at 6 weeks post injection and the brains analyzed. Infiltrative tumors that closely resembled human glioblas-tomas developed in 100% of the animals by 10 days post injection. The tumors were composed of a mixture of retrovirus infected (GFP1) and unin-fected (GFP–) cells. To further explore the recruitment phenomenon, we co-injected retroviruses that express PDGF-IRES-DsRed and GFP. The rats formed tumors that contained a mixture of DsRed and GFP1 cells (with a small population of co-infected cells). The majority of cells in both popula-tions expressed markers characteristic of oligodendrocyte progenitor cells (OPCs). To test the tumorigenic potential of the recruited progenitors, we separated the DsRed1 and GFP1 populations by FACS and transplanted them into neonatal rats. Most rats (6 of 9) injected with the DsRed1 cells formed tumors whereas most rats (8/9) injected with GFP1 cells did not form tumors, but had a small population of mature appearing GFP1 glia near the injection site. One rat injected with GFP1 cells formed a tumor that contained both GFP1 and DsRed1 cells, indicating that some DsRed1 cells had contaminated the sorted population of GFP1 cells. These results show that the recruited progenitors will continue to proliferate if transplanted with PDGF expressing cells, but will stop proliferating and differentiate if they are separated from the PDGF expressing cells. These results show that: (1) Constitutive expression of PDGF leads to the rapid formation of tumors that closely resemble human glioblastomas in neonatal rat brain. (2) Non-infected progenitors are recruited to the tumor via paracrine signaling. (3) Recruited progenitors retain the capacity to differentiate when removed from the tumor environment.

MO-09. A STATISTICAL MODEL FOR WATER DIFFUSION OFFERS A MORE SENSITIVE MARKER OF TREATMENT RESPONSE THAN STANDARD ADC MEASURES IN A MURINE MODEL OF MALIGNANT GLIOMASarah Jost,1 Dmitriy Yablonskiy,2 David Gutmann,3 and Joel Garbow2; 1Neurological Surgery, Washington University in St. Louis, St. Louis, MO, USA; 2Radiology, Washington University in St. Louis, St. Louis, MO, USA; 3Neurology, Washington University in St. Louis, St. Louis, MO, USA

High-grade glioma is the most common primary brain tumor in adults. Even after multimodal therapy, outcomes remain poor, with a median sur-vival of approximately one year. Presently, a patient’s response to therapy is limited to survival analysis and serial measurements of tumor growth using Magnetic Resonance Imaging (MRI). Whereas standard MR images are unable to provide information about cellular changes that occur within the tumor in response to therapy, diffusion MR imaging is highly sensitive to the microstructure of biological tissue on a scale of 1 micron and, as such, has the capacity to reflect these changes. Detection of these changes, which occur shortly after the initiation of therapy, would allow for an earlier identification of treatment response. In a patient population whose median survival is less than one year, temporal improvements in the detec-tion of treatment response would be significant. Initial studies have sug-gested that changes in the apparent diffusion coefficient (ADC) of water correlate with tumor response and changes in cell density in rat models of glioma. We have recently developed a statistical model of restricted dif-fusion that allows for evaluation of the microscopic variability in tissue characteristics resulting from the variations in cell types, sizes, geometries, orientations, and membrane permeabilities that occur within each imaging voxel. This model describes diffusion as a continuous distribution of appar-ent diffusion coefficients (ADCs) within each voxel, characterized by the position of distribution maxima (D) and the distribution width (sigma). We hypothesize that this statistical model will more accurately reflect changes in glioma cells early after initiation of treatment, but prior to measurable changes in tumor volume, and will provide predictive information about a tumor’s response to therapeutic intervention. We have employed the DBT malignant glioma intracranial explant model to detect early changes in diffusion following standard cytotoxic therapy. Eight animals bearing intracranial DBT cell tumors were serially imaged before, during, and fol-lowing administration of two doses of 25 mg/kg BCNU at 12 and 15 days post-implantation. T1-weighted, gradient-echo multi-slice images were col-lected following gadolinium contrast administration, and diffusion data collected using standard spin-echo sequences with the addition of diffu-sion sensitizing gradients with multiple b-values. Response to therapy was quantitated using a standard T1-weighted gradient-echo sequence. At 21 days post-implantation, standard ADC, Sigma, and D were all significantly

higher (p , 0.05) than their respective pre-treatment values. However, at 18 days post-implantation, measurements of diffusion using the statistical model for ADC (D) were significantly higher in tumors that responded to therapy (tumors shrank or stabilized) than in those that did not (0.648 vs. 0.489; p 5 0.02). At this same time point, standard ADC values were not significantly different in these two groups (0.871 vs. 0.913; p 5 0.67). These observations suggest that this new statistical model for diffusion may be a better method for an early assessment of treatment response than standard measures of water diffusion.

MO-10. RESOLVING INCONSISTENCY OF SYNERGY DETECTION AT DIFFERENT TIME POINTSMing Zhang,1 Chandra Das,2 Dolly Aguilera,3 Neeta Kang,2 Vidya Gopalakrishnan,1 and Johannes Wolff4; 1University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Pediatric, University of Texas M.D. Anderson Cancer Center, TX, USA; 3University of Texas M.D. Anderson Cancer Center, Pearland, TX, USA; 4Houston, TX, USA

Synergism (or antagonism) occurs when the effect of two or more drugs used in combination is greater (or less) than the additive effect of the drugs used separately. A problem for drug synergy detection is that the conclusions can be inconsistent at different time points. The LN18 tumor cells were treated with etoposide and Valproic acid (VPA) at 24hr, 48hr, 96hr, and 144hr separately and in combination. The concentration of VPA is 1.5 mM and the concentration of etoposide is 0.00028 mM. The cell viability (in percentage of control) numbers treated by VPA, etoposide, and in combina-tion at the four time points were recorded. These viability numbers from each treatment were fitted to a two-term tumor regrowth model y(t) 5 a 3 exp(–b 3 t) 1 c 3 exp(d 3 t) where the first term describes contribution to tumor cell number from a dying cell sub-population, the second term from a growing sub-population and all the parameters a, b, c, and d are posi-tive. The cell viability numbers treated by VPA at the four time points are 0.9235, 0.9255, 0.7806, and 0.9448; the numbers treated by etoposide are 0.9944, 0.8390, 0.8275, and 0.5082; the numbers treated by both drugs are 1.0000, 0.7680, 0.6341, and 0.2689. Using the fraction product method, the viability numbers should be 0.9183, 0.7765, 0.6460, and 0.4801 at the four time points if the two drugs are additive. Thus, the synergy conclusions are that the two drugs are additive at 24 hr, 48 hr, and 96 hr, but synergistic at 144 hr. Using the two-term regrowth model we define drug efficacy based on the percentages of the growing sub-population (over the control). These percentages for VPA, etoposide, and two drugs together are 0.9293, 0.4847, and 0.2292. The fraction product is 0.4504 if the two drugs are additive. Therefore the two drugs are synergistic. Other concentrations of the drugs have been tested and the results were similar. By removing the contribution from the dying sub-population to the total tumor cell number and defining drug efficacy using the growing sub-population, the inconsistency on syn-ergy detection conclusions is resolved.

MO-11. BIOLUMINESCENT IMAGING AND THERAPEUTIC RESPONSE IN AN EXPERIMENTAL RODENT BRAINSTEM TUMOR MODELRintaro Hashizume,1 Tomoko Ozawa,1 Eduard Dinca,1 Michael Prados,2 Anu Banerjee,2 David James,2 and Nalin Gupta2; 1Brain Tumor Research Center, University of California, San Francisco, San Francisco, CA, USA; 2University of California, San Francisco, San Francisco, CA, USA

Most infiltrative brainstem tumors in children are malignant gliomas and patients usually die within 2 years after initial diagnosis. Because of their anatomic location within the pons, brainstem tumors are not ame-nable to surgical resection. In addition, systemically administered agents have limited distribution to the brainstem. In order to evaluate the effi-cacy of directly delivered agents using new techniques such as convection- enhanced delivery, a robust brainstem tumor model is required. In this study, we demonstate the histologic features and growth kinetics of an orthotopic brainstem tumor model in rats. Tumor growth was further char-acterized by in vivo bioluminescence imaging (BLI). Finally, we used oral temozolomide to show efficacy against human U87 tumor cells. U87MG glioblastoma cells were implanted into the pontine tegmentum of athymic rats using an implantable guide-screw system. This cell line was modified to constitutively express luciferase by lentivirus infection. Survival time, loca-tion of the tumor, and time to development of symptoms were measured. In vivo BLI was performed using the Xenogen Lumina Imaging Station (Cali-per Life Sciences) coupled to a data-acquisition PC running Livingimage software. BLI showed progressive tumor growth in all animals, with symp-toms indicative of tumor burden between 26 and 31 days. Histopathologic analysis revealed tumor growth within the pons in all rats. BLI correlated quantitatively with tumor volume calculated by three-dimensional measure-ments from serial histologic sections. For efficacy experiments, rats with brainstem tumors received temozolomide delivered by oral gavage (50 mg/kg for 5 consecutive days). Control animals were euthanized at the onset



of symptoms between 26 and 28 days. Animals treated with temozolomide survived more than 50 days after implantation. Longitudinal BLI revealed a sustained decrease in luminescence of temozolomide-treated animals. This decrease was consistent with temozolomide anti-tumor activity as indicated by the increased survival of treated animals. This orthotopic brainstem tumor model system allows a reproducible assessment of survival defined by time to development of symptoms and will greatly facilitate testing of pre-clinical therapeutic agents. Tumor response to therapeutic agents can be non-invasively measured using BLI.

MO-12. DEVELOPMENT OF AN INTRACRANIAL MODEL OF INVASIVE CNS LYMPHOMA USING BIOLUMINESCENCE IMAGINGRamona Voicu,1 Eduard Dinca,2 Juliana Karrim,3 Cigall Kadoch,3 C. David James,4 and James Rubenstein4; 1Dept. Neurological Surgery, Brain Tumor Research Center, University of California, San Francisco, San Francisco, CA, USA; 2Brain Tumor Research Center, University of California, San Francisco, San Francisco, CA, USA; 3Medicine, University of California, San Francisco, San Francisco, CA, USA; 4University of California, San Francisco, San Francisco, CA, USA

Primary central nervous system lymphoma (PCNSL) is an aggressive form of non-Hodgkin’s lymphoma typically associated with a prognosis worse than other localized extranodal lymphomas with similar histology. The defining feature of PCNSL is its confinement to the central nervous system (CNS), with proclivity for widespread dissemination as well as growth within the leptomeningeal as well as intraocular compartments. PCNSL rarely disseminates outside of the CNS. The majority of PCNSL patients succumb to progressive disease and/or treatment-related toxicities associated with the standard, genotoxic therapeutic strategies used against this type of neoplasm. We have previously demonstrated that Pim 1 and 2, serine-threonine kinases which regulate survival signaling, are highly expressed in both brain parenchymal and leptomeningeal CNS lymphoma tumors, suggesting that these oncogenes may represent rational therapeu-tic targets. We have developed an orthotopic intracranial mouse model of CNS lymphoma using human Raji B-lymphoma cells which express high basal levels of Pim kinases (Pim-1 and Pim-2). B-lymphoma cells were modi-fied for bioluminescence and reproducibly yielded intracranial tumors with onset of neurologic symptoms within ten days post-implantation. Biolumi-nescence was detected within three days post-implantation and reflects the rapid intracranial tumor growth rate observed. Tumor cells exhibited an immunophenotype consistent with primary CNS lymphoma in that these tumors were positive for CD20, bcl-6, and MUM-1. Histopathologic analy-sis demonstrated a highly infiltrative growth pattern, with characteristic angiotropism as well as evidence for leptomeningeal dissemination. Studies are in progress to utilize this model system to evaluate the in vivo response to chemotherapy and to modulate this response using strategies that attenu-ate relevant survival signaling pathways. Results will be presented at the meeting.

MO-13. ABILITY OF GLIOBLASTOMAS TO GENERATE EXPANDABLE NEUROSPHERE CULTURES IS PREDICTED BY COPY NUMBER CHANGES ON CHROMOSOMES 7 AND 10Heidi Phillips,1 Ruihuan Chen,1 Katrin Lamszus,2 Stephanie Bumbaca,3 Steve Guerrero,3 Hauke Gunther,4 Nils-Ole Schmidt,4 Samir Kharbanda,3 Robert Soriano,3 Celina Sanchez Rivers,3 Manfred Westphal,4 William Weiss,6 David James,3 Cynthia Cowdrey,3 Scott VandenBerg,5 Michael Prados,5 and Zora Modrusan3; 1Genentech, SSF, CA, USA; 2University Medical Center, Hamburg, Germany; 3CA, USA; 4Germany; 5University of California, San Francisco, San Francisco, CA, USA

Neurosphere cultures of glioblastoma (GBM) have gained popularity as a model for testing of novel therapeutic approaches, but most investiga-tors report that expandable long-term cultures result from only a subset of GBM samples that are placed in neurosphere conditions. One important issue that has not been adequately resolved is whether there is a bias in the molecular subtypes of GBMs that generate long-term neurosphere cultures. We report here the results of two parallel investigations into the molecular nature of tumors that generate successful long-term neurosphere cultures. A series of 19 GBMs cultured in Hamburg yielded 9 long-term expandable cultures and 10 cultures which failed to expand. A second set of 31 high-grade gliomas was cultured in South San Francisco. 16 tumors (all GBMs) generated expandable neurosphere cultures, 8 cultures failed to generate propagable long-term cultures (6 GBMs and 2 grade III tumors), and 7 cul-tures are indeterminate to date. Specimens utilized to generate cultures were profiled on Affymetrix 100K or 500K SNP chips. Comparisons of copy number changes between tumors generating expandable cultures vs. those yielding failed cultures were performed separately for the two investiga-tions. Both studies revealed that tumors generating expandable neurosphere cultures showed relative copy number gains on chromosome 7 and losses on

chromosome 10 compared to tumors yielding failed cultures. Relative copy number differences were widespread across both chromosomes, indicating the likelihood that most GBMs generating successful cultures exhibit tri-somy 7 and monosomy 10. These results indicate that panels of neurosphere lines derived from GBMs may overrespresent certain molecular subtypes of tumor and that many tumors lacking copy number alterations on chromo-somes 7 and 10 may not be amenable to modeling by culture under standard neurosphere conditions.

MO-14. INTRACRANIAL GRAFTS OF GBM-DERIVED NEUROSPHERES REVEAL A BIAS TOWARD EXPRESSING MARKERS OF PRONEURAL AND PROLIFERATIVE TUMOR SUBTYPESSamir Kharbanda,1 Merry Nishimura,2 Katrin Lamszus,3 Hauke Gunther,3 Nils-Ole Schmidt,4 Robert Soriano,1 Zora Modrusan,1 Ruihuan Chen,1 Manfred Westphal,4 and Heidi Phillips1; 1Genentech, Inc., South San Francisco, CA, USA; 2CA, USA; 3University Medical Center, Hamburg, Germany; 4University Medical Center, Germany

The development of therapeutic treatments for glioblastoma (GBM) is hampered by the inability of conventional cancer cell lines grown under standard serum-containing medium to fully recapitulate the gene expression pattern and in vivo behavior of human GBMs. We have recently defined three prognostically significant molecular subtypes of primary human glio-blastoma termed Proneural, Proliferative, and Mesenchymal. Microarray analysis of a panel of GBM lines propagated in serum-containing medium revealed that none of the cell lines expressed the class of markers that defines the Proneural GBM tumor subtype. In contrast to conventional GBM lines, some long-term neurosphere cultures of GBMs displayed expression of Proneural markers such as Brevican, Olig 2, and NCAM. Intracranial implantation of two neurosphere lines derived from primary tumors dis-playing a mixture of Proneural and Mesenchymal markers showed invasive growth but lacked microvascular proliferation and geographic necrosis. Relative to their respective primary tumors, orthotopic grafts of both neu-rosphere lines demonstrated enhancement of Proneural and Proliferative marker expression at the expense of Mesenchymal markers. The Proneural markers, included elements of the Notch pathway, such as DLL1, DLL3, Hey2, and ASCL1. Expression profiling and immunohistochemistry also revealed that tumor phenotype was remarkably stable across 5 serial in vivo passages. No evidence for the emergence of microvascular prolifera-tion or geographic necrosis after serial in vivo passaging was noted and the lesions failed to display contrast enhancement on MR imaging. The neurosphere GBM models investigated thus capture the invasive feature of human GBMs and may provide a good model system for therapeutic agents targeting markers of the Proneural GBM subtype, including members of the Notch pathway.

MO-15. CANINE GLIOMAS OVER-EXPRESS IL-13RALPHA2, EPHA2, AND FRA-1 IN COMMON WITH HUMAN HIGH-GRADE ASTROCYTOMASWaldemar Debinski,1 Denise Gibo,2 Jill Wykosky,2 Constance Stanton,3 John Rossmeisl,4 and John Robertson4; 1Neurosurgery, Wake Forest University, Winston-Salem, NC, USA; 2Neurosurgery, Wake Forest University School of Medicine, Winston-Salem, NC, USA; 3Pathology, Wake Forest University, Winston-Salem, NC, USA; 4The Center for Comparative Oncology, Virginia Tech College of Veterinary Medicine, VA, USA

Canine gliomas are increasingly considered as human counterparts, since they are spontaneous, relatively common and exhibit similar clini-cal and pathobiological characteristics. We have previously identified three proteins that are over-expressed in a majority of human patients with high-grade astrocytomas: (i) interleukin 13 receptor alpha2 (IL-13R), a cancer-testis tumor-like antigen, (ii) EphA2, a tyrosine kinase receptor, and (iii) fos-related antigen 1 (Fra-1), an AP-1 transcription factor. In an attempt to investigate the similarity of human and canine gliomas on a molecular level, we have investigated the expression of these three proteins in various types of canine brain tumors. Immunohistochemistry was performed using speci-mens from 18 dogs with grades II–IV astrocytoma and oligodendroglioma, some with an astrocytic component. Staining for GFAP was also performed. 9 and 14 out of 15 high-grade astrocytoma and mixed oligoastrocytoma specimens were positive for IL13R and EphA2, respectively, and 6 out of 13 stained positive for Fra-1. Normal brain did not exhibit significant immu-noreactivity for any of the studied factors. Oligodendrogliomas were most frequently stained for EphA2 and less for IL13R and Fra-1. Of interest, tumor cells invading normal brain parenchyma stained prominently for these proteins. This is the first attempt to analyze concomitantly IL-13R, EphA2, and Fra-1 in canine gliomas. These three factors appear to be molecular markers/therapeutic targets not only of human high-grade astro-cytomas, but they may also represent common molecular denominators of



canine gliomas, which is currently under further investigation. These data provide yet another line of evidence for a close similarity between canine and human gliomas, suggesting that the canines offer a unique, faithful pre-clinical model of human disease.

MO-16. A SUBSET OF SPONTANEOUS CANINE OLIGODENDROGLIAL TUMORS SHARE GENETIC SIMILARITIES WITH HUMAN OLIGODENDROGLIOMASAmanda Rynearson,1 Caterina Giannini,1 Fausto Rodriguez,1 Sandra Passe,1 Terrell Blanchard,2 Terry McElwain,3 Ronald Marler,4 and Robert Jenkins1; 1Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, USA; 2Veterinary Pathology, Walter Reed Medical Center/AFIP, DC, USA; 3College of Veterinary Medicine, Washington State University, WA, USA; 4Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, AZ, USA

Human oligodendrogliomas are characterized cytogenetically by recur-rent losses of chromosome arms 1p and 19q. This co-deletion has been associated with a better prognosis and an increased response to treatment. Morphologically similar tumors also arise spontaneously in dogs. Twenty four such cases were identified in the files of the Armed Forces Institute of Pathology (AFIP) with sufficient material for further studies. All avail-able H&E slides were examined by a neuropathologist. Fluorescent in situ hybridization (FISH) studies were performed on paraffin embedded tissues with probes targeting the following syntenic gene regions: HDAC1 (human 1p35.1, canine 2), GLTSCR1 (human 19q13.3, canine 1), and CAMTA1 (human 1p36.3, canine 5). Each FISH probe consisted of three bacterial artificial chromosomes (BACs) which flanked the gene region of interest. Each probe was validated on metaphases obtained from peripheral blood in normal canine controls. A total of 200 non-overlapping nuclei were scored by two independent observers. A third observer evaluated cases with dis-crepancies. Histologic evaluation revealed that most tumors were high grade and had moderate to high cellularity. Classic oligodendroglial features were present in the majority (n 5 18), while the remaining resembled oligoastro-cytomas or small cell astrocytomas (n 5 6). The following features were present in decreasing frequency: endothelial hypertrophy (n 5 18), mucoid background (n 5 16), increased mitotic activity (n 5 15), necrosis (n 5 10), and hemorrhage (n 5 8). All cases lacked microscopic calcification. Loss of the region of interest was defined as a percentage of monosomy signal of . 60%. Of the 24 cases, 3 (13%) showed loss of GLTSCR1, 2 (8%) showed loss of CAMTA1, and 1 (4%) case showed loss of both GLTSCR1 and CAMTA1. This study suggests that genetic alterations involving chromo-somes 1 and 19 previously described in human oligodendrogliomas occur in the syntenic regions of a subset of morphologically similar canine tumors.

MEDICAL THERAPIES, PEDIATRIC

MP-01. A CASE REPORT OF A COMPLETE RESPONSE AND 20-YEAR SURVIVAL OF A PATIENT WITH A RECURRENT DIFFUSE INTRINSIC BRAINSTEM ANAPLASTIC ASTROCYTOMAStanislaw Burzynski, Robert Weaver, and Barbara Szymkowski; Burzynski Clinic, Houston, TX, USA

High-grade diffuse intrinsic gliomas of the brainstem (HDBSG) that recur after radiation therapy carry a grave prognosis and have a median sur-vival of less than 7 months. This presentation describes an unusually lengthy complete response (CR) of a 36-year-old female with HDBSG treated with Atengenal and Astugenal (ANP) which consist of synthetic analogs of natu-rally occurring phenylacetylglutamine, phenylacetylisoglutamine, and phe-nylacetate. The patient was diagnosed with a pontine glioma in June 1987 and biopsy performed on July 25, 1987, at UCSF confirmed an anaplastic astrocytoma. She was treated with hyperfractionated radiotherapy for a total dose of 76 Gy, which was completed on October 9, 1987. In February 1988, she developed diplopia, right facial paralysis and vertigo. A MRI of the head on February 25, 1988, showed interval enlargement of the tumor with extension to the right anterior pons and middle cerebral peduncle. No further therapy was recommended at UCSF due to her tumor progression, and on May 23, 1988, she began treatment with ANP. On May 29, 1988, a low dose of methotrexate 7.5 mg PO daily (0.1 mg/kg/d) in five days on/off courses was added to ANP. On July 12, 1988, methotrexate was dis-continued and she continued intravenous infusions of ANP only (the maxi-mum dosage of Atengenal was 0.8g/kg/d and of Astugenal was 0.2g/kg/d). Intravenous ANP were discontinued on August 10, 1989 and replaced by oral ANP (the maximum dosages were 0.14g/kg/d). All treatment was dis-continued on January 21, 1990. Follow-up MRIs revealed gradual decrease and eventual disappearance of the tumor on January 23, 1989. Numerous

subsequent MRIs confirmed CR. ANP was well tolerated with only minor reversible side effects including a skin rash, fever and slight leukopenia from which she recovered completely except for a mild right facial nerve paresis, which occurred after the initial tumor biopsy. She is currently doing well and has not suffered from any chronic toxicity related to ANP. Periodical repeat MRIs of the head (the last on April 28, 2007) have shown no tumor recurrence. The National Cancer Institute (NCI) confirmed her diagnosis and CR during a site visit. ANP is a multitargeted therapy, which is cur-rently pending use in Phase III trials in newly diagnosed diffuse intrinsic brainstem gliomas.

MP-02. VALPROIC ACID IN THE TREATMENT OF MALIGNANT GLIOMA IN CHILDRENPablo Hernaiz Driever,1 Jindrich Cinatl,2 Sabine Wagner,3 and Johannes Wolff4; 1Department of Pediatric Oncology/Hematology, Berlin, Germany; 2Institute of Medical Virology, Frankfurt am Main, AE (Europe), Germany; 3Germany; 4Houston, TX, USA

Several differentiation inducing drugs are currently tested for their anti-tumoral activity considering that tumor cells resemble immature precursor cells that can be prompted either to differentiate or to become apoptotic. Previously, we were able to show in preclinical studies that VPA changes the biology of neuroectodermal tumor cells towards differentiation, cytostasis, reduced metastatic capacity and increased immunogenicity. We used the his-tone deacetylase inhibitor valproic acid (VPA) in the HIT-GBM-C protocol for the treatment of malignant glioma in children. Continuous oral intake of VPA after combined chemotherapy and irradiation for malignant glioma aimed at further reduction of tumor volume or at maintaining the previously achieved remission status after postsurgical combined radiochemotherapy ensuring quality of life. 53 children (32:21 m:f) were treated with VPA. 4 children were treated with VPA for epilepsy and 10 children received VPA despite progressive disease. Median daily dose was 33 mg/kg/day (range: 9.8–78) allowing median trough serum levels of 95 mg/l (range: 24–168). 46 children were evaluable for response to VPA after 6–8 weeks of treat-ment. 15 patients showed stable disease and 3 children even showed partial remission. Toxicity was mild and patients with tumors responding to VPA treatment had an increased event-free survival. This is the first report show-ing that the histone deacetylase inhibitor VPA is effective in the treatment of malignant glioma at tolerable drug levels. Thus, we strongly suggest that VPA should be further tested as an antitumoral drug in children.

MP-03. STABILIZATION FOR 9 MONTHS OF RAPIDLY PROGRESSIVE MALIGNANT GLIOMA USING BEVACIZUMAB, IRINOTECAN, AND TEMOZOLOMIDE IN A PREVIOUSLY HEAVILY TREATED 15-YEAR-OLD FEMALEAlejandro Torres-Trejo1 and John Collins2; 1Neurology, West Virginia University, Morgantown, WV, USA; 2Neurosurgery, West Virginia University, Morgantown, WV, USA

A 15-year-old female who presented in October 2002 with hydrocepha-lus and mild left hemiparesis, was found to have a right thalamic area mass for which she underwent a resection. The pathology revealed a glioblas-toma multiforme. She also had Gliadel wafers implantation at the time of resection. Her treatments consisted of radiation followed by stereotactic radiosurgery to residual tumor. Chemotherapy treatment consisted initially of temozolomide for 12 months. In September 2005 she developed drop metastasis to the spine for which she had spinal radiation which decreased the tumor size. In January 2006 she initiated irinotecan and cetuximab as part of a clinical trial. In April 2006 there was evidence of progression for which she was enrolled in another clinical trial and she received oxaliplatin in combination with 5-fluorouracil. In June 2006 further progression was identified and at that time Avastin and CPT-11 were started, which stabi-lized her disease. In November 2006, temozolomide was added with the goal to reduce tumor burden. With this regimen her disease was stabilized for almost 9 months with relative good quality of life. This outcome suggests that using agents with different mechanisms may arrest tumor growth.



MP-04. APOPTOSIS IN ALPHA INTERFERON (IFN-a) INTRATUMORAL CHEMOTHERAPY FOR CYSTIC CRANIOPHARYNGIOMASDaniela Filippini Ierardi,1 Maria Jose Da Silva Fernandes,2 Iara Ribeiro Silva,2 Juliana Thomazini-Gouveia,3 Patricia Dastoli,4 Andréa Maria Cappellano,4 Nasjla Saba Silva,4 Silvia Regina Caminada Toledo,5 and Sergio Cavalheiro2; 1Neurology and Neurosurgery Department, Pediatric Oncology Institute-IOP/GRAACC, Federal University of São Paulo, São Paulo, Brazil; 2Neurology and Neurosurgery Department, Federal University of São Paulo, São Paulo, Brazil; 3Pediatric Oncology Institute—IOP/GRAACC, Federal University of São Paulo, São Paulo, Brazil; 4Pediatrics, Pediatric Oncology Institute—IOP/GRAACC, São Paulo, Brazil; 5Pediatric Oncology Institute—IOP/GRAACC, São Paulo, Brazil

Craniopharyngiomas is the most common of childhood neoplasms originated in hypothalamic and pituitary-gland region and most common non-glial primary intracranial tumor. Although craniopharyngiomas have a benign nature, they have a malignant clinic evolution. Many therapeutics modalities have been tried in the aim to eliminate the tumor or at least to reduce the tumor size to minimize the secondary effects. Cavalheiro et al. (2005) described that the use of alpha interferon (IFN-a) as an intracystic chemotherapy was able to reduce craniopharyngiomas size by an unknown mechanism. Recent studies suggest a link between IFN-a and Fas-mediated apoptosis. Objectives: In this way, the aim of this study was to verify whether intracystic injections of IFN-alpha were able to activate the Fas/FasL apop-totic pathway while reduce the tumor size. The soluble FasL (sFasL) was determined by Elisa assay in samples of tumor fluid taken before and during and after IFN-a injections from eight patients of the Pediatric Oncology Institute (IOP/GRAACC) of the Federal University of São Paulo, Brazil. The tumor size before and after the treatment were monitored by magnetic resonance imaging (MRI) and computed tomography (CT). Results and The concentration of sFasL was markedly increased during the IFN-a injections. Concomitantly, we observed the tumor size reduction in all patients. Our data showed that the IFN-a was able to induce Fas-mediated apoptosis while reduce the craniopharyngioma size. This study open a new window to investigate mechanisms involved with craniopharyngioma therapy.

MP-05. ATYPICAL TERATOID/RHABDOID TUMOR OF THE CENTRAL NERVOUS SYSTEM: A SINGLE INSTITUTION EXPERIENCETheodore Nicolaides,1 Tarik Tihan,2 Biljana Horn,1 Jaclyn Biegel,3 Michael Prados,1 and Anuradha Banerjee4; 1University of California, San Francisco, San Francisco, CA, USA; 2Pathology, University of California, San Francisco, CA, USA; 3Children’s Hospital of Philadelphia, PA, USA; 4Pediatric Oncology, University of California, San Francisco, San Francisco, CA, USA

Atypical teratoid/ rhabdoid tumors (AT/RT) of the brain are rare but aggressive tumors of childhood. Recent molecular characterizations have given us insights into the pathogenesis of these tumors. In particular, inac-tivation of the INI1 gene appears to be a sensitive marker for this disease. Despite our increased knowledge, however, treatment outcomes remain poor. We report on nine patients treated at our institution from 1997–2007. All diagnoses were confirmed by either the presence of an INI1 mutation or absence of INI1(BAF47) nuclear staining on immunohistochemistry. All patients underwent surgical resection followed by adjuvant chemotherapy. Chemotherapy consisted of cisplatin, etoposide, vincristine, cyclophosph-amide, and for four patients, high dose methotrexate. Those patients achiev-ing a complete response (CR) underwent high dose chemotherapy with Autologous Stem Cell Transplant (ASCT). The mean age of diagnosis was 22 months (Range 6–49 months). Six out of the nine patients underwent a gross total resection (GTR), while three had either a subtotal resection (STR) or a biopsy. Six patients were considered a CR at the end of induction chemotherapy and went on to ASCT. There are two long-term survivors (. 5 years) who both had GTRs. There were no toxic deaths. There were three deaths due to progressive disease during induction chemotherapy and three deaths due to progressive disease after ASCT. Patients with AT/RT have a poor prognosis. Progressive disease is common during treatment, despite intensive therapy. Gross total resection and ASCT may be associated with longer survival.

MP-06. TYPE OF THERAPY STRONGLY INFLUENCES OUTCOME IN PATIENTS WITH ATYPICAL TERATOID RHABDOID TUMORS OF THE CNS—EXPERIENCE IN 13 CONSECUTIVE CHILDRENIrene Slavc,1 Thomas Czech,2 Christine Haberler,3 Andreas Peyrl,1 Georg Widhalm,2 and Karin Dieckmann4; 1Department of Pediatrics, Vienna, Austria; 2Department of Neurosurgery, Vienna, Austria; 3Institut of Neurology, Vienna, Austria; 4Department of Radiation Oncology, Vienna, Austria

Atypical teratoid/rhabdoid tumor (AT/RT) of the CNS is a rare and aggressive tumor of early childhood. Before immunohistochemistry with an antibody to INI1 was described as a specific means to distinguish AT/RT from other CNS tumors these tumors were often misdiagnosed as medullo-blastoma or supratentorial PNETs because of the sometimes indeterminate histologic features. We report on the difference in outcome of 13 consecu-tive patients with AT/RT of the CNS depending on initial diagnosis and treatment received. A retrospective analysis applying the INI1 antibody to all highly malignant pediatric brain tumors treated at the University of Vienna between 1992 and 2007 disclosed 13 patients with AT/RT. Only six patients were originally diagnosed correctly. Diagnoses of the remaining seven patients included medulloblastoma, supratentorial PNET, ependymo-blastoma and Ewing sarcoma. Metastatic disease was present at diagnosis in five, and not known in two. The six patients diagnosed initially as AT/RT were treated with an intensive, multiagent chemotherapy including cyclo-phosphamide, vincristine, cisplatin, ifosfamide, etoposide, methotrexate, and adriamycin followed by high-dose chemotherapy with autologous stem cell rescue (modified Finlay protocol) and local radiotherapy. Five of these six patients are alive, four of which in continous complete remission for 21, 50, 56, and 120 months after diagnosis, respectively, and one is too early to be evaluated. The seven patients originally misdiagnosed were treated according to the HIT SKK 92 or HIT 91, and Ewing sarcoma protocols of the German Society of Pediatric Hematology and Oncology. Of this initially misdiagnosed cohort only one 9-year-old patient became a long-term sur-vivor. Because of the poor outcome and a median survival of 6–19 months with conventional infant brain tumor therapy proper diagnosis of AT/RT is essential.

MP-07. VALPROIC ACID AND CHEMOTHERAPY IN PEDIATRIC HIGH-GRADE GLIOMA: RETROSPECTIVE CHART REVIEWAmirhadi Masoudi, Joann L. Ater, Vidya Gopalakrishnan, Elham Amini, Margaret E. Nagel, Marily S. Elopre, and Johannes Wolff; University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Cytotoxic chemotherapy has some but limited effects on high grade glioma (HGG) such as anaplastic astrocytoma and glioblastoma multiform. Valproic acid (VPA) is a branched chain fatty acid, used as anitepleptic drug and mood stabilizer which is now also known as a histone deacetylase inhib-itor and can induce tumor cell differentiation and apoptosis in HGG cells. Combining this drug with standard chemotherapy might increase the effect of chemotherapy but might also increase adverse effects. In preparation of systematic clinical trials we did a retrospective chart review of pediatric HGG patients treated at MD Anderson Cancer Center. 66 patients with histologically proven World Health Organization (WHO) Grade IV glio-blastoma multiforme (n 5 40) or WHO Grade III anaplastic astrocytoma (n 5 26) between the ages of 1 years and 19 years were available for analysis. The tumor locations were supratentorial in 46 patients, and infratentorial in 20 patients. Tumor surgeries were biopsy in 15 patients, partial resec-tion in 11 patients, subtotal resection in 25 patients, and macroscopic total resection in 15 patients. Among these patients we have identified 9 patients that have received VPA for their seizure in addition to their radiochemo-therapy. The response, best response and potential adverse effects of VPA have been analyzed. One of the patients that received VPA in addition to his radiochemotherapy showed deep venous thrombosis with pulmonary embolism that was the severest side effect. 33% of patients showed hemato-logic toxicity and they received transfusion without any significant problem. We did not observe any hepatotoxicity that was our main concern. There were no severe dermatological and gastrointestinal side effects, laboratory changes, or infection problems. Response to the combined treatment after 8 weeks was: CR:0, PR:3, SD:4, and PD:2. Some of the SD responded later resulting in best response: CR:0, PR:5, SD:2, and PD:2. VPA in addition to radiochemotherapy was well tolerated and feasible in a limited number of patients available. Response was encouraging. We will start a formalized prospective trial for pediatric HGG.



MP-08. HIGH-DOSE METHOTREXATE FOR PEDIATRIC HIGH-GRADE GLIOMA—PROMISING SURVIVAL DATA IN THE SUBGROUP OF COMPLETELY RESECTED TUMORSJohannes Wolff,1 Sabine Wagner,2 Rolf Kortmann,3 Torsten Pietsch,4 Norbert Graf,5 Ove Peters,2 Hansjoerg Schmid,6 Stefan Rutkowski,7 Monika Warmuth-Metz,7 and Christoph Kramm8; 1University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2University of Regensburg, Germany; 3University of Leipzig, Germany; 4University of Bonn, Germany; 5University of Hamburg, Germany; 6University of Hannover, Germany; 7University of Wuerzburg, Germany; 8Pediatric Oncology Halle, Germany

Based upon promising data from a the multiagent protocol “HIT-91S” (Cancer 2002; 94:264–271) a pilot study has been conducted to test one of these drugs, Methotrexate (5g/m2), as single drug in a phase II window prior to radiation for histological proven WHO grade III or grade IV tumors (HGG), and radiological proven gliomatosis cerebri and diffuse intrinsic pontine glioma (DIPG) Favorable preliminary response (Anticancer Res 25: 2583–2588) prompted the randomized protocol “HIT-GBM-D” testing the phase II window in comparison to standard treatment. This completed accrual in April 2007, now allowing the first look at the follow data of the pilot study with survival data updated until 2006. Thirty one patients (20 male, mean age 9.9, range 3–18) from 20 hospitals fulfilled the eligi-bility criteria of being in enrolled in the pilot study and having received two cycles of methotrexate. Tumor locations were: cerebral hemispheres: 10, basal ganglia: 3, pons: 7, brain stem outside of pons: 2, cerebellum: 2, spinal cord: 2, and overlapping brain areas/gliomatosis cerebri: 5. Once the HGG occurred after acute lymphoblastic leukemia, twice after a previous low-grade glioma. Two patients had metastases. Tumor resection was clas-sified as gross total resection: 6, subtotal: 6, partial: 6, biopsy: 11, and no surgery: 3. WHO grading of the histology was: IV: 11, III: 12, II: 3, and I: 1. Patients without biopsy or with low-grade glioma histology fulfilled the radiological criteria for DIPG or gliomatosis cerebri. Methotrexate 5 g/m2 was given in 24-hour infusions on day 1 and 15 followed by leucovorine rescue at hour 42. 54 Gy radiation 5 fractions per week of 1.8 Gy was given simultaneously with chemotherapy: cisplatin (CDDP) 20 mg/m2 3 5 days 1 etoposide (VP16) 100 mg/m2 3 5 days 1 vincristine (VCR) 1.5 mg/m2 once followed by weekly vincristine and one more cycle of CDDP 1 VP16 1 VCR 1 ifosfamide 1.5g/ m2 3 3 in the last week of radiation. Maintenance chemotherapy consisted of VCR day 1, lomustine (CCNU) 75 mg/m2 on day 2, prednisone 40 mg/kg day 1–17 (tapering the dose during last days). Eight of those cycles were planned every six week. Known protocol deviations included: no ifosfamide (1), substituting cisplatin with carboplatin (2), and no vincristine during radiation (2) Survival: In the whole group the event free survival (EFS) rate after 6 months, 1 year, 2 years, and 3 years was: 84%, 35%, 16%, and 9%, respectively. Overall survival was 97%, 77%, 44%, and 9%, respectively. Median EFS time (mEFS) 0.59 1 SE 0.17 years, mOS: 1.7 1 0.37 years. In subgroup analyses, those patients with complete resection differed significantly (p 5 0.04) from the others with EFS/OS of 100/100, 67/100, 50/83, and 50/67, after 6 months, 1 year, 2 years, and 3 years, respectively. The promising early data of the pilot study translated in favorable long-term survival only for the small subgroup of patients with complete resection. Two cycles of methotrexate might be too short of a treat-ment. The data of the randomized trial will answer more questions.

MP-09. CHOROID PLEXUS TUMORS: A PLANET-WIDE APPROACH WITH NO AGE LIMITS—INTERIM REPORT 03–2007Johannes Wolff,1 Brigitte Wrede,2 Ove Peters,3 Vidya Gopalakrishnan,4 Anita Mahajan,4 Jeffrey Weinberg,5 Werner Paulus,6 Xumei Wang,4 and Peter Thall7; 1Pediatrics, M.D. Anderson Cancer Center, Houston, TX, USA; 2Pediatrics, Krankenhaus Barmherzige Brueder, Regensburg, Bavaria, Germany; 3Pediatrics, University of Regensburg, Regensburg, Bavaria, Germany; 4University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 5New York, NY, USA; 6Neuropathology, University of Muenster, Muenster, Nord-Rehine Westfalen, Germany; 7Biostatistics, MDAnderson Cancer Center, Houston, TX, USA

Choroid Plexus tumors are too rare to conduct conventional large scale standard national cooperative clinical trials. The international society for Pediatric oncology SIOP therefore started in 2000 the experiment to allow worldwide registration of both adults and children into a combined regis-try for all choroid plexus tumors and an open labeled randomized trial for those patients requiring postoperative treatments “CPT-SIOP 2000.” Chor-oid plexus papillomas (CPP) are generally believed to be cured by complete resection. Atypical choroid plexus papillomas (APP) show increased mitotic activity requiring further treatment if residual tumor or metastases are pres-ent. Choroid plexus carcinoma (CPC) are treated by maximal possible sur-gery, postoperative chemotherapy, 8 weeks delayed irradiation for patients . 3 years, and further chemotherapy. A total of 6 blocks of chemotherapy with VP16 100 mg/m2 3 5 days, VCR 1.5 mg/m2, and either carboplatinum 350 mg/m2 3 2d or cyclophosphamide 1g/ m2 3 2d is intended. After two

cycles response is evaluated. 102 patients were registered since 2000 from 21 nations. 51% were male, the median age was 2.1 years, and the histol-ogy was 39 CPC, 25 APP, and 38 CPP. Response data are available from 33 patients: CR:2, PR:12, SD 15, and PD:4. For survival, histology was a sig-nificant prognostic factor with no death among CPP. The 5-year event-free survival for CPC was 43% and 76% for APP (p , 0.001). In CPC patients treated according to the protocol (n 5 32). Irradiation was linked to better survival (2-year event-free-survival 90% versus 25%, b 11SD; p , 0.0006) independently of the degree of resection, but numbers are still low. Of 54 patients treated according to the protocol, 13 relapses were seen (3/22 APP; 10/32 CPC). Distant metastases were present in 62% and local relapse in 77% of these patients. The treatment arms are still blinded. In contrast with previous reports, these tumors respond to chemotherapy. Irradiation might improve survival. It is too early to obtain confirmatory results.

MP-10. SHRINKAGE OF GERMINOMA BY DEXAMETHASONE ONLY: CASE REPORTElham Amini, Amirhadi Masoudi, Gregory Fuller, Leena Ketonen, Frederick Lang, and Johannes Wolff; University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Central nervous system (CNS) germinomas presumably originate from primordial germ cell rests and characteristically exhibit a biphasic cel-lular histology, with one population of large, neoplastic undifferentiated germ cells and a second population of small reactive lymphocytes. Within the CNS, germinomas most commonly arise in the pineal and suprasellar regions. Pineal germinomas present predominantly in males during the first two decades of life. Without treatment, the natural history of intracranial germinoma is one of rapid growth accompanied by extensive dissemina-tion throughout the CNS via the cerebrospinal fluid pathways. However, germinomas usually respond well to both irradiation and chemotherapy. We report a case of a 17-year-old male with a pineal germinoma who pre-sented with decrease in visual acuity, visual field deficit, severe bilateral papilledema, and headache. MR imaging revealed a pineal region mass accompanied by hydrocephalus. The patient received dexamethasone and was transferred to our hospital for further evaluation and treatment. He was admitted to the ICU and received supportive care in addition to dexametha-sone (8mg IV q6h). An external ventricular drain was placed to decompress the hydrocephalus. Subsequent serial MRI and CT studies performed over the next 15 days showed a striking reduction in tumor size, with the tumor losing 92% of its initial volume (shrinking from 35.6 3 38.1 mm to 15.6 3 17.2 mm). Fourteen days after presentation, open biopsy was performed for definitive diagnosis. Histopathologic examination revealed germinoma with the characteristic immunophenotypic profile of strong expression of c-KIT and OCT4, and no reactivity for CD30, AFP, or hCG. The patient was treated with radiation (45 Gy to the primary tumor bed, 30 Gy to the ventricular system, and 21 Gy to the craniospinal axis) without significant complications. No chemotherapy was administered. The tumor responded further to this treatment and the patient is now free of recurrence 11 months after therapy as documented by serial MRI scans and cerebrospinal fluid studies. There has been only minimal recovery in the patient’s vision. Dra-matically rapid tumor shrinkage in response to steroid therapy is commonly seen with B-cell lymphoma and Langerhans cell histiocytosis. To the best of our knowledge, this is the first case of this phenomenon reported for intracranial germinoma. This experience suggests that caution be exercised in the interpretation of dramatic tumor size reduction in response to thera-peutic intervention.

MP-11. EFFECTIVENESS AND TOLERABILITY OF LEVETIRACETAM AS AN ANTICONVULSANT IN CHILDREN WITH BRAIN TUMORS AND OTHER CANCERSSonia Partap1 and Paul Fisher2; 1Neurology, Stanford University, Palo Alto, CA, USA; 2Neurology, Pediatrics, Neurosurgery, and Human Biology, Stanford University, Palo Alto, CA, USA.

Our objective was to assess efficacy and tolerability of levetiracetam (LEV), a non-cytochrome P450 metabolized drug, as an anticonvulsant in children with primary brain tumors and other cancers. We identified at our institution all oncology patients , 18 years (yr) treated with LEV for seizures. Records were reviewed for seizure type, LEV efficacy and duration of therapy, adverse effects, and other factors. Attention was paid to concur-rently administered enzyme-inducing drugs (EID). SPSS (version 14.0) was used for descriptive and analytic statistical analysis, with non-parametric methods employed. 23 children were identified: median age 12.6 yr 1 stan-dard deviation (SD) 5.4 yr; 11 males; 19 brain tumors, 4 systemic cancers; 19 complex partial seizures, 4 generalized tonic-clonic seizures or both. 17 subjects were on LEV monotherapy and 6 as add-on treatment; 73.9% of children were administered a P450 inducer (9 chemotherapeutics, 8 other). Mean time on LEV was 1.15 6SD 1.24 yr (median 0.70 yr). Final mean LEV dosage for all subjects was 1303.9 6SD 784.4 mg (median 1000 mg); final



mean LEV dosage for children , 12 yr was 31.3 mg/kg (range 17.5–53.6 mg/kg). Over 95% of patients had fewer seizures, with 65.2% becoming sei-zure-free; 14 probably improved from LEV and 8 possibly. Seizure improve-ment did not differ among patients on chemotherapy (p 5 0.22) or an EID (p 5 0.42), although there was a trend toward improvement with craniotomy (p 5 0.08). Only 1 patient experienced an adverse reaction demonstrated by behavioral changes, leading to LEV discontinuation. No patients experi-enced clinically significant depression of blood cell counts or dose-limiting sedation. LEV was well tolerated and effective as an antiepileptic drug, even as monotherapy, in children with brain tumors and other cancers, who are often on multiple enzyme-inducing drugs.

MP-12. THE TREATMENT OF YOUNG CHILDREN WITH MALIGNANT GLIOMAS AND DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPG): RESULTS OF AN IRRADIATION-AVOIDING STRATEGY, THE “HEAD START” PROTOCOLS 1991–2006Jonathan Finlay,1 Ronald Tang,2 Kelley Haley,2 Melissa Siegel,3 Sharon Gardner,4 Jeffrey Allen,4 Anna Butturini,2 Diane Puccetti,5 Albert Cornelius,6 Joseph Torkildson,7 Lingyun Ji,2 and Richard Sposto8; 1University of Southern California, Los Angeles, CA, USA; 2Childrens Hospital Los Angeles, CA, USA; 3Childrens Hospital Los Angeles, New Brunswick, USA; 4New York University, New York, NY, USA; 5University of Wisconsin-Madison, Madison, WI, USA; 6DeVos Childrens Hospital, MI, USA; 7Childrens Hospital of Oakland, CA, USA; 8University of Southern California, CA, USA

The outcome for young children with malignant gliomas and DIPG, in terms of both survival and quality of life, remains poor. The “Head Start” protocols were developed to improve the survival and quality of life of young children with malignant brain tumors, through intensive chemotherapy and avoidance/ minimization of irradiation. Fifty-two children less than 9.5 years of age at diagnosis (mean5 4.3 yrs) of either DIPG (n 5 16) or other malignant gliomas (n 5 36) were enrolled on “Head Start” I, II, and III protocols between 1991 and 2006. The treatment plan included intensive induction chemotherapy, followed by consolidation with a single cycle of marrow ablative chemotherapy (thiotepa, carboplatin 1/– etoposide) with autologous hematopoietic progenitor cell rescue (AuHPCR). Three distinct induction regimens were employed: In “Head Start” I, Regimen A with vin-cristine, cisplatin, cyclophosphamide, and etoposide 3 5 cycles. In “Head Start” II, an initial Regimen B with vincristine, procarbazine, and carbo-platin 3 4 cycles, was replaced by vincristine, temozolomide, and carboplatin 3 4 cycles (Regimen C), which was continued in “Head Start” III. Eighteen children enrolled on the “Head Start” I protocol, (glioblastoma multiforme [GBM] 5 8, anaplastic astrocytoma [AA] 5 6, DIPG 5 4). Twelve children developed progressive disease prior to consolidation. Six children achieved minimal disease and underwent myeloablative chemotherapy; one remains a long-term survivor, 13 years from diagnosis of GBM, without irradiation. All 4 children with DIPG developed tumor progression prior to transplant. Five children were enrolled on “Head Start” II Regimen B, (DIPG 5 3, GBM 5 2). There were no responses and none proceeded with myeloablative chemotherapy; the toxicity of this regimen was substantial. Twenty-eight children were enrolled on Regimen C on either “Head Start” II or III, (DIPG 5 9, GBM 5 9, AA 5 10). Fourteen children attained minimal residual dis-ease, underwent myeloablative chemotherapy and AuHPCR. Eleven of these children survive following myeloablative therapy, three with AA, two with anaplastic oligo-astrocytic tumors, four with GBM, one with a malignant glioneuronal tumor, and one with DIPG, currently 12 to 78 months (mean 5 35 months) from diagnosis. Five of the 11 have received irradiation as per protocol (residual tumor or . 6 yo). Nine children developed progressive disease prior to myeloablative chemotherapy; none survived. One child died of respiratory failure and sepsis during induction, one child died of shunt malfunction and brain stem herniation during induction, and one child was lost to follow beyond 15 months following diagnosis. The prognosis of chil-dren with malignant gliomas and DIPG remains poor. Children with DIPG do not benefit from this irradiation-avoiding intensive chemotherapy strat-egy. A proportion of young children with malignant gliomas may benefit provided they can achieve minimal tumor burden, through either induction chemotherapy, or surgical resection, prior to myeloablative chemotherapy with AuHPCR.

MP-13. HIGH-DOSE CHEMOTHERAPY AND AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANT (HDC/AHSCT) IN CHILDREN WITH PROGRESSIVE OR RELAPSED MEDULLOBLASTOMA OR SUPRATENTORIAL PRIMITIVE NEURO-ECTODERMAL TUMORS (ST-PNET)Anna Butturini,1 Jennifer Aguayo,1 Alexis Teplick,1 Rima Jubran,1 Judy Villablanca,1 Anat Erdreich-Epstein,2 Robert Lavey,1 and Jonathan Finlay1; 1Childrens Hospital of Los Angeles, Los Angeles, CA, USA; 2HemOnc, Childrens Hospital of Los Angeles/University of Southern California, Los Angeles, CA, USA

From 1992 to 2006, 17 patients with medulloblastoma or ST-PNET received HDC/AHSCT after progression. Nine children had and eight had not received radiotherapy as part of the initial treatment. Their age at diag-nosis was 7.2 61.3 and 3.8 61 years; age at transplant was 9.6 61.5 and 5.8 61.3, respectively. HDC at relapse was given as a single cycle in 12 children and multiple cycles in five. The graft was bone marrow in seven patients, peripheral blood HSC in eight and both in two. Radiotherapy was part of the treatment of relapse in five children who were not previously radiated. After transplant, three children died of toxicity and six relapsed. All toxic deaths and four of the six relapses occurred in children who had received radiotherapy as part of their initial therapy. Overall survival and event-free survival (EFS) 3-year post-transplant were 42 615% and 39 612% respec-tively. Survival was better in children who did not receive radiotherapy as part of the initial treatment (87 611% versus 26 616%, p 5 0.01) and in those who received radiotherapy around the time of HDC/AHSCT (100% versus 33 615%, p 5 0.02). EFS was better in children who received radio-therapy at the time of HDC/AHSCT (100% versus 10 69%, p 5 0.006). Age at diagnosis or transplant or type of transplant (single vs. multiple cycles) or HSC source (bone marrow vs. blood) did not affect outcome. In conclusion, HDC/AHSCT is effective predominantly in children who relapsed without having received prior radiotherapy. In such children the use of radiotherapy at time of transplant is beneficial.

MP-14. CHEMORADIOTHERAPY WITH TEMOZOLOMIDE IN THE TREATMENT OF DIFFUSE INTRINSIC PONTINE GLIOMAS OF CHILDHOOD: RESULTS OF THE CHILDREN’S ONCOLOGY GROUP (COG) ACNS0126 TRIALKenneth Cohen,1 Richard Heideman,2 Tianni Zhou,3 Emi Holmes,3 and Ian Pollack4; 1Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA; 2University of New Mexico SOM, Albuquerque, NM, USA; 3Children’s Oncology Group, Arcadia, CA, USA; 4University of Pittsburgh, Pittsburgh, PA, USA

The prognosis for children diagnosed with a Diffuse Intrinsic Pon-tine Glioma (DIPG) remains dismal with most children dying between 6 months and two years from the time of diagnosis. Based on evidence sug-gesting that temozolomide might be efficacious in the treatment of fibrillary astrocytomas, the Children’s Oncology Group (COG) evaluated the utility of chemoradiotherapy with temozolomide (54.0 Gy / 90 mg/m2/day 3 42 days) followed by adjuvant temozolomide (200 mg/m2/day 3 5 days for a maximum of 10 cycles). The results were compared to the historical control cohort on CCG-9941 (n 5 63) in which 32 patients with newly diagnosed brainstem gliomas were randomly assigned to receive three courses of carbo-platinum, etoposide, and vincristine or three courses of cisplatin, etoposide, cyclophosphamide, and vincristine followed by hyperfractionated external-beam radiotherapy to a dose of 72 Gy. 61 children newly diagnosed with DIPG were enrolled on ACNS0126. No significant difference was found between the two studies. The one-year EFS is 15% 65% for ACNS0126 compared to 21% 65% for CCG-9941 (logrank p 5 0.16); the one-year OS is 39% 66% for ACNS0126 compared to 32% 66% for CCG-9941 (logrank p 5 0.3). Thirty-seven patients (61%) died within 12 months and 54 patients (89%) died within 18 months. In contrast to the patients diag-nosed with a supratentorial high-grade glioma on ACNS0126, no biopsy was required for enrollment on the DIPG stratum. For this reason, analyses for methylguanine-methyltransferase (MGMT) or other predictive factors for sensitive to temozolomide could not be undertaken. Chemoradiotherapy with temozolomide followed by adjuvant temozolomide does not improve the outcome for children with DIPG and alternate treatment approaches should be pursued.



MP-15. EVALUATION OF THE EFFICACY OF HIGH-DOSE CHEMOTHERAPY (HDC) 1 AUTOLOGOUS STEM CELL RESCUE (ASCR) 1/– RADIOTHERAPY (RT) OR STANDARD SALVAGE THERAPY IN CHILDREN WITH RECURRENT MEDULLOBLASTOMA (MBL)Sridharan Gururangan,1 Jeanne Krauser,1 Melody Watral,1 Timothy Driscoll,2 Nicole Larrier,3 Herbert Fuchs,4 David Reardon,1 James Vredenburgh,1 Jennifer Quinn,1 Annick Desjardins,1 Jeremy Rich,1 and Henry Friedman1; 1The Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC, USA; 2Division of Blood and Marrow Transplantation, Duke University Medical Center, NC, USA; 3Department of Radiation Oncology, Duke University Medical Center, NC, USA; 4Division of Neurosurgery, Duke University Medical Center, NC, USA

Evaluate the efficacy of HDC 1 ASCR in patients (pts) with recurrent MBL who did or did not receive prior radiotherapy (RT) as compared to those who received standard salvage therapy. Retrospective chart review of pts with recurrent MBL diagnosed between 1995–2005 and who had undergone HDC 1 ASCR 1/– RT (n 5 19) or standard salvage therapy (n 5 11) at our institution. A total of 30 pts were diagnosed with recurrent MBL following standard RT alone or chemotherapy 1/– RT (local relapse 11 pts, metastatic 14, local 1 metastatic, 5). Nineteen pts (median age, 7 yrs [range, 2–12]; Group A, pts who received no RT before recurrence [n 5 7]; Group B, received definitive RT before recurrence [n512]) underwent surgery and/or cyclophosphamide (CTX) based induction chemotherapy followed by HDC 1 ASCR with CTX 1 melphalan (n 5 11), busulfan 1 melphalan (n 5 6), carboplatin 1 etoposide (n 5 1), and carboplatin 1 thiotepa 1 etoposide (n 5 1) regimens. Six of 7 Group A pts also received RT (Focal [56 Gy] 1/– craniospinal [36–39.6 Gy]) just prior to or post recovery from HDC. Group B pts were either given none or only stereotactic focal RT post HDC. At a median follow-up of 33 months (range, 6–116), 2 of 6 pts in Group A are alive and disease-free at 1101 and 1161 months post-HDC respectively. One additional pt in Group A with Gorlin’s syndrome and local relapse of MBL did not receive any RT post-HDC and is disease-free at 281 months from HDC. All pts in Group B have died of further disease progression at a median of 11 months (range, 4–24) from HDC. All 11 pts (median age 6 yrs, range, 2–24) who underwent standard salvage therapy at recurrence have died of disease at a median of 26 months (range, 3–112) from relapse. The 3-year progression-free survival for Group A pts is 14% (95% CI, 0%–30%). HDC1ASCR or standard salvage therapy does not appear to be effective in our patients with recurrent MBL when they have had definitive RT before progression. While 2 pts are long-term survivors following HDC, the impact of the strategy on the long-term disease control of these pts is unclear since they also received adjuvant definitive RT fol-lowing recurrence.

MP-16. ARE PONTINE GLIOMAS DIFFERENT? ANALYSIS OF THE HIT-GBM-DATABASEJohannes Wolff,1 Carl Classen,2 Rolf-Dieter Kortmann,2 Torsten Pietsch,3 Stefan Rutkowski,4 Astrid Gnekow,2 Shan Palla,2 Pablo Hernaiz Driever,5 and Christoph Kramm2; 1M.D. Anderson Cancer Center, Houston, TX, USA; 2Germany; 3University of Bonn, Germany; 4University of Wuerzburg, Germany; 5Berlin, Germany

In the pediatric population, malignant gliomas represent a heteroge-neous group of tumors. The HIT-GBM study concept consists of a num-ber of five sequential treatment protocols, and also included observation patients as tumor registry. This publication describes a prognostic factor analysis based upon the patients registered in the first three protocols (HIT-GBM-A, -B, and -C). Inclusion criteria were either histologically confirmed high-grade glial tumors (HGG), or radiologically confirmed diffuse intrinsic pontine gliomas (DIPG). 310 patients (173 male, median age 10.0 years, 3.2–18.0 years) were enrolled. The primary tumor location were cerebral cortex and white matter in 80, thalamus and basal ganglia in 38, pons in 134, brain stem outside of pons in 14, cerebellum in 14, spinal in 8, and overlapping areas in 22 cases. Surgery was complete resection in 49, subtotal in 35, partial in 58, biopsy in 99, and no surgery in 69 cases. Histologically, 123 cases were WHO grade IV tumors, 101 grade III, and 15 grade I/II. For 71 of 123 pontine gliomas no histology was obtained. 228 patients could be evaluated for response after eight weeks of treatment, CR was found in 8, PR in 32, SD in 116, and PD in 72 cases. Median overall survival (OS) time was 1.03 years (standard error: 0.05), and median event-free survival (EFS) time was 0.54 years (standard error 0.03). 5-year OS-rate was 10.3% 1 2.1%. Possible prognostic factors were not distributed homogeneously. Tumors in the pons differed from tumors of the cerebral hemispheres, con-cerning the frequency of previous diseases such as leukemia (pons 1.6%, cortex 15.4%), the age at diagnosis (median age pons 7.9 years, cortex 11.4), and the frequency of Grade III versus Grade IV (III:IV 5 1.6 for pons, and 0.7 for cortex). In the total database tumor location, grading, and surgical resection were prognostic factors, but their relevance differed in various subgroups of locations with no relevance for sex, grading or surgical resec-

tion for pontine tumors. The biology of pontine glioma differs from other poor prognostic glioma. Future clinical protocols should be separate for pontine and for non-pontine tumors using different treatment protocols and different primary endpoints.

MP-17. A RECURRENT NON-LIPOMATOUS CEREBELLAR NEUROCYTOMA IN A YOUNG BOYGregory T. Ray1 and Roger McLendon2; 1Duke University, Durham, NC, USA; 2Pediatric Brain Tumor Foundation Institute, Duke University, Durham, NC, USA

Central neurocytomas, first described in 1982 by Hassoun et al., typically grow from the foramen of Monro or the septum pellucidum and involve the lateral or third ventricle; often with calcification. This benign tumor affects young adults and comprises less than 1% of all brain tumors. We present a case of a 7-year old boy who originally presented with a cer-ebellar lesion. The MRI report described a complex posterior fossa mass with predominantly T1 darkness and a nidus of enhancement. The mass was impinging upon the posterior aspect of the fourth ventricle. This original resection was followed by a second resection approximately 2 years later and a third resection approximately 4 years after the first resection. In the original resection specimen, this neoplasm is characterized by a monomor-phic population of small round cells with dispersed chromatin. There are many acellular neuropil-rich formations. Mitotic figures, microvascular pro-liferation, and necrosis are not seen. Rosenthal fibers, eosinophilic granular bodies, and a biphasic appearance are not identified. The tumor cells are positive for synaptophysin and negative for glial fibrillary acidic protein. In the most recent resection specimen, the tumor has similar morphology and the tumor cells were positive for synaptophysin and S-100 but negative for Neu-N, neurofilament protein, epithelial membrane antigen, chromogranin, and GFAP. The Ki67 labeling index is 1%. Histologically, the tumor is a non-lipomatous extra-ventricular neurocytoma. Neurocytic lesions in the cerebellum have only recently been reported and are uncommon. A major-ity of the neurocytic lesions in the cerebellum are liponeurocytomas and contain neuronal and glial differentiation with lipomatous areas. To our knowledge, there have been only three reports of neurocytomas (without lipomatous areas) in the cerebellum (including the present case) exist. This unusual variant must be distinguished from the pilocytic astrocytoma and the medulloblastoma with extensive nodularity.

MP-18. PROGNOSTIC FACTORS IN PONTINE GLIOMAFlorian Segerer,1 Leena Ketonen,2 Ming Zhang,2 Margaret Nagel,2 and Johannes Wolff2; 1University of Regensburg, Regensburg, Bavaria, Germany; 2University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Pontine gliomas have a poor prognosis, and little is known about their biology, since tissue is rarely available for analysis. With this retrospective chart review (IRB: RCR05–0728), we intended to use clinical and imaging data to determine, how much a stem cell population in the tumors con-tributes to the prognosis. We hypnotized that a treatment resistant tumor cell population will continue to grow while other tumor parts disappear in response to the treatment. If this is the case, overall survival is determined by different parameters then tumor response. We analyzed the MR-images of 27 patients with confirmed diagnosis of pontine glioma, who were treated with radiation and were seen at M.D. Anderson Cancer Center. The devel-opment of the tumors’ volumes was compared to the patients’ age and over-all survival. In this population, the overall survival was independent from response (SD n 5 17, survival 151 1 47days; PR n 5 10, survival 382 1 46 days, n.s.), the maximal shrinking-rate (slow shrinking n 5 14, survival 364 1 43 days, fast shrinking n 5 13, survival 367 1 57 days, n.s.) or the time of relapse free survival (rfs) (short rfs, n 5 13, survival 338 1 43 days; long rfs n 5 14, survival 407 1 60 days, n.s.) seen on standard MRIs. The survival was related to the tumor’s size at the beginning of radiation (small n 5 13, survival 464 1 49 days, big n 5 14, survival 271 1 28 days, significance 5 0.0022). In different age groups, the tumors showed different behavior: Children younger than 7 years responded better to radiotherapy (linear regression, m 5 –3%/year, significance 5 0.047) and girls in this group sur-vived longer than boys (age , 7 years, male n 5 8, survival 228 1 30 days, female n 5 4, survival 430 1 73 days, significance 5 0.038), which was not the case in older children (age . 7 years, male n 5 5, survival 369 1 65 days, female n 5 10, survival 427 1 59 days, n.s.). The initial tumor volume was the only prognostic factor for the overall survival in these pontine glioma patients. The independence of survival from response-parameters may be due to heterogeneity of the tumor cells, consisting of resistant tumor stem cells and a second radio-sensible tumor cell population. Pontine gliomas in young children behaved different than in older children.



PATHOLOGY AND PROGNOSTIC FACTORS

PA-01. GENETIC MARKERS PREDICTIVE OF CHEMOSENSITIVITY AND OUTCOME IN GLIOMATOSIS CEREBRIMarc Sanson,1 Gentian Kaloshi,2 Florence Laigle-Donadey,2 François Ducray,2 Ahmed Idbaih,2 Sibille Everhard,3 Yannick Marie,3 Soledad Navarro,4 Karima Mokhtari,5 Joelle Thillet,3 Khe Hoang-Xuan,2 and Jean-Yves Delattre2; 1Neurology, Paris, France; 2Neurology, France; 3INSERM U711, France; 4Neurosurgery, France; 5Neurolopathology, France

Patients with gliomatosis cerebri (GC) have an extremely variable prog-nosis. So far no predictive and prognostic markers have been identified for this rare disease. Several studies have suggested that methylation of methyl-guanine methyl transferase promoter (MGMTP) and chromosome 1p 19q codeletion were correlated with an increased response rate (RR), progression free survival (PFS) and overall survival after treatment with nitrosoureas or temozolomide (TMZ) in patients with gliomas. With regard to these data we performed a retrospective analysis of a cohort of patients with GC previ-ously treated with TMZ to assess the predictive value of 1p19q codeletion and MGMTP status on the RR and PFS. Tumor samples of 25 patients with GC treated with Temozolomide were analyzed to detect 1p19q deletions by heterozygosity technique. Status of MGMT methylation was determined in 19 out of 25 patients. Nine out of 25 patients presented 1p19q codeletion. Codeletion of 1p19q was associated with a higher response rate (8/9 5 88 % vs. 4/16 5 25 %, p 5 0.002) compared with patients without 1p19q codele-tion. Similar results were observed for the PFS and overall survival (OS), patients with 1p19q codeletion had longer PFS, 24.5 months (m) versus 13.7 m, (p 5 0.017), their OS was improved 66.8 m vs. 15.2m (p 5 0.011). Four-teen out of 19 evaluable tumors for MGMTP status were methylated. There was an association between MGMTP methylation and 1p/19q codeletion (p 5 0.045). Patients with unmethylated MGMTP tended to have shorter PFS and higher rate of progressive disease. In this cohort chromosome 1p19q codeletion seems to be the most important prognostic marker and molecular predictor of the response to treatment with TMZ. Nevertheless the impact of MGMTP methylation status with respect to the TMZ sensitivity is still unsettled in this population.

PA-02. IQGAP1 AND IGFBP2: VALUABLE BIOMARKERS FOR DETERMINING PROGNOSIS IN GLIOMA PATIENTSKerrie McDonald,1 Maree O’Sullivan,2 Jonathon Parkinson,1 Jan Shaw,1 Cathy Payne,1 Janice Brewer,3 Helen Wheeler,4 Raymond Cook,5 Michael Biggs,5 Nicholas Little,5 Charles Teo,6 Glenn Stone,2 and Bruce Robinson7; 1Cancer Genetics, Kolling Institute of Medical Research, St Leonards, New South Wales, Australia; 2Mathematical and Information Sciences, CSIRO, New South Wales, Australia; 3Department of Anatomical Pathology, Royal North Shore Hospital, New South Wales, Australia; 4Department of Oncology, Royal North Shore Hospital, New South Wales, Australia; 5Department of Neurosurgery, Royal North Shore Hospital, New South Wales, Australia; 6Centre for Minimally Invasive Neurosurgery, Prince of Wales Hospital, New South Wales, Australia; 7Faculty of Medicine, University of Sydney, New South Wales, Australia

Developing reliable prognostic markers for patients diagnosed with high grade gliomas has been challenging, despite overwhelming evidence that wide variability exists in the clinical behavior, and thus in survival times, of morphologically identical tumors. We used microarray analysis to identify biomarkers that could be used as an adjunct to the WHO 2000 grading sys-tem to improve prognostic accuracy in glioma. To identify markers strongly associated with the most aggressive biological behavior, we classified all gliomas into a modified grading scheme based on the histologic features typically associated with poor survival, namely, necrosis and microvascular proliferation (MVP). These two groups were designated NMVP positive (Necrosis and MVP present in tumor) and NMVP negative (Necrosis and MVP absent in tumor). This partitioning was done, in addition to their original WHO classifications. Two multivariate analysis methods, Gene-Rave and SDDA were used to identify small gene sets with high predictive accuracy. The candidate gene sets were validated using real time PCR and protein expression was investigated by immunohistochemistry. The over-expression of IQGAP1 and IGFBP2, at both the gene and protein levels, was highly correlated with gliomas exhibiting necrosis and MVP. Protein expression of IQGAP1 and IGFBP2 were observed in over 80 percent of NMVP positive gliomas and were associated with significantly shorter adjusted median survival. When either of one of these markers was used in conjunction with the WHO system, both IQGAP1 and IGFBP2 readily iden-tified a subgroup of AIII, OdgIII, and OAIII whose prognosis was relatively poor. These results suggest that IQGAP1 and IGFBP2 are clinically useful prognostic markers and provide additional information to the WHO sys-tem. Interestingly, a sub-group of patients who exhibited long term survival

(. 3 year survival post-GBM diagnosis) did not express IGFBP2 nor IQGAP1. The absence of IQGAP1 and IGFBP2 may prove to be prognosti-cally useful as histological marker of long-term survival. We have shown that the protein expression of IQGAP1 and IGFBP2 is strongly associated with poor survival in astrocytoma and oligodendroglioma. Although the association of IGFBP2 has previously been described with poor prognosis, the association of IQGAP1 is a novel finding. The findings of our study are significant as IQGAP1 and IGFBP2 protein expression markers could com-plement the WHO classification system to permit more precise delineation of the existing tumor grades and identify potentially more aggressive glioma subtypes. These markers could also improve accuracy in the prediction of biological grade and aggressive potential, particularly in small biopsies of gliomas that are surgically inoperable.

PA-03. NEUROTROPHINS AND CHEMOKINES IN BRAIN METASTASESBianca Pollo, Emanuela Maderna, Chiara Calatozzolo, Paola Gaviani, Graziella Filippini, and Andrea Salmaggi; Istituto Neurologico C. Besta, Milano, Italy

Neurotrophins (NTs) are growth factors that play an important role in tumorigenesis in non-neural tissues, their expression have been investigated in several non-neuronal carcinomas as markers in the clinic. Moreover NTs are important brain-invasion stimulating factors and can promote invasion of responsive tumor cells through the blood-brain barrier by enhancing the production of basement-membrane-degradative enzymes. Different tumors showed different tendencies to brain metastatization, this depend on prop-erties of primary tumor and on brain-specific features, among these the interaction of chemokines with their receptors may play a role in homing of neoplastic cells from the primary site to the target organ. Recent works underscore the potential relevance of CXCL12/CXCR4 interaction in the metastatization process in a large number of tumors. NTs are a family of secreted proteins consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5). These molecules bind two types of cell surface receptors: the Trk receptors and the common neurotrophin receptor p75. The Trk family of receptors has tyrosine kinase activity and is made up of three members, each with a different specificity: Trk-A binds NGF, Trk-B binds BDNF and NT-4/5, and Trk-C binds NT-3. Firstly we performed a retrospective study on the presence and distribution of some NTs and their receptors in 63 sur-gically resected brain metastasis from different types of malignant tumor, with the following origin: lung (20), breast (20), kidney (10), colon (5), ovary (5), prostate (2), and thyroid (1). A clinico-histological study was performed correlating molecular expression to clinical outcome (time to tumor pro-gression-TTP, survival time-ST, KPS) and to the pattern of nervous tissue infiltration, to evaluate a possible clinical role of NTs and CXCL12. The specificity of antibodies was determined by Western blot analysis. In our study, most of brain metastases from different origin displayed high level of high-affinity tirosin-kinase receptors Trk-A, Trk B, and Trk-C and low expression of p75. As concern the neurotrophins expression, NT-3 showed moderate expression in all the cases except in ovary carcinomas. NGF and BDNF displayed a variable distribution: NGF was higher in breast, BDNF was higher in lung, colon and kidney. Immunoreactivity for CXCL12 was detected in more differentiated subtype of lung metastases and not in “small cells” carcinomas with worse outcome. Colon metastases showed CXCL12 positivity in more differentiated types. Highest expression of CXCL12 was observed in breast metastasis while ovary metastases showed lowest expres-sion. A partial correlation was found between the immunohistochemical expression and the pattern of nervous tissue infiltration, also with NTs expression in reactive astrocytes in some cases. The overall survival of the patients was evaluated and the correspondence with the NTs, NT recep-tors and CXCL12 expression showed that TrkA/NGF and BDNF positivity seems to be related to shorter ST, while TrkB, TrkC, and CXCL12 could be predictive of a prolonged survival. Our results suggest that NTs and chemokines may be involved in growth and invasion of several brain metas-tases and may have a clinical significance as possible prognostic factors and new therapeutic target.

PA-04. MONITORING TRANSCRIPTIONAL ACTIVATION OF SPARC GENE IN GLIOMAS USING INDICATOR INFECTIOUS BAC (iiBAC) TECHNOLOGYHiroshi Nakashima,1 Yoshinaga Saeki,2 and E.Antonio Chiocca2; 1Neurosurgery, Ohio State University, Columbus, OH, USA; 2Ohio State University, Columbus, OH, USA

Malignant gliomas are characterized by heterogeneous regions of necro-sis, apoptosis, proliferation, invasion, and angiogenesis. Understanding dif-ferences in gene expression between normal brain and malignant glioma tissue is useful for diagnosing and treating gliomas. Recently, serial analysis of gene expression (SAGE) data for malignant gliomas have become avail-



able through the Cancer Genome Anatomy Project (CGAP), and several genes have been identified that are overexpressed in gliomas but not in nor-mal brain. Some have been postulated to correlate with a particular glioma phenotype, such as invasion or angiogenesis. We propose to use a set of technologies to translate knowledge obtained from CGAP in imaging the transcriptional activation of the glioma-expressed genes and to correlate such activation with the phenotypic heterogeneity observed in in vitro and in vivo glioma models. We have devised a simple and efficient methodology to convert any BAC or PAC library clone into a herpes simplex virus (HSV)-based amplicon vector and demonstrated that genomic loci of up to 150 kb can be delivered intact into and expressed in target cells. We plan to combine this technology, designated infectious BAC (iBAC), with bioluminescence imaging and/or magnetic resonance imaging to monitor and visualize tran-scriptional activation of glioma-expressed genes in glioma models. Among the genes upregulated in human malignant gliomas, we selected SPARC (secreted protein, acidic and rich in cysteine) as a model gene to study. SPARC is known to be involved in remodeling of the extracellular matrix (ECM) and is associated with tumor invasion. To monitor transcriptional activity of the SPARC gene, we first identified a BAC clone that contains the 120 kb of 5' flanking region and the first exon of this gene. By homologous recombination in Escherichia coli, we inserted into the BAC clone a bicis-tronic reporter cassette that contained firefly luciferase and internal ribo-some entry site (IRES)-green fluorescent protein (GFP) within the first exon of the SPARC gene. Both reporter genes are thus expressed under the con-trol of the SPARC promoter and its native regulatory sequences. To deliver the indicator BAC efficiently into glioma cells, the BAC clone was retrofitted with HSV amplicon elements (oriS and pac) using the Cre-loxP system. The engineered SPARC-indicator iBAC (iiBAC) clone was then packaged into HSV virions using the helper virus-free HSV amplicon packaging system. When the packaged SPARC-iiBAC was inoculated into human glioma cell lines, we observed levels of luciferase expression that increased in an MOI-dependent manner. This result confirmed that the HSV amplicon-delivered 5' flanking region and promoter of the SPARC gene is functional in human glioma cells. We are evaluating if the levels of reporter expression from the SPARC-iiBAC recapitulate endogenous levels of SPARC expression in vari-ous cell lines or culturing conditions. We believe that this methodology will also provide a new means to identify transcriptional elements (promoters and enhancers) that can target specific cell types and conditions, which will be useful tools to develop disease-selective gene therapies.

PA-05. A UNIQUE PRESENTATION OF LYMPHOMATOSIS CEREBRIMatthew Rendel,1 Ania Pollack,2 Sarah Taylor,3 and Newell Kathy4; 1University of Kansas Medical Center, Kansas City, KS, USA; 2Neurosurgery, University of Kansas Medical Center, Kansas City, KS, USA; 3Hematology/Oncology, University of Kansas Medical Center, Kansas City, KS, USA; 4Pathology, University of Kansas Medical Center, Kansas City, KS, USA

Lymphomatosis cerebri (LC) is a unique and rare form of primary central nervous system lymphoma (PCNSL) that presents as diffuse wide-spread infiltration of lymphoma cells throughout the white matter tracts of the cerebral hemispheres. In contrast to lymphomatosis cerebri, PCNSL typically presents in the immunocompetent patient as a single, discrete, contrast-enhancing mass lesion found adjacent to the CSF spaces. Symptom presentation of PCNSL includes focal neurological deficits consistent with mass effect. Lymphomatosis cerebri presents more insidiously with neu-robehavioral changes related to the disruption of the white matter tracts, a syndrome called white matter dementia. Focal deficits have occurred later in the disease course. We present a 73-year-old immunocompetent male who initially presented with left hand weakness. The patient denied cog-nitive changes. But more extensive interview with his wife indicated dif-ficulty with attention, decision-making, executive function, and significant personality changes including irritability. Physical examination revealed a mild left hand weakness as well as anxiety, behavioral impulsiveness, and emotional lability. Memory, speech, and language were clinically intact. MRI was consistent with an infiltrative process. Several contrast-enhancing areas within the white matter of the bilateral frontal lobes were present with extension across the corpus callosum. Cortex was not involved and there was not appreciable mass effect. FLAIR imaging showed a diffuse involve-ment throughout the white matter of the centrum semiovale. An astrocytic process was suspected and a stereotactic needle biopsy was performed with tissue cores taken from the enhancing areas and non-enhancing areas of increased FLAIR signal on the MRI. Histopathology showed a diffuse, infiltrating large B-cell lymphoma within the white matter, consistent with the description of lymphomatosis cerebri. Immunoreactivity to CD45, CD20, and bcl-2 confirmed B-cell origin. The patient was started on high-dose methotrexate therapy within one week of diagnosis. He is currently in remission approximately one year from diagnosis. Imaging follow-up shows resolution of contrast-enhancing areas, with no signal changes detected on FLAIR images. He has shown improvement neurologically and his person-ality and cognition have returned to their baseline. There are only a few

cases of lymphomatosis cerebri present in the literature. Previous reports describe a diffuse infiltration of neoplasm throughout the bilateral white matter visualized with MRI T2-weighted and FLAIR image sequences. Contrast enhancement has not been described. Additionally, current clini-cal descriptions characterize lymphomatosis cerebri as a cause of white matter dementia, with less prominent focal findings. However, the clinical description of LC is still in its infancy. This patient’s presentation shares common features of the reported cases of LC, but varies by presenting with a pronounced focal deficit and including the presence of enhancing lesions on MRI. This case expands on the known presentations of LC and repre-sents a variation in the clinical and radiographic manifestations of the same disease process.

PA-06. AQP1 IS ASSOCIATED WITH GBM SURVIVAL AND MAY MEDIATE AGGRESSIVENESS THROUGH INCREASED CELL MOTILITYEzra Mirvish,1 Erik Sulman,2 Anjan Misra,1 Janice Nigro,3 Ivan Smirnov,4 Leon Kapp,4 J. Matthew McDonald,2 Lihong Long,2 Sonya Popoff,2 Howard Colman,2 Michael Berens,5 Kenneth Aldape,2 and Burt Feuerstein6; 1Barrow Neurological Institute, St. Joseph Hospital and Medical Center, Phoenix, AZ, USA; 2University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3Bergen, Norway; 4CA, USA; 5Translational Genomics Research Institute, Phoenix, AZ, USA; 6Neurology, Barrow Neurological Institute, St. Joseph Hospital and Medical Center, Phoenix, AZ, USA

Despite aggressive surgery and adjuvant chemo- and radiotherapy, glio-blastoma multiforme (GBM) median survival remains near one year. We used high-throughput DNA and expression microarrays to identify three genetic subgroups—mesenchymal, proliferative, and proneural—strongly associated with survival (Phillips et al., Cancer Cell 2006;9[3]: 157–173; Nigro et al., Cancer Res 2005;65[5]: 1678–1686). Our analysis suggested genes, including the water channel Aquaporin1 (AQP1), that predict patient survival. Array data suggests that tumors with high AQP1 expression are more likely to fall within the mesenchymal and proliferative subgroups (p 5 0.03) and have poor survival (p , 0.0001). IHC analysis of 177 tumors for AQP1 protein expression and Real-Time, Quantitative Reverse Tran-scriptase PCR (qRT-PCR) further suggest that long-term GBM survivors have approximately three-fold lower AQP1 expression than do short-term survivors. We developed a stable model of AQP1 over-expression in the U251 cell line and validated by qRT-PCR and Western blotting. We used this model, to investigate cell growth, survival, and migration using growth curve, colony forming efficiency, and wound healing assays. Our data sug-gest that AQP1 overexpression does not affect cell growth and survival, but it does increase cell migration. This confirms previous data that suggest AQP1 regulates cell motility. Since GBMs often present with an invasive phenotype, and fail locally, a direct relationship between AQP1 expression and GBM cell motility will argue that AQP1 predicts GBM outcome and is a therapeutic target. Supported by the NIH (NS42927 and CA85799), National Brain Tumor Foundation, the Barrow Neurological Foundation, and the Brain Tumor Resource and Information Network.

PA-07. THE PROGNOSTIC SIGNIFICANCE OF PTEN PROMOTER HYPERMETHYLATION IN LOW-GRADE GLIOMASSean M. McBride, Justin S. Smith, Shichun Zheng, Edward F. Chang, Kathleen R. Lamborn, John Wiencke, Susan Chang, Michael D. Prados, Mitchel S. Berger, David Stokoe, and Daphne Haas-Kogan; University of California, San Francisco School of Medicine, San Francisco, CA, USA

Methylation of DNA promoter sequences can result in the transcrip-tional silencing of tumor suppressor genes and thus promote tumorigen-esis. Recent data have demonstrated that the putative 5'-promoter region of the tumor suppressor PTEN is hypermethylated in approximately 50% of low-grade (Grade II) gliomas (LGGs). We sought to define the incidence of PTEN promoter methylation and to determine its prognostic significance in LGGs in order to determine the potential usefulness of rapamycin for the treatment of patients with LGGs. Patients and Forty-three LGG (grade II) tumor specimens from newly diagnosed patients were analyzed for methy-lation of the putative 5'-promoter region of the PTEN promoter using gel-based methylation specific PCR (MS-PCR). Genomic DNA was isolated from frozen tissue samples and then bisulfite treated using the CpGenome DNA Modification Kit (Chemicon). To assess PTEN promoter methyla-tion we used methylation-specific primers. Female to male ratio was 21:22. Twenty-one patients had astrocytomas (49%), fifteen oligodendrogliomas (35%), and 7 mixed histology (16%). Median age at diagnosis was 37 years (range 20–64). Overall survival (OS) and progression-free survival (PFS) were assessed using Kaplan-Meier methods and log-rank test. P values are two-tailed, unless otherwise specified. Median follow-up for surviving



patients in the unmethylated group was 5.1 years (range 0.01–11.5) and 6.2 years (range 0.01–11.69) in the methylated group. Of the 43 patients included in the study, 26 had methylation of the PTEN promoter (60%). There was no statistically significant relationship between PTEN methyla-tion status and PFS (p 5 0.32). Nine patients progressed in each of the meth-ylated and unmethylated groups. There was a trend toward a relationship between PTEN promoter methylation and overall survival (OS). Relative to the unmethylated group, patients with PTEN promoter methylation had shorter survival times that approached statistical significance (p 5 0.11, one tailed). Estimated five-year survival was 93% for patients without methyla-tion vs. 83% for patients with methylation. Estimated six-year survival in the unmethylated group remained 93% but dropped to 79% for the methy-lated group. Seven of 26 patients in the methylated group died versus 1 of 17 patients in the unmethylated group. Patients with PTEN tumor methylation show a trend towards decreased OS relative to those without methylation. Continued follow-up of the current cohort and evaluation of additional specimens will provide improved estimates. PTEN promoter methylation did not correlate with PFS, likely due to ambiguities in conclusively estab-lishing progression of LGGs and lack of stringent schedules for follow-up imaging in this cohort. Correlating PTEN promoter methylation with both PTEN expression and the activity of downstream effectors such as PKB/Akt, p70S6K, 4E-BP1, and S6, will help ascertain the biological significance of the observed trend.

PA-08. MALIGNANT GLIOMAS WITH NEUROBLASTIC (PNET-LIKE COMPONENTS (GBM-PNET): A CLINICOPATHOLOGIC AND GENETIC STUDY OF 28 CASESArie Perry,1 Ryan Miller,2 Meena Gujrati,3 Bernd Scheithauer,4 Sarah Jost,5 Ravi Raghavan,6 Eric Holland,7 Peter Burger,8 and Marc Rosenblum9; 1Pathology, Washington University in St. Louis, St. Louis, MO, USA; 2Pathology, University of North Carolina School of Medicine, Chapel Hill, NC, USA; 3Pathology, University of Illinois College of Medicine at Peoria, Peoria, IL, USA; 4Pathology, Mayo Foundation for Medical Education and Research, Rochester, MN, USA; 5Neurosurgery, Washington University in St. Louis, St. Louis, MO, USA; 6Pathology, Loma Linda University Medical Center, Loma Linda, CA, USA; 7Cancer Biology & Genetics, Surgery (Neurosurgery), and Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA; 8Pathology, Johns Hopkins University, Baltimore, MD, USA; 9Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

Although rare examples of glioblastoma (GBM), gliosarcoma (GS), and other malignant gliomas combined with CNS primitive neuroectodermal tumor (GBM-PNET) have been published, such tumors remain poorly char-acterized. In fact, GBM and PNET are widely considered separate, non-overlapping entities with distinct biologic and therapeutic implications. Thus, their occurrence in combination is not only a histogenetic curiosity, but poses significant diagnostic and treatment dilemmas. Herein, we present the largest series of GBM-PNET reported to date, including 28 patients (16 male, 12 female), ranging in age from 11 to 79 (median, 50) years. Every case presented as an enhancing, cerebral hemispheric mass and the histol-ogy contained regions of classic GBM or GS, often with additional foci resembling a lower grade glioma (17 astrocytomas, 5 oligoastrocytomas, 1 oligodendroglioma); in 3 cases a diagnosis of WHO grade II glioma was made 2 to 7 years prior to their GBM-PNET presentation. The PNET component usually appeared sharply demarcated from the glioma and fea-tured marked cellularity, elements of neuronal differentiation (neuroblastic rosettes, neuropil, streaming, nodular/desmoplastic pattern, cell wrapping), and immunoreactivity for one or more neuronal markers (synaptophysin, Neu-N, chromogranin, NSE). Anaplasia as seen in some medulloblastomas was noted in 17 (61%). In two-thirds of cases, the Ki-67 proliferation label-ing index was . 70% in neuroblastic foci. Immunostains for p53 protein were positive in 80% of cases, 56% showing strong, diffuse staining. FISH studies revealed PNET-like MYC gene amplifications (mostly N-myc) in 42%, being more common in anaplastic (60%) than non-anaplastic (18%) neuroblastic components (p 5 0.05). Glioma-associated alterations included chromosome 10q loss (62%), EGFR amplification (12%), and 1p/19q code-letion (9%), with some cases featuring both PNET-like and glioma-like patterns. Most patients were treated with radiation and temozolamide che-motherapy. Cerebrospinal or soft tissue dissemination of the neuroblastic element developed in 5 of 12 patients, a favorable response to PNET-like therapy occurring in at least 2. At last follow-up, 18 patients had died of dis-ease and 6 were alive, their overall survival ranging from 1 week to 3.3 years (median, 8 months). We conclude that the neuroblastic component of the GBM-PNET: (1) arises within a pre-existing malignant glioma, most often a secondary GBM or GS; (2) may represent a metaplastic process or expansion of a tumor stem/progenitor-cell clone; (3) often shows histologic anaplasia and N-myc amplification; (4) has a capacity to seed the cerebrospinal space; and (5) may respond to PNET-like therapeutic regimens.

PA-09. MICRODISSECTION MGMT METHYLATION QUANTIFICATION IN INDIVIDUAL GBM YIELDS MARKED HETEROGENEITYLara Kunschner,1 Deepinder Singh,1 and Syd Finkelstein2; 1Neurology, Allegheny General Hospital, Pittsburgh, PA, USA; 2RedPath Integrated Pathology, Inc., Pittsburgh, PA, USA

MGMT (O6–methylguanine–DNA methyltransferase) DNA-repair gene promoter methylation has been shown to correlate with prolonged survival in glioblastoma multiforme (GBM) treated with alkylating chemotherapy. MGMT methylation has previously been determined by methylation spe-cific PCR analysis on denatured DNA from paraffin sections. This provides qualitative analysis (yes/no) for promoter status, but not quantitative data. Sequential surgical specimens meeting histological criteria for GBM were selected for analysis. The most representative sections from the fixative treated paraffin tissue were selected. A series of not one, but multiple micro-scopic targets are analyzed, thereby incorporating heterogeneity analysis in the overall molecular genotyping. DNA is not extracted as typically done by others, but rather the microdissection target itself undergoes direct bisulfite modification of non-methylated cytosines. This enables us to utilize far less total tissue and allows us to determine with precision the methylation state at multiple microscopic sites within each glioma. Instead of methylation specific primer PCR to determine the presence or absence of DNA methyla-tion of the 5' region of MGMT, we carry out bisulfite genomic sequencing affording the ability to evaluate each CpG potential sites of methylation and permitting a quantitative determination of the extent of methylation at each CpG island and at each microdissected site. qPCR is performed using consensus methylation primers which will lead to a determination of the efficiency of bisulfite conversion. DNA sequencing is accomplished by cycle sequencing and fluorescent labeled dideoxy chain termination. Each patient analysis is performed prospectively prior to treatment. Patients receive oral temozolomide chemotherapy and fractionated radiotherapy as per the standard protocol as described by Stupp et al. or as a participant in the RTOG 0525 phase III clinical trial. Seventeen consecutive patients with newly diagnosed Glioblastoma Multiforme have been analyzed to date using the novel microdissection technique. The cohort consists of 10 men and 7 women, mean age 60 (range 29–83). Molecular genotyping has been performed . MGMT methylation analysis was non-informative in 1 patient. Twelve patients exhibited positivity at any microdissection site; 3 at 100% of sites, 6 at 51%–99%, 3 at 1%–50%. More precise determination of MGMT methylation quantification is possible and will be analyzed statisi-cally as the cohort size expands. Four patients had completely unmethylated MGMT. Molecular genotyping was performed at common sites of LOH for malignant glioma. All patients are currently receiving or have completed definitive treatment with radiation and concurrent oral temozolomide. One patient received Gamma knife radiosurgery prior to chemoradiation. There has been 1 death due to GBM. Five patients have developed tumor progres-sion. We report a new technique to determine MGMT promoter methyla-tion status by means of tissue microdissection at multiple sites. Marked heterogeneity of MGMT methylation within an individual GBM specimen is demonstrated. We will detail progression-free and overall survival for this recently diagnosed cohort at the time of presentation. The relation-ship between total or partial methylation and clinical parameters will be described. Data collection is ongoing.

PA-10. COMPREHENSIVE ANALYSIS OF TELOMERE MAINTENANCE UNRAVELS ITS ROLE IN EPENDYMOMA PROGRESSION AND RESPONSE TO THERAPY IN VIVOUri Tabori, Jing Ma, James Rutka, Eric Bouffet, Ute Bartels, David Malkin, and Cynthia Hawkins; The Hospital for Sick Children, Toronto, Ontario, Canada

The role of telomere maintenance in pediatric brain tumors is largely unknown. We used a template of ependymoma, a tumor in which mul-tiple resections are performed, to examine this role in tumor progression and survival in vivo. We analyzed 133 ependymomas from 83 pediatric patients for telomere length, dysfunction, hTERT expression and telom-erase activity. Telomere dysfunction was determined by H2AX expression. We correlated these with markers of proliferation, anaplasia, and cytoge-netic abnormalities on available tissues. Thirty-one patients had multiple resections which enabled us to examine tumor progression and response to therapy. hTERT expression was associated with proliferative markers and tumor grade (p 5 0.001). Although variable telomere length was found, there was lack of alternative lengthening of telomeres and no correlation between telomere length, dysfunction and chromosomal abnormalities in our cohort. Multiple resections revealed that hTERT(–) tumors could not progress while hTERT(1) tumors progressed but eventually exhibited evi-dence of telomere dysfunction corresponding to their slow growth pattern. Surprisingly, radiation and chemotherapy did not affect hTERT or ×H2AX expression indicating resistance of recurring tumors to such modalities in vivo. Inverse correlation between hTERT expression and telomere dysfunc-tion was noted (p 5 0.016). This translated into prognostic importance as combining ×H2AX and hTERT expression could segregate tumors to four



different survival groups (p , 0.0001). This study further emphasizes the importance of telomere maintenance as a prognostic and therapeutic target for pediatric ependymoma. Following tumor progression in vivo is a novel aspect of studying tumor biology in humans.

PA-11. RHABDOMYOSARCOMA (RMS) OF EXTREMITY AND CEREBRAL GLIOBLASTOMA MULTIFORME (GBM) IN A CHILD WITH LI-FRAUMENI SYNDROME AND GERMLINE TP53 SPLICE MUTATIONKristin Page,1 Joanna Wiszniewska,2 Monica Basehore,2 Melody Watral,3 Christine Eng,2 and Sridharan Gururangan3; 1Division of Pediatric Hematology-Oncology, Duke University Medical Center, Durham, NC, USA; 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA; 3The Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC, USA

Li-Fraumeni Syndrome is a familial predisposition to cancer syndrome associated with sarcomas, leukemias, adrenocortical carcinoma (ACC), and CNS tumors including malignant glioma. It results mostly from germ-line missense mutations in the p53 tumor suppressor gene and rarely from mutations affecting splicing of the TP53 mRNA. We present the clini-cal, radiographic, pathologic, and molecular genetic analysis of a patient with malignant glioma diagnosed 17 months after completing therapy for non-metastatic RMS. Patient originally presented at 3 years of age with embryonal RMS of the right thigh successfully treated with surgery, VAC chemotherapy and focal radiotherapy (XRT). Seventeen months later, he developed frontal headache and left upper extremity weakness. Neuroimag-ing revealed a large right parietal lobe mass. Pathology was consistent with GBM. Family history was positive for paternal grandmother who died of lung cancer at age 40 years and father’s maternally related half siblings who reportedly had ACC (died age 19) and brain tumor (died age 6). Of note their father had bone cancer. Patient’s genomic DNA analyzed by direct sequence analysis of all exons and exon/intron boundaries of the TP53 gene revealed heterozygous G to A change in the last nucleotide of exon 4 that does not result in an amino acid substitution (T125T), but has been described to affect mRNA splicing. Testing of first degree family members revealed this mutation in the pt’s father who is currently 40 years old and has not been diagnosed with any cancer. Variable penetrance of the T125T mutation in this family may result from differences in splicing capacity determined by individual genetic background.

PA-12. UTILITY OF CHROMOGENIC IN SITU HYBRIDIZATION FOR DETECTION OF EGFR AMPLIFICATION IN GLIOBLASTOMA: COMPARISON WITH FLUORESCENCE IN SITU HYBRIDIZATIONIngeborg Fischer,1 Clarissa De La Cruz,2 Robert B Jenkins,3 and Kenneth Aldape2; 1New York University, New York, NY, USA; 2Pathology, M.D. Anderson Cancer Center, Houston, TX, USA; 3Mayo Clinic College of Medicine, Rochester, MN, USA

Our objective was to validate chromogenic in-situ hybridization (CISH) as a method to assess epidermal growth factor receptor (EGFR) gene ampli-fication in glioblastoma. Amplification of the EGFR leading to increased EGFR signaling is frequent in glioblastoma multiforme and instrumental to tumor growth. With the advent of therapies targeting this signaling path-way, assessment of EGFR amplification status is of increasing importance. Currently, fluorescence in situ hybridization (FISH) is the gold standard for detection of EGFR amplification. Chromogenic in situ hybridization (CISH) is a potential alternative testing method. In contrast to FISH, CISH allows for concomitant assessment of tumor morphology by light micros-copy, facilitating result interpretation. Furthermore, the signal produced by CISH lasts over an extended period of time, while fluorescence produced by FISH fades. To evaluate its reliability, EGFR amplification status by CISH was assessed in 71 cases of glioblastoma and compared to FISH results. For CISH, a commercially available digoxigenin labeled EGFR DNA probe was used on whole GBM sections. Sections were then examined with a bright-field microscope using a 40× dry objective. Presence of up to 5 EGFR signals per nucleus was defined as no amplification and 6 or more EGFR signals per nucleus as positive amplification. Sections from 71 tumors were first examined by two independent primary observers. In cases of interob-server disagreement, a third independent observer scored the CISH slide and the scoring result of 2/3 observers was used as the definitive result. The results were compared to those of FISH previously performed on the same tumors on a set of 3 tissue arrays. Immunohistochemistry (IHC) was performed using a monoclonal antibody (clone 528) for EGFR as scored semiquantitatively according to intensity of staining. FISH was scored as EGFR-amplified in 37/71 tumors and CISH was scored as amplified in

35/71 tumors. The CISH and FISH results were in agreement in 65/71 cases (91%). 4 glioblastomas were scored as amplified by FISH, but not amplified by CISH; whereas amplification was detected in 2 tumors by CISH that were not amplified using FISH. There was a strong correlation between EGFR amplification status by CISH and EGFR expression by IHC: all but one case with EGFR amplification also had strong EGFR expression. 6 cases that were not EGFR amplified displayed strong EGFR protein expres-sion. The high concordance of CISH and FISH for the assessment of EGFR gene amplification status suggests that CISH is a viable alternative to FISH for the detection of EGFR gene amplification in glioblastoma. Discordance between FISH and CISH results were likely due to decreased sensitivity of CISH compared to FISH (in 4 cases which were FISH-positive but FISH-negative), while the FISH negative results positive by CISH in our study were likely due to intratumoral heterogeneity and resulting sampling error in the tissue arrays used for FISH. Since CISH is detected using ordinary light microscopy, its routine use may be more feasible for the detection of EGFR amplification in many pathology laboratories.

PA-13. CENTRAL NERVOUS SYSTEM TUMORS IN PATIENTS WITH NEUROFIBROMATOSIS TYPE 1: CLINICOPATHOLOGIC STUDY OF 100 PATIENTSFausto Rodriguez,1 Arie Perry,2 David Gutmann,3 Brian O’Neill,4 Jeffrey Leonard,5 Sandra Bryant,6 and Caterina Giannini1; 1Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, USA; 2Division of Neuropathology, Washington University in St. Louis, MO, USA; 3Washington University in St. Louis, St. Louis, MO, USA; 4Neurology, Mayo Clinic College of Medicine, Rochester, MN, USA; 5Neurosurgery, Washington University in St. Louis, MO, USA; 6Biostatistics, Mayo Clinic College of Medicine, MN, USA

Neurofibromatosis type 1 (NF1) is a tumor predisposition syndrome in which affected children and adults develop tumors affecting the peripheral and central nervous systems. Pathologic studies of central nervous system tumors in this patient population are scant. We searched the pathologic records of the Mayo Clinic Rochester, MN, and Washington University, St Louis, MO, for patients that satisfied established clinical criteria for NF1 and had a tumor of the central nervous system with available pathologic material for review. We identified a total of 100 patients (M:F 54:45) with a median age at surgery of 13 years (range 2–68). Overall 34% of patients were less than 10 years of age at time of diagnosis. The pathologic mate-rial available included first primaries (n 5 96), recurrent tumors (n 5 14), second primaries (n 5 2), and autopsies (n 5 4). Locations of the tumors included hemispheric (35%), optic pathways (24%), brainstem (16%), cer-ebellum (9%), intraventricular (6%), spinal cord (5%), and tectum (2%). Histologic types were pilocytic astrocytomas (PA) (51%), infiltrating astro-cytomas (28%) including WHO grade II (5%), III (16%) and IV (7%), and low-grade astrocytoma subtype indeterminate (LGSI)(16%). Rare subtypes included desmoplastic infantile ganglioglioma, ganglioglioma and pilomyx-oid astrocytoma (1 each). Some low grade tumors (n 5 6) demonstrated an unusual morphology with stout cytoplasmic processes and prominent nucleoli, but no Rosenthal fibers or other features classic of PA. Tumors in this group were immunoreactive for GFAP, lacked immunoreactivity for neuronal markers, and were hemispheric in location. There was a trend for better overall survival in patients with PA in the following categories: gross total resection and age less than 10 years. However, the number of events was too low to allow a robust statistical comparison. No obvious prognos-tic differences were noted among PA according to site, tumor size, mitotic activity, p53 immunostaining, or MIB-1 labeling index. When comparing the PA and LGSI with the infiltrating astrocytoma group, the latter showed a decreased overall survival and increased recurrence (p , 0.001). The spec-trum of central nervous system tumors in patients with NF1 is wide, with PA being the most common histologic group. No obvious prognostic differ-ences were noted between PA and LGSI in this study. Infiltrating astrocy-tomas have a worse behavior, similar to those that arise sporadically.

PA-14. HER2 STATUS PREDICTS SURVIVAL AFTER DIAGNOSIS OF BRAIN METASTASIS IN BREAST CANCERApril Eichler,1 Irene Kuter,2 Paula Ryan,2 Lidia Schapira,2 Jerry Younger,2 and John Henson1; 1Pappas Center for Neuro-Oncology, Massachusetts General Hospital, Boston, MA, USA; 2Gillette Center for Women’s Cancers, Massachusetts General Hospital, Boston, MA, USA

Brain metastases (BM) are the most common intracranial tumors in adults, and breast cancer is the second leading cause of BM. Established prognostic factors for survival after diagnosis of BM do not include Her2 status, but this may have increasing relevance in the trastuzumab era. We identified 83 patients with breast cancer who developed new parenchymal



BM between January 1, 2001, and December 31, 2005, and were treated longitudinally at Massachusetts General Hospital. Overall survival (OS) was measured from the date of BM diagnosis to the date of death or last follow-up and estimated using the Kaplan-Meier method. Survival curves were compared using the log-rank test. A Cox proportional hazards model was used to determine independent predictors of survival after diagnosis of BM. The median age at diagnosis of breast cancer and BM was 47 years (range 28–83) and 53 years (range 32–85), respectively. Her2 status was known in 96% of patients. 38% of patients with BM were Her2-positive (Her21) as defined by 31 overexpression by immunohistochemistry and/or amplification by fluorescent in situ hybridization. 52% were ER-positive, 51% were PR-positive, and 26% were triple negative. Median time from breast cancer diagnosis to BM was 44 mo. (range 0–273). Patients with triple negative disease had significantly shorter time to first BM (25 mo. vs. 60 mo., p 5 0.02), compared to patients with Her21 and/or endocrine sensi-tive tumors; median time to BM did not vary significantly by Her2 status. Brain was the first site of metastasis in 20/83 (24%). 76% (16/21) of Her21 patients with known metastatic disease were receiving trastuzumab at the time of first BM. There was no difference in the number or distribution of BM based on Her2 status. Asymptomatic detection rate was 20% and 29% in Her1 and Her2– patients, respectively. The proportion of patients treated with surgery, SRS and whole brain radiation did not vary significantly by Her2 status. Median follow up was 8 months. 93% of patients died, and median OS was 8.0 mo. [95% CI 4, 11]. Cause of death could be determined in 60/77 and was attributed to isolated central nervous system (CNS) pro-gression in 15%, combined CNS and systemic in 43%, and isolated systemic progression in 38%. Her21 patients had significantly longer OS from time of BM compared with Her2– patients (17.0 mo. vs. 4.5 mo., p 5 0.001). Endocrine insensitive tumors, multiple BM and active local disease were associated with significantly decreased OS. After adjusting for age, local and systemic disease control, hormone receptor status and number of BM, Her21 status remained a significant predictor of improved survival (HR 0.40, 95% CI 0.24, 0.69). Her2 status is an independent predictor of sur-vival after diagnosis of BM in the trastuzumab era. Her21 and triple nega-tive tumors appear to be over-represented in breast cancer patients with BM. In patients with favorable prognostic factors, aggressive local and systemic therapy is warranted, but improved understanding of the delayed effects of treatment is also needed.

PA-15. LOW-GRADE GLIOMAS IN OLDER PATIENTS—A MALIGNANT TUMOR?Andrew Chi and Tracy T. Batchelor; Neurology and Neuro-Oncology, Massachusetts General Hospital Cancer Center, Boston, MA, USA

Management of low-grade gliomas remains a controversial topic in neu-ro-oncology, especially in older patients where the incidence is rare. Con-ventional wisdom is that low-grade gliomas behave like malignant gliomas and that these patients do poorly. Older age has been shown to be a poor prognostic factor in clinical trials. However, there are few studies focused specifically on the clinical features of older patients with low-grade gliomas. We retrospectively reviewed 16 subjects > age 55 at our institution who were diagnosed with supratentorial WHO grade II gliomas to determine clinical outcomes and factors that may be associated with survival in these patients. Features examined included type of surgery, histology, location, contrast-enhancement on MRI, MIB-1 proliferative index, and the use of adjuvant treatment. There were 5 astrocytomas, 4 oligoastrocytomas, and 7 oligodendrogliomas. The median age was 58 (range 55–77). Nine patients had at least a partial resection and seven patients had biopsy only. Ten patients were given adjuvant treatment consisting of various regimens of radiation, temozolomide, or PCV chemotherapy. The median progres-sion-free survival (mPFS) for the whole group was 17.5 months. However, 5 patients with oligodendrogliomas have not yet progressed with at least 30 months of followup (range 30–73 months). Excluding these stable patients, the mPFS was 11 months. The mPFS of oligodendroglioma patients was 49 months, whereas the mPFS for the other tumor types together was 14 months. For 6 tumors with a MIB-1 index of less than 5%, the mPFS was 49 months, while 6 tumors with a MIB-1 index of . 5% had a mPFS of 11 months. Resection, adjuvant treatment, and contrast-enhancement on MRI did not affect survival in this cohort of patients. These results sug-gest that low-grade gliomas behave like malignant tumors in older patients. However, oligodendrogliomas and tumors with low proliferative indexes may have significantly longer progression-free survivals even in this older population of patients. Although the sample size in this study is small, these preliminary observations suggest that aggressive management may be warranted for older patients diagnosed with low-grade astrocytoma or oligoastrocytoma.

PA-16. PANEL REVIEW OF ANAPLASTIC OLIGODENDROGLIOMA FROM EORTC TRIAL 26951: CONSENSUS IN DIAGNOSIS AND INFLUENCE OF 1P/19Q LOSS ON OUTCOMEJohan Kros,1 Thierry Gorlia,2 and Martin Van Den Bent3; 1Erasmus Medical Center, Rotterdam, Netherlands; 2EORTC DataCenter, Brussels, Belgium; 3Rotterdam, Netherlands

The diagnosis of an anaplastic oligodendroglioma (AOD) or oligoas-trocytoma (AOA) is subject to interobserver variation. We estimated the consensus in typing and grading of oligodendroglial tumors by using tumor material collected in a large prospective randomized phase III study, viz., EORTC 26951. The consensus diagnosis were correlated with the 1p/19q status of the tumors and the clinical outcome. The available pathology mate-rial of the first 150 patients randomized into this trial was reviewed by an independent panel of 9 neuropathologists. The presence of deletions of 1p and 19q were assessed by fluorescence in situ hybridization (FISH) with locus specific probes. The panel reached consensus on the diagnosis of AOD in 52% of the tumors which had been diagnosed AOD by the local patholo-gists, while only 8% of the local diagnosis of AOA was confirmed with consensus. The concordance on the panel diagnosis of AOD was high (Intra class correlation; ICC 5 86%). The survival curves for AOD with 1p/19q loss, AOD without these losses and AOA without 1p/19q loss ran sepa-rately in this order. The absence of necrosis and the presence of endothelial abnormalities are correlated with better outcomes. In multivariate analysis, patients’ age, 1p/19q loss and necrosis were identified as independent prog-nostic factors.

PA-17. GENETIC ABERRATIONS IN SERUM DNA OF PATIENTS WITH GLIAL TUMORS DETECTED SOME TIME AFTER TISSUE DIAGNOSIS OR INITIAL THERAPYIris Lavon,1 Jose Goldmann,2 Bracha Zelikovitch,1 Miriam Rfael,1 and Tali Siegal1; 1Gaffin Center for Neuro-Oncology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel; 2Neurosurgery Department, The Hebrew University-Hadassah Medical School, Jerusalem, Israel

Genetic and epigenetic aberrations, like loss of chromosomal heth-erozygosity (LOH) and hypermethylation of gene promoters, are frequently observed in glial tumors. Detection of these aberrations has a potential diagnostic and prognostic relevance. For instance, oligodendrogliomas with combined 1p/19q LOH respond better to chemotherapy and have favorable prognosis, whereas LOH on chromosome 10q is a marker for worse outcome. In glioblastoma multiforme (GBM) hypermethylation of the promoter of O6-methylguanine-methyl-transferase (MGMT) gene is associ-ated with improved response to alkylating agent and with longer overall survival. As sample materials for diagnosis should be easily accessible by a minimally invasive procedure, there has been much interest in the potential use of nucleic acid markers in the blood of patients with cancer. Objective: To find whether genetic aberrations in serum DNA of glial tumors can be detected at time distant from surgical intervention and to evaluate its con-cordance with tumor DNA. DNA was extracted from the serum and the paraffin embedded tumor sections of 35 patients with GBM and 23 patients with oligodebdrogliomas (18 grade II; 5 grade III). The methylation status of MGMT promoter and chromosomal 10q LOH were evaluated in DNA samples obtained from both the serum and tumor sections. In GBM, the incidence of MGMT promoter methylation was 43% in the tumor and 31% in serum while 10q LOH was found in 64% of tissue samples and 43% of serum DNA. In oligodendrogliomas, the incidence of MGMT promoter methylation was 70% in the tissue and only 17% in serum. The concordance between the results obtained from tumor DNA and serum DNA for both MGMT methylation status and 10q LOH was 71% for GBM. In oligo-dendrogliomas the concordance of the results for MGMT was 48%. These results imply that serum DNA which is obtained at various times following tissue diagnosis or initial treatment often represents the tumor DNA. The concordance between serum and tumor DNA is better for GBM than for oli-godendrogliomas. It is possible that higher proportion of GBM patients had an active tumor at time of serum sampling as compared to patients treated for oligodendrogliomas. It can not be excluded that high-grade tumors shed more DNA into the bloodstream which explain the better concordance observed in GBM. We are currently correlating results of paired serum-tumor samples with clinical and imaging details of each patient.



PA-18. EORTC 26951: PROGNOSIS OF OLIGODENDROGLIAL TUMORS IS AN INTERPLAY OF MOLECULAR, HISTOLOGICAL, AND CLINICAL CHARACTERISTICSMathilde Kouwenhoven,1 Thierry Gorlia,2 Johan Kros,1 Antoine Carpentier,3 Alba Brandes,4 Marc Sanson,3 Martin Taphoorn,5 Hans Teepen,6 Hans Bernsen,7 Marc Frenay,8 Cees Tijssen,6 Wolfgang Grisold,9 László Sipos,10 Roger Stupp,11 Charles Vecht,5 Anouk Allgeier,2 Denis Lacombe,2 and Martin Van Den Bent12; 1Erasmus MC, Rotterdam, ZH, Netherlands; 2EORTC DataCenter, Brussels, Belgium; 3Department of Neurology, CHU Pitié-Salpétrière, Paris, France; 4Azienda Ospedale-Università-Istituto Oncologico Veneto, Padova, Italy; 5Medical Center Haaglanden/Westeinde Ziekenhuis, The Hague, Netherlands; 6St Elisabeth Hospital, Tilburg, Netherlands; 7Canisius Wilhemina Ziekenhuis, Nijmegen, Netherlands; 8Centre Antoine Lacassagne, Nice, France; 9LBI-Neurooncology and Kaiser Franz Josef Spital, Vienna, Austria; 10National Institute of Neurosurgery, Budapest, Hungary; 11University of Lausanne Hospitals, Lausanne, Switzerland; 12Daniel den Hoed Oncology Center/Erasmus MC, Rotterdam, Netherlands

Prognosis of high-grade oligodendroglial tumors differs considerably. We used prospective EORTC 26951 study (Van den Bent et al., J Clin Oncol. 2006;24:2715–22) on the effects of adjuvant PCV in oligodendroglial tumors, to evaluate molecular characteristics of oligodendroglial tumors. We also evaluated clinical, histological, and molecular characteristics factors associated with overall survival (OS). Additionally, survival of patients with oligodendroglial tumors with specific molecular aberrations was compared to OS of GBM patients treated in a prospective study with radiotherapy only (EORTC 26981 [Mirimanoff et al., J Clin Oncol. 2006;24:2563–9.2]). 326 patients included in EORTC 26951 with histological confirmed newly diag-nosed anaplastic oligodendroglioma (AOD) or anaplastic oligoastrocytoma (AOA) with at least 25% oligodendral elements with sufficient material for translational research were included. 1p, 19q, EGFR, and 10q as well as copy numbers of chromosome 7 and 10 were investigated with Fluorescent In Situ Hybridization (FISH). For pathological diagnosis the diagnosis made at central review was used. EGFR amplification (EGFRamp) was inversely correlated with combined 1p/19q loss (1ploss19qloss; r 5 –0.25). Presence of EGFRamp was associated with presence of polysomy chromosome (chr.) 7 (7poly; r 5 0.40), 10qloss (r 5 0.26) and monosomy 10 (10loss; r 5 0.48). Tumors with 1ploss19qloss were mainly anaplastic oligodendroglioma (AOD; p , 5*10–7) or tumors located in the frontal lobe (p , 0.0005). EGFRamp was observed in 42% of anaplastic oligoastrocytoma (AOA; p , 10–4) and were not localized in the frontal lobe (p , 0.0005). Still, 18% of AOD had EGFRamp. In univariate analysis, survival was improved in patients with 1ploss19qloss (p , 0.0001; median survival not reached vs. 19 months [mo]). Presence of EGFRamp (p , 0.0001; 17.2 vs. 59.3 mo), 7poly (p , 0.0001; 19.1 vs. 59.3 mo), 10qloss (p 5 0.098; 21.6 vs. 49.9 mo) and of 10loss (p , 0.0001; 18 vs. 49.9 mo) were associated with decreased survival. Multivariate Cox PH analysis identified presence of 1ploss19qloss (p , 0.0001, HR 5 0.13), 7poly (p 5 0.0045, HR 5 1.76), endothelial abnormalities (p 5 0.002, HR 5 3.66), necrosis (p 5 0.022, HR 5 2.09), extent of surgery (resection vs. biopsy, p 5 0.0068, HR 5 0.483), perfor-mance status (PS0,1 or 2; p 5 0.0011, HR 5 1.62) and age (.50 vs. < 50; p 5 0.0009, HR 5 1.88) as independent prognostic factors. In compari-son to GBM patients treated with RT only, patients with oligodendroglial tumors with 10loss (p 5 0.24) or EGFRamp (p 5 0.26) had similar survival times. Conclusion: Oligodendrogliomas with 1ploss19qloss generally have AOD phenotype, frontal localization, and a favorable prognosis. Tumors with EGFRamp and/or 10loss have more frequently the AOA phenotype, are not located in the frontal lobe and their survival is similar survival to GBM. Our findings show that molecular features add significantly to the histological classification of these tumors, and should be used to classify some oligodendroglial tumors as GBM.

PA-19. WELL-DIFFERENTIATED EXTRAVENTRICULAR NEUROCYTOMA WITH 1P AND 19Q LOSS IN DIFFERENT CELL CLONESFausto Rodriguez,1 Caterina Giannini,1 Hilary Blair,1 Kevin Wu,2 Dennis Dickson,2 Kent New,3 and Robert Jenkins1; 1Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, USA; 2Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Jacksonville, FL, USA; 3Neurosurgery, Mayo Clinic College of Medicine, Jacksonville, FL, USA

Concurrent deletion of chromosome arms 1p and 19q is frequently encountered in oligodendrogliomas. Most recently this cytogenetic altera-tion has been reported to be mediated by t(1;19)(q10;p10), an unbalanced translocation. Codeletion of 1p19q has also been described in a minority of extraventricular neurocytomas, rare neoplasms with neuronal differentia-tion that morphologically resemble oligodendrogliomas at the light micros-copy level. We recently encountered the case of a 27-year-old male who underwent resection of a right frontal lobe mass. Preoperative magnetic resonance imaging (MRI) demonstrated a well circumscribed cyst with an enhancing mural nodule. Histologically the tumor was composed of monot-

onous cells with round nuclei and perinuclear halos sharply demarcated from surrounding brain parenchyma. Prominent vascular hyalinization and calcification were also noted, as well as focal Rosenthal fiber accumulation. Immunohistochemical stains performed with antibodies directed against synaptophysin, chromogranin and neurofilament protein labeled neoplas-tic cells. MIB-1 labeling index was low. Fluorescent in situ hybridization (FISH) studies were performed using probes targeting 1p36 (Spectrum Orange;Vysis, Downers Groove, IL, USA), 1q24 (Spectrum Green, Vysis), 19q13 (Spectrum Orange, Vysis), and 19p13 (Spectrum Green, Vysis), to evaluate for 1p19q loss, as well as chromosome 1a satellite (CEP1, Spectrum Orange, Vysis) and 19p12 (Spectrum Green, Home brew) to assess for the translocation. Threshold for deletion was defined as a red to green signal ratio , 0.8. The threshold for 1q and 19p fusion was set to at least 60% of cells showing red and green signals within 2 signal widths from each other. Loss of 19q was noted in multiple regions within the tumor, with most cells demonstrating 1 red and 2 green signals. However, in a focus of tumor there was a relative loss of 1p with many cells showing 2 red signals but 3 or more green signals. In the latter area 19q was intact (not deleted). There was no evidence of t(1;19)(q10;p10). These results suggest that there may be mechanisms for 1p19q loss other than t(1;19)(q10;p10). Studies of this phenomenon in an independent series of extraventricular neurocytomas are in progress.

PA-20. IL-13R ALPHA2, EphA2, AND FRA-1 AS MOLECULAR DENOMINATORS OF HIGH-GRADE ASTROCYTOMASJill Wykosky,1 Denise Gibo,1 Constance Stanton,2 and Waldemar Debinski1; 1Neurosurgery, Wake Forest University School of Medicine, Winston-Salem, NC, USA; 2Pathology, Wake Forest University, Winston-Salem, NC, USA

We have previously identified three proteins which are over-expressed in a majority of, but not all patients with GBM: (i) interleukin 13 recep-tor alpha2 (IL-13R), a cancer-testis tumor-like antigen (Clin. Cancer Res. 1999;5:985–990), (ii) EphA2, a member of the largest family of tyrosine kinase receptors (Mol Cancer Res. 2005;3:541–51), and (iii) fos-related anti-gen 1 (Fra-1), an AP-1 transcription factor (Mol Cancer Res. 2005;3:237–249). All three proteins have been found to play a significant role in GBM pathobiology and to be regulated by signaling pathways downstream of receptors such as epidermal growth factor receptor (EGFR) and EGFRvIII, involving ERK as well as PI3-K, among others. Here, we investigated the expression of these proteins in a panel of the same tumor specimens in order to establish the rationale for novel combinatorial therapeutic approaches that would be applicable to all patients with GBM. Immunohistochemistry was performed on tissue microarrays containing specimens from 30 patients with grades II–III astrocytoma, 46 with GBM, and 9 normal brain controls. Staining was scored based on the percentage of positively stained tumor cells within a section (0%–10%, 10%–50%, and .50%), and on overall staining intensity of tumor cells (negative, weak, moderate, or strong). With respect to the frequency of positive-staining cells within tumor sections, 5 (11%), 5 (11%), and 36 (78%) grade IV (GBM) tumors had 0%–10%, 10%–50%, and .50% of tumor cells positive for IL-13R, respectively. Similarly, 1 (2%), 3 (7%), and 42 (91%) demonstrated positive staining for EphA2, while 7 (15%), 6 (13%), and 33 (72%) were positive for Fra-1. In addition, 25 (56%), 7 (15%), and 13 (29%) were positive for EGFR, which is consistent with the data reported in the literature describing the expression of this receptor in GBM. The intensity of expression of all three proteins was statistically significantly higher in GBM compared to normal brain and low-grade astro-cytomas. In a combinatorial analysis, 95% of GBM patient tumors were moderate or strongly positive for 2 of the 3 markers. A similar pattern of expression was observed for all three markers by Western blotting of lysates obtained from human GBM tumor tissue, human GBM xenograft tissues and cell lines, and primary GBM explant cells. Thus, IL-13R, EphA2, and Fra-1 are attractive molecular markers/therapeutic targets representing com-mon molecular denominators of high-grade astrocytomas due to their high frequency and intensity of expression. Each individual patient with GBM over-expresses at least one of these proteins and almost all patients over-express two of them. Our data provide the basis for rational combinatorial targeted therapies/diagnostics of GBM based on the three studied factors.

PA-21. COINCIDENCE OR ASSOCIATION? TWO 19Q DISORDERS IN THE SAME PATIENT: 1P/19Q CO-DELETED OLIGODENDROGLIOMA AND MYOTONIC DYSTROPHY TYPE 1Jai Grewal, Merja Kallio, Sudhakar Tummala, and Vinay Puduvalli; Neuro-Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

The discovery of 1p and 19q co-deletions in oligodendrogliomas is an important milestone that has influenced therapeutic approaches for this tumor. However, the precise mechanism by which these co-deletions might



be involved in the pathogenesis and clinical behavior of oligodendrogliomas is unknown. In this case report, we present a 34-year-old Indian patient with two 19q disorders, oligodendroglioma, and myotonic dystrophy type 1. The patient presented with an increasing frequency of complex partial seizures over 13 years and was found to have a left medial temporal non-enhancing mass on MRI. Stereotactic biopsy confirmed a grade 2 oligoden-droglioma with classic pathologic features, with molecular analysis showing 1p/19q co-deletion. Due to her age and the location and extent of tumor, neither further surgery nor radiation therapy were considered viable first-line options. She was treated with 11 cycles of temozolomide (150mg/m2, days 1–5 of a 28-day cycle) which was discontinued due to myelotoxicity. Her tumor has remained stable since diagnosis (22 months). Neurological examination at the time of tumor diagnosis revealed percussion myotonia of the tongue and thenar eminence that led to further workup, including electromyography, which were highly suggestive of myotonic dystrophy type 1. She was also noted to have other manifestations of myotonic dystrophy including multiple endocrinopathies (including diabetes and infertility), cataracts, and gastrointestinal dysfunction. Genetic testing of peripheral mononuclear cells by PCR (Athena Diagnostics, Inc.) confirmed the diag-nosis with 377 CTG repeats in the myotonic dystrophy protein kinase gene on chromosome 19q13.3. The 19q region, along with 1p, appears to be important in the clinical behavior, and possibly pathogenesis, of oligoden-drogliomas although the relevant genetic and epigenetic factors associated with these regions remain to be determined. Triplet repeats are known to alter adjacent chromatin structure (Otten et al., 1995) and may potentially increase genomic instability. Whether such changes can induce chromo-somal breakpoints at locations other than at the site of triplet repeat expan-sion is unknown. The co-occurence of 19q GTC repeat expansion disease and a 19q-deleted brain tumor in our patient raises the intriguing question of a potential relationship between the two genetic abnormalities.

PA-22. GENE EXPRESSION ANALYSIS OF PRIMARY AND RECURRENT MENINGIOMASPolly Foureman,1 William P. Hendricks,1 Phillip Stafford,2 Stephen W. Coons,3 and Adrienne C. Scheck1; 1Neuro-Oncology Research, Barrow Neurological Institute, Phoenix, AZ, USA; 2AZ Biodesign, Center for Innovations in Medicine, Arizona State University, Tempe, AZ, USA; 3Neuropathology, Barrow Neurological Institute, Phoenix, AZ, USA

Meningiomas are among the most common intracranial tumors. Although the majority of these tumors are benign, a significant number recur and require additional treatment. If those tumors most likely to recur could be identified on initial diagnosis, adjuvant treatment could be started sooner, thus reducing or even eliminating the need for subsequent more invasive treatments. As part of a study to identify factors associated with aggression and recurrence in meningiomas, cDNA microarray analysis (Affymetrix, Inc., Santa Clara, CA, USA) was performed on total RNA from 57 primary (24 grade I, 32 grade II and one grade III) and 24 recurrent meningiomas (7 grade I, 8 grade II, and 5 grade III). Analysis was performed using GeneSpring software (Agilent Technologies, Inc., Santa Clara, CA, USA). Initial analysis identified 122 oligos differentially expressed in pri-mary versus recurrent tumors. Initial pathway analysis (www.biorag.org, University of Arizona, Tucson, AZ, USA) of the 59 genes over-expressed in recurrent tumors compared to primary tumors identified a group associ-ated with the WNT signaling pathway. These included genes localized to chromosome 17q (PSCD1, MGC111019) and 9p24 (ANKRD15, SLC1A1). Chromosome 17q is commonly amplified in meningiomas, particularly in those that are more aggressive. We also demonstrated under-expression of secreted frizzled protein (SFRP1; 8p12–p11.1), a negative inhibitor of the WNT signaling pathway. Additional support for alteration of the WNT signaling pathway in a subset of recurrent meningiomas includes the over-expression of genes encoding seven in absentia homolog 1 (SIAH1; 16q12) and an under-expression of sprouty4 (SPRY4; 5q31.3). While there have been some reports associating the WNT signaling pathway with menin-giomas, this report demonstrates a more comprehensive and confident asso-ciation of genes with altered expression identified with the WNT signaling pathway. Furthermore, this is the first report of alterations in the secreted frizzled proteins. Under-expression of these proteins due to hypermethyla-tion of CpG islands have been described in a variety of neoplasms, including colon cancer and prostate cancer. A second cluster of genes over-expressed in recurrent tumors was identified at 1q21–24. This region includes can-didate genes EFNA3, PIP5K1A, and SKI. Among genes under-expressed in recurrent tumors versus primary tumors were those localized to 1p31 and 14q, both regions commonly associated with LOH in meningiomas. Reverse transcription-polymerase chain reaction (RT-PCR), methylation-specific PCR and immunohistochemistry is being used to further elucidate gene expression pathways associated with meningioma aggression and recurrence. The ultimate goal of this work is to use gene expression profile analysis to define molecular classifiers and to suggest novel treatments based on underlying molecular characteristics.

PA-23. EXPRESSION PROFILING IDENTIFIES SURVIVAL-RELATED GENES OF THE PHOSPHATIDYLINOSITOL 3'-KINASE SIGNALING PATHWAY IN GLIOBLASTOMA MULTIFORMETeresa Ribalta,1 Yolanda Ruano,2 Manuela Mollejo,3 Concepción Fiaño,4 Elena Gomez,5 Jose L. Hernandez-Moneo,3 and Barbara Melendez2; 1Pathology (Neuropathology), Hospital Clinic, University of Barcelona, Barcelona, Spain; 2Genetics, Hospital Virgen de la Salud, Toledo, Spain; 3Pathology, Hospital Virgen de la Salud, Toledo, Spain; 4Pathology, Hospital Xeral Cies, Vigo, Spain; 5Genomics, Centro Nacional de Investigaciones Cientificas, Madrid, Spain

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults and remains extremely lethal despite the continuous technical improvements and recent therapeutic advances. High-throughput analyses of gene expression can identify new key genes, networks and path-ways which may be clinically relevant in patients with this highly malignant tumor. We investigated the pattern of gene expression in 20 primary GBMs using cDNA microarrays containing 27,454 cDNA clones, including 9,900 known genes and uncharacterized tumorigenesis-related ESTs. Differentially expressed genes detected by cDNA array analysis were further evaluated by RT-PCR, tissue microarray (TMA) immunohistochemistry, FISH, and Western blot techniques. Statistical analysis revealed 345 deregulated genes (159 upregulated and 186 downregulated). A gene ontology-based study showed that the most strongly deregulated pathways in our series were those of MAPK and actin cytoskeleton, followed by cell cycle, GnRH and calcium signaling pathways. Several genes of the PI3K pathway were also included in the signature of our GBM series. Microarray analyses showed strong upregulation of ANXA1 in 90% (18/20) of GBMs. Immunohistochemical analysis of protein expression demonstrated strong expression of ANXA1 in 86% (102/118) of the GBM in our TMA, and in 70% (129/182) of the astro-cytomas (all grades), 5% (1/20) of oligodendrogliomas, and 17% (3/17) of oligoastrocytomas included in a separated TMA (ANOVA, p , 0.05). Dis-tinct molecular subsets of GBM showing different gene expression patterns were detected. Survival analysis using functional clusters of genes revealed an association between the PI3K-signaling pathway and clinical outcome. TIAF1, BAX, RPS6KB2 (p70s6k), and BAD were among the genes whose expression was significantly associated with survival. Some candidate target genes, including ANXA1, BAX, USP7 (HAUSP), and STIM2 were further evaluated in GBM tumors by TMA immunohistochemistry and quantifica-tion of the expression levels of STIM2 and USP7 was tested by RT-PCR. We also analyzed the activation status of Akt and GSK3ß and demonstrated that PI3K/Akt pathway is activated in our series of GBM. Our high-throughput studies of gene expression patterns, complemented by immunohistochem-istry, RT-PCR, and other validation studies confirm the distinctive expres-sion profile of some PI3K pathway survival-related genes, which may play a significant role as prognostic markers in GBM.

PA-24. IDENTIFICATION OF EXPRESSED GENES CHARACTERIZING LONG-TERM SURVIVAL IN MALIGNANT GLIOMA PATIENTSRyuya Yamanaka1 and Kazuto Nishio2; 1Research Center for Innovative Cancer Therapy, Kurume, Fukuoka, Japan; 2Osaka, Japan

Better understanding of the underlying biology of malignant gliomas is critical for the development of early detection strategies, and new therapeu-tics. This study aimed to define genes associated with survival. We investi-gated whether genes coupled with a class prediction model, could be used to define subgroups of high-grade gliomas in a more objective manner than standard pathology. RNAs from 29 malignant gliomas were analyzed using Agilent microarrays. We identified 21 genes whose expression was most strongly and consistently related to patient survival based on univariate pro-portional hazards models. In six out of 10 genes, changes in gene expression were validated by quantitative real-time PCR. After adjusting for clinical co-variates based on a multivariate analysis, we finally obtained a statistical significance level for DDR1, DYRK3 and KSP37. In independent samples, it was confirmed that DDR1 protein expression was also correlated to the prognosis of glioma patients detected by immunohistochemical staining. Furthermore, we analyzed the efficacy of the siRNA-mediated inhibition of DDR1 mRNA synthesis in glioma cell lines. Cell proliferation and invasion were significantly suppressed by siRNA against DDR1. Thus, DDR1 can be a novel molecular target of therapy as well as an important predictive marker for survival in patients with glioma. Our method was effective at classifying high-grade gliomas objectively, and provided a more accurate predictor of prognosis than histological grading.



PA-25. CSF IGH GENE REARRANGEMENT ANALYSIS IN EBV-RELATED LYMPHOPROLIFERATIVE DISORDER OF THE CENTRAL NERVOUS SYSTEMJoachim Baehring,1 Dennis Cooper,2 Pei Hui,3 and Serguei Bannykh3; 1Neurology and Neurosurgery, Yale University, New Haven, CT, USA; 2Medicine, Yale University, New Haven, CT, USA; 3Pathology, Yale University, New Haven, CT, USA

Isolated lymphoprolifertive disorder (LD) of the central nervous system in the setting of newly acquired or reactivated infection by the Epstein-Barr virus (EBV) most commonly occurs in patients with the acquired immuno-deficiency syndrome (AIDS). In non-AIDS patients, it is exceedingly rare and its diagnosis can be challenging. Risk factors for EBV-related LD (ERLD) are immunosuppression after solid organ transplantation or for systemic inflammatory disorders. Immunoglobulin heavy chain gene rearrangement analysis (IGHR) has been accepted as a valuable adjunct to classical meth-ods of diagnosis in LD outside the nervous system but its sensitivity and specificity is unknown in paucicellular specimens of CSF. We present two cases of isolated ERLD in whom IGHR analysis of CSF was instrumen-tal in establishing the diagnosis. Case Reports: Patient 1: A 66-year-old woman presented with nausea, fatigue, and progressive imbalance seven months after kidney transplantation. Her medication included mycopheno-late mofetil, sirolimus, and prednisone. Examination was notable for leth-argy, right-sided Bell’s palsy, dysmetria, and intention tremor on the right side and a right hemiparesis. Brain MRI revealed a right cerebellar mass lesion and multiple parenchymal and meningeal signal abnormalities. CSF analysis showed atypical lymphocytes. Flow cytometry was negative. IGHR identified a single B-cell clone. EBV polymerase chain reaction was positive. Biopsy of the cerebellar mass revealed large B-cell lymphoma. Patient 2: A 62-year-old woman with a 40-year history of rheumatoid arthritis treated with prednisone, methotrexate, and infliximab complained of headache, nausea, and double vision. She had bilateral third nerve palsies. Brain MRI revealed enhancement of both oculomotor nerves. CSF was remarkable for lymphocytic pleocytosis. Flow cytometry identified a clonal population of B-cells. IGHR confirmed presence of a B-cell clone on the background of an oligoclonal background. EBV PCR was positive. Sufficient data on the role of CSF analysis in ERLD are unavailable. Morphological analysis is sub-ject to considerable inter-observer variability, and flow cytometry requires large cell numbers that often cannot be retrieved from the spinal fluid as in patient 1. A peculiar molecular feature of ERLD has to be considered in the interpretation of IGHR. IGHR of spatially separated lesions reveals their origin from different EBV-transformed B-cell clones. A temporal evolution from a polyclonal to an oligoclonal and ultimately a clonal process has been described outside the nervous system, correlating with the morphological spectrum ranging from “early” lesions characterized by relatively well dif-ferentiated lymphoid proliferations to polymorphic and monomorphic neo-plasms. It is conceivable, that polyclonal or oligoclonal stages of ERLD can be recognized using CSF IGHR. Patient 1 may be an example of “advanced” monoclonal ERLD, whereas patient 2 may represent an earlier, oligoclonal stage. Further study of IGHR is warranted in this setting in order to deter-mine its overall diagnostic value.

PA-26. ASSOCIATION OF PROGRESSION-FREE SURVIVAL FOLLOWING ADJUVANT THERAPY WITH THE DNA REPAIR ACTIVITIES APURINIC ENDONUCLEASE AND O6-METHYLGUANINE-DNA METHYLTRANSFERASE (MGMT) IN GLIOBLASTOMAJohn Silber,1 Michael Bobola,2 Robert Rostomily,1 Daniel Silbergeld,1 John Chen,3 and Mitchel Berger4; 1University of Washington, Seattle, WA, USA; 2Neurological Surgery, University of Washington, Seattle, WA, USA; 3Neurosurgery, Taipei Medical College, Taipei, Taiwan; 4University of California, San Francisco, San Francisco, CA, USA

Apurinic endonuclease (Ap endo) is a key repair enzyme that cleaves DNA at cytotoxic abasic sites caused by alkylating agents and radiation. Here we use Cox proportional hazards regression models adjusted for age and extent of resection to examine the association of Ap endo activity in glioblastomas with progression-free survival (PFS) after radiotherapy (RT) alone, and after RT followed by alkylating agent-based chemotherapy. No association was observed between PFS following RT alone in 54 glioblas-tomas or between PFS following RT and alkylating agents in 48 glioblas-tomas. However, analysis of patients less than 50 revealed emerging trends between activity and TTP after RT alone in 18 glioblastomas (HR 5 1.152; CI 5 [1.031, 1.287]; p < 0.013), and after RT and alkylators in 30 tumors (HR 5 1.191; CI 5 [1.037, 1.368]; p < 0.013). A sub-set of 15 of the glio-blastomas receiving alkylating agents for which we currently have MGMT activity data also shows an inverse association between Ap endo and PFS when MGMT activity is included in the model as a continuous variable (HR 5 1.382; CI 5 [1.00, 1.91]; p < 0.05), suggesting that MGMT strengthens the association between Ap endo and PFS. For this sub-set, the relative risk for progression differed by ca. 25-fold between the tumors with the lowest and highest Ap endo activity. Interestingly, the model also revealed a strong

inverse trend between MGMT activity and PFS (HR 5 1.107; p < 0.065). Further analysis revealed a significant association between Ap endo and MGMT activity in patients younger than 50 (R 5 0.44; p < 0.027) that was absent in older patients (R 5 0.04; p < 0.82). Our data suggest (1) Ap endo activity mediates resistance to alkylating agents and radiation and may be a useful predictor of progression following adjuvant therapy in a sub-set of glioblastomas, and (2) greater responsiveness associated with absent MGMT activity may also reflect inability to efficiently repair abasic sites.

PA-27. ANAPLASTIC OLIGODENDROGLIAL TUMORS: REFINING THE CORRELATION AMONG HISTOPATHOLOGY, 1P 19Q DELETION, AND CLINICAL OUTCOME IN INTERGROUP RADIATION THERAPY ONCOLOGY GROUP TRIAL 9402Caterina Giannini,1 Peter Burger,2 Minhee Won,3 Gregory Cairncross,4 Robert B. Jenkins,1 Minesh Mehta,5 Walter Curran,6 and Kenneth Aldape7; 1Mayo Clinic College of Medicine, Rochester, MN, USA; 2Johns Hopkins University, Baltimore, MD, USA; 3Radiology Therapy Oncology Group, Philadelphia, PA, USA; 4Foothill Hospital, University of Calgary, Calgary, Alberta, Canada; 5University of Wisconsin-Madison, Madison, WI, USA; 6Thomas Jefferson University, Philadelphia, PA, USA; 7M.D. Anderson Cancer Center, Houston, TX, USA

RTOG 9402 study, a phase III trial of chemotherapy plus radiotherapy (PCV-plus-RT) versus radiotherapy alone for anaplastic oligodendroglioma and oligoastrocytomas confirmed the prognostic significance of 1p 19q co-deletion and showed that only progression-free survival (PFS) was prolonged in PCV-plus-RT treated patients and only in association with 1p 19q dele-tion. We critically reviewed tumor histopathology, separating 115 “classic” oligodendroglial tumors (COT) from 132 lacking the classic features of oli-godendroglioma (NCFO) and evaluated the relationship of histopathology and 1p 19q to treatment and outcome. Overall survival (OS) of patients with COT was significantly longer than for patients with NCFO (COT 6necrosis vs. NCFO with necrosis: log-rank p , 0.0001 and COT 6necro-sis vs. NCFO without necrosis: log-rank p 5 0.002) and was not affected by necrosis. Median OS for patients with COT with and without necrosis was respectively 6.6 and 6.3 years (OS log-rank p 5 ns), in contrast to NCFO showing 1.9 and 3.3 years (OS log-rank p 5 0.014). Classic oligodendroglial morphology was highly associated with 1p 19q deletion, present in 80% of COT and only in 13% of NCFO. On multivariate analysis, both classic oligodendroglial morphology and 1p 19q deletion remained significantly associated with PFS and OS. Patients with COT treated with PCV-plus-RT showed a trend toward increased survival compared to COT treated with RT (p 5 0.08): median OS was not reached in the PCV-plus-RT group and 6.3 years in RT group. These findings suggest that the combination of clas-sic oligodendroglial morphology with 1p 19q deletion may in the future be predictive of chemotherapeutic response and survival.

PA-28. VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) AND MATRIX METALLOPROTEASE (MMP) CEREBROSPINAL FLUID (CSF) BIOMARKERS IN LYMPHOMATOUS MENINGITISMin Yu,1 Susan Song,1 Songklha Nguyen,1 Suriya Jeyapalan,1 Morris Groves,2 Kenneth Swanson,3 Shiva Gautam,4 and Eric Wong1; 1Brain Tumor Center and Neuro-Oncology Unit, Beth Israel Deaconess Medical Center, Boston, MA, USA; 2University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3Cancer Biology, Beth Israel Deaconess Medical Center, Boston, MA, USA; 4Biostatistics, Beth Israel Deaconess Medical Center, Boston, MA, USA

A diagnosis of lymphomatous meningitis based on CSF cytology and flow cytometry has low sensitivity and specificity. We hypothesize that CSF VEGF and MMPs could be used as reliable biomarkers for central nervous system lymphomas (CNSL) due to their putative roles in angiogenesis and invasion. Using Quantikine ELISA for human VEGF165 (R&D Systems) and a fluorimetric substrate cleavage assay for MMP-9 (AnaSpec), we ana-lyzed 122 longitudinal CSF samples taken from 42 subjects with biopsy-proven CNSL. The resulting data were correlated to the presence or absence of lymphoma cells in CSF detected by cytology using generalized estimating equations and mixed linear model methods. Median white blood cell (WBC) count was 11 cells/mL for positive and 1 cell/mL for negative CSF cytology (p 5 0.005), while the median VEGF165 level was 4.72 pg/mL for positive and 1.02 pg/mL for negative CSF cytology (p 5 0.009). Normalized median MMP-9 level was 1.01 for positive versus 1.01 for negative CSF cytology (p 5 0.42). To our knowledge, this is the largest series of CSF analysis from patients with CNSL, and our findings suggest that VEGF165 is a reliable biomarker for disease activity. The fluorimetric substrate cleavage assay may not be as sensitive as our previously reported MMP-9 CSF analysis using zymography.



PA-29. QUANTITATIVE TUMOR CELL DETECTION ASSAYS FOR DIAGNOSIS OF LEPTOMENINGEAL DISEASEMichelle Melisko,1 Janet Scott,2 Hope Rugo,2 Robert McCormack,3 and John Park2; 1Medicine, University of California, San Francisco, San Francisco, CA, USA; 2University of California, San Francisco, San Francisco, CA, USA; 3Veridex LLC, Warren, NJ, USA

Leptomeningeal disease (LMD) is a devastating event in the course of metastatic breast cancer and other solid tumors, leading to progressive neu-rologic disability and death in most patients. As patients live longer with metastatic cancer and undergo more frequent and sensitive imaging, there appears to be an increasing incidence in the diagnosis of LMD, particularly among breast cancer patients. Traditionally, diagnosis of LMD is estab-lished by CSF cytology, which is insensitive and non-quantitative, or by MRI, which is frequently non-specific. Although a positive CSF cytology is considered the gold standard for this diagnosis, many patients with clinical or radiographic findings suspicious for LMD do not have malignant cells detected in their CSF even after multiple lumbar punctures. We investigated the feasibility of using CTC-based assays for detection and quantitative enumeration of tumor cells in CSF in patients for whom there was a clini-cal or radiographic suspicion of LMD. Methods included a modified Cell SearchTM assay and immunomagnetic enrichment/flow cytometry (IE/FC) to test for the presence of tumor cells in CSF. CSF was collected from either a routine lumbar puncture or through an Omaya reservoir. Between 0.5 and 4 mls of CSF were run on the control channel of the Cell Search system, and IE/FC was performed on 2–4 ml CSF samples. At least 8 mls of CSF were sent concurrently for standard cytology to compare the results from this routine evaluation. Forty three sets of CSF samples were analyzed by CellSearch and/or IE/FC as well as standard cytology. CellSearch results in cells/ml were a mean of 179.1, a median of 6.5, and range of 0–2927. IE/FC results were a mean of 70.1, a median of 6.9, and range of 0–528. CellSearch and IE/FC results were highly correlated. Both CTC assays appeared more sensitive than standard cytology with 77% of the IE/FC and 85% of the CellSearch samples positive compared to 32% positive and an additional 16% described as containing atypical cells by standard cytology. Quanti-tative CSF testing appears to correlate with clinical course and outcome. One patient receiving ongoing intrathecal (IT) chemotherapy experienced a decrease in tumor cell number from 2927 to 11 cells/ml by the CellSearch assay, at which time standard CSF cytology was interpreted as negative. A second patient receiving IT chemotherapy had a decrease in tumor cell number from 108 to 14.1 cells/ml by IE/FC, corresponding with resolution of meningeal enhancement on MRI. Detection of tumor cells in the CSF using modified CTC assays (Cell Search, IE/FC) is feasible, and can provide quantitative analysis of tumor cells over time. This approach appears to be more sensitive than standard cytology. Changes in cell number may be associated with response to treatment in some patients. Additional samples are being collected in both solid tumor patients and controls to continue evaluating the sensitivity and specificity of this assay compared to standard CSF cytology.

PA-30. JOINT NCCTG AND NABTC PROGNOSTIC FACTORS ANALYSIS FOR HIGH-GRADE RECURRENT GLIOMAWenting Wu,1 Kathleen Lamborn,2 Jan Buckner,1 Kurt Jaeckle,3 Susan Chang,2 Paul Novotny,1 and Michael Prados2; 1Mayo Foundation for Medical Education and Research, Rochester, MN, USA; 2University of California, San Francisco, San Francisco, CA, USA; 3Mayo Foundation for Medical Education and Research, Jacksonville, FL, USA

Multiple variables may impact endpoints used to assess therapeutic efficacy for patients with recurrent gliomas. To identify prognostic factors for each of 3 outcome variables, a master analysis file was constructed by pooling datasets summarizing key baseline factors and outcomes obtained in 15 North Central Cancer Treatment Group (NCCTG) trials (n 5 583, 1980–2004) and 12 North American Brain Tumor Consortium (NABTC) trials (n 5 596, 1998–2002). In addition to traditional variables (Gender, Age, Performance Score [PS], Race, Extent of Primary Resection, Year of Study Entry, Last Known Histology, Time Since Initial Diagnosis, Base-line Steroid Use, Prior Chemotherapy [Y/N], Prior Nitrosoureas, Baseline Anticonvulsant Use), we investigated Prior Temozolomide (TMZ), Current TMZ , Number of Relapses, Academic vs. Community Site and Low Grade at Initial Diagnosis. Outcome variables were: (1) Overall Survival (OS), (2) Progression Free Survival (PFS) and (3) PFS6 5 proportion of patients alive and progression-free 6 months after start of treatment. For all 3 endpoints, Classification and Regression Tree (CART) models, Cox and logistic regres-sion with bootstrap samples were used to identify prognostic variables and to look for evidence of interactions among the independent variables affect-ing outcome. In addition to completing analyses on all data combined, the data set from each of the two groups was used as an independent sample to validate the results from the other. For all outcomes the initial CART analysis selected Last Known Grade (4 vs. 3) as the most important vari-able. Additional factors selected as predicting poor prognosis differed with grade and outcome measure and included Prior TMZ, longer Time Since

Primary Diagnosis, older Age and poor PS. Community vs. Academic was not a strong predictor in any of the models nor was Low Grade at Ini-tial Diagnosis. There were differences in the variables selected based on the two data sets. The ability of the models to predict results for the other will be presented. It was determined that adjustment was required to take into account whether a protocol therapy was determined to be effective. Without this adjustment it was not possible to separate variables that rep-resented intrinsic patient prognostic factors from treatment related factors. The results from analyses to date are consistent with current thinking that Last Known Histology (Grade) is an important factor. The variability in factors selected between the two groups together with the observed impact of treatment re-emphasizes the need to be cautious in using small or poorly characterized data sets as the basis for determining prognostic factors for use in subsequent trials.

PA-31. CEREBROSPINAL FLUID (CSF) O6-METHYLGUANINE-METHYLTRANSFERASE (MGMT) FROM PATIENTS WITH GLIOBLASTOMA MULTIFORMEMin Yu,1 Susan Song,1 Songklha Nguyen,1 Haihua Gu,2 and Eric Wong3; 1Brain Tumor Center and Neuro-Oncology Unit, Beth Israel Deaconess Medical Center, Boston, MA, USA; 2Hematology and Oncology, Beth Israel Deaconess Medical Center, Boston, MA, USA; 3Beth Israel Deaconess Medical Center, Boston, MA, USA

MGMT, a DNA repair enzyme, is a strong prognostic marker for glio-blastomas, predicting response to temozolomide treatment, progression-free survival, and overall survival. The assay to evaluate MGMT status involves polymerase chain reaction (PCR) of promoter methylation performed on fresh frozen tissue by biopsy or resection. However, this is impractical for longitudinal follow up of MGMT status. Since analyzing MGMT levels in CSF may be performed repeatedly in patients with malignant gliomas, we investigate whether MGMT could be detected in the CSF by PCR and Western blot analysis. We therefore treated genomic DNA isolated from the CSF of 22 glioblastoma patients with sodium bisulfite, which converts unmethylated cytosines to uracils while not altering 5-methylcytosines. The resulting samples were subjected to PCR analysis using primers against the altered and unaltered MGMT promoter sequences, allowing analysis of their methylation status. Western blot analysis of CSF using a monoclonal antibody directed against MGMT detected either high or low MGMT levels; 60% of samples that showed low MGMT level by western blot had methy-lated but no unmethylated promoters, while 70% of samples that showed high MGMT levels by western blot had unmethylated and no methylated promoters. Cox regression analysis showed a trend in increased survival in patients with low CSF MGMT (median survival 51.0 months) versus those with high MGMT (median survival 23.5 months). Thus, MGMT-promoter methylation status and MGMT protein can be detected in CSF and may correlate with patient survival.

PA-32. CORRELATION OF IMMUNOHISTOCHEMISTRY STUDIES WITH TUMOR PROGRESSION AMONG INCOMPLETELY RESECTED PILOCYTIC ASTROCYTOMASDaniel Bowers,1 Lynn Gargan,2 Nirupa Raghunathan,1 and Linda Margraf3; 1University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA; 2Neuro-Oncology, Children’s Medical Center of Dallas, Dallas, TX, USA; 3Pathology, University of Texas Southwestern Medical School at Dallas, Dallas, TX, USA

Other than tumor location and extent of surgical resection, there are few prognostic factors for children with Pilocytic Astrocytomas (PAs). This study examines PAs with several immunohistochemistry antibodies, includ-ing MIB-1, MGMT, PHH3, and MCM2, for an association with tumor progression free survival (PFS). Immunohistochemistry for MIB-1 (n 5 83), MGMT (n 5 83), PHH3 (n 5 31), and MCM2 (n 5 13) were performed according to standard methods on PAs from children who had an incom-plete resection. Immunohistochemistry results were compared with PFS by log-rank analysis. The mean age of all patients (n 5 83) at diagnosis for all patients was 7.9 years. Follow-up interval was 7.0 years. 44 patients (53%) of patients had experienced tumor progression at a mean of 1.3 years after diagnosis. MIB-1 (,2.3 vs. >2.3) was associated with a shortened PFS (p 5 0.014). MGMT (<0.5 vs. 0.5–25 vs. >25) was not associated with PFS (p 5 0.6). PHH3 (,1 vs. >1) was not associated with PFS (p 5 0.89). MCM2 (,1 vs. >1) was not associated with PFS (p 5 0.34). For children with incompletely resected PAs, an elevated MIB-1 was associated with a shortened PFS. However, MGMT, PHH3, and MCM2 were not associated with PFS. Prospective clinical trials of adjuvant therapy for PAs should include an evaluation of the MIB-1 labeling index.



PA-33. METHYLATION OF DISCRETE CPG SITES WITHIN THE MGMT PROMOTER PROVIDE A SUPERIOR PREDICTOR OF TEMOZOLOMIDE SENSITIVITY IN THE MAYO GBM XENOGRAFT PANELJann Sarkaria,1 Gaspar Kitange,1 Brett Carlson,1 Ann Mladek,1 Paul Decker,1 Mark Schroeder,1 Wenting Wu,1 Patrick Grogan,1 Caterina Giannini,1 Karla Ballman,1 Jan Buckner,1 and David James2; 1Mayo Clinic, Rochester, MN, USA; 2University of California, San Francisco, San Francisco, CA, USA

CpG methylation within the O6-methylguanine methyltransferase (MGMT) promoter is associated with enhanced survival of glioblastoma multiforme (GBM) patients treated with temozolomide (TMZ). However, the methylation-specific PCR (MS-PCR) assay routinely used to evaluate MGMT promoter methylation evaluates a limited number of CpG sites within the CpG island. To evaluate whether methylation at other CpG sites influences TMZ sensitivity, TMZ responsiveness of 13 GBM xenograft lines was correlated with detailed CpG methylation sequencing across the MGMT promoter. Mice with established intracranial xenografts were treated with vehicle control or TMZ (200 mg/m2 3 5 days), and TMZ response was defined as relative prolongation in median survival for TMZ-treated vs. control-treated mice. Genomic DNA from each line was bisulfite treated and subjected to methylation-specific sequencing and MS-PCR. Classifi-cation and regression tree (CART) analysis was used to identify specific CpG methylation sites that are predictive for TMZ response. Proportional hazards regression was used to evaluate the association of CpG sites identi-fied in the CART analysis and MGMT MS-PCR on survival, and Akaike’s information criteria (AIC) was used to evaluate the goodness of fit of these models. There was a broad range in TMZ sensitivity in the xenografts with the prolongation in median survival ranging from 21% to 579%. When assessing methylation status by MS-PCR, there was a trend toward greater survival prolongation with TMZ treatment in the MGMT hypermethylated tumors (median survival benefit 253%, range 82%–579%) relative to non-methylated tumors (median survival benefit 65%, range 21%–558%; rank sum test, p 5 0.13). CpG methylation sequence analysis demonstrated sig-nificant heterogeneity in methylation status across the CpG island, ranging from 2 of 96 CpGs methylated in GBM10 to 75 of 96 CpGs being methylated in GBM12. However, this crude measure of the extent of CpG methylation had no relationship to TMZ responsiveness (p 5 0.747). CART analysis of the data identified methylation at 4 CpG sites as being highly predictive of TMZ response. The hazard ratios associated with methylation at these sites were: CpG89—HR 0.21 (p , 0.001), CpG58—HR 3.26 (p 5 0.05), CpG 66—HR 11.55 (p , 0.001), CpG 8—HR 0.32 (p , 0.027). In comparison, the hazard ratio for the MS-PCR assay was 0.77 (p 5 0.241), which suggests that the 4 site methylation model (AIC 5 596.1) provides a more robust model of TMZ sensitivity as compared to MS-PCR (AIC 5 727.9). In a separate analysis of sequence data limited to the 9 CpG sites (CpGs 78–82 and 86–89) queried by MS-PCR, methylation status defined by sequencing was discordant in at least 1 CpG site in 7 of 13 xenograft lines. CpG89 methylation was the most divergent between the MS-PCR and sequencing assessment, and methylation at CpG89 was highly correlated with TMZ sensitivity: median survival benefit of 277% (range 82%–579%) for tumors with CpG89 methylation as compared to a survival benefit of 60% (range 21%–110%) for tumors without methylation at CpG89 (p 5 0.004). These data support the idea that MGMT promoter methylation is an important determinant of TMZ sensitivity and suggest that more precise delineation of MGMT promoter methylation may provide greater predictive accuracy.

PA-34. STAT3 CONSTITUTIVE ACTIVATION IS ASSOCIATED WITH GLIOMA TUMOR GRADE: PATHWAY PROFILING AND THERAPYHui-Wen Lo, Xinyu Cao, Hu Zhu, and Francis Ali-Osman; Department of Surgery, Duke University, Durham, NC, USA

Malignant gliomas result in approximately 40% of the brain tumor death in adults and the median survival of these patients is less than 12 months. A better understanding of the biology of malignant gliomas is thus urgently needed to improve the current therapy and to facilitate the future development of more effective agents. Deregulation of cell signaling pathways is a major characteristic of malignant gliomas leading to tumori-genesis, proliferation, migration and poor patient survival. Consequently, hyper-active growth-stimulatory pathways can serve as an indicator for poor prognosis. Targeted therapy that aims to restore normal status of cell signaling is of significant interest and is under tremendous preclinical and clinical development. In search for an attractive target for glioma prog-nosis and therapy, we focused on STAT3, an oncogenic transcription fac-tor that activates the expression of growth-stimulatory genes (cyclin D1, c-myc, c-fos) and anti-apoptotic genes (bcl-xl and Fas). In addition, STAT3 activates expression of angiogenic factors, such as, VEGF. Despite a critical role of STAT3 in several cancers has been well established, its involvement in malignant gliomas remains elusive. Here, we report that STAT3 consti-tutive activation (STAT3 phosphorylated at Y705; p-STAT3) was detected in 40% of primary gliomas (n 5 55) in which gliomas with p-STAT3

. 20% were regarded as those with constitutive activated p-STAT3. In nor-mal adjacent tissues, only 20% (1/5) contained STAT3 constitutive activa-tion. Specifically, 27.5% of grade I astrocytomas were found to have con-stitutively activated STAT3 and 29.2%, 57.1%, and 66.7% in grades II, III, and IV astrocytomas, respectively. Regression analysis further indicated a significant positive correlation between STAT3 constitutive activation and glioma tumor grade (r 5 0.949, p 5 0.0136). We further performed studies to profile the STAT3 pathway in cultured glioma cells and found that high levels of p-STAT3 expression were present in almost all the brain tumor cell lines but not in the normal astrocytes that we analyzed. Consistently, STAT3-mediated downstream genes, VEGF and cyclin D1, were concur-rently elevated in glioma cells with hyperactive STAT3. Next, we examined whether targeting STAT3 led to glioma cell death. Anti-JAK2/STATs agent, Cucurbitacin I/JSI-124, suppressed expression of cyclin D1 and VEGF and robustly killed glioma cells but not normal astrocytes. The IC-50 values for JSI-124 were determined to range between 10 and 100 nM. We also tested other anti-STAT3 agents such as AG490 and an inhibitory peptide and found them to be less effective in targeting glioma cells compared to JSI-24. Moreover, JSI-124 sensitized resistant glioma cells to temozolomide, BCNU, cisplatin, and Iressa, an EGFR tyrosine kinase inhibitor. Further median-effect analysis indicated a synergistic killing effect when JSI-124 was used in combination with cisplatin or Iressa. Together, we provide rationales to regard STAT3 as a predictive factor for glioma progression and to consider STAT3 as a novel major therapeutic target for malignant gliomas. This study is supported by the Duke Comprehensive Cancer Center, Duke Brain Tumor Center and NIH, K01 CA118423–01 to H.-W. Lo.

PA-35. LEPTOMENINGEAL DISEASE FROM OLIGODENDROGLIOMA: CLINICAL AND MOLECULAR ANALYSISGloria Roldán,1 James Scott,2 David George,3 Jacob Easaw,4 Elizabeth Yan,4 Ian Parney,5 Gregory Cairncross,1 and Peter Forsyth4; 1Dept. Clinical Neurosciences, University of Calgary, Tom Baker Cancer Centre, Clark Smith Integrated Brain Tumor Research Centre, Calgary, Alberta, Canada; 2Dept. Clinical Neurosciences, Dept. Diagnostic Imaging, University of Calgary, Alberta, Alberta, Canada; 3Dept. Pathology & Lab Medicine, University of Calgary, Alberta, Canada; 4Dept. Oncology, Tom Baker Cancer Centre, Clark Smith Integrated Brain tumor Research Centre., Alberta, Canada; 5Dept. Oncology, Tom Baker Cancer Centre, Dept. Clinical Neurosciences, University of Calgary, Clark Smith Integrated Brain Tumor Research Centre., Alberta, Canada

Leptomeningeal disease (LMD) is a late complication of malignant glioma, mostly of glioblastoma, that usually responds poorly to treatment and is rapidly fatal. A long surviving case led us to review our experience with LMD in patients with oligodendrogliomas. A 15-year retrospective chart review was performed. Patients with both oligodendroglial tumors and LMD were identified. A single neuro-pathologist reviewed all histological sections, a single neuro-radiologist reviewed all available images and 1p/19q status was assessed. Seven out of 145 patients with oligodendroglioma were diagnosed with LMD. Six were male. Median age at tumor diagnosis was 41 years (range, 28–50). None had radiographic or pathological evidence of leptomeningeal or subependymal tumor at initial diagnosis. Most patients had pure anaplastic oligodendrogliomas (4/7); 6/7 had 1p/19q co-deletion. The median time to first relapse was 41 months (range, 19–127). The median time to LMD was 76 months (range, 19–151) from initial diagnosis and 28 months (range, 0–36) from first relapse, respectively. LMD treat-ments included spinal radiation and intrathecal and systemic chemotherapy. After progression, some patients with LMD remained stable clinically. The median survival from initial diagnosis was 104 months (range, 19–183) and from LMD diagnosis was 32 months (range, 2–43). LMD is a complica-tion of oligodendroglioma that may occur preferentially in long surviving patients with 1p/19q co-deletion. LMD in patients with oligodendrogliomas appears to be relatively indolent which may have implications for their treat-ment and be related to 1p/19q status.

PA-36. INTRAVENTRICULAR MYXOID MENINGIOMA IN A 7-YEAR-OLD FEMALEMohanpal Singh Dulai,1 Abdul-Majid Khan,2 Michael S. B. Edwards,3 and Hannes Vogel1; 1Neuropathology, Stanford University, Stanford, CA, USA; 2Radiology, Stanford University, Stanford, CA, USA; 3Neurosurgery, Stanford University, Stanford, CA, USA

Meningiomas are rare in childhood and display important differences from adult forms. We report a pediatric meningioma which is character-istic of childhood meningiomas in some respects but unique in others. A previously healthy 7-year-old female presented with a 3-week history of progressive headaches and diplopia. She also had a single episode of nausea and vomiting. There were no clinical signs or family history of neurofibro-matosis type II. An MRI of the brain showed a 4.8 cm, intensely enhancing,



partially cystic lesion centered within the left ventricle with a significant amount of surrounding vasogenic edema. The lesion was resected and noted to be intimately associated with the choroid plexus. Histologic evaluation revealed a neoplasm composed of cells with oval nuclei and a small amount of eosinophilic cytoplasm. The neoplastic cells were separated by abundant myxoid and mucinous stroma. No significant mitotic activity, necrosis, inflammation, or vascular proliferation was identified. The neoplastic cells showed weak immunoreactivity for epithelial membrane antigen (EMA), S-100 protein, and vimentin. No glial fibrillary acidic protein (GFAP) immunoreactivity was seen. Ultrastructural analysis revealed microvilli, complex intercellular interdigitations and tight junctions in the neoplastic cells, and a diagnosis of myxoid (metaplastic) meningioma was rendered. Metaplastic variants such as myxoid meningioma have been reported in childhood. The differential diagnosis otherwise included chordoid glioma, pilocytic astrocytoma, and other myxoid mesenchymal tumors. Notwith-standing the unusual nature of this tumor, it reflects the much higher inci-dence of intraventricular meningiomas in childhood as compared with in adults, and the greater incidence in general of intraventricular meningiomas in the left lateral ventricle. Some reports describe a higher incidence of chor-doid meningiomas in childhood series. Recognition of the Grade I myxoid meningioma in this case, as distinct from the histologically similar chordoid meningioma, is important since chordoid meningiomas are of a higher grade and worse prognosis.

PA-37. PODOPLANIN IS AN INDEPENDENT PROGNOSTIC MARKER OF SURVIVAL IN MALIGNANT GLIOMAS AND IS CO-EXPRESSED WITH CD133 ON TUMOR-DERIVED STEM CELLSErik Sulman, Howard Colman, Christopher Pelloski, and Kenneth Aldape; University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Few molecular markers are capable of discriminating long-term survivors of high-grade gliomas. Data from multiple expression profiling experiments suggested that expression of the mucin-type transmembrane glycoprotein podoplanin (PDPN) might be prognostic for survival. We sought to validate this finding in an independent cohort of patients. A set of 112 tumors from patients with glioblastoma (GBM) was examined for PDPN expression by immunohistochemical (IHC) analysis of formalin-fixed, paraffin embedded tissue, with expression scored as negative, intermediate, or high. Median survival in the PDPN-expressing group (immediate plus high) was 60 wks vs. 148 wks in the non-expressing group (p 5 0.008, log-rank test). This result was validated using an independent set of 94 GBMs. Median and 2-year survival among all 206 GBMs (median follow-up of 55 weeks) for PDPN expression vs. non-expression was 54 wks vs. 148 wks and 15% vs. 56%, respectively (p , 0.0001). IHC results were further validated using real-time RT-PCR for 71 GBMs. In multivariate analysis, PDPN expression was an independent predictor of survival after adjustment for age and extent of surgical resection (HR 2.5, 95% CI 1.4–4.7). The presence of PDPN expression was also associated with poorer radiation response among the 74 patients with GBM who underwent sub-total tumor resection. In addi-tion, we examined PDPN expression in a panel of 93 WHO grade II and III astrocytomas and observed a correlation to survival. The estimated 5-year survival for negative-, intermediate-, and high-PDPN levels was 82%, 65%, and 20%, respectively (p , 0.001). PDPN level was independent of age and grade among the astrocytomas in predicting overall survival (HR 3.9, 95% CI 1.4–13.7). Based on its prognostic significance, we sought to identify a role for PDPN in tumor formation. Previous reports have demonstrated PDPN as a marker of murine neural stem cells. We hypothesized that this prognostic protein may be a marker of glioma stem cells, a subpopulation of tumor cells believed to be responsible for tumor initiation and treatment resistance. Immunoblot analysis of PDPN revealed high-level expression in 9 of 11 glioma stem-like cell lines (GSCs) cultured from primary GBMs. Expression was also examined by flow cytometry for 10 GSCs. Significant levels of PDPN were detected in 9 of 10 GSCs (range 11.5%–99.9% of cells). The neural stem cell marker CD133 was also examined and the same 9 of 10 GSCs expressed significant levels of this marker (range 4.9%–82.4% of cells). Co-expression of both markers was seen in 8 of 10 GSCs (range 4.9%–78.7%). While most of the GSC lines contained large percentages of cells which were PDPN-positive/CD133-negative (range 9.6%–95%), cells that were CD133-positive co-expressed PDPN at a proportion much greater than would be expected by chance alone. The results suggest that PDPN, an independent prognostic marker of survival in malignant astrocytomas, is, in conjunction with CD133, a putative marker of tumor-derived neural stem cells. Further examination of the tumor initiating ability of the PDPN-expressing, CD133-positive GSCs and the functional role of PDPN in tumor development is currently underway.

PA-38. SOX2 IS EXPRESSED IN GLIOMAS BUT NOT IN NEURONAL TUMORSJennifer Eschbacher and Stephen Coons; Neuropathology, Barrow Neurological Institute, Phoenix, AZ, USA

SOX2 is a transcription factor which plays a fundamental role in mam-malian embryonic CNS development and in adult neurogenesis. As SOX2 functions to maintain pluripotency in these stem cell populations, and as increasing evidence points towards a stem cell or progenitor cell origin for gliomas, we sought to establish differential expression of SOX2 in primary brain tumors. Using immunohistochemical techniques, we analyzed SOX2 expression in 85 primary brain tumors, including 68 gliomas (astrocytomas, oligodendrogliomas, oligoastrocytomas, ependymomas) and 17 nonglial CNS tumors (medulloblastomas, atypical teratoid rhabdoid tumors, cen-tral neurocytomas, pineal parenchymal tumors). We found 65 of 68 gliomas to demonstrate diffuse nuclear immunopositivity for SOX2, while 16/17 nonglial tumors were completely negative or exhibited very limited focal positivity. The differential expression of SOX2 in tumors of glial origin as opposed to tumors of neuronal origin may provide further evidence to the lineage of the neoplastic cells as well as prove to be of diagnostic benefit in the clinical setting.

PA-39. INTRACRANIAL EXTRANODAL MARGINAL ZONE B-CELL MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT) LYMPHOMAAdam Gaban, Ali Raja, Murat Gokden, and Ali Krisht; University of Arkansas for Medical Sciences, Little Rock, AR, USA

Primary extranodal marginal zone B-cell mucosa-associated lymphoid tissue (MALT) lymphomas are infrequently encountered tumors of the cen-tral nervous system. They are most commonly seen as dural-based tumors, but other intracranial locations have also been described. Although, now a clearly defined disease entity with several case reports in the literature, little is known about their clinical behavior. The presentation, radiological appearance, and clinical course can be misleading due to similarities with other slow growing, more frequently found tumors like meningiomas. Due to the distinct natural history and management of this disease, familiarity with these tumors and their early diagnosis is of paramount clinical signifi-cance. A comprehensive review of these tumors including clinical, radiologi-cal, histopathological, and immunohistochemical diagnosis are presented along with their treatment.

PA-40. PLEOMORPHIC PINEAL PARENCHYMAL TUMOR OF INTERMEDIATE DIFFERENTIATIONGregory Fuller,1 Jai Grewal,2 Merja Kallio,2 Monica Loghin,2 Matthew Debnam,3 and Ian McCutcheon4; 1Pathology (Neuropathology), University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Neuro-Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3Neuroradiology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 4Neurosurgery, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Pineal parenchymal tumors (PPTs) are subdivided by the World Health Organization Classification (WHO 2007) as follows: pineocytoma (grade I); pineal parenchymal tumor of intermediate differentiation (PPTID; grades II–III), and pineoblastoma (grade IV). All PPT subtypes are composed of a relatively uniform, monotonous tumor cell population, with only mild-to-moderate overall pleomorphism and very rarely containing occasional giant cells. We present here a case of PPTID characterized by marked pleo-morphism and large numbers of multinucleated giant cells. A 47-year-old woman presented with a 2-month history of increasing headaches, nausea and emesis. MR imaging revealed a circumscribed mass (3.5 3 2.4 3 2.2 cm) in the posterior third ventricle and pineal region that was hypointense on T1-weighted images, hyperintense on T2-weighted images, and showed homogeneous enhancement following contrast administration. The patient underwent biopsy followed by surgical resection. Microscopic examina-tion of the resected tumor showed a pleomorphic neoplasm with prominent multinucleated giant cells, increased mitotic activity with up to 11 mitoses per 10 high-power fields, and a high Ki-67 antigen (MIB-1) labeling index of 23%. Immunohistochemical studies revealed strong tumor cell expression of synaptophysin and neurofilament protein, and ultrastructural examina-tion showed dense-core, neurosecretory-type vesicles. A diagnosis of PPTID was rendered and the patient subsequently received craniospinal irradiation (30.6 Gy in 17 fractions). Conclusion: The current standard histopathologic descriptions and classification scheme for pineal parenchymal tumors fail to account for the complete spectrum of phenotypic variation that can be encountered in this distinctive class of CNS tumor. Further refinement of the classification and grading criteria for PPTs warrant additional study.



PA-41. ATYPICAL PSEUDOPAPILLARY EXTRAVENTRICULAR NEUROCYTOMAJianyi Li,1 Gregory Fuller,2 and Min Wang3; 1Department of Pathology, The Methodist Hospital, Houston, TX, USA; 2Department of Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3Department of Pathology, The University of Texas Health Science Center, Houston Medical School, Houston, TX, USA

Extraventricular neurocytoma (EVNC) comprises a recently described class of neurocytic neoplasm that has been formally codified in the revised 2007 WHO Classification of Tumours of the Central Nervous System, in which EVNCs are recognized as an entity distinct from central neuro-cytoma. EVNCs are relatively circumscribed, noninfiltrative, frequently cys-tic and calcified low-grade tumors that are composed of a variable mixture of small uniform neurocytes, large ganglion cells, and intermediate-sized ganglioid cells. The constituent cells are often arrayed in sheets or lobules within a fibrillar background. Mitoses and other atypical features are rare, and the behavior and criteria for atypical forms have yet to be established. We report a case of mitotically active extraventricular neurocytoma char-acterized by pseudopapillary histological architecture. A 10-year-old boy presented with a 2-year history of headache with recent onset of nausea and vomiting. There were no speech abnormalities, behavioral changes or localizing neurologic findings. MRI studies revealed a 9.5 3 6.3 3 5.5 cm multicystic mass within the right temporo-parietal cerebral cortex, accom-panied by midline shift and compression of the right lateral ventricle. The mass was hypointense on T1-weighted sequences, hyperintense on T2, and exhibited irregular peripheral and internal enhancement following contrast administration. Gross total resection was performed. H&E-stained cyto-logical smear preparations showed a monotonous population of tumor cells with uniform, round, bland nuclei and scant eosinophilic cytoplasm along with prominent patches of acellular, bluish-purple mucoid material. Tis-sue sections revealed a neurocytic neoplasm with large areas displaying a pseudopapillary architecture in which tumor cells were circumferentially arrayed around often hyalinized blood vessels. Occasional large, dysmor-phic-appearing ganglion cells were also present. The prominent pseudo-papillary and perivascular rosetted pattern raised the differential diagnosis of choroid plexus papilloma, papillary meningioma, and astroblastoma. Areas of more compact tumor were also seen, in which the majority of cells were small, uniform neurocytes. The surrounding brain parenchyma showed reactive changes, including astrogliosis and scattered granular bod-ies. The tumor cells were strongly immunopositive for synaptophysin, and negative for GFAP and EMA. Mitotic figures were readily identified on H&E-stained tissue sections, and quantitation facilitated by the mitosis-specific immunomarker phosphohistone-H3 yielded a mitotic index of 6 per 10 high-power fields. The Ki-67 antigen (MIB1) labeling index was 8%. The phenotypic spectrum and range of behavior for the recently recognized entity of extraventricular neurocytoma continues to evolve with additional clinical experience. Characterization of forms that exhibit atypical features, such as increased proliferative activity, warrants further investigation.

PEDIATRIC BASIC SCIENCE

PB-01. RAC1-REGULATED SIGNALING AS A NOVEL THERAPEUTIC TARGET FOR MEDULLOBLASTOMASalvatore Zavarella,1 Shawn Belverud,2 Amanda Chan,1 Bettie Steinberg,3 Mark Mittler,4 Steven Schneider,4 and Marc Symons1; 1The Feinstein Institute for Medical Research, Manhasset, NY, USA; 2Department of Neurosurgery, North Shore University Hospital, Manhasset, NY, USA; 3New Hyde Park, NY, USA; 4Division of Pediatric Neurosurgery, Schneider Children’s Hospital, New Hyde Park, NY, USA

Medulloblastoma, accounting for 25% of pediatric brain tumors, is the most common solid primary tumor of childhood. These tumors display invasion of individual cells into adjacent parenchyma, and subsequently the CSF, causing metastasis and poor prognosis. The Rac1 GTPase has been shown to be essential for the invasive behavior of several tumor types. We therefore investigated the role of Rac1 and Rac1-controlled signaling ele-ments in medulloblastoma invasion. We found that siRNA-mediated deple-tion of Rac1 strongly inhibits hepatocyte growth factor-stimulated invasion of Daoy medulloblastoma cells through Matrigel. Interestingly, depletion of Rac1 enhances Daoy cell proliferation, but promotes cell death induced by ionizing radiation. Thus, these results are in line with the go or grow hypothesis. Rac1 promotes cell invasion by remodeling the actin cytoskel-eton, and we showed that depletion of Rac1 strongly inhibits HGF-stimu-lated formation of lamellipodia, an actin-based structure that is important for cell migration. PAK serine-threonine kinases are critical effectors that regulate actin dynamics downstream of Rac1. We found that depletion of either PAK1 or PAK2 significantly inhibits invasion, suggesting that PAK1 and PAK2 deliver non-redundant contributions to invasion downstream of Rac1. In other cell types, Rac1 has been shown to regulate cyclooxygenase-2 (COX-2) levels and COX-2 levels are known to be elevated in medulloblas-

toma. We therefore examined the role of COX-2 in medulloblastoma inva-sion using the COX-2 selective inhibitor celecoxib. Celecoxib at 5 mM, a concentration achievable in patient serum, inhibits invasion over 50%. In summary, our results indicate that Rac1 contributes to medulloblastoma invasion and metastasis. Thus, targeting signaling components controlled by Rac1, including PAK kinases and COX-2, present novel therapeutic avenues for advanced medulloblastoma.

PB-02. NOVEL ANGIOGENIC FACTORS LINKED TO AKT2 OVER-EXPRESSION IN PEDIATRIC BRAIN TUMORSTimothy Van Meter,1 Catherine Dumur,2 William Broaddus,2 and Gary Tye2; 1Virginia Commonwealth University, Richmond, VA, USA; 2Richmond, VA, USA

Elevated AKT kinase activity is a feature of several advanced cancers including adult brain cancers such as gliomas. Much less is known about AKT signaling pathways in pediatric brain tumors. We have character-ized AKT isotype expression and activity in 50 pediatric brain tumors (11 medulloblastoma, 15 ependymoma, 11 pilocytic astrocytomas, 3 anaplas-tic astrocytomas, and 10 oligodendrogliomas) and compared these to our previous data in adult high grade gliomas. Protein and activity levels for AKT1,2,3 and signaling targets were assessed by triplicate Western blots and mean values compared with normal brain. Transcript levels of the three AKT isotypes were also assessed using triplicate Taqman assays of total RNA extracts from the same snap frozen tumor specimens. In both PNET and in ependymoma, AKT2 levels were elevated compared to age-matched non-tumor “normal” brain tissues. This profile is in contrast to data derived previously from adult gliomas, wherein heightened expression of AKT1 was more prevalent. In an attempt to further characterize the role of heightened AKT isotype expression in oncogenesis of individual tumor types, we first focused on ependymomas in which AKT2 over-expression was prevalent, and compared gene expression profiles with tumors bearing AKT2 expres-sion levels which were not significantly different from normal brain. Using Affymetrix high density oligonucleotide microarrays with 26,262 probe sets, we identified 213 genes whose expression level differed between the AKT2 high samples and the AKT2 low samples. Represented in this gene set were several genes with greater than 2- to 3-fold increase in expression, that have potential impact on the cell cycle or invasiveness/extracellular matrix remodeling. To our knowledge, none of these genes have previously been described as having a functional role in ependymoma tumorigenesis. Genes implicated in angiogenesis were also found to be elevated, including Angio-poietins and f-Spondin. To validate the relationship of this gene subset with AKT2 expression/activity, we performed further quantitative PCR assays and correlated mRNA expression level of each gene with AKT2 expression level. Results of these studies suggest novel mediators of AKT signaling and angiogenesis in pediatric brain tumors.

PB-03. THE ROLE OF RAC SIGNALING IN PDGF-MEDIATED CELL MOTILITY IN MEDULLOBLASTOMALiangping Yuan and Tobey MacDonald; Center for Cancer and Immunology, Children’s National Medical Center, Washington, DC, USA

We previously showed that PDGFR is highly overexpressed in meta-static medulloblastoma (MB) and is related to the promotion of cell migra-tion and invasion, but the mechanism is still poorly understood. In this study, we investigated the roles of the small GTPases Rac/Rho, which have been implicated in cancer metastasis, to determine whether PDGFR medi-ates pro-migratory cell signaling via Rac in MB cells. By microarray analy-sis, we found Rac mRNA was expressed in 89% metastatic MB and only 57% of non-metastatic MB. Immnunohistochemistry of tissue microarrays containing 205 MB showed Rac1 was detectable in over 90% of tumors. Furthermore, we found that PDGF promotes the activation of Ras-Rac, phosphorylation of MEK-ERK, and pro-migratory actin cytoskeletal rear-rangements, while inhibiting activation of Rho in Daoy MB cells. Finally, we show that PDGF promotes Daoy MB cell migration via a Rac-dependent manner. Our investigation supports the hypothesis that Rac plays a critical role in PDGF-mediated migration in medulloblastoma .

PB-04. INTERACTIONS BETWEEN THE c-MET PATHWAY AND FOCAL ADHESION KINASE (FAK) FAMILY MEMBERS IN MEDULLOBLASTOMAFadila Guessous, Elizabeth Johnson, Yunqing Li, David Schiff, and Roger Abounader; Neurology, University of Virginia, Charlottesville, VA, USA

The molecular mechanisms of progression and metastasis of medullo-blastoma, the most common primary brain tumor of childhood, are not well understood. We recently demonstrated the involvement of hepatocyte



growth factor (HGF) and its tyrosine kinase receptor c-Met in medullo-blastoma malignancy. The nonreceptor tyrosine kinases, focal adhesion kinase (FAK) and the proline-rich tyrosine kinase 2 (Pyk2), have emerged as key players in the progression of different cancers. FAK is a multidomain protein with multiple tyrosine and serine sites of phosphorylation. FAK localizes to regions of the cell membrane that attach to the extracellular matrix. Pyk2 has a conserved domain and significant sequence identity with FAK. FAK and Pyk2 are important signaling effectors linking integrin and growth factor signaling to cell invasion, proliferation, migration, survival, and apoptosis in many cancers. However, their role in medulloblastoma remains unexplored. In the present study, we demonstrate for the first time that HGF/c-Met activates FAK and Pyk2 and that FAK and Pyk2 mediate various malignant functions of HGF/c-Met in medullobalstoma. Three dif-ferent medullobalstoma cell lines, D425, DAOY, and ONS-76, were treated with HGF for various times and the phosphorylation of regulatory tyrosines (Tyr) and serines (Ser) on FAK and Pyk2 were assessed by immunoblotting. HGF treatment induced a strong phosphorylation of FAK at Tyr 397 and Tyr 925, which are important for the maximal adhesion-induced activation of FAK and signaling to downstream effectors. HGF also induced phos-phorylation of FAK at Ser 910, which is involved in modulating binding/stability of downstream signaling proteins. Similarly, cell treatment with HGF resulted in a strong and fast phosphorylation of Pyk2 at Tyr 402, reported to promote invasion and migration. The above described phos-phorylations of FAK and Pyk2 were observed in the settings of activated as well as inactivated integrins, indicating that c-Met also cooperates with integrins in activating FAK family members. To determine if FAK and Pyk2 mediate HGF/c-Met malignant functions in medulloblastoma, FAK or Pyk2 expressions were inhibited with siRNA and the effects of these inhibitions on various malignancy parameters in medulloblastoma cells were studied. The results show that inhibition of FAK leads to a significant inhibition of basal and HGF-induced medulloblastoma cell invasion as assessed by Boy-den Chamber invasion assays. Inhibition of FAK or Pyk2 expressions also led to inhibition of basal and HGF-induced medulloblastoma tumor cell migration and proliferation as assessed by migration assays and cell count-ing, respectively. We are currently testing the effects of FAK and Pyk2 on HGF-induced medulloblastoma cell cycle progression, cytoprotection and in vivo tumor growth. We are also examining possible direct physical interac-tions between c-Met and FAK/Pyk2 and plan to study potential correlations between HGF/c-Met and FAK/Pyk2 expressions in human medulloblastoma tissue samples. In conclusion, our study shows for the first time that HGF regulates FAK and Pyk2 activation and that FAK and Pyk2 mediate basal as well as HGF-induced medulloblastoma malignancy. These findings provide new insights in medulloblastoma malignancy and open new avenues for developing novel therapeutic strategies targeting focal adhesion kinases in conjunction with HGF/c-Met.

PB-05. MODELING MEDULLOBLASTOMA DISSEMINATION IN THE DEVELOPING NERVOUS SYSTEMTene Cage, Arturo Alvarez-Buylla, Nalin Gupta, and Jeanette Hyer; Brain Tumor Research Center, University of California, San Francisco, San Francisco, CA, USA

Medulloblastomas (MB) are primary malignant brain tumors that belong to a group of embryonal tumors and occur mostly in children and young adults. MBs are thought to arise from neoplastic transformation of granule cell precursors in the cerebellum. About 50% of medulloblastoma patients will present with widespread leptomeningeal dissemination either initially or at relapse. The molecular mechanisms behind MB dissemination are poorly understood. We propose that MB dissemination is guided by specific cell-cell interactions between tumor and non-neoplastic tissue medi-ated by two signaling molecules, Hepatocyte Growth Factor (HGF) and Stromal Cell-Derived Factor 1 (SDF1). Both molecules have important roles in the normal migration of the cerebellar precursors during development. They may also play a role in establishing a favorable tumor microenviron-ment through cell chemotaxis, directed homing recruitment of stem cells, and promotion of cell proliferation. We have established an avian-human xenograft system to study migration and growth of medulloblastoma cells in the developing nervous system. The cell lines chosen were originally derived from metastatic MB (D283), desmoplastic MB (Daoy), and invasive astro-cytoma (U87) human tumors. These cell lines were stably transduced with a GFP-expressing vector and then implanted into the developing central nervous system (CNS) of embryonic day 2 chick embryos. Cell migration was followed by conventional and confocal microscopy. Response of these cell lines to HGF and SDF1 in vitro was also studied. When introduced into the embryonic CNS, the three cell lines closely recapitulate behavior seen in human disease. Metastatic MB cells (D283) integrate into the developing hindbrain and migrate away from their site of introduction in a rostral-to-caudal pattern. This is similar to the pattern of leptomeningeal dissemina-tion observed in humans. In contrast, Daoy cells incorporate into the host tissue but remain as a solid aggregate which is similar to the solid cerebel-lar mass formed in the human desmoplastic counterpart. Finally, U87 cells integrate in the developing avian brain in a radial pattern. This behavior

recapitulates the non-directional incorporation of infiltrative astrocytomas. In addition, during dissemination, D283 cells assume a cell morphology characterized by an elongated cell body and extension of neurites. Finally, we also observed a location-specific pattern of tumor cell incorporation along the CNS. Astrocytoma and desmoplastic MB cell lines show similar patterns of growth regardless of the specific site of implantation in the devel-oping CNS (e.g., forebrain, midbrain, and hindbrain). D283 cells, however, do not integrate and recapitulate their cranial-caudal pattern of invasion when implanted outside the hindbrain. This suggests that a permissive interaction between the developing hindbrain and MB cells is necessary to promote a directed invasive phenotype. Additional in vitro results suggest that HGF and SDF1 signaling may be involved in altering directed tumor cell migration. Using a xenograft-avian model system for disseminated MB, we have begun to define patterns of migration that are dependent upon spatial cues in the developing CNS and potential signaling molecules that underlie this behavior.

PB-06. A9B1 INTEGRINS MEDIATE ADHESION OF D283 MEDULLOBLASTOMA CELLS TO A GLIOMA-DERIVED EXTRACELLULAR MATRIX AND ACTIVATE PATHWAYS ASSOCIATED WITH SURVIVAL, PROLIFERATION, AND RESISTANCE TO TREATMENTSidney Croul,1 Paul Fiorilli,2 Izabela Staniszewska,2 Jin Ying Wang,2 Maja Grabaka,3 Cezary Marcinkiewicz,2 Krzysztof Reiss,2 and Kamel Khalili2; 1Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; 2PA, USA; 3Poland

Medulloblastoma metastasizes by leptomeningeal dissemination rather than by the tissue infiltration that characterizes the spread of many central nervous system tumors. Combining a previously successful approach to adhesion with these current assays, this study represents an initial attempt to define the molecular interactions between medulloblastoma cells and the leptomenings that result in adhesion and survival of the metastases. The results of this study demonstrate in vitro adhesion of a medulloblastoma cell line (D283) to a glial extracellular matrix (ECM). Within this context, a9 and b1 integrin subunits were expressed at significantly greater levels than any of the other integrin subunits assayed. Following blockade of the D283 a9and b1integrin subunits, binding to the ECM did not occur. The ECM was enriched in only one of the proteins, tenascin, known to serve as a binding partner for the a9b1 heterodimer. The results of this study also demonstrate enhanced medulloblastoma cell survival following engagement of the ECM. Adherent D283 cells grown in serum free medium demon-strated increased survival and proliferation relative to non-adherent cells. Western blots performed on adherent cells demonstrated marked activation of MAP kinase p42/44 (Erk1/2). These findings suggest that a glial ECM can provide a surface to which medulloblastoma cells will adhere, survive and proliferate. This combination of ECM with medulloblastoma cell lines may serve as a model for the molecular events that underlie leptomeningeal dissemination.

PB-07. AMPA-TYPE GLUTAMATE RECEPTOR EXPRESSION AND ACTIVITY IN ADULT HIGH-GRADE GLIOMA AND CHILDHOOD MEDULLOBLASTOMADannis Van Vuurden,1 Maryam Yazdani,1 Rogier Min,2 Rhiannon Meredith,2 Nail Burnashev,2 Ingeborg Bosma,3 Tjeerd Postma,3 Jan Heimans,3 Paul Van Der Valk,4 Eleonora Aronica,5 Richard Broekhuizen,1 Gertjan Kaspers,1 and Jacqueline Cloos1; 1Department of Pediatric Oncology/Hematology, Free University Hospital Amsterdam, Amsterdam, Netherlands; 2Department of Experimental Neurophysiology, Free University of Amsterdam, Center for Neurogenomics and Cognitive Research, Amsterdam, Netherlands; 3Department of Neurology, Free University Hospital Amsterdam, Amsterdam, Netherlands; 4Department of Pathology, Free University Hospital Amsterdam, Amsterdam, Netherlands; 5Department of Pathology, Academic Medical Center, Amsterdam, Netherlands

Calcium influx through ionotropic, Na1- and Ca21-permeable AMPA-type glutamate receptors (AMPARs) is thought to play a role in several pro-survival, proliferation and anti-apoptosis pathways in glioma cells. AMPARs mediate fast neurotransmission in the central nervous system. Posttranscrip-tional RNA-editing of the GluR2 AMPAR subunit leads to a Ca21-imper-meable receptor. Expression of AMPA-type subunits and unedited GluR2 (GluR2Q)-subunits results in calcium-permeable AMPARs in glioblastoma cells. Little is known about AMPAR expression in childhood brain tumors, such as medulloblastoma. We aimed to measure AMPAR expression, as potential treatment targets, in glioblastoma and medulloblastoma cell lines and glioblastoma patient material. GluR1, 2, and 4 expression (protein and RNA) and GluR2 RNA editing were measured in glioblastoma and medulloblastoma cells from primary patient material and established cell



lines and correlated to receptor functionality, using whole-cell patch-clamp techniques and calcium imaging following glutamate stimulation. AMPAR-antagonist NBQX was used in a growth inhibition assay on cells with dif-ferential GluR2Q expression. Although AMPAR mRNA and protein are heterogeneously expressed in the cells used in our study, we could not detect any AMPAR-mediated current in patch-clamp recordings. Calcium imaging revealed relative intracellular calcium changes between the nucleus and cyto-plasm, rather than an influx of calcium from the extracellular space upon glutamate stimulation. AMPAR-inhibition with NBQX resulted in growth inhibition in cells with higher GluR2Q expression. Underexpression and/or afunctionality of AMPARs might enable glioblastoma and medulloblas-toma cells to survive in a “high glutamate environment” without going into excitotoxic cell death. AMPARs could function as cell-surface signal trans-ducers via non-calciuminflux dependent tyrosine phosphorylation leading to downward signaling to oncogenic PI3K/Akt and Ras/MAPK pathways. Inhibitors of AMPARs might have anti-tumor properties by inhibition of downward signaling to these pathways.

PB-08. MULTIPLE RECURRENT GENETIC EVENTS CONVERGE ON PROPER CONTROL OF HISTONE 3 LYSINE 9 METHYLATION IN PEDIATRIC MEDULLOBLASTOMAPaul Northcott,1 Yukiko Nakahara,1 John Peacock,1 David Ellison,2 Sidney Croul,3 Lars Feuk,1 Paul Kongkham,1 Karen Zilberberg,1 Stephen Mack,1 Steve Scherer,1 Sunil Rao,4 Wieslawa Grajkowska,5 Richard Grundy,6 Ian Pollack,7 Ronald Hamilton,7 Boleslaw Lach,8 Yancey Gillespie,9 Timothy Van Meter,10 Darrell Bigner,11 Carlos Carlotti,12 Rick Boop,13 Richard Gilbertson,2 James Rutka,1 and Michael Taylor1; 1Hospital for Sick Children, Toronto, Ontario, Canada; 2St Jude Children’s Research Hospital, TN, USA; 3University Hospital Network, Toronto, Ontario, Canada; 4Case Western Reserve University, OH, USA; 5CMHI, Poland; 6University of Nottingham, Turks and Caicos Islands; 7University of Pittsburgh, Pittsburgh, PA, USA; 8McMaster University, Ontario, Canada; 9University of Alabama, AL, USA; 10Medical College of Virginia, VA, USA; 11Duke University, NC, USA; 12Ribeirao Preto School of Medicine, Brazil; 13University of Tennessee, TN, USA

Proper control of histone lysine methylation is critical for transcriptional regulation and plays a critical role in the differentiation of embryonic stem cells, and progenitor cell populations. Genotyping of 212 medulloblastomas using high resolution SNP arrays identified mutations in multiple genes criti-cal for the post-translational modification of histone 3, lysine 9 (H3K9). We identified two tumors with focal homozygous deletions of EHMT1 on 9q34, whose protein product is a histone lysine methyltransferase for H3K9. Similarly, we found focal deletion of SMYD4, another histone lysine meth-yltransferase at 17p13 (3 tumors). Conversely, we found amplification of JMJD2C (7 tumors) and JMJD2B (5 tumors) through the combined analysis of our SNP array data as well as FISH probing of a medulloblastoma tis-sue microarray. The JMJD2 family genes are H3K9 demethylases, with the opposite effect of EHMT1 on H3K9. The ability of EHMT1 to methylate K3K9 is blocked by H3K9 acetylation. We found amplification of MYST3 (4 tumors), a histone 3 lysine acetyltransferase. We also defined deletions in polycomb genes that interpret the state of H3K9 methylation includ-ing L3MBTL3 (1 tumor), L3MBTL2 (4 tumors), and SCML2 (3 tumors). These proteins all contain a malignant brain tumor domain (MBT domain); so named as drosophila with a mutation in l3mbt develop neuronal brain tumors. Mutations in EHMT1, SMYD4, L3MBTL2, LMBTL3, SCML2, JMJD2C, JMJD2B, and MYST3 are mutually exclusive in our cohort of 212 medulloblastomas studied by high resolution SNP array. In addition, medulloblastomas with monosomy at the EHMT1 locus show greatly decreased expression of EHMT1 suggesting that EHMT1 may function as a haploinsufficient tumor suppressor gene. Collectively, these mutations in genes that write, erase, block, and read the methylation state of H3K9 account for 25% of medulloblastomas. EHMT1, L3MBTL, and SCML2 are found in a polyprotein complex with E2F6. This complex is critical for silencing transcription from MYC and E2F dependant promoters, thereby promoting transition from G1 to the G0 phase of the cell cycle. Both rb –/–, and GFAP-E2F1 mice develop medulloblastoma, and MYCC is a well known medulloblastoma oncogene. Another member of the E2F6 polyprotein com-plex is the stem cell factor BMI1, which is over-expressed in medulloblas-tomas. BMI1 knockout mice have a hypoplastic cerebellum due to loss of granule cells. Mice with mutations in l3mbtl3 and myst have abnormalities in the differentiation of progenitor cell populations. We demonstrate that the peak of EHMT1 expression in the external granule cell layer peaks at the same time as p27Kip1. We show that levels of H3K9 methylation are low in the outer, mitotic EGL where cells retain a progenitor state, but drasti-cally increase in the inner, post-mitotic EGL after the cell fate decision to differentiate has been made. Our genetic and functional data are consistent with a model in which proper control of H3K9 methylation with subsequent silencing of MYC and E2F dependant promoters in the cerebellar EGL is critical for the differentiation of progenitor cells; and suggest that muta-tion of genes that promote H3K9 methylation in medulloblastoma results in failure to silence transcription from E2F and MYC dependant promoters, ongoing progression through the cell cycle, and eventual tumorigenesis.

PB-09. IDENTIFICATION OF KNOWN AND NOVEL RECURRENT AMPLIFIED ONCOGENES IN PEDIATRIC MEDULLOBLASTOMAPaul Northcott,1 Yukiko Nakahara,1 John Peacock,1 David Ellison,2 Sidney Croul,3 Lars Feuk,1 Paul Kongkham,1 Stephen Mack,1 Karen Zilberberg,1 Steve Scherer,1 Sunil Rao,4 Wiesia Grajkowska,5 Richard Grundy,6 Ian Pollack,7 Ronald Hamilton,7 Boleslaw Lach,8 Yancey Gillespie,9 Timothy Van Meter,10 Darrell Bigner,11 Carlos Carlotti,12 Rick Boop,13 Richard Gilbertson,2 James Rutka,1 and Michael Taylor1; 1Hospital for Sick Children, Toronto, Ontario, Canada; 2St Jude Children’s Research Hospital, TN, USA; 3University Hospital Network, Toronto, Ontario, Canada; 4Case Western Reserve University, OH, USA; 5CMHI, Poland; 6University of Nottingham, Turks and Caicos Islands; 7University of Pittsburgh, Pittsburgh, PA, USA; 8McMaster University, Ontario, Canada; 9University of Alabama, AL, USA; 10Medical College of Virginia, VA, USA; 11Duke University, NC, USA; 12Ribeirao Preto School of Medicine, Brazil; 13Univerisity of Tennessee, TN, USA

We genotyped 212 human medulloblastomas using Affymetrix high resolution SNP genotyping (100K or 500K) in order to identify regions of recurrent amplification. We defined a high level amplification as having a copy number of greater than 5 by SNP array as predicted by dChip-SNP and CNAG. We identified 12 distinct regions of the genome where there was high level amplification in at least 2 tumors. Focal gain was defined as a region with increased copy number (3.0–4.9) that is less than 5 megabases in size. As expected, we found amplification (15 tumors) and focal gain (4 tumors) at the MYCC locus, and the MYCN locus (amplified 4, focal gain 10). Amplification of MYCC was seen much more commonly in medullo-blastoma cell lines than in primary tumors. MYCC amplicons were highly variable in their size and copy number. We also found high level amplifica-tion of OTX2 in two medulloblastoma cell lines, D425 and D458, but not in any primary medulloblastomas. We did however see extremely focal, highly recurrent gain that encompasses only the OTX2 locus in 22 medulloblas-tomas, making gain of OTX2 more common than gain of MYCC. As SNP array analysis is done on bulk tumor, this data is compatible with higher level amplification of OTX2, but in a subclone of cells, suggesting that amplification of OTX2 is a progression event. We also observed amplifica-tion of PDGFRA in 3 medulloblastomas. This very large cohort of medullo-blastomas, studied at extremely high resolution allows an accurate estimate of the incidence of amplification in these known medulloblastoma onco-genes. We observed high level amplification of the Hedgehog effector GLI2 (copy number .50) in two tumors, and amplification of GLI1 in one tumor. Along with previously identified mutations in HSUFU, these mutations show that anti-Hedgehog therapies targeted at the level of the membrane (SMO) will not be effective for all medulloblastomas with high levels of hedgehog signaling. We also detected high level amplification of the known oncogene IRS2 (insulin receptor substrate 2) in two tumors. These novel medulloblas-toma oncogenes represent targets for future therapy. Gain of chromosome 7 is common in medulloblastoma (30% of tumors). We observed amplification (3 tumors) and focal gain (4 tumors) of a single poorly characterized gene: ZNF679 on chromosome 7. We suggest that the recurrent gain of chromo-some 7 in medulloblastoma may be driven by ZNF679, and that ZNF679 is a medulloblastoma candidate oncogene and an excellent candidate for development of future therapy. We also observed amplification limited to the ATP1B1 gene (1q24.2) in two tumors. Recurrent amplifications that include more than more one gene include examples on chromosome 2q14.2 (2 tumors, genes 5 DBI, MARCO, TSAP6, SCTR), 8p11.21 (4 tumors, genes 5 ANK1, MYST3, AP3M2, PLAT, IKBKD, POLB, DKK4, VDA3, SLCOA2), and 18q21.31 (3 tumors, genes 5 FECH, NARS, ATP8B1). We present a large series of human medulloblastoma genotyped at an unprec-edented resolution in which we have identified a number of novel candidate oncogenes, known oncogenes, and known medulloblastoma oncogenes as amplified in medulloblastoma.

PB-10. PEPTIDOMIC ANALYSIS OF CEREBROSPINAL FLUID FROM CHILDREN WITH COMMON BRAIN TUMORSMeena Rajagopal,1 Yetrib Hathout,2 Tobey MacDonald,1 and Brian Rood1; 1Center for Cancer and Immunology Research, Children’s National Medical Center, Washington, DC, USA; 2Center for Genetic Medicine, Children’s National Medical Center, Washington, DC, USA

Peptidomics is the technology that involves identification of low molecu-lar weight peptides in biological fluids using various enrichment/separation techniques coupled to mass spectrometry. Presence of peptides in cerebro-spinal fluid (CSF) of patients suffering from a brain tumor could indicate activation of inherent proteolytic mechanisms. Therefore, identification of endogenous peptides in cerebrospinal fluid from brain tumor patients may provide valuable information regarding cancer biology. Alternatively, iden-tification of differentially expressed peptides between various cancer-types could prove valuable as clinical diagnostic markers. In this current work, a “top down” peptidomic approach was carried out using linear ion trap mass spectrometry after off-line enrichment of peptides by solid-phase extraction (SPE) to identify peptide biomarkers for pediatric brain tumors using ante-



mortem CSF. In a pilot study, cerebrospinal fluid samples from patients diagnosed with grade I glioma, medulloblastoma and germinoma were col-lected by lumbar puncture. Endogenous CSF peptides were extracted using the Oasis HLB resin which is a reverse phase polymer (poly (divivinylben-zene-co-N-vinylpyrrolidone)) that has both lipophilic and hydrophilic bal-ance. Briefly, HLB cartridges were washed with methanol, 75% acetonitrile (ACN)/0.1% formic acid (FA) and 2% ACN/0.1% FA. A volume of CSF corresponding to 500 microgram of total CSF protein was diluted 1/10 with 2% ACN/0.1% FA and applied to HLB cartridge. Cartridges were washed with 2% ACN/0.1% FA and peptides were eluted using 15% ACN/0.1% FA. Mass spectrometric identification of extracted CSF peptides was carried out using Thermo Electron’s LTQ, a linear ion trap mass spectrometer equipped with a nanospray source. Six microliters of extracted peptides were injected into a C18 column (75 mm i.d., 15 cm length) by an isocratic flow of solvent A (5% ACN/0.1% FA) for 35 min and eluted using a gradient of increasing concentrations of solvent B (95% ACN/0.1% FA) from 5% to 35% in 60 minutes at a flow rate of approximately 140 nL/min. Peptide mass spectra were searched against Swiss-Prot human database using Bioworks software (version 3.2). A number of novel endogenous peptides were identified using this top-down proteomics approach. The majority of identified peptides were fragments of larger proteins indicative of protease activity in cancer tissue. Peptides from chromagranin A, thymosin beta-4, prothrombin and two other neuronal proteins were specific to germinoma. On the other hand, peptides from alpha-2HS-glycoprotein and complement C4–A were unique to glioma while peptides from fibrinogen beta chain were identified in medulloblastoma. Peptides from two proteins namely, Testican-1 and Secretogranin-1 were observed in both germinoma and medulloblastoma samples. The successful isolation of endogenous peptides from CSF using a top-down approach represents a novel advance in cancer proteomics. The intriguing observation of different peptidomes in different cancer types has the potential to contribute to the generation of cancer-type specific CSF peptide markers. Future efforts will expand the current study to include a larger training set and validation of our results in blinded samples.

PB-11. NT-3/TrkC-REGULATED PROTEINS IN THE BIOLOGY OF MEDULLOBLASTOMAMariella Gruber-Olipitz,1 Thomas Strobel,2 Franz Quehenberger,3 Michael Grotzer,4 Gert Lubec,5 and Irene Slavc5; 1Department of Pediatrics, Medical University of Vienna, 1090, Austria; 2Institute of Neurology, Medical University of Vienna, Austria; 3Institute of Medical Informatics, Statistics and Documentation, Medical University of Graz, Austria; 4Department of Pediatrics, University Children’s Hospital of Zurich, Switzerland; 5Department of Pediatrics, Medical University of Vienna, Austria

Medulloblastoma (MB) is the most common malignant childhood brain tumor. Because of its high risk of leptomeningeal dissemination, standard postoperative treatment includes craniospinal irradiation and chemother-apy. Such treatment produces significant sequelae including neurocognitive dysfunction, endocrine and growth disturbances. These therapy-induced morbidities warrant a novel less toxic approach possibly targeting key mol-ecules within specific pathways of medulloblastoma biology. Recent reports identified high neurotrophin receptor TrkC mRNA expression as a power-ful independent predictor of a favorable survival outcome in MB patients. However, the role of activated TrkC receptors in the development and biol-ogy of MB remains unclear. To determine downstream effector proteins of TrkC signaling, the medulloblastoma cell line DAOY was stably transfected with a vector containing the full-length TrkC cDNA sequence or an empty vector control. Accounting for the complexity of ligand-induced changes in cellular pathways and effector proteins, we investigated proteomic changes at multiple time points for up to 48 hrs following neurotrophin-3 induced TrkC receptor activation. We identified 18 proteins differentially expressed: Caldesmon; Cathepsin D; Metastasis inhibition factor nm23; Multidrug resistance-associated protein Mgr1-Ag; Vimentin; Vinculin; Superoxide dismutase (Mn); Glutathione S-transferase P; Stathmin, Lamin A/C, Valosin-containing protein, Annexin A1; ULIP protein; DJ-1 protein; Fascin; Mitofilin; Heterogeneous nuclear ribonucleoprotein H and K. The proteins affected play substantial roles in differentiation, migration, inva-sion, proliferation, apoptosis, and drug resistance. Almost all of the proteins have been described as being essential in the pathogenesis of different solid tumors, but have not been related to MB pathogenesis so far.

PB-12. DEREGULATION OF AN EXTRAORDINARILY IMPORTANT DEVELOPMENTAL GENE PROMOTES MEDULLOBLASTOMA TUMORIGENESISHai Yan, Qun Shi, Chunhui Di, Chris Duncan, and Darell Bigner; Duke University, Durham, NC, USA

Medulloblastoma is highly invasive with a tendency to metastasize throughout the neuroaxis. Despite advances in therapy, 30% to 40% of the children afflicted with medulloblastoma die of the disease, and aggressive treatment of children results in impaired intellectual development, growth deficits, and endocrine dysfunction in the survivors. Consequently, consider-able efforts are being made to identify patients who can be cured with less intensive therapy and also to develop more effective treatments for children who have therapy-resistant disease. Utilizing novel whole-genome gene- dosage screens at unprecedented resolution, we have identified a develop-mentally regulated gene, orthodenticle homolog 2 or OTX2, to be ampli-fied in medulloblastomas. We have performed genetic studies on medullo-blastoma specimens and identified OTX2 amplification and demonstrated its overexpression in medulloblastoma associated with tumor anaplasia. In addition, epigenetic studies revealed that OTX2 promoter methyla-tion plays an important role in regulating OTX2 expression in a subset of medulloblastoma cells. We have shown that knockdown of OTX2 by small interfering RNA reduced medulloblastoma cell growth. When medulloblas-toma cells were transfected with pSR-OTX2–shRNA and injected into the cerebral hemisphere of nude mice, we observed that knockdown of OTX2 expression significantly increased the survival of the animals. Moreover, the cells with high levels of OTX2 expression were sensitive to treatment with retinoic acid. OTX2 is a transcription factor with a bicoid-like homeodo-main (HD), binds to genomic DNA to control gene activity. We have inves-tigated the OTX2 molecular signaling pathway and identified the direct downstream genes of OTX2. Both our genetic and biological studies have further supported our hypothesis that OTX2 genetic alteration is a reliable biomarker for molecular classification of medulloblastomas and the target for molecular-based therapies.

PB-13. IDENTIFICATION OF NOVEL CHROMOSOMAL ALTERATIONS SPECIFIC TO PEDIATRIC GLIOBLASTOMAKarine Jacob,1 Huiqi Qu,1 Alexandre Montpetit,2 Constantin Polychronakos,1 and Nada Jabado1; 1McGill University, Montreal, Quebec, Canada; 2Centre d’Innovation Genome Quebec, Montreal, Quebec, Canada

Brain tumors are the largest group of solid neoplasm in children and are currently the leading cause of cancer-related mortality and morbidity in the pediatric years. Pediatric glioblastoma (pGBM) is a rare, but devastating brain tumor. Several studies suggest that pGBM may be biologically distinct from their adult counterparts, despite being histologically identical. In con-trast to GBM in adults (aGBM), relatively little is known in children about the molecular mechanisms underlying its development and progression, and the low number of clinical samples available has resulted in very few studies of this cancer. In pGBM, only 25% of samples show an abnormal karyotype in contrast to adult tumors that show a markedly altered karyotype for most samples. Moreover, even if there are few similarities, most abnormalities found in primary aGBM are absent in pGBM. The only study using com-parative genomic hybridization (CGH) on otherwise normal karyotypes was performed on 9pGBM and showed aberrations specific to children that included 11p, 12q, 122q, –6q, and –16q (16%–25%). This study had no central review and used low-resolution arrays. Our hypothesis is that there are novel candidate genes/pathways/genetic abnormalities involved in GBM formation and specific to pediatric GBM. Our aim is to identify, narrow the genetic interval, outline chromosomal regions of interest and better characterize the chromosomal abnormalities in pGBM. Here, we have ana-lyzed 19 pGBM and 6 aGBM using the Illumina 100K single-nucleotide polymorphism (SNP) arrays. The Illumina Sentrix Human-1 Genotyping BeadChip covers 109 365 gene-centric SNPs loci distributed evenly over the genome with a mean intermarker distance of 26 kb. All samples were assayed with the Infinium I whole genome genotyping (WGG). SNP arrays detect simultaneously copy number alterations and losses of heterozygosity. Our data was also confirmed by BAC arrays. Our results show an unex-pected low frequency of genetic amplification/homozygous deletion and a high frequency of LOH and copy neutral changes in pGBM. They further confirm that pediatric and adult GBM are genetically distinct tumors and identify novel alterations. The most common copy number alterations where LOH on chromosome 10q22, 15q15, 17p13, and 22q12. Futhermore, we identified genes in the restricted regions that can potentially represent tumor supressor genes or oncogenes. We also detected other previously reported regions of deletions and amplifications further validating our data. We will confirm their expression status of candidate genes by qRT-PCR and by Western blot analysis. We are now comparing those genomic results with previous results obtained through a transcriptional analysis. This study, which is the first using high resolution SNP arrays in pGBM, generated an important set of data that should help to improve our understanding of this rare tumor. pGBM are molecularly distinct from aGBM and should



be studied separately. Our findings suggest that alterations in tumor sup-pressor genes involved in the regulation of the cell cycle are major events in pHGA pathogenesis and may ultimately lead to more specific molecular therapeutic targets.

PB-14. PROTEOMIC STUDIES IN CSF OF PATIENTS WITH MEDULLOBLASTOMAMaria Lüth,1 Andreas Kurtz,2 Günter Henze,3 and Pablo Hernaiz Driever4; 1Department of Pediatric Oncology/Hematology, Charité Universitätsmedizin, Berlin, Germany; 2Robert-Koch-Institut Berlin, Germany; 3Dept. for Pediatric Oncology and Hematology, Charite-Universitätsmedizin Berlin, Germany; 4Charité Universitätsmedizin Berlin, AE (Europe)

Medulloblastoma is the most common malignant brain tumor in child-hood that often spreads via the cerebrospinal fluid (CSF). Mechanisms underpinning the malignant biology in medulloblastoma remain poorly understood. Proteomic studies offer a comprehensive bird’s eye view to ana-lyze CSF at the protein level. The aim of the present study was to examine the protein profile of CSF from patients with medulloblastoma. Thus, we investigated whether proteome analysis based on two-dimensional gel elec-trophoresis (2DGE) and nano-liquid- chromatography-electrospray ionisa-tion-mass spectrometry (nano-LC-ESI-MS) is able to identify differences in protein levels in CSF between tumor patients and healthy controls. In order to detect alterations of protein patterns during the course of chemotherapy we further analyzed the CSF of patients at different times of treatment. So far, CSF samples of 4 medulloblastoma patients and 3 healthy controls were compared. Among the proteins most highly expressed in CSF of tumor patients we found complement C3 precursor protein, phosphate-glycerol kinase and, glyceraldehyde-3–phosphate dehydrogenase (GAPDH). Inter-estingly, recent studies provided evidence that GAPDH plays a role not only as a catalytic enzyme but also in neuronal apoptosis. GAPDH levels were increased in CSF of medulloblastoma patients while barely or not detectable in healthy controls. The highest protein levels were found prior to chemo-therapy whereas levels of GAPDH decreased during the course of therapy, which may indicate response to treatment of medulloblastoma.

PB-15. ASPM EXPRESSION IN MEDULLOBLASTOMATânia Maria Vulcani-Freitas,1 Nasjla Saba-Silva,2 Andréia Maria Cappellano,3 Sergio Cavalheiro,4 and Sílvia Regina Caminada De Toledo5; 1Federal University of São Paulo, São Paulo, Brazil; 2Pediatrics, Pediatric Oncology Institute-GRAACC, Brazil; 3Pediatrics, Pediatric Oncology Institute-GRAACC, São Paulo, Brazil; 4Neurology and Neurosurgery Department, Federal University of São paulo, São Paulo, Brazil; 5Genetic, Pediatric Oncology Institute-GRAACC, Brazil

Medulloblastomas are the most common malignant tumors of central nervous system in the childhood. The incidence is about 19%–20% between children youngest than 16 years old, and with peak of incidence among 2 and 7 years old. The 5-year survival rates are around 50%–60%. Despite its sensibility to no-specific therapy like chemotherapy and radiotherapy, the treatment is very aggressive and frequently results in neurological develop-ment and growth deficit and endocrine dysfunction. Hence, new treatment approaches are needed such as molecular targeted therapies. In our study, we validate ASPM (abnormal spindle-like microcephaly–associated) gene expression as this gene was overexpressed in microarrays investigations. Studies of glioblastoma demonstrated that ASPM gene was overexpressed when compared to normal brain and ASPM inhibition by siRNA-mediated inhibits tumor cell proliferation and neural stem cell proliferation, support-ing ASPM gene as a potential molecular target in glioblastoma. Therefore the aim of our study was to study ASPM expression in 12 medulloblas-toma tumor fragments and 1 control of normal brain. ASPM mRNA was measured by quantitative real-time RT-PCR. All samples overexpressed the ASPM gene, suggesting that this gene could be a good molecular target for glioblastoma therapy. Functional studies will be necessary to confirm these results and to sustain this propose.

PB-16. GENOMIC ANALYSES OF PEDIATRIC SUPRATENTORIAL PRIMITIVE NEUROECTODERMAL BRAIN TUMORSKyle Lee,1 Meihua Li,1 Charles Eberhart,2 Scott Pomeroy,3 Michael Taylor,4 James Rutka,4 Cynthia Hawkins,5 and Annie Huang1; 1Division of Hematology Oncology, SickKids Hospital, Toronto, Ontario, Canada; 2Neuropathology, John Hopkins University School of Medicine, Baltimore, WA, USA; 3Dana Farber Cancer Centre, Boston, MA, USA; 4Neurosurgery, SickKids Hospital, Ontario, Canada; 5Pediatric Laboratory Medicine, SickKids Hospital, Canada

Primitive neuro-ectodermal tumors (PNET) of the supratentorial region are rare, highly invasive malignant brain tumors affecting young children. Although supratentorial PNETs (sPNET) are histologically indistinguish-able from the infra-tentorial PNET/ medulloblastoma, they are character-ized by more aggressive clinical phenotypes which suggest sPNET represent distinct biological entities from medulloblastoma. In contrast to consider-able progress in understanding of signaling pathways involved in medullo-blastoma development, little is known about the biology of sPNET and the nature of genes which contribute to sPNET development and/or tumor progression. Prior studies with low resolution CGH (comparative genomic hybridization) techniques indicate sPNET have frequent genomic imbal-ances and copy number aberrations (CNAs). Our recent studies indicate that CNAs involving several different components of the CDK/cyclin D complex can result in deregulation of the pRB tumor suppressor pathway in sPNET. Collectively these observations point to CNAs as important mechanisms in development and/or progression of sPNET. In order to better delineate genetic aberrations underlying sPNET development and tumor progression, we undertook transcriptional profiling combined with high resolution geno-typing analyses to map genes underlying recurrent CNAs in primary and recurrent pediatric sPNET in this study. Genomic DNA from 30 primary and 6 recurrent sPNET samples were analysed using the Affymetrix 500K SNP (single nucleotide polymorphism) genotyping microarrays and oligo-nucleotide expression arrays. The ultra-high gene resolution of the 500K SNP which permits the mapping of focal regions of allelic loss as well as CNA (copy number alterations) have been successfully used to map etiologic loci in the complex genomes of an increasing number of human maligancies. All tumors with available material were tested for expression of the INI1 protein to exclude tumors that may represent rhaboid tumors. Preliminary data analysis reveal frequent CNA across the sPNET genome, which encom-pass both large and focal chromosome segments. Primary sPNET samples had an average of 7.44 CNA/tumor, with only two tumors containing very few CNA and three tumors acquiring over 20 CNA. This data corroborate previous suggestions of frequent genomic imbalances and possibly genomic instability in sPNET. In addition, as reported in prior studies, we observed a lack of isochromosome 17q in all sPNET, an abnormality that characterize up to 1/3 of medulloblastoma. We observed large regions of copy number gains on 1q (25% of samples), 8q (18%) 18q (12%), 12, 15q24–26, 4q, and 11q (6% for each), and focal gains in 19q13.42 (18%), 4q25, 5p15, 11q13.3, and 12q14.1 (6% for each). Regions with larger regions of DNA copy number losses in sPNET included 22q (25%), 6q (18%), 13q (12%), 16q (12%), 2q, 4p, 5q, 11p, and 14q (6%), as well as focal loss at 21q21.1 (12%), 2q33–37, and 17q11.2 (6%). Hierarchical cluster analysis of high resolution genotypes indicate that there are at least 3 genetic sub-groups of sPNET that may be distinguished by changes on chr2, chr 19q, and chr 22. Candidate genes within such recurrent regions of focal change and with cor-responding gene expression changes on transcriptional profiles are currently being further characterized using quantitative real time PCR, sequencing and fluorescence in situ hybridization. In conclusion, our study identify at least 3 different genetic subtypes of sPNET and indicate that genomic imbalances is a frequent feature of sPNET. We have also identified several regions of focal CNAs that point to candidate sPNET oncogenes and tumor suppressor genes. Further characterization of these regions will help clarify mechanisms of sPNET pathogenesis and may direct future developments in therapy.

PB-17. OVER-EXPRESSION OF SEMAPHORIN 3E AND 5A IN JUVENILE PILOCYTIC ASTROCYTOMASYvonne Tsang,1 Zhouying Yu,2 Ching Lau,3 and Kwong-Kwok Wong1; 1University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Gynecologic Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3Pediatrics, Baylor College of Medicine, Houston, TX, USA

Juvenile pilocytic astrocytoma (JPA) is the most common brain tumor in children. The outcome of patients is good if complete resection of the tumor is achievable. However, approximately one third of patients have tumors that cannot be totally resected and the outcome of this patient with residue tumor is poor. Unfortunately, the etiology of this tumor is still unclear and detail molecular study on this tumor is limited. The semaphorin family of proteins plays an essential role in the development of the central nervous system (CNS) and was originally characterized in the nervous system as



axonal guidance molecules. From the expression profiles of 21 JPA and 3 normal cerebella, we analyzed the expression of 17 genes of the sema-phorin family. Our result indicated that Sema3E and Sema5A are the only two over-expressed semaphorin genes in JPA. The over-expression of both Sema3E and Sema5A transcripts were further confirmed by quantitative real-time RT-PCR. Polyclonal antibodies were raised against both SEMA3E and SEMA5A proteins. Immunohistochemistry and Western blot analysis further demonstrated the over-expression of both SEMA3E and SEMA5A proteins in JPA. Recent studies using mouse knockout have indicated that both sema5A and Sema3E gene are essential for the formation of cranial vascular system and vascular patterning during embryonic development. Since glomeruloid vasculature is common in JPA, we speculate that over-expression of SEMA3E and SEMA5A are responsible for angiogenesis in JPA. These semaphorins could be potential therapeutic targets. Experiments are in process to understand the role of these semaphorins in the pathogen-esis of JPA.

PB-18. UTILIZATION OF MULTIPLE ANALYTIC PLATFORMS TO DISTINGUISH THE MOLECULAR PHENOTYPES OF CHOROID PLEXUS TUMORSUri Tabori, Adam Shlien, Margaret Pienkowska, Harriet Druker, Sonia Nanda, Berivan Baskin, Peter Ray, Cynthia Hawkins, and David Malkin; The Hospital for Sick Children, Toronto, Ontario, Canada

Choroid plexus carcinomas (CPC) and papillomas (CPP) are rare pedi-atric brain neoplasms with variable clinical course. Insufficient understand-ing of the biological processes governing behavior of these tumors leads to inconsistent approaches to therapy and poor survival outcomes. CPC are frequently observed in Li-Fraumeni syndrome (LFS) families in which germline mutations in the TP53 tumor suppressor gene is associated with a high rate of multiple early onset cancers. In an attempt to identify mark-ers of potential biological and therapeutic significance for choroid plexus tumors, we used high throughput microarray platforms to examine the complex molecular alterations in these tumors. TP53 mutation and MDM2 SNP309 polymorphism analysis, was performed on both tumors and paired germline (blood) samples. SNP array was applied to available frozen samples using the Affymetrix 250K GeneChip to explore genome-wide alterations. Twenty-two tumors and germline analysis revealed that all TP53 mutated tumors belonged to families with full LFS phenotype. All CPC patients without LFS phenotype were TP53 wild type in the germline. The same was true for all CPP (n 5 5). Interestingly, all tumors negative for TP53 muta-tion harbored the combination of the MDM2 SNP309 and TP53 codon 72 polymorphisms suggesting an alternative mechanism of p53 dysfunction. SNP analysis revealed high copy number variation in LFS related tumors compared to wild type tumors and CPP which had a less variable profile. Only one CPC revealed homozygous deletion of 22q associated with rhab-doid tumors, but this tumor was immunopositive for INI1, further sup-porting the distinction between these two tumor types. In summary, our results revealed that while germline TP53 mutation was not consistently observed in CPC, alternative mechanisms of p53 functional abnormalities contributed to CPC tumorigenesis. Copy number variability may possibly differentiate molecular and phenotypic subtypes of this family of tumors. The use of multiplex molecular genotyping may eventually lead to novel prognostic and therapeutic markers for choroid plexus tumors.

PB-19. TARGETED EXPRESSION OF MYCN CAUSES MEDULLOBLASTOMA IN TRANSGENIC MICEWilliam Weiss,1 Matthew Grimmer,1 David Goldenberg,1 Selma Masic,1 Qi-Wen Fan,1 Mai Nagaoka,2 Kohichi Tanaka,2 Xiaoning Zhe,1 Rodney Collins,1 Tarik Tihan,1 David James,1 and Louis Chesler1; 1University of California, San Francisco, CA, USA; 2Tokyo Medical and Dental University, Bunkyo-Ku, Tokyo, Japan

Medulloblastoma, a tumor composed of both neuronal and glial ele-ments, is the most common primary pediatric brain tumor. Aberrant sig-naling through the Sonic Hedgehog (SHH) and Wnt signaling pathways is associated with medulloblastoma and drives transformation, likely involv-ing cerebellar granule cell precursors (cGNP). MYCN is a major regulator of cGNP proliferation and differentiation during normal development, and is an indirect target of the SHH pathway. The MYCN family member c-myc is also know to be a critical mediator of transformation through the Wnt pathway. In addition, a recent study suggests that Mycn is expressed in the majority of medulloblastoma tumors, and not in adjacent normal cerebel-lar tissue. To characterize the importance of MYCN in the pathogenesis of medulloblastoma, we targeted MYCN to neuroectodermal cells of the cerebellum using the tetracycline (Tet) system to conditionally express both MYCN and luciferase transgenes. We targeted expression of the tetracy-cline transactivator protein (tTA) to brain astrocytes using the murine Glt-1 (glutamate transporter-1) promoter. We then generated a bi-directional Tet-

response element (TRE) driving expression of both human MYCN and fire-fly luciferase (Luc). The tTA molecule is active in the absence of tetracycline derivatives, allowing transcription of MYCN and Luc from the TRE. In the presence of tetracycline derivatives, TRE expression is suppressed. Animals doubly transgenic for Glt1–tTA and TRE-MYCN/Luc were serially screened for tumor formation using luciferase imaging. Some animals with tumors were treated with doxycycline. Luciferase bioluminescence and subsequent pathology were analyzed. We characterized expression of the Glt1 transgene in the brain, demonstrating high levels of expression in astrocytes, with maximal expression in the cerebellum. We then crossed Glt1-tTA mice with TRE-MYCN/Luc mice. Bio-imaging of Luc expression in doubly transgenic animals revealed increased hindbrain signal intensity, allowing us to track tumor growth and potentially response to therapeutic interventions over time. Animals sacrificed at 2–4 months of age showed focal tumors aris-ing in the cerebellum. Histology was characteristic of large-cell, anaplastic medulloblastoma. Expression of Mycn protein was confirmed by immu-nohistochemistry, along with expression of the astrocytic marker GFAP, and the neuronal markers synaptophysin and neuron specific enolase. Lev-els of angiogenesis, apoptosis, and proliferation were characterized. Pen-etrance and survival data, and response to doxycycline will be presented. Significance: This model validates the functional importance of MYCN in medulloblastoma, and generates a new model for anaplastic medulloblas-toma. The high penetrance of tumors in this model suggests that dysregula-tion of MYCN may be a common feature of medulloblastomas, and could conceivably cooperate with mutations in Wnt or SHH signaling pathways, a hypothesis currently being tested. This model is amenable to non-invasive imaging of malignant progression and to measurement of real-time thera-peutic responses. Furthermore, the regulable nature of MYCN expression, when combined with luciferase imaging, lends this model to temporal stud-ies of tumor initiation and oncogene dependence.

PB-20. INVESTIGATING THE ANTITUMOR ACTIVITY OF FIBROBLAST GROWTH FACTOR (FGF) IN MEDULLOBLASTOMAMarie Fogarty, Eugene Hwang, Brian Emmenegger, John Dutton, and Robert Wechsler-Reya; Duke University, Durham, NC, USA

Medulloblastoma, the most common malignant pediatric brain tumor, arises when precursor cells in the developing cerebellum fail to differen-tiate and instead continue to divide indefinitely. Since signaling pathways influencing cerebellar growth and differentiation are commonly dysregu-lated in medulloblastoma, it seems likely that a greater understanding of the mechanisms governing normal cerebellar signaling will provide insight into the molecular basis of this tumor and lend valuable insights in to potential hedgehog pathway-specific therapies. For example, the finding that Sonic hedgehog (Shh) controls granule precursor cell (GCP) proliferation has shed light on why mutations in the Shh pathway result in human cerebellar tumors. Our recent studies have established that FGF potently inhibits Shh-mediated proliferation and induces differentiation of normal GCPs. FGF also inhibits transcription of hedgehog target genes including gli1 indicat-ing that the mechanism by which FGF blocks proliferation is specific to the hedgehog pathway. Furthermore, FGF potently inhibits in vitro prolifera-tion of tumor cells derived from mouse models of human medulloblastoma in which one or both copies of patched are mutated. In order to test the effects of FGF on tumor growth in vivo, a xenograft model was employed—transplanting tumor cells isolated from ptch1/– mice into the cerebella of SCID-beige mice. 0/10 mice that received tumor cells treated in vitro for 36 hours with FGF developed tumors. In contrast, 10/10 mice that received untreated tumor cells developed tumors within 3 months of transplant. These results suggest that FGF suppresses tumor growth in vivo and may be a novel therapy for medulloblastoma treatment as well as other hedgehog-dependent tumors. Ongoing studies seek to optimize delivery of FGF in vivo via convection-enhanced delivery (CED), with the goal of treating endog-enous tumors. In addition, a luciferase imaging system is being developed so that the effects of FGF on tumor growth can readily be visualized in vivo. Development of these systems is likely to have broader implications for test-ing brain tumor therapies in vivo.

PB-21. DIFFERENTIATION AND MIGRATION SUBVERT MEDULLOBLASTOMA IN PATCHED MUTANT MICEJessica Kessler and Robert Wechsler-Reya; Duke University, Durham, NC, USA

Medulloblastoma is an aggressive cerebellar tumor that is the leading cause of cancer deaths in children. Therefore, it is essential to understand the molecular mechanisms that govern the onset of this tumor in order to better target and treat the disease. One important model for the study of medulloblastoma is the patched mutant mouse, which develops spontaneous medulloblastomas in approximately 15% of the heterozygous population



(ptc1/–). Interestingly, 70%–80% of these mice have pre-neoplastic lesions in their cerebellum between 3–8 weeks of age, several weeks before any of them develop tumors. Since the majority of pre-neoplastic lesions are tran-sient and do not give rise to tumors, they represent a unique opportunity to study the early stages of tumorigenesis. To determine the fate of pre-neoplastic cells that do not form tumors, we generated a transgenic inducible reporter mouse to label these cells in vivo. Using this reporter mouse, we have shown that the majority of pre-neoplastic lesions can undergo differ-entiation and migration. In addition, we have transplanted pre-neoplastic cells and tumor cells into the cerebellum of immunocompromised hosts. Whereas 100% of the tumor transplants have generated tumors, to date we have not observed any tumors arising from pre-neoplastic cells. These results demonstrate that pre-neoplastic cells are not fully transformed and that dysregulation of normal differentiation and migration in these cells may be necessary for tumorigenesis. This work was supported by a pre-doctoral fellowship from the National Cancer Center.

PB-22. A NOVEL TRANSGENIC MOUSE MODEL OF MEDULLOBLASTOMA WITH LEPTOMENINGEAL METASTASES DRIVEN BY THE SLEEPING BEAUTY TRANSPOSON IDENTIFIES GENES AND PATHWAYS IMPORTANT FOR TUMOR INITIATION, MAINTENANCE, AND PROGRESSIONXiaochong Wu,1 Lara Collier,2 Jessica Mcleod,3 Paul Northcott,1 Sidney Croul,4 Jeff MacDonald,3 Junjun Zhang,3 Steve Scherer,3 David Largaespada,5 and Michael Taylor1; 1Hospital for Sick Children, Toronto, Ontario, Canada; 2University of Minnesota, MN, USA; 3Hospital for Sick Children, Ontario, Canada; 4University Health Network, Toronto, Ontario, Canada; 5Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA

The sleeping beauty (SB) transposon, a novel functional genomics tool was recently shown to be a powerful tool for cancer gene discovery. In the presence of its transposase (enzyme), the sleeping beauty transposon is cut out of the genomic DNA, and is pasted into a new position in the genome. Transposition sites for SB require only the presence of a TA dinucleotide, therefore most of the genome is accessible to SB mutagenesis. Mice that har-bored both the enzyme (transposase, SB11) and the transposon donor con-catemer developed hematopoetic malignancies, and rare medulloblastomas. Tumorigenesis is caused by activation of nearby oncogenes by the MSCV-LTR contained in the transposon (activation of oncogenes), and by gene trapping using poly-A traps in either direction at the ends of the transposon (loss of tumor suppressor gene function). Rapid analysis of sleeping beauty insertion sites allows the identification of genes important for the initiation, maintenance, and progression of cancer. We have modified the Sleeping Beauty transposon system by expressing the SB11 transposase selectively in the murine cerebellar EGL using the Math1 enhancer/promoter (J2Q-SB11). These mice do not develop hematopoetic malignancies. However, whereas 10%–20% of ptch 1/– mice develop localized medulloblastoma by eight months of age, 100% of J2Q-SB11 ptch 1/– mice that also carry the donor transposon concatemer develop medulloblastoma with leptom-eningeal metastases by 10 weeks of age. Ptch 1/– mice that lack either the transposase, or the transposon concatemer develop non-metastatic medullo-blastoma at the background rate. Leptomeningeal metastases are seen in both the peri-cerebral, and peri-spinal cord sub-arachnoid space. Sleeping beauty transposition sites can be determined through a relatively simple PCR based technique followed by cloning and sequencing. We generated .20,000 sequences of .4700 insertions sites from a series of 25 sleeping beauty induced medulloblastomas using deep sequencing technology (454 pyrosequencing). We identified common insertion sites (CIS) using statisti-cal techniques derived from MMLV and MMTV insertional mutagenesis. CIS from medulloblastoma include rspo2 (WNT signaling), nfia (neural stem cell factor), cdh20 (adhesion molecule), inhbb (TGF-Beta signaling), cdc2-related protein kinase 7, glrx, agxt2, and lama3. Analysis of the clonal insertion patterns in primary medulloblastoma versus spinal leptomeningeal metastases shows that the metastases carry all of the insertion sites seen in the primary tumor, as well as additional clonal insertions that presumably have driven tumor progression. Comparison of CIS in metastases versus the matching primary tumor should allow the identification of genes impor-tant in leptomeningeal dissemination of medulloblastomas. Our new model of medulloblastoma with leptomeningeal spread is ideal for translational research as it is fully penetrant (100% incidence), has a short latency (10 weeks), has leptomeningeal disease faithful to the human disease, occurs on an immune competent background, and involves random, easily discernible secondary genetic events. Analysis of the SB insertion sites in a larger cohort of these mice will allow rapid dissection of the genetic events important for the initiation, maintenance, and progression of medulloblastoma.

PB-23. HISTONE ACETYLATION RESULTING IN RESISTANCE TO METHOTREXATE IN CHOROID PLEXUS CELLSPreethi Prasad,1 Hernan Vasquez,2 Vidya Gopalakrishnan,3 and Johannes Wolff2; 1Biochemistry and Cell Biology, Rice University, Houston, TX, USA; 2M.D. Anderson Cancer Center, Houston, TX, USA; 3University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Choroid Plexus Carcinoma are rare infant brain tumors. Complete resection (Cancer 2002 87:1086) and radiation (Lancet 1999 353:2126) are important prognostic factors. Clinical evidence for chemotherapy is dif-ficult to generate with small patient numbers (Med Ped Oncol. 2002 39:76). Preclinical models are necessary. In glioma cells, we recently showed that histone deacetylase inhibitors (HDACi) increase the chemotherapy efficacy (Das J Neurooncol 2007). However, in choroid plexus cells, we saw antago-nistic effects between the HDACi valproate and methotrexate (MTX NeuroOncology 2006 8: 469). Another HDACi, MS-275, was used to confirm that that it was indeed the HDACi effect of valproate that had caused the metho-trexate resistance. Z310 Choroid plexus cells from Sprague Dawley rats where obtained from Zheng (Brain Res 2002 958: 371), mouse SV11 cells from Schell (J Virol, 1999 73: 5981). These cells have been transformed by addition of SV40 large T-antigen. Human choroid plexus tumor cells were a gift of Takahashi (J Cardiovasc Pharmacol 1998;31 Suppl 1:S367). MS-275 (also called SNDX-275) is a synthetic benzamide derivative designed as an HDACi (Poc Natl Acad Sci U S A. 1999 96:4592) is commercially available for experimental use (Alexis). MTT (thiazolyl blue tetrazolium bromide) test were by plating cells at 3000 cells/well on 96 well plates, adding drug one day later. After various time periods the medium was removed, and 100 ml MTT 1mg/mL was added. Both rodent cell lines grow fast (doubling time Z310: 10hours, SV11: 12 hours) while the human cell line is slow (doubling time 4 weeks). MTT tests could only be performed with the rodent cells. As single agents, all the drugs kill the cells in a dose and time dependent fash-ion. For instance, the IC50s in Z310 cells after 48 hours of incubation were 0.3 mM for MTX, 1.25 mM for MS-275, and 2mM for valproate. Addi-tional incubation time increased the toxicity of MTX significantly (IC50 36 h: 2.5 mM, 48h 0.3 mM, 72h: 0.1 mM). SV11 showed the same pattern but was in general less sensitive to all the drugs. In drug combinations of 1.5 mM MS-275 with various MTX concentrations, we found antagonism of the methotrexate effect. For instance, 48 hours of incubation with 5 mM of MTX were survived by 20% of SV11 cells, while the same concentration was survived by 50%, when MTX was combined with 1.5 mM MS-275. This antagonism was only found when the incubations were longer than 36 hours. In Z310 cells the antagonism was weaker but still significant. The addition of VPA resulted in the same type of antagonism, but it was stronger in Z310 than in SV11 cells. Histone deacetylase inhibition causes resistance to methotrexate in Z310 and SV11 cells. We are assessing the expression of cellular molecules related to the folate pathway to elucidate the mechanism further.

PB-24. ANALYSIS OF INTRACEREBELLAR HUMAN MEDULLOBLASTOMA ENGRAFTMENT AND GROWTH IN ATHYMIC MICE USING QUANTITATIVE BILOLUMINESCENCE IMAGINGRintaro Hashizume, Tomoko Ozawa, Lily Hu, Nalin Gupta, and C. David James; Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA

The development of relevant preclinical models of medulloblastoma is essential for evaluating the therapeutic potential of novel treatments for this pediatric cancer. Towards this purpose, we have developed a refined ortho-topic xenograft model in which human tumor cells modified for quantita-tive bioluminescence imaging (qBLI) are propagated in the cerebellum of athymic mice. 5-week-old, female athymic mice were injected with 1 3 10e5 luciferase-modified D283-MED human medulloblastoma cells in the right cerebellum. Mice were monitored twice weekly by bioluminescence imag-ing betweeen day 7 and 28 after implantation. qBLI revealed a progressive increase in signal for each mouse receiving an intracerebellar injection of tumor cells, indicating successful tumor cell engraftment in all cases. His-topathological analysis shows that intracerebellar xenograft tumors have features characteristic of human medulloblastoma. Detailed histopathology, immunohistochemistry, and survival data in comparison to corresponding qBLI results will be presented at the meeting. We have developed an intrac-erebellar orthotopic model of medulloblastoma, the growth of which can be accurately monitored by bioluminescence imaging. This refined orthotopic model is more appropriate for preclinical evaluations of new therapies for medulloblastoma. Furthermore, these data also suggest that the local CNS environment influences the pattern of tumor growth.



PB-25. A VERSATILE NEW MOUSE MODEL FOR MEDULLOBLASTOMAXiang-He Shi, Anat Erdreich-Epstein, Ignacio Gonzalez-Gomez, Shahab Asgharzadeh, Rex A. Moats, Michael Rosol, Xiao-Hui Peng, Ying Wang, Robert C. Seeger, and Gregory Shackleford; Childrens Hospital Los Angeles, Los Angeles, CA, USA

Animal models that mimic cancers at the molecular level are an impor-tant part of research into the scientific understanding and treatment of these diseases. The goals for this project are to (1) identify a medulloblastoma-associated gene promoter and (2) use it to make a versatile mouse model of medulloblastoma, the most common malignant brain tumor in children. We used a bioinformatics approach (Serial Analysis of Gene Expression) to identify a gene, Barhl1, which is expressed almost exclusively in human medulloblastomas. Barhl1 has been shown recently to be highly specific for the external granular layer (EGL) cells of the developing cerebellum, the cells from which medulloblastomas arise. As expected, our RT-PCR results show that Barhl1 was expressed most strongly in the first week after birth in the mouse cerebellum, and it was expressed in four of five medulloblastoma cell lines but not in glioblastoma or other brain tumor lines. We isolated a 4.3-kb potential Barhl1 promoter fragment and found, as anticipated, that it drove expression only in medulloblastoma cell lines and not in any non-medulloblastoma cells tested. We have used this promoter fragment to build a transgene for a versatile “platform” transgenic mouse into which almost any oncogene (or RNAi gene) can be introduced. The transgene expresses the receptor (called Tva) for a replication-competent avian retroviral vec-tor. The transgene also expresses an eGFP-luciferase fusion protein for the noninvasive imaging of tumor growth, regression and metastasis in vivo. Transgenic founders have been produced and their offspring are being char-acterized for expression patterns in the brain. This model is distinct from other Tva models in that it is designed to express Tva in EGL cells with higher specificity and contains the eGFP-luciferase fusion protein for imag-ing tumor behavior. The Barhl1-driven transgene should allow EGL cells, but not other cells, to be infected in the first postnatal week by injection of avian retroviral vector stocks or virus producer cells into the cerebellum. Thus, this single “platform” Barhl1-Tva transgenic is expected to enable: (1) multiple candidate oncogenes to be quickly evaluated in vivo, (2) tumors to be monitored noninvasively with imaging technologies, and (3) new targeted therapies to be tested in multiple defined oncogenic backgrounds.

PB-26. MEDULLOBLASTOMAS FROM PATCHED MUTANT MICE ARE PROPAGATED BY A CD151 CD133– NEURAL PROGENITORRobert Wechsler-Reya1 and Tracy-Ann Read2; 1Pharmacology & Cancer Biology, Duke University Medical Center, Durham, NC, USA; 2Department of Biomedicine, University of Bergen, Bergen, Norway

The long-term growth of many tumors is thought to depend on a subset of tumor cells with an extensive capacity for self-renewal, often called can-cer stem cells (CSCs). In human glioblastoma, cells that express the neural stem cell marker CD133 have been reported to function as CSCs, and have been shown to play critical roles in angiogenesis and in resistance to radia-tion and chemotherapy. CD1331 cells have also been described in human medulloblastoma, but the importance of these cells in tumor initiation and maintenance has not been studied extensively. We sought to isolate CSCs from a widely used model of medulloblastoma, the patched mutant mouse. Although CD1331 cells could be detected in many patched-mutant tumors, these cells were never able to initiate tumors following transplantation into the cerebellum of immunocompromised mice; in contrast, CD133-negative cells from the same animals reproducibly propagated tumors. To identify other possible markers of CSCs, we screened a number of antibodies that have been shown to mark subsets of neural progenitors. We found that tumor cells expressing the granule neuron precursor (GNP) marker Math1 and the carbohydrate antigen CD15 (also called Lewis 3 or stage-specific embryonic antigen 1) were highly enriched for tumor-propagation: trans-plantation of Math11CD151 cells caused tumors in 100% of recipients, whereas Math1– and CD15– cells never caused tumors. CD151 medullo-blastoma cells showed increased proliferative capacity in vitro, but unlike CD1331 CSCs, they could not form neurospheres or undergo multi-lineage differentiation. These studies suggest that not all CSCs express phenotypic and functional characteristics of neural stem cells, and that cells resembling neural progenitors can also mediate long-term growth and propagation of brain tumors. Preliminary studies reveal expression of CD15 in a subset of human medulloblastomas, and we are currently investigating the functional importance of these cells in tumor propagation.

PB-27. THE CELLULAR ORIGIN OF PATCHED ASSOCIATED MEDULLOBLASTOMASZengjie Yang,1 Tammy Ellis,2 Shirley Markant,3 Melissa Bourboulas,4 Karen McCue,4 Tracy-Ann Read,5 Jessica Kessler,6 Brandon Wainwright,7 and Robert Wechsler-Reya1; 1Duke University, Durham, NC, USA; 2The University of Queensland, Queensland, Australia; 3Duke University Medical Center, NC, USA; 4Queensland, Australia; 5Department of Biomedicine, University of Bergen, Bergen, Norway; 6NC, USA; 7Brisbane, Queensland, Australia

Medulloblastoma (MB) is the most common malignant brain tumor in children. Although it is thought to originate from immature cells in the cerebellum, the identity of these cells remains unclear: while some studies have suggested that the tumors arise from lineage-restricted granule neu-ron precursors (GNPs), others have shown that MBs can express stem cell markers and can differentiate into both neurons and glia, raising the pos-sibility that they originate from multipotent neural stem cells (NSCs). Here we examine the cell of origin for MBs resulting from mutations in the Sonic hedgehog (Shh)-Patched (Ptc) signaling pathway. Using conditional knock-out mice, we show that deletion of ptc in GNPs results in expansion of the external germinal layer (EGL) where granule cells develop. Although some ptc-deficient GNPs eventually stop dividing and differentiate into neurons, in each animal a cohort of GNPs continues to proliferate, and by 3 months of age, all mice develop tumors. Deletion of ptc in multipotent stem cells also results in MB; notably, NSC-derived tumors grow much more rapidly, leading to death of all animals by 3 weeks of age. We now are investigating the functional difference between the tumors coming from GNPs and NSCs. These studies indicate that the capacity for self-renewal and oncogenic transformation is not limited to neural stem cells, but can also be induced in lineage restricted neural progenitors. Since tumors initiated in progenitors and stem cells may have distinct growth characteristics, identifying the cell of origin for specific types of cancer may be critical in developing effective methods for therapy.

PB-28. IMMUNOTHERAPY FOR HER2-POSITIVE MEDULLOBLASTOMA: HER2-REDIRECTED T CELLS MIGRATE TO TUMOR SITES WITHIN THE CNS AND INDUCE REGRESSION OF EXPERIMENTAL TUMORS IN VIVONabil Ahmed,1 Vita S Salsman,1 Huseyin Kadikoy,2 Meena Bhattacharjee,3 Heidi L Weiss,4 Cliona M Rooney,5 Helen Heslop,6 and Stephen Gottschalk5; 1Pediatrics, Center for Cell and Gene Therapy, Texas Children’s Cancer Center, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX, USA; 2Baylor College of Medicine, Houston, TX, USA; 3Pathology, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX, USA; 4Medicine, Baylor College of Medicine, Houston, TX, USA; 5Pediatrics and Immunology, Center for Cell and Gene Therapy, Texas Children’s Cancer Center, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX, USA; 6Pediatrics, Medicine and Immunology, Center for Cell and Gene Therapy, Texas Children’s Cancer Center, Texas Children’s Hospital, The Methodist Hospital, Baylor College of Medicine, Houston, TX, USA

The objective of this project is to eradicate medulloblastoma, the most common malignant brain tumor of childhood, using genetically modified antigen-specific T cells. We propose to use T lymphocytes to target the human epidermal growth factor receptor 2 (HER2), a surface antigen that is overexpressed in 40% of medulloblastomas. Our preliminary studies have validated this target by showing that genetically modified T cells express-ing engineered HER2–specific chimeric antigen receptors (CARs; HER2–T cells) induce regression of HER2-positive human medulloblastomas grow-ing in the central nervous system (CNS) of mice after intratumoral injection. The aim of this study was to determine if HER2–T cells migrate within the CNS to tumor sites and eradicate established medulloblastomas. Mice with established human medulloblastoma xenografts (Daoy) were stereotactically injected into the contra-lateral hemisphere with HER2–T cells expressing the firefly luciferase (luc) gene to determine T-cell migration and expansion in vivo using bioluminesence imaging. To determine the anti-tumor activ-ity of T cells, unmodified HER2–T cells were injected into mice bearing luciferase expressing Daoy tumors in the contra-lateral hemisphere. The chemokine expression profile of Daoy cells was determined using chemokine arrays and ELISAs. HER2–T cells migrated to medulloblastoma xenografts implanted in the contra-lateral hemisphere as judged by bioluminescence imaging. Within 48 hours of injection HER2–T cells clustered at the tumor site and proliferated as judged by an increase in light signal over the tumor site. These imaging results were confirmed by histology. Chemokine expres-sion analysis of Daoy cells revealed the presence of CXCL-8 (IL-8), Ser-pin E1 (PAI-1), and CXCL-1 (GRO-a), and we are currently determining which chemokine is responsible for the active migration of HER2–T cells to tumor sites in vivo. HER2–T cells had potent anti-tumor activity after injection into the contra-lateral hemisphere of tumor bearing mice, inducing regression, which resulted in a survival advantage in treated animals. This indicates that these T cells not only migrate to tumor sites but also retain



their cytolytic properties and expand at distant tumor sites. We have shown that HER2–T cells migrate to tumor sites within the CNS, expand at tumor sites, and induce regression of medulloblastoma xenografts in vivo. Hence, the adoptive transfer of HER2–T cells represents a promising immunothera-peutic approach to control loco-regionally advanced, incompletely resected and/or metastatic medulloblastoma.

PB-29. GLI2 ZINC FINGER PROTEIN AS A PROGNOSTIC MARKER IN PEDIATRIC MEDULLOBLASTOMAPawel Buczkowicz, Jing Ma, Lauren Solomon, and Cynthia Hawkins; Hospital for Sick Children, Toronto, Ontario, Canada

The Sonic hedgehog (SHH) signaling pathway has been implicated in the maintenance of medulloblastoma (MB). However, the number of tumors involved and the particular pathway member responsible for SHH pathway deregulation in MB has not been well delineated, although PTCH, SMO, SUFU, and GLI proteins are among the potential candidates. We investi-gated whether SUFU, GLI2, and GLI3 proteins may be used as prognostic markers in pediatric MBs. GLI proteins are zinc finger transcription factors in the SHH signaling pathway that may undergo proteolytic cleavage, as well as exon splicing, resulting in various isoforms that play both repressive and/or activating roles in the signaling pathway. SUFU is a member of a complex that binds and inhibits GLI transcriptional activity in the cytoplasm. Clini-cal data was gathered for 133 medulloblastoma patients that had under-gone surgery at the Hospital for Sick Children (Toronto, Ontario, Canada). All morphologic sub-types based on degrees of nodularity/desmoplasia were represented in the sample set (74% none, 5% slight, 5% moderate, 4% extensive, 5% widespread). Tumor samples on tissue microarrays (TMAs) were assayed by immunohistochemical staining and were scored using a four point scale by two independent observers, after which the data was analyzed using SPSS software. 72% of medulloblastoma exhibited moderate-strong, diffuse staining of SUFU in the cytoplasm, but positivity did not correlate to clinical outcome. 54% of tumors stained for GLI3 had nuclear positivity and 53% showed staining in the cytoplasm. However, neither was signifi-cant when correlated to clinical outcome. Of the 74 tumor samples assayed for GLI2 nuclear positivity, 13 (17.5%) scored positive. The absence of GLI2 was found to be a predictor of improved survival (p 5 0.05). This correla-tion was predominantly evident in patients over the age of three. Overall survival of GLI2 negative patients was 10.70 60.84 years versus 6.98 62.00 years for GLI2 positive patients. Patients negative for SUFU but positive for GLI2 had worse clinical outcome then patients with no SUFU and no GLI2 positivity (p 5 0.045). There was no correlation between patient outcome and morphologic subtypes of MB based on GLI2 expression. In this study, we demonstrate that GLI2 may be a good biological prognostic marker for survival of MB patients. Phenotyping MBs for SHH pathway expression may point to subgroups of patients where SHH signaling blockade may serve as a useful therapeutic option.

PB-30. PROGNOSTIC SIGNIFICANCE OF GENE EXPRESSION PROFILES OF MEDULLOBLASTOMASShahab Asgharzadeh,1 Yue-Xian Tu,1 Ignacio Gonzalez-Gomez,1 Jonathan Finlay,2 Robert Seeger,1 Lingyun Ji,1 and Richard Sposto3; 1Children’s Hospital of Los Angeles, Los Angeles, CA, USA; 2University of Southern California, Los Angeles, CA, USA; 3University of Southern California, CA, USA

Standard and high-risk groups of patients with medulloblastoma are identified to plan therapy using risk factors that include clinical stage and age at diagnosis. Therapeutic studies performed by the Childrens Oncology Group have shown that the prognosis remains poor for young children with medulloblastoma, and treatment involving irradiation of the craniospinal axis results in significant neurological sequelae for the survivors. “Head Start” and other clinical trials utilizing intensive chemotherapy aimed at avoiding or delaying radiation therapy have shown a significant portion of young children can be cured of their disease and have normal neurop-sychological performance. Improved prediction of outcome, especially for patients diagnosed less than 10 years of age at diagnosis, will aid in identify-ing tumors that can be eradicated by chemotherapy-only regimens and thus reducing the use of craniospinal irradiation with its long-term effects. We hypothesize that molecular profiles derived from high-density oligonucle-otide microarray analyses of RNA from untreated primary medulloblasto-mas will define clinically relevant biologic subgroups that have either excel-lent or poor outcomes and improve current risk stratification. In our initial study, we have profiled 25 tumors (n 5 11 desmoplastic, n 5 14 classic his-tology) along with 4 medulloblastoma cell lines using Affymetrix U133 plus 2.0 GeneChips. Unsupervised principal component analysis revealed cluster formation of three main groups of medulloblastomas. One cluster, which clearly separated from others, included the highest proportion of patients with progression of disease. Supervised analysis using diagonal linear dis-criminant analysis (DLDA) provided a gene expression signature in which a

member of the forkhead family of genes, FoxG1, was the top ranking gene where high expression predicted for high risk. FoxG1 results were confirmed for these tumors using TaqMan RT-PCR on the frozen tumor samples and immunohistochemistry on paraffin embedded tissue. FoxG1 level also was elevated in medulloblastoma cell lines as compared to glioma or normal white blood cells. The cluster of patients with event free survival who did not receive irradiation therapy had tumors negative for FoxG1 RNA and protein expression. In summary, this promising preliminary data reveals that an accurate classifier using gene profiling may be used to identify patients that have chemotherapy curable medulloblastoma.

PB-31. TARGETING CONSTITUTIVELY ACTIVATED STAT3 SENSITIZES MEDULLOBLASTOMAS TO CHEMOTHERAPEUTIC AGENTSHui-Wen Lo, Xinyu Cao, Hu Zhu, and Francis Ali-Osman; Department of Surgery, Duke University, Durham, NC, USA

Primary brain tumors are the most common solid tumors found in chil-dren, in which medulloblastomas are the most frequent type and constitute approximately 20% of all pediatric brain malignancies. Although advances have been made in understanding the genetic alterations and malignant biology of medulloblastomas, recurrence and metastasis are common and patients have a 5-yr survival rate of 50%–60%. Unfortunately, toxicity is a major setback of not only radiation but also high-dose chemotherapy and the effects can be long-term and devastating. A better understanding of these tumors is, therefore, needed to facilitate the development of more effective and less toxic therapy, prolong patients’ life span, and to improve their quality of life. A potentially more effective and less toxic chemotherapy is the targeted therapy. In this regard, STAT3 is an oncogenic transcription factor that is constitutively activated in many cancers and is regarded as an attractive anti-cancer target. The role of STAT3 in medulloblastoma biology and therapy, however, is poorly defined. The goal of this study is, therefore, to understand the extent of STAT3 constitutive activation in childhood medulloblastomas and its involvement in the malignant biology of these tumors. Here, we reported that STAT3 is constitutively activated in human medulloblastoma cells and primary medulloblastomas. Consistent with its role as a transcription factor, activated STAT3 is primarily localized in tumor nuclei, as shown by the confocal microscope and immunofluorescent staining method. In contrast, normal astrocytes do not contain detectable levels of STAT3. Increased STAT3 activity is associated with high expression levels of the STAT3 target genes, cyclin D1 and VEGF, a positive G1 regula-tor and angiogenic growth factor, respectively. Consistently, STAT3 targeted promoters, cyclin D1 and GAS, were activated in medulloblastoma cells but not in normal astrocytes. Next, we explored whether STAT3 inhibition would lead to medulloblastoma cell death. Indeed, STAT3 inhibitors JSI-124 and AG490 significantly killed medulloblastoma cells as indicated by the 96–well survival and clonogenic growth assays. Notably, JSI-124 resulted much lower IC50 values, at nMs, in the tumor cells compared to the normal astrocytes. Moreover, JSI-124 treatment induced significant apoptosis in medulloblastomas cells. Importantly, we found that the human medullo-blastoma cells were highly resistant to chemotherapeutic agents, such as cisplatin, temozolomide and BCNU, and that JSI-124 sensitized these tumor cells to all three agents. Median-effect analysis further indicated a treatment synergy between JSI-124 and cisplatin. Together, our findings defined an important role of STAT3 constitutive activation in medulloblastomas and provided rationales to regard STAT3 as a novel target for medulloblastoma therapy. This study is supported by the Duke Comprehensive Cancer Center, Duke Brain Tumor Center and NIH, K01 CA118423–01 to H.-W. Lo.

PB-32. TARGETING HEDGEHOG SIGNALING IN MEDULLOBLASTOMAJiangbo Wang,1 Renshui Liu,1 Michael Bond,1 Minyong Chen,1 Simandra Singh,2 Francis Ali-Osman,2 Robert J Lefkowitz,3 Anna Mae Diehl,1 H. Kim Lyerly,4 Lawrence Barak,5 and Wei Chen1; 1Medicine, Duke University Medical Center, Durham, NC, USA; 2Surgery, Duke University Medical Center, Durham, NC, USA; 3Duke University Medical Center, Durham, NC, USA; 4Duke Comprehensive Cancer Center, Durham, NC, USA; 5Cell Biology, Duke University Medical Center, Durham, NC, USA

In children, malignant brain tumors themselves are a cause of devastat-ing illnesses and even if cured they leave with long term morbidities. The most common form of malignant childhood brain cancer is medulloblas-toma. These tumors usually originate in the cerebellum, but they can metas-tasize to other central nervous system (CNS) sites and bone. Up-regulation of the Hedgehog (Hh) signaling pathway plays a key role in the pathogenesis of medulloblastoma as well as a variety of other deadly malignancies includ-ing pancreatic cancer. In recognizing the need to develop new therapies we have taken advantage of our laboratory discoveries (Chen, W. et al., Science 306:2257–60) to identify novel compounds with therapeutic potential that block Hedgehog signaling. We have created a state-of-the-art drug discovery



infrastructure based upon our research to systematically search for com-pounds with anti-cancer properties. Recently, as part of our ongoing screen-ing project, we have identified by primary and secondary screening several potent inhibitors of Hedgehog signaling. We are now performing studies that will provide the biological rationale and preclinical information to help guide the clinical development of Hedgehog inhibitors that have desirable developability characteristics (e.g., linear pharmacokinetics, poor inducer or inhibitor of P450 enzymes). Although the initial target tumor type will be medulloblastoma, Smo antagonists may also be effective in cancers of the breast, pancreas, prostate, skin, and liver.

PB-33. TELOMERASE INHIBITION AS A NOVEL THERAPY FOR PEDIATRIC EPENDYMOMASVincent Wong, Jing Ma, and Cynthia Hawkins; Hospital for Sick Children, Toronto, Ontario, Canada

Central nervous system neoplasms constitute the most common solid tumor of childhood. Although there has been great improvement in the treat-ment and prognosis in many pediatric tumor types, this has not occurred in most brain tumors, which are currently the major cause of mortality as well as long-term morbidity in pediatric oncology. One of the hallmarks of cancer is its limitless replicative potential. This could be attributed to the maintenance of telomere length by telomerase, which is expressed in high levels in more than 85% of all human malignancies. Previously, our group has shown that hTERT expression in pediatric ependymomas cor-relates with a decrease in overall and progression free survival. This led us to hypothesize that ependymomas with functional telomerase may behave more aggressively and that these ependymoma patients may benefit from anti-telomerase therapy. The aim of this study is to test the efficacy of telomerase inhibition as a potential therapy for pediatric ependymomas. Primary ependymoma cells, gliomas cell lines (U87, U373, and U343) as well as hTERT-immortalized normal human astrocytes (NHA/hTERT1) were characterized for GFAP and hTERT expression, initial telomere length and telomerase activity. The commercially available telomerase inhibitor, MST-312, was used for short-term for 72 hours at concentrations rang-ing from 0M to 5M. After 72 hours of treatment, MTT assay was used to determine cell viability. All glioma cell lines, except U343 (p 5 0.2), showed a significant dose-dependent decrease in viability (p , 0.005) compared to vehicle treated cells. This was also observed in primary ependymoma cells (p , 0.001). MST-312 treated U87 cells were stained for H2AX, a marker for DNA damage, and cleaved caspase-3. DNA damage was detected in 20% (n 5 100) of the U87 treated with 2 M MST-312 for 72 hours and up to 69% (n 51 00) of U87 in long-term MST-312 treatment. No DNA dam-age were detected at lower MST-312 dose or vehicle treatment. Apoptosis was not detected in all treatments. A non-lethal concentration of 0.5 M was determined from short-term treatment and U87 cells were cultured in the presence of MST-312 for 60 days at 0.5 M. Long-term MST-312 treat-ment of U87 cells resulted in an increase in doubling time as well as a 30% shortening of telomere length after 35 days. However, there was evidence of resistance to the inhibitor after 20 days. H2AX staining showed DNA damage in these cells and no apoptosis was detected. The observation that telomerase inhibition results in decreased cell viability by 72 hours, before telomere shortening actually occurs, suggests a role for telomerase in cancer cell survival beyond telomere maintenance. The data also suggests that inhi-bition of telomerase results in DNA damages which might lead to decrease in viability. These in vitro data support a role for telomerase inhibition as a potential therapy for pediatric ependymoma.

PB-34. THE TRANSFERRIN RECEPTOR (CD-71) AS A POTENTIAL THERAPEUTIC TARGET IN MEDULLOBLASTOMADuncan Stearns,1 Andrew Donson,2 Nicholas Foreman,2 and Charles Eberhart3; 1Pediatrics, Drexel University, Philadelphia, PA, USA; 2CO, USA; 3Johns Hopkins University, Baltimore, MD, USA

Medulloblastoma is the most common malignant brain tumor of child-hood. Tumor anaplasia, defined histopathologically, has been identified as a poor prognostic feature. Novel therapeutics targeting high risk tumors are needed to improve patient outcomes. We have recently published a causative link between MYC overexpression and tumor anaplasia in a model sys-tem. As part of the ongoing study of the role of MYC in medulloblastoma, an analysis of the effects of MYC on global gene expression was carried out. Total RNA from primary medulloblastomas and mouse flank tumors derived from medulloblastoma cell lines was used to probe Affymetrix U133 gene chips and the results processed using the Genespring GX expres-sion analysis program. Genes that were .2-fold differentially expressed in tumors with high levels of MYC were identified. One of the genes with elevated expression was the transferrin receptor (TFRC1, CD71). TFRC1 expression levels were increased 2.3- to 2.8-fold in the high MYC subset of tumors. The increase was confirmed using RT-PCR methodology. This

is the first evidence of TFRC1 upregulation by MYC in medulloblastoma/neuroectodermal tissue, although this phenomenon has been seen in other cell types. The upregulation was not limited to mRNA; western blot analysis of protein extracts derived from xenografts revealed 3.5-fold higher levels of TFRC1 protein expression in high-MYC tumors as compared to the par-ent line. Fluorescent immunocytochemical analysis for TFRC1 expression in cell culture showed membrane-specific staining in all medulloblastoma cell lines tested, with some suggestion of increased signal in the lines with higher levels of MYC. Immunohistochemical analysis of flank and intrac-ranial xenografts showed intense staining of the outer membrane of tumor cells as compared to surrounding normal tissue. To assess whether primary medulloblastoma tumors express TFRC1 at high levels, a tissue microarray was analyzed. Of 80 tumors, 73 were evaluable, of which 63 were medullo-blastomas. Of the 63 tumors, only 2 were negative. There was a statistically significant association of increased staining (21) in tumors with anaplasia (p 5 0.01 by one way ANOVA). These data indicate that the transferrin receptor is expressed in medulloblastoma and at high levels in more aggres-sive tumors. Given that normal neural tissue expresses TFRC1 at relatively low levels, targeting this molecule may provide therapeutic benefit with reduced toxicity. Preliminary results utilizing in vitro assays with a novel transferrin-toxin conjugate (TfCRM107) are ongoing and encouraging.

PB-35. THE HISTONE DEACETYLASE INHIBITOR, MS-275 UP-REGULATES FAS, DR4, AND DR5 EXPRESSION AND ACTIVATES THE DEATH RECEPTOR PATHWAY IN MEDULLOBLASTOMA CELLSDolly Aguilera,1 Neeta Sinnappah-Kang,2 Johannes Wolff,2 and Vidya Gopalakrishnan1; 1University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Pediatrics, University of Texas MD Anderson Cancer Center, Houston, TX, USA

Medulloblastoma is a malignant pediatric brain tumor that mostly occurs in the cerebellum. While current therapies gave greatly improved survival, relapse still occurs giving these patients very poor prognosis. Addi-tionally, survivors often face quality of life issues as a result of treatment-re-lated damage to the developing brain. Thus, therapies that target aberrations in tumors with minimal toxicity to the normal brain tissue are necessary. Recent studies have demonstrated a role for epigenetic deregulation of gene expression in medulloblastomas. Specifically, genes that promote apoptosis appear to be epigenetically silenced in tumor tissues, suggesting that evasion of cell death may contribute to tumor survival. Since histone deacetylation is a key epigenetic mechanism by which gene expression can be repressed, we have examined the ability of the histone deacetylase inhibitor, MS-275, to reverse the aberrant silencing of genes that control both the death recep-tor or extrinsic and the mitochondrial or intrinsic pathways, and promote apoptosis in medulloblastoma cells in vitro. Using MTT assays, we have demonstrated that Daoy and D283 medulloblastoma cells lines are sensitive to MS-275 in a concentration- and time of exposure-dependent manner. Flow cytometric analyses revealed that MS-275 induced accumulation of cells with sub-G1 DNA content, and was further confirmed by an increase in cleaved-PARP by western blotting analysis. These results suggested that MS-275 induced apoptosis in medulloblastoma cells. MS-275 promoted an early increase in the activity of Caspase-8, the effector caspase in the extrinsic pathway by up-regulating the expression of the CASP8 gene. An increase in the gene expression of the death receptors, FAS, DR4 and DR5 and a subsequent cell surface accumulation of the gene products was also observed upon exposure to MS-275. Interestingly, expression of the FASL was not detected in both untreated and MS-275 treated cells, indicating that apoptosis in the presence of the drug most likely occurs in a ligand-independent manner. However, TRAIL expression was demonstrated in the untreated cells, and increased to higher levels upon exposure to MS-275. Cell death observed in the presence of MS-275 could be rescued by expres-sion of a dominant negative mutant of FADD, confirming the transduction of the death signal through the death domain containing adaptor protein FADD. Surprisingly, a rapid decrease in Caspase-8 activity was observed at later time points and involves MS-275–dependent up-regulation of FLIP, a negative regulator of Caspase-8. However, since apoptosis is observed even upon down-regulation of Caspase-8 activity, we asked if the intrinsic path-way involving Caspase-9 is activated by MS-275 treatment. Indeed, drug treatment results in activation of the mitochondrial pathway and involves in part, an increase in the expression of CASP9 gene. Additionally, the intrinsic pathway also appears to amplify the death signal initiated by the extrinsic pathway, since cross-talk between the two pathways involving BID-cleavage could be demonstrated in MS-275 treated cells. Our study provides the first detailed understanding of the cellular apoptotic machinery activated in response to MS-275 and lays the basis for future studies with animal models and clinical trials for medulloblastomas.



PB-36. METRONOMIC THERAPY WITH TEMOZOLOMIDE AND 13–CIS-RETINOIC ACID IS EFFECTIVE AGAINST MEDULLOBLASTOMA CELL LINES IN VITRO AND IN VIVOElizabeth Melendez, Shun-Ping Huang, Melissa Millard, Goar Smbatyan, Ira Harutyunyan, Michael Rosol, Rex Moats, Ignacio Gonzalez-Gomez, and Anat Erdreich-Epstein; Childrens Hospital Los Angeles, Los Angeles, CA, USA

Despite remission by MRI at end of therapy for medulloblastoma (a malignant brain tumor in children), tumor will recur in 30%–50% of chil-dren treated on the Head Start protocols. This indicates presence of micro-scopic/minimal residual disease (MRD). We sought to develop a non-toxic biologically rational therapy targeting this MRD. We tested temozolomide (TMZ; alkylating agent effective against gliomas) and 13–cis-retinoic acid (cRA; retinoid effective against gliomas and in its trans-form, in vitro against medulloblastoma cell lines expressing OTX-2). In cell culture, TMZ (5–25 mg/ml) and/or cRA (0.1–10 mM) decreased cell number of two medullo-blastoma cell lines (D425MED, D487MED) in a dose dependent manner (up to 97%, trypan blue exclusion). Interestingly, although VW-228, an OTX-2–negative line, was resistant to TMZ (22% inhibition) or cRA (19% inhibition) when used alone, it was more sensitive to their combination (80% inhibition, MTT assay). D283MED medulloblastoma cells (OTX-2 expressing) underwent apoptosis that increased with TMZ1cRA compared to each alone (PARP cleavage, Western blot). Interestingly, Human Brain Microvascular Endothelial Cells (HBMEC) were also sensitive to TMZ and/or cRA (up to 97% inhibition), suggesting a possible anti-angiogenic compo-nent to the activity of these drugs. In vivo, the combination TMZ1cRA was effective against intracranial cell line-derived D425MED medulloblastomas in nude mice (100,000 cells, n 5 5 per group). Mice were treated with either vehicle, cRA (3mg/kg/day BID, 14 consecutive days of every 28 day cycle), TMZ (10mg/kg/day Mon-Friday 5 metronomic dosing, i.e., daily low dose), fenretinide (4HRP, synthetic retinoid; 3mg active drug/kg/day BID Mon-Friday), TMZ1cRA or TMZ14HPR. Drugs were hand fed to the mice in peanut butter/peanut oil (9:1). Mice were followed by MRI and daily obser-vation and sacrificed when they showed signs of distress. All mice treated either with vehicle, cRA, or 4HPR were sacrificed due to tumor by day 51. At the time of submission of this abstract (day 122), none of the cRA1TMZ mice have developed tumors (one mouse died on day 79 without detectable tumor). Ongoing experiments are expanding this work to more mice and to additional medulloblastoma cell lines. In summary, metronomic TMZ and cRA have anti-tumor activity against medulloblastoma in vitro and in vivo. The combination TMZ1cRA seems more effective than each drug alone. Studies are ongoing to determine the effect of this therapy on angiogenesis and the tumor microenvironment.

PB-37. TREATMENT OF MEDULLOBLASTOMA WITH MODIFIED MEASLES VIRUSCorey Raffel,1 Evanthia Galanis,2 Caterina Giannini,2 Cole Kreofsky,2 and Stephen Russell2; 1Neurologic Surgery, Mayo Clinic, Rochester, MN, USA; 2Mayo Clinic, MN, USA

While the treatment of medulloblastoma has improved, at least 30% of patients with this tumor die of progressive disease. Patients with CSF dissemination at any time in their course have an especially grim prognosis. We present data here on the treatment of medulloblastoma with a modified measles virus. Tumor cell killing is accomplished in vitro on adherent cells and on cells growing in suspension. Five medulloblastoma cells lines were treated in vitro with a modified measles virus. Infections were monitored, infectious virus production was measured and cell kill was assessed. Expres-sion of CD46, the measles virus receptor, was assessed in tumor samples by immunohistochemistry. All five cell lines were shown to express the measles virus receptor, CD46, by FACS analysis. All cell lines were infectable with the measles virus at MOI as low as 0.1 by day five after infection. Measles virus was produced by infected cells until all cells were killed. Syncitia for-mation was observed during infection. All five lines were effectively killed by the virus at MOI as low as 0.1. Cell killing occurred in cell lines regard-less of whether the cells grew as adherent cells or suspension cells. CD46 expression was demonstrated in 13 of 13 medulloblastoma tissue specimens by immunohistochemistry. We have demonstrated that a modified measles virus is capable of infecting and killing medulloblastoma cells in vitro. Effective killing occurs in both adherent and suspension cell lines. Tumor samples from patients express the measles virus receptor, suggesting that this therapy may be applicable to the clinic. Experiments on xenografts, using both intracerebral and subacrachnoid, models are underway.

PB-38. DEVELOPMENTAL ORIGINS OF MEDULLOBLASTOMA FROM REGIONALLY RESTRICTED MULTIPOTENT PROGENITORS IN THE MAMMALIAN CENTRAL NERVOUS SYSTEMDavid Rowitch; University of California, San Francisco, San Francisco, CA, USA

Self-renewing, multipotent stem cells have been proposed as therapeu-tic targets in human brain cancer. Recently, such populations have been reported in medulloblastoma, the most common pediatric brain tumor, challenging the generally held view of medulloblastoma formation from unipotent neuronal progenitors. I will present data that medulloblastoma is generated from multipotent progenitors related to the cerebellar granule neuron precursor (CGNP) lineage in vivo. First, by targeting CGNP with an oncogenic Hedgehog pathway mutant allele (SmoM2–YFP), we show that CGNP are transformed into multipotent self-renewing tumor cells, analo-gous to those observed in human medulloblastoma. Secondly, we identify populations of apparently distinct hGfap- and Olig2-expressing multipotent glial progenitor populations for CGNP in the rhombic lip of the developing hindbrain. SmoM2 activation in these cells readily generated medulloblas-toma equivalent to tumors derived from CGNPs themselves. In contrast, SmoM2 activation in multipotent GFAP-expressing precursors, Olig2- and Gli1-positive cells in the forebrain failed to produce glioma or primitive neuroectodermal tumors. These findings indicate stem cells for medulloblas-toma derive from region-restricted progenitors relating to several stages of CGNP lineage progression in the mammalian central nervous system.

PB-39. BMI1-INDEPENDENT PROLIFERATION OF TUMOR STEM CELLS DERIVED FROM HEDGEHOG PATHWAY-INITIATED MEDULLOBLASTOMARod Feddersen,1 Katherine Galvin,2 Hong Ye,3 and Cynthia Wetmore3; 1Pediatric Oncology, Mayo Clinic, Rochester, MN, USA; 2Program in Neuroscience, Mayo Clinic, MN, USA; 3Pediatric Hematology/Oncology, Mayo Clinic, Rochester, MN, USA

Integration of extrinsic and intrinsic signals is critical for the prolif-eration and differentiation of multipotent neural stem cells (NSCs) dur-ing normal nervous system development. Sonic hedgehog (Shh), a secreted mitogen and morphogen essential for normal development, has been found to maintain progenitor and stem cell populations in the brain and other tissues. Aberrant activation of the Shh pathway has been found in a subset of medulloblastomas and may be a mechanism by which normal stem cells are induced to proliferate, undergo malignant transformation and initiate tumor formation. Bmi1, a member of the polycomb group of transcriptional repressors, is critical to the self-renewal capacity of stem cells. Bmi1 has also been found to be important for cerebellar development, is induced with Shh pathway stimulation and is highly expressed in medulloblastoma (Leung et al. 2004). However, it is not well understood whether Bmi1 expression contributes to the self-renewal of tumor-derived stem cells or is simply a gene highly expressed by this rapidly proliferating population of cells. Nor is it understood whether Shh pathway activation is mediated, in part, through Bmi1. Data from our laboratory and others have demonstrated that haploinsufficiency of Patched (Ptc), a component of the Shh receptor com-plex, promotes spontaneous tumor development in the murine cerebellum (medulloblastoma). We have found that these tumors contain a population of stem-like cells (TSCs) that retain the ability to generate cells of all three lineages (astrocytes, oligodendrocytes and neurons) and initiate new tumor formation in the brains of naïve mice. We have found that both TSCs and NSCs express Bmi1, however Bmi1 expression does not correlate with levels of Shh pathway activation in TSCs. Additionally, we have found that deple-tion of Bmi1 reduces proliferation of normal NSCs but does not reduce pro-liferation of malignant TSCs, suggesting that the tumor cells have “escaped” Bmi1 dependence and are able to continue to self-renew despite Bmi1 ideple-tion. Our efforts to better understand the divergence of Shh signal transduc-tion in TSCs and normal neural progenitors, and the role of Bmi1 in tumor stem cell self-renewal may provide a novel molecular mechanism to target future therapeutic intervention for the cure of medulloblastoma. Research supported by Sontag Foundation, Waterman Family Foundation, Mayo Clinic College of Medicine and Hope Street Kids Foundation.



PHARMACOLOGY

PH-01. NOVEL COMPOUNDS DESIGNED USING IN SILICO PATHWAY ANALYSES IMPAIR RECURRENCE OF INTRACRANIAL HUMAN GLIOBLASTOMA XENOGRAFTSFredric Gorin,1 William Harley,2 Tamara Dunn,2 Leela Maitreyi,3 Joachim Schnier,4 Shireen Vali,3 and Michael Nantz5; 1Neurology and Center for Neuroscience, UC Davis School of Medicine, Davis, CA, USA; 2Neurology, UC Davis School of Medicine, Davis, CA, USA; 3Cellworksgroup Inc, Bangalore, India; 4Neurology, University of California, Davis, Davis, CA, USA; 5Department Chemistry, University of Louisville, Davis, KY, USA

Malignant gliomas possess several biological properties making them particularly resistant to definitive therapy, such as local cellular invasion, poorly defined infiltrative borders, and extensive dissemination along white matter tracts. The urokinase-plasminogen activation (uPA) pathway is increased in grade 3 and 4 grade malignant gliomas with expression of uPA or its receptor (uPAR) closely corresponding with increasing tumor grade. Osteopontin (OPN), a glycophosphoprotein associated with the extracellular matrix, enhances the invasive potential of high grade malig-nant gliomas by activating constituents of the uPA pathway. Increased OPN expression corresponds with increased invasiveness of malignant gliomas, pediatric atypical teratoid/rhabdoid tumors of CNS, and of some types of invasive breast, renal cell, prostate, and lung cancers. A pathway model of the uPA-OPN pathway was kinetically constructed utilizing math modeling methodologies originally developed for semiconductor circuit design. Based upon experimental data and using this uPA-OPN autocrine pathway model, several compounds were synthesized. UCD007 is a selective and reversible inhibitor of uPA in human malignant glioma cells. UCD007 interferes with U87 glioma cell adherence to the extracellular matrix and with prolifera-tion. Human malignant glioma cells implanted intracerebrally in athymic rats failed to establish tumors or demonstrated marked size reduction with only local invasion when animals were pretreated with UCD007 for 3 days and subsequently ×1/daily during a 10d treatment period. UCD007 was combined with RNA inactivation experiments to elucidate how inhibition of intracellular uPA-OPN impairs glioma cell adherence, proliferation, and restricts local intracranial invasion.

PH-02. THE L84F VARIANT OF O6-METHYLGUANINE-DNA METHYLTRANSFERASE EXHIBITS ELEVATED O6-BENZYLGUANINE-INDUCED DEGRADATION COMPARED TO WILD-TYPEMaya Remington, Timothy Cloughesy, and Albert Lai; Neurology, University of California Los Angeles, Los Angeles, CA, USA

First line therapy for glioblastoma multiforme (GBM) includes treat-ment with temozolomide (TMZ), an alkylating agent. Low expression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is associated with increased sensitivity of glioma cells to TMZ. A strategy to sensitize glioma cells to TMZ is to deplete MGMT using O6-benzylguanine (O6-BG), a synthetic substrate of MGMT. Several MGMT coding region polymorphisms (L84F and I143V/K178R) commonly exist in the general population and appear to affect the risk of developing a variety of cancers. To determine whether the L84F and I143V/K178R polymorphisms influ-ence resistance to TMZ and/or O6-BG, we stably expressed enhanced green fluorescent protein (eGFP)-tagged wild-type (WT) and polymorphic variant MGMT constructs in U87MG glioma cell lines. We confirmed that the WT eGFP-MGMT protein is properly localized within the nucleus and confers resistance to TMZ. We found that the L84F and I143V/K178R eGFP-MGMT variants exhibit the same nuclear localization pattern as WT. The L84F and I143V/K178R variants also confer resistance of U87MG cells to TMZ in clonogenic and MTT growth assays. When exposed to O6-BG, the L84F variant is degraded more rapidly than WT or I143V/K178R. This observed O6-BG induced degradation of both WT and L84F is completely reversed by bortezomib, suggesting that this degradation is proteasome-dependent. We are conducting MTT growth assays to explore the possibility that the L84F variant is more sensitive to the combination of O6-BG and TMZ than WT.

PH-03. PHARMACOKINETIC ANALYSIS OF IMATINIB AND CGP74588 IN PATIENTS WITH RECURRENT ANAPLASTIC OLIGODENDROGLIOMA: NORTH CENTRAL CANCER TREATMENT GROUP (NCCTG) N0272Kurt Jaeckle,1 Merrill Egorin,2 S. Keith Anderson,3 Wenting Wu,3 Jann Sarkaria,4 Evanthia Galanis,5 Paul Brown,6 Anthony Murgo,7 and Jan Buckner5; 1Neurology and Oncology, Mayo Foundation for Medical Education and Research, Jacksonville, FL, USA; 2Medicine and Cancer Pharmacology, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA; 3Cancer Center Statistics, Mayo Foundation for Medical Education and Research, Rochester, MN, USA; 4Mayo Foundation for Medical Education and Research, Rochester, MN, USA; 5Hematology/Oncology, Mayo Foundation for Medical Education and Research, Rochester, MN, USA; 6Radiation Oncology, Mayo Foundation for Medical Education and Research, Rochester, MN, USA; 7National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

NCCTG N0272 is an ongoing Phase I-II study of imatinib in treat-ment of patients (pts) with recurrent anaplastic oligodendroglioma, a tumor known to overexpress PDGFR. Prior studies have shown that PDGFR phos-phorylation and PDGF-dependent MAP-K activation are inhibited at ima-tinib concentrations of 0.1–1 mM (59–590 ng/ml), and proliferation of U-87 and U-343 cell lines is observed at 1.5 uM. At imatinib doses in mice (50mg/kg/D) that produce plasma concentrations of 13 uM (7670 ng/ml), prolonged survival has been observed in U87 and U-343 xenografts. Plasma from base-line and steady-state (days 28 and 56 of treatment) was assayed (M.Egorin) for imatinib and the metabolite CGP74588 in two patient groups: those not receiving enzyme-inducing anticonvulsants (non-EIAC; treated with 600 mg/D imatinib), and those receiving EIAC (1000 mg/D imatinib). Data were correlated with steroid therapy (yes vs. no) and toxicity of grade 3 or greater (CTC 2.0) (yes vs. no), and compared using Wilcoxon-Rank-Sum or Wilcox-on-Signed-Rank tests. To date, data are available from 13 non-EIAC and 4 EIAC pts. Ten pts had at least 2 steady-state samples. The tables show the mean steady-state concentrations of imatinib and CGP74588 in non-EIAC and EIAC pts at day 28, day 56, the combined change from day 28 to 56. Although the mean imatinib and CGP74588 concentrations at were lower in EIAC pts at 28 and 56 days, these differences are not statistically significant. There was a trend for concentrations to be lower at day 56 as compared with day 28, but these changes did not reach statistical significance. In non-EIAC patients, there was no difference in the day 28 concentrations of imatinib or CGP74588 between pts receiving or not receiving steroids at baseline (p 5 0.287 and 0.573, respectively). Similarly, there was no significant dif-ference in day 28 plasma concentrations of imatinib or CGP74588 between pts experiencing or not experiencing at least one toxicity of CTC grade 3 or greater (p 5 0.148 and 0.414 respectively). The doses of imatinib uti-lized produced plasma levels in non-EIAC and EIAC patients within the lower range of that reported to show activity in human glioma cell lines and murine glioma xenografts. Plasma steady-state levels remained relatively constant over time in both EIAC and non-EIAC patients, and did not appear to be negatively influenced by concomitant steroid administration. To date, steady-state concentrations of imatinib have not shown significant correla-tion with grade 3 or greater toxicity. Given the marginal blood-brain barrier permeability of imatinib, it will be important to correlate plasma concentra-tions with target bioactivity of imatinib in tumor tissue, which is currently under evaluation in a subgroup of patients on this study.

QUALITY OF LIFE

QL-01. IMPROVEMENT OF NEUROLOGICAL FUNCTION AFTER CONCURRENT TREATMENT WITH INTRAVENOUS IMMUNOGLOBULIN AND CHEMOTHERAPY WITH DOXORUBICIN AND CYCLOPHOSPHAMIDE IN A PATIENT WITH CEREBELLAR DEGENERATION PARANEOPLASTIC SYNDROMEJay-Jiguang Zhu,1 Xuemei Li,2 Thomas Hedges,3 and Katie Wakley4; 1Neurology, Hem/Onc, Tufts-New England Medical Center, Boston, MA, USA; 2Internal Medicine, Berkshire Medical Center, Pittsfield, MA, USA; 3Ophthalmology, Tufts-New England Medical Center, Boston, MA, USA; 4OBGYN Oncology, Tufts-New England Medical Center, Boston, MA, USA

Paraneoplastic syndrome is a rare manifestation of systemic cancers, often associated with lung or ovarian malignancy. Paraneoplastic cerebellar degeneration (PCD) is one of the common presentations of paraneoplastic syndrome. Clinically, patients present with cerebellar symptoms including dysarthria, gaze evoked nystagmus as well as gait ataxia. Frequently, anti-Purkinje cell antibodies (anti-Yo) are detected in the serum and cerebrospi-nal fluid. There is no effective treatment for such conditions. Intravenous immunoglobulin (IVIG) is one of commonly used therapies, but with limited success. We reported a 55-year-old woman with ovarian cancer who was



diagnosed with paraneoplastic cerebellar degeneration with clinical mani-festation of cerebellar syndrome and positive anti-Yo antibody (1:1500 dilu-tion) in CSF only and mild elevation of tumor markers for ovarian cancer, CA-125. She presented with one and a half months history of sub-acute onset of dysarthria, gait ataxia and intermittent blurry vision with upright position. Exam showed downbeat nystagmus and alternating skew devia-tion, dysmetria as well as gait ataxia. Incidentally, her symptoms were sig-nificantly improved one week after a routine influenza vaccine injection. However, it did not last long and the same symptoms recurred 2–3 weeks later. Magnetic resonance imaging (MRI) head with and with contrast at the time of diagnosis did not reveal cerebellar atrophy. She received IVIG at 0.4 gm/kg/day 3 5 days every 4 weeks with concurrent chemotherapy with intravenous doxorubicin (Doxil) and cyclophosphamide (Cytoxan) monthly. Three months after such treatment, her speech and dysmetria have markedly improved. She will continue to receive the current treatment monthly pro-vided she tolerated them well. Updated outcome of this patient and review of current treatment options of paraneoplastic syndromes will be reported at the meeting.

QL-02. OBJECTIVE AND SUBJECTIVE SLEEP QUALITY OF LOW-GRADE GLIOMA PATIENTSDebbie Van Tilburg,1 Eus Jw Van Someren,2 Jaap C. Reijneveld,3 Cornelis J. Stam,4 Ingeborg Bosma,3 Jan J. Heimans,3 and Martin Klein1; 1Medical Psychology, VU University Medical Center, Amsterdam, Netherlands; 2Netherlands Institute for Neuroscience, Amsterdam, Netherlands; 3Neurology, VU University Medical Center, Amsterdam, Netherlands; 4Clinical Neurophysiology, VU University Medical Center, Amsterdam, Netherlands

Although sleep disorders are a prominent problem in cancer patients, it has never been thoroughly studied in glioma patients. Insomnia, as well as disturbed sleep patterns, can negatively affect daytime patterns of cognitive functioning, a hallmark of many glioma patients. Actigraphy or polysom-nography recordings indicate that sleep and circadian rhythms in cancer patients differ significantly from healthy subjects. However, these record-ings also show a discrepancy between objective and subjective sleep experi-ence, a phenomenon known as sleep state misperception. This study aims at determining (1) the objective and subjective sleep and circadian rhythms in low-grade glioma patients, and (2) the potential discrepancies between these ratings. Objective and subjective sleep quality was recorded in 11 low-grade glioma patients (8 astrocytomas, 2 oligodendrogliomas type B, 1 oligoastrocytoma, six males, mean age 43.9 610.5 years and 11 age- and gender-matched healthy controls (six males, mean age 44.1 610.9 years). Patients and healthy controls were recruited from the VU University Medi-cal Center, the Academic Medical Center Amsterdam, and the Netherlands Institute for Neuroscience. Four patients had a biopsy and 7 patients had a resection, 3 radiotherapy, and 1 chemotherapy. All patients suffered from epileptic seizures and received antiepileptic drugs. Objective sleep record-ings were carried out by actigraphy. An actigraph has the size and shape of a watch and is wrist-worn. By registration of arm movements, rest-activity patterns and thus circadian rhythms were recorded continuously for 7 days and nights. Using a software program, sleep estimates were obtained from the 1-minute activity epochs. Subjective sleep indices were obtained using the Pittsburgh Sleep Quality Index (PSQI), specifically designed to mea-sure sleep quality, the Athens Insomnia Scale (AIS), which is designed for quantifying sleep difficulty based on ICD-10 criteria, and the Sleep Disor-ders Questionnaire (SDQ) designed to diagnose the presence of common sleep disorders. Compared to matched healthy controls, low-grade glioma patients spent more time in bed and had longer nocturnal awakenings than healthy controls (Student’s t-test: p 5 0.036 and p 5 0.01, respectively). Low-grade glioma patients also had a tendency to have less assumed sleep (p 5 0.059). In addition, their circadian rhythm showed a tendency towards more nocturnal activity (p 5 0.052). Subjectively, patients qualified their sleep as lower (PSQI: p 5 0.009) and more difficult (AIS: p 5 0.001), than healthy controls. Patients also had significantly higher ratings on the SDQ clusters indexing excessive daytime sleepiness (p 5 0.005), narcolepsy (p 5 0.020), psychiatric problems (p 5 0.01), and a tendency for insomnia (p 5 0.057). In conclusion, low-grade glioma patients suffer from distur-bances in sleep patterns and circadian rhythms that, except for insomnia, are largely unrelated to their subjective ratings. Although it is acknowledged that cognitive deficits in low-grade glioma patients can be caused by the tumor, by tumor-related epilepsy, by tumor treatment, and by psychological distress, disorders in sleep-wake rhythms as a separate potential source of cognitive compromise may play an important role as well.

QL-03. FATIGUE IN LOW-GRADE GLIOMA PATIENTSK. Struik,1 M. Klein,2 J.J. Heimans,3 M.F. Gielissen,4 G. Bleijenberg,4 M.J. Taphoorn,5 J.C. Reijneveld,3 and T.J. Postma3; 1VU University Medical Center, Amsterdam, Netherlands; 2Medical Psychology, VU University Medical Center, Amsterdam, Netherlands; 3Neurology, VU University Medical Center, Amsterdam, Netherlands; 4Expert Center Chronic Fatigue, University Medical Center Nijmegen, Nijmegen, Netherlands; 5Neurology, Medical Center Haaglanden, The Hague, Netherlands

The objective of this study was to determine the prevalence and severity of fatigue in long-term survivors with a low-grade glioma (LGG), and to analyze the relationship between fatigue and demographic variables, disease duration, tumor characteristics, former treatment modalities, self-reported concentration, motivation, and activity. Fifty-four LGG patients with stable disease (mean age 48 years, range 25–73 years, 28 male, 39 astrocytoma II, 10 oligodendroglioma II, 5 oligoastrocytoma II) who were diagnosed more than 8 years ago were included in this study. Fatigue was analysed with the Checklist Individual Strength (CIS). The CIS is a multidimensional fatigue scale; it measures four aspects of fatigue during the previous 2 weeks, namely: fatigue severity (eight items), concentration problems (five items), reduced motivation (four items), and reduced activity (three items). Thirty-nine percent of the LGG patients were severely fatigued, with older patients being most affected (Mann-Whitney, p 5 0.012). Fatigue was associated with prior antiepileptic drug (AED) use (Mann-Whitney, p 5 0.013), and with reduced self-reported concentration, motivation and activity (Mann-Whitney, p , 0.001). No relation was found between fatigue and gender, histology, tumor laterality, disease duration, type of neurosurgical inter-vention, and radiation treatment. Fatigue is a severe, long-term problem in a large proportion of LGG patients. Assessment and treatment of fatigue in LGG patients need to be further investigated, potentially resulting in improvement of quality of life.

QL-04. LATE-DELAYED RADIATION INJURY AND NEUROCOGNITIVE FUNCTIONING IN VERY LONG-TERM LOW-GRADE GLIOMA SURVIVORSJosje Van Den Heuvel,1 Jaap C Reijneveld,2 Selene Ssa Fagel,3 Martin J.B. Taphoorn,4 Jan J. Heimans,2 and Martin Klein1; 1Department of Medical Psychology, VU University Medical Center, Amsterdam, Netherlands; 2Department of Neurology, VU University Medical Center, Amsterdam, Netherlands; 3Department of Clinical Child and Adolescent Studies, Leiden University, Leiden, Netherlands; 4Department of Neurology, Medical Center Haaglanden, The Hague, Netherlands

In a recent neurocognitive follow-up study in low-grade glioma (LGG) survivors with stable disease diagnosed on average 13 years earlier and who had neurocognitive baseline assessment six years previously, we found that irradiated LGG patients (n 5 34) show a neurocognitive decline during this interval in contrast with non-irradiated LGG patients (n 5 33). The present study aimed at determining whether (1) irradiated LGG patients demon-strate more cortical atrophy and white matter hyperintensities compared with non-irradiated LGG patients, and whether (2) the cognitive deteriora-tion found in irradiated LGG patients is correlated with those abnormali-ties. Using MR scans, generalized cortical atrophy (GCA) and white matter hyperintensities (WMH) of irradiated and non-irradiated LGG survivors with stable disease out of the initially included 195 low-grade glioma patients, were rated. Owing to the large differences in scanning protocols and MRI/CT use between participating centers and over time, complete GCA and WMH baseline and follow-up pairs could be evaluated in 13 irra-diated LGG patients and 9 non-irradiated LGG patients. Radiological out-come was related to changes between baseline and follow-up in irradiated LGG patients in the cognitive domains of (1) information processing speed, (2) psychomotor function, (3) attentional functioning, (4) verbal memory, (5) working memory, and (6) executive functioning. The average time between baseline and follow-up MR scans was 5.5 years. At baseline (6 years fol-lowing diagnosis) no differences between irradiated and non-irradiated patients were found in GCA (t-test: p 5 0.549) or WMH (t-test: p 5 0.992). At follow-up (13 years following diagnosis), however, irradiated patients displayed an increase both in GCA (paired t-test: p 5 0.020) and in WMH (p 5 0.016), while radiological characteristics of the unirradiated patients remained stable over time (GCA: p 5 1.000 and WMH, p 5 0.834, respec-tively). The radiotherapy 3 time interaction (p 5 0.034) in WMH indicates that radiological abnormalities increase in irradiated patients beyond 6 years following diagnosis. To determine whether an increase in radiologi-cal abnormalities in irradiated patients was associated with a decrease in neurocognitive functioning, a general linear repeated measures model was employed. Increasing WMH was associated with lower speed of informa-tion processing (p 5 0.012), attentional functioning (p 5 0.035), work-ing memory (p 5 0.037), and executive functioning (p 5 0.042). Although the available MR data are limited, we conclude that radiotherapy in LGG patients at the higher end of the survival curve leads to cortical atrophy and white matter hyperintensities and that these radiological abnormalities are correlated with neurocognitive decline.



QL-05. FINANCES AND QUALITY OF LIFE FOR BRAIN TUMOR PATIENTS: RESULTS FROM A NATIONAL SURVEYHarriet Patterson and Robert Tufel; National Brain Tumor Foundation, San Francisco, CA, USA

The negative impact of cancer treatment on patient financial well-being has been documented. Furthermore, financial well-being has been shown to be closely tied to self-reported quality of life. Brain tumor patients are at greater risk of devastating financial situations because cognitive effects of tumors and treatment impairs ability to return to work, limits functional status, and may not improve. This is the first study to document the finan-cial impact that brain tumor patients and families experience. In the fall of 2006, a national patient advocacy organization conducted an online survey about the financial impact of brain tumors on patients and families utilizing an online survey tool. A total of 550 patients and caregivers completed the closed-ended multiple choice survey. In addition, respondents had the option of completing an open-ended comment box. 300 respondents provided qual-itative comment data which was analyzed thematically and using keywords. The surveys included questions on demographic information, tumor type, time since diagnosis, employment history, health insurance coverage, out of pocket costs, and financial burden. The survey results describe a bleak situ-ation for brain tumor patients and their families. Though 91% of respon-dents had health insurance, 54% of caregiver report spending over $250 per month out of pocket for medical expenses and 27% reported spending over $1000 per month. Prescription drug costs were the highest category reported. Employment status changes were also prevalent. Though 91% of patients were working full-time at diagnosis, only a third continued work-ing, creating a significant decrease in household income across all income levels. Those reporting income of less than $15,000 per year increased by 300% after diagnosis. Those reporting income of $75,000 per year dropped by a third after diagnosis. In order to meet high costs of treatment and make up for income losses families reported making cut backs to house-hold expenses but also made long terms sacrifices to make ends meet. 47% incurred credit card debt, 42% had to borrow money from family or friends, 15% took out a second or third mortgage, 8% cashed in life insurance poli-cies, and 7% declared bankruptcy. In some cases, families lost their homes and became homeless. Many of these actions continued to plague families beyond the treatment period, with over 60% of respondents indicating that brain tumor related expenses were a major problem and four out of five respondents indicating their financial situation had changed for the worse since their loved one was diagnosed. This study describes a clear picture of downward mobility among respondents who were working full-time, owned their homes, were insured, and were largely middle class and above. The result on quality of life for patients in active treatment, long term survivors, and their families has not been adequately understood until now.

QL-06. FERTILITY ISSUES IN ADULTS TREATED WITH TEMOZOLOMIDEKathleen Mogensen; Neuro-Oncology, Roswell Park Cancer Institute, Dent Neurologic Institute, Buffalo, NY, USA

With the increasing use of temozolomide (TMZ) in the treatment of lower grade astrocytomas, oligodendrogliomas, and mixed gliomas, issues of fertility and childbearing ability in patients arise. The acute toxicities of TMZ are comparatively mild, however the long term effects have yet to be defined. Patients of childbearing age receiving TMZ for these tumors are often working, living a full life, and requesting information about fertility. Published accounts of fertility in cancer patients deal primarily with breast cancer, germ cell tumors, leukemia, and lymphomas. Reports of infertil-ity range from 10% to more than 60% depending on the drug used, and duration of treatment. Chemotherapies such as lomustine, carmustine, and matulane used for primary gliomas are associated with a high incidence of low or absent sperm count in males, ammenorrhea and infertility in females. It appears TMZ may have less reproductive toxicity, but this has yet to be documented in the literature. Patients at our center are counseled on the risk of infertility, while at the same time advised to take effective measures to prevent pregnancy. Males are encouraged to use sperm banking if they might desire children following treatment. While in no way advocating patients attempt a pregnancy when on active treatment, the case studies of three couples are shared. A women in her 30s, already the mother of 7, received radiation and 12 months of TMZ for an anaplastic astrocytoma. She was advised to wait at least 6 months to attempt a pregnancy, however she became pregnant in less than 2 months. She delivered a full term, healthy infant. One year later she delivered a second healthy, post TMZ therapy child. Prior to treatment of a mixed oligo/astrocytoma with TMZ, a male patient and his wife were counseled about the risk of lowered sperm count, sperm banking options, as well as advised to prevent pregnancy. They chose not to follow recommendations; the wife became pregnant in the midst of his treatment. At age 12 months, their child has achieved the expected devel-opmental milestones. Another male proceeded with sperm banking prior to starting TMZ, and used adequate birth control. After his wife had 2 unsuccessful IVF attempts, he had analysis of live sperm. After more than

12 cycles of TMZ, still on active therapy, he was found to have more than adequate sperm count with good motility. They hope for a pregnancy after completion of treatment. The number of adults of child bearing age treated with TMZ for lower and intermediate gliomas is increasing. It is important to document and share information on the effect of TMZ on fertility and childbirth.

QL-07. PALLIATIVE CARE IN NEURO-ONCOLOGYMichael Salacz; Saint Luke’s Cancer Institute, Kansas City, MO, USA

Palliative care physicians have continued to partner with medical oncologists to improve care for general cancer patients—a full 39 abstracts involving palliative care appeared at the recent American Society of Clinical Oncology annual meeting in 2006. But what is palliative medicine and how can these professionals help in the care of patients with brain tumors? In this concurrent session, we will review the development of palliative medicine as a medical subspecialty. Then we will discuss recent outcomes data demon-strating the benefits of a medical oncology/palliative medicine partnership to improve patient care. Finally, we will focus on potential opportunities for neuro-oncology/palliative medicine collaboration.

QL-08. THE NEURO-ONCOLOGY ADVANCED PRACTICE NURSES’ ROLE AND EXPERIENCE IN THE COLLABORATIVE MANAGEMENT OF PATIENTS WITH PRIMARY BRAIN TUMOREva Lu Lee and Terri Armstrong; Neuro-Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

The collaborative practice model between the advanced practice nurse and the physician is a comprehensive approach to health care management that has particular application in complex disease processes such as pri-mary brain tumors. In the Department of Neuro-Oncology at The Uni-versity of Texas M.D. Anderson Cancer Center, physicians work directly with advanced practice nurses (APNs) to collaboratively manage patients in both the outpatient and inpatient settings. Written protocols and guidelines have been established to direct the practice. APNs work collaboratively with physicians to provide initial care and assessment of new patients, follow-up care of returning patients, and acute care of patients in the inpatient setting. APNs working with the physicians have a multifaceted role given the com-plexity of care required in this patient population, whose tumors and treat-ment frequently have a devastating impact on quality of life. Primary brain tumors are a heterogeneous group of neoplasms that arise from the con-stituent elements of the central nervous system (CNS). Patients commonly present with headaches, nausea, vomiting, seizures, altered mental status, or focal deficits such as weakness. Focal symptoms vary by tumor location. Not only does the tumor itself produce symptoms, but also the therapies result in multiple side effects, including worsening neurologic function or constitutional symptoms such as fatigue, nausea, or pancytopenia. Treat-ment modalities such as surgical resection, radiation, and chemotherapy are used concurrently or sequentially. Despite a concerted effort, tumors often recur quickly and life expectancy may be short. As a consequence, palliation and end-of-life care is an important component of the collaborative practice. Management of these patients by the collaborative method and successful patient outcome demand a mutual understanding, agreement, and commu-nication between physician and APN. Patients with the diagnosis of brain tumor are provided a deeper understanding of the nature of the disease, the goal of therapy, and the complications that can develop from the tumor or therapy. APNs, who are in continual contact with the patient, have the advantage of being able to educate, manage symptoms, and enforce health behaviors to enhance a positive patient experience and outcome. Through enhanced patient education, symptom management, and reinforcement of positive health behaviors, collaborative practice between APNs and physi-cians in the neuro-oncology setting provides comprehensive care to patients with primary brain tumors and helps lead to optimal outcomes.

QL-09. FAMILIAL APPRAISAL OF PROVIDING CARE TO PATIENTS WITH BRAIN CANCERStephen Keir and Henry Friedman; The Preston Robert Tisch Brain Tumor Center, The Tug McGraw Research Center, Durham, NC, USA

Brain tumor caregivers are unique in that they provide care to a relative with a potentially short terminal disease trajectory that is often times associ-ated with severe functional, neurocognitive, and neuropsychiatric sequalae. Understanding the impact of caregiving is particularly important for this population in that these caregivers are frequently called upon to help make difficult decisions regarding the patient for whom they provide care. The



goal of this study was to document caregiver appraisal of providing care to persons with glioblastomas. Using the Family Appraisal of Caregiving Questionnaire (FACQ), caregiver levels of distress, strain, positive caregiv-ing, and family wellbeing were assessed in 75 adult familial caregivers. In addition, the relationship between caregiver appraisal and patient quality of life (FACT-BR) and levels of perceived stress (PSS-10) was explored. To assess whether demographic variables were associated with differences in caregivers’ FACQ scores, Pearson’s correlations were calculated between caregivers’ age and length of care, and patients’ score on the FACT-BR and PSS-10. In addition, a t-test was performed to evaluate whether caregiver functioning differed according to gender. To control for family-wise error rate, the alpha level for each correlation within a set of analyses was adjusted using a Hochberg modified bonferroni method. Respondents indicated high levels of positive caregiver appraisal (M 5 4.2, SD 0.53) and low levels of strain (M 5 2.73, SD 0.88). Caregiver age was associated with positive appraisals such that older caregivers were more likely to report a positive view of providing care. However, length of caregiving was not related to any domains of the FACQ. With regard to gender differences, male care-givers reported significantly greater positive appraisals than did females (t5 –2.11, p , 0.05), and there was a trend for males to report greater positive views of their family (t 5 –1.87, p 5 0.07). Of the four domains assessed by the FACQ, only caregiver distress correlated (r 5 –0.245, p 5 0.04) with patients’ overall FACT-BR score. When looking at domains within FACT-BR, patients’ functional well-being scores were significantly and negatively correlated with caregivers’ distress (r 5 –0.276, p 5 0.02). Moreover, there was a trend for distress scores to be negatively associated with patients’ social well-being (r 5 –0.254, p 5 0.03) and additional brain tumor concerns (r 5 0.25, p50.04). Similarly, caregiver strain was signifi-cantly associated with patients’ reports of poor social well-being (r 5 –0.29, p50.02), functional well-being (r 5 –0.31, p 5 0.01), and additional con-cerns (r 5 0.36, p 5 0.02). Positive caregiver appraisal was associated with patients’ social well-being (r 5 0.38, p 5 0.001). Caregiver family wellbeing was negatively associated with additional concerns (r 5 –0.25, p 5 0.037) with a positive trend for patients’ physical well-being (r 5 0.24, p 5 0.042). Patients’ PSS-10 scores were significantly correlated with caregiver strain (r 5 0.34, p 5 0.004) and trended toward association with caregiver distress (r 5 0.26, p 5 0.033). Additionally, caregivers’ family well-being scores were significantly associated with patients’ lower PSS scores (r 5 –0.29, p 5 0.015) and there was a trend for positive appraisal (r 5 –26, p 5 0.033). There is a clear relationship between caregiver appraisal and patient quality of life and more so regarding patient stress; future studies are warranted and should look to further document this relationship and explore it longitudi-nally across the caregiving trajectory.

QL-10. PHASE II TRIAL OF ZOLEDRONIC ACID (ZA) FOR BONE LOSS PREVENTION IN GLIOMA PATIENTSMary Lou Affronti,1 Cindy Bohlin,1 Karen Bronson,1 Lee Jones,1 James Herndon,2 Jennifer Quinn,3 David Reardon,1 Annick Desjardins,4 Jeremy Rich,3 Henry Friedman,5 and James Vredenburgh3; 1Surgery, Duke University, Durham, NC, USA; 2Biostatistics and Bioinformatics, Duke University, Durham, NC, USA; 3Medicine, Surgery, Duke University, Durham, NC, USA; 4Medicine, Duke University, Durham, NC, USA; 5Medicine, Surgery, Pathology, Duke University, Durham, NC, USA

Glioma patients are at risk for osteopenia/osteoporosis due to neurologi-cal deficits and use of anticonvulsants and glucocorticoids. We reported that 38% of glioma patients demonstrate osteopenia (defined by bone mineral density t-score , –1) and 16% osteoporosis (defined by t- score , –2: spine; t-score , –2.5: femur) compared to 10% in the general population. We conducted a Phase II trial to determine the effect of prophylactic q3 month ZA on bone mineral density (BMD) and skeletal complications. Primary endpoint was defined as the percentage of patients with worsening BMD (defined by a negative change in t-score of .0.5) at the 6-month and one year evaluation. Eligibility criteria included: (1) histologically confirmed diagnosis of a primary glioma, (2) age . 18 years, (3) KPS . 60%, (4) treated with valproate or an enzyme inducing anticonvulsant (EIAC) and/or on more than physiologic replacement steroid therapy, (5) creatinine , 2.0 mg/dl and calculated creatinine clearance of .60 ml/min, (6) bilirubin , 1.5 3 nl, LFTs , 2.5 3 nl, (7) recovery from surgery, (8) life expectancy .12 wks, and (9) written informed consent. Exclusion criteria: Previously diagnosed with osteoporosis requiring oral bisphosphonates. 50 subjects are required to have 80% power to demonstrate a reduction from 50% to 25% of patients with worsening BMD using a 1 sample binomial test (a of 0.05). Skeletal-related complications and toxicities were tabulated. Of the 48 patients who were eligible for baseline BMD analysis, 42% of glioma patients demonstrated osteopenia, and 17% osteoporosis. 76% were diag-nosed with a glioblastoma. Mean age, 55.8 (SD 9.6; range 32–76). 66% were male and 92% were Caucasian. 46% had a KPS of .90: 31% KPS .70%. AC use: 62% phenytoin; 11% valproate; 8% phenobarbital; 4% oxcarbazepine; 6% levetiracetam; and 6% none. 37% were on dexametha-sone, 4% other steroids, 28% off steroids, and 8% unknown. Mean baseline N-telopeptide (a bone resorption marker) was 14.4 (SD 12.8; range 5.6–74), reference range of 5.4–24.2. No skeletal complications were documented.

Majority of toxicities were grade 1–2. (1 patient was hospitalized for grade 3 fever; 2 were discontinued for grade 2 non-osteonecrosis of the jaw dental changes). Without the use of ZA it was expected (based on clinical review) that 50% of glioma patients who continued on steroids or anticonvulsants would demonstrate deterioration in their BMD over time. However, to date all 10 patients who have completed a 6-mo evaluation have stable BMD. No patient thus far, has demonstrated worsening bone density and in fact 20% of patients have improvement in BMD (defined by a positive change in t-score of .0.5). In addition to an analysis of predictors (immobility, steroids and anticonvulsants) of BMD, an updated analysis of N-telopeptide and BMD outcomes will be presented. ZA treatment was well tolerated and improved the bone health of glioma patients. A phase III trial of ZA for glioma patients is warranted.

QL-11. LEVETIRACETAM MONOTHERAPY IN PATIENTS WITH MALIGNANT GLIOMAPhioanh Nghiemphu,1 Lily Chattopadhyay,2 Carrie Graham,1 Mady Stovall,1 Nanette Fong,1 Albert Lai,1 and Timothy Cloughesy1; 1Neurology, University of California Los Angeles, Los Angeles, CA, USA; 2University of California, Los Angeles, CA, USA

Enzyme-inducing antiepileptic drugs such as Phenytoin are usually the first choice for seizure management in patients with primary brain tumor, especially at the time of surgery. However, these types of medications can interfere with the metabolism of many chemotherapies and molecular treat-ments for high grade gliomas. At our institution, we frequently transition patients on Phenytoin to Levetiracetam to avoid potential interactions with future treatments. This retrospective study reviews our experience with using Levetiracetam as a monotherapy for seizure prevention. From our brain tumor patient database, we identified 66 patients with glioblastoma who have been placed on Levetiracetam monotherapy after tapering off phenytoin. Their medical records were analyzed for length of treatment, dosage, seizure frequency, side effects, and additional antiepileptic drugs. Most patients (45 out of 66) were first maintained at 1500–2000 mg total dose per day. Of these patients, 39 patients remained seizure free through out their course of treatment. For those who had seizures while on levetirac-etam alone, 18 patients required an increase in dosage, and only 9 patients actually required additional medications after levetiracetam has been maxi-mized. Most common complaints on Levetiracetam included fatigue, cog-nitive slowing, dizziness, and personality changes. Levetiracetam appears to prevent seizures as a monotherapy in patients with glioblastoma and is well-tolerated. This review warrants further prospective studies of Leve-tiracetam as a monotherapy to prevent recurrent seizures in patients with primary brain tumors.

QL-12. FUNCTIONAL CONNECTIVITY CHANGES AND CONSEQUENCES FOR COGNITIVE DISABILITY IN LOW-GRADE GLIOMA PATIENTS: COMPARING TWO METHODS OF MEG SIGNAL ANALYSISIngeborg Bosma,1 Cornelis J. Stam,2 Martin Klein,3 Linda Douw,3 Fabrice Bartolomei,4 Jan J Heimans,1 Bob W. Van Dijk,5 and Jaap C. Reijneveld6; 1Neurology, VU University Medical Center, Amsterdam, Netherlands; 2Clinical Neurophysiology, VU University Medical Center, Amsterdam, Netherlands; 3Medical Psychology, VU University Medical Center, Amsterdam, Netherlands; 4Clinical Neurophysiology and Epileptology, CHU Timone, Marseille, France; 5MEG Center, VU University Center, Amsterdam, Netherlands; 6Neurology, VU University Medical Center and Academic Medical Center, Amsterdam, Netherlands

Low-grade glioma (LGG) patients may suffer from severe (global) cognitive disturbances with serious negative impact on the quality of life. It is difficult to understand the nature of this global cognitive decline. In a previous study, in which we used magnetoencephalography (MEG) in LGG patients, we found large-scale changes in functional brain networks. Moreover, we found significant correlations between neurocognitive perfor-mance and changes in functional connectivity in various frequency bands and across multiple brain areas, suggesting that the neurocognitive deficits seen in these patients are correlated to changes in functional connectivity due to the tumor itself, or to tumor treatment. In our previous study we used the synchronization likelihood (SL) as a measure of functional coupling between brain areas. Since it is possible that the results obtained by using the SL are influenced by volume conduction and power differences, we intro-duced the Phase Lag Index (PLI) to quantify phase synchronization, without bias due to volume conduction. This study aims to evaluate functional con-nectivity in LGG patients and healthy controls by using the PLI and to com-pare these results with the previous used SL. Seventeen LGG patients and 17 healthy controls, matched for age, sex and education were included. Four artifact-free epochs of 13 seconds of MEG recordings during an eyes-closed resting-state were carefully selected by visual analysis. Subsequently, the signals were filtered in seven frequency bands, and PLI scores were calcu-



lated between all channels and then averaged to a number of short-distance (within area) and long-distance (between area) scores. LGG patients showed significant differences in functional connectivity compared to the matched healthy controls. In the theta band a higher long- and short-distance syn-chronization was found in the LGG patient group compared to the healthy controls. In the upper alpha and lower gamma band, LGG patients showed a decline in short distance synchronization compared to these healthy con-trols. By using the SL (as we did in our previous study), more differences were found. In respectively the delta, theta and beta frequency band, LGG patients showed significantly higher long-distance synchronization scores than controls. In contrast, patients displayed a decline in long-distance SL in the alpha and the upper gamma frequency ranges. Our findings suggest that by using the PLI, to bypass the effects of volume conduction and power differences, we were still able to find significant differences between the LGG patients compared to the healthy controls. Especially the observed differences in the theta band are robust and not likely to be influenced by methodological limitations. As we have already showed that a focal lesion (i.e., a low-grade glioma) can cause serious disturbances in the brain net-work which correlated with cognitive decline, our future perspective will be to correlate neurocognitive function with the observed differences in PLI and to evaluate the influence of various treatment modalities (surgery, radio-therapy, chemotherapy, and antiepileptic drugs) on network disturbances in order to improve the quality of life in glioma patients.

QL-13. REFRACTORY EPILEPSY IN GLIOMA PATIENTSJon Glass1 and James Ko2; 1Fox Chase Cancer Center, Philadelphia, PA, USA; 2Thomas Jefferson University, Philadelphia, PA, USA

Refractory epilepsy (RE) is associated with increased morbidity and sudden death. Approximately 20% to 40% of glioma patients have had at least one seizure at the time the tumor is diagnosed. An additional 20% to 45% eventually develop seizures. The risk of RE in glioma patients has been poorly defined. A retrospective chart review was performed on consecu-tive patients with supratentorial gliomas seen between 3/1/00 and 7/1/05. Patients with phakomatoses, active systemic cancer, and infratentorial tumors were excluded. RE was defined as more than one partial or gen-eralized seizure per week and failure of three or more anti-epileptic drugs (AEDs). Attention was directed to patterns of treatment of the seizure disor-der. Patients were considered improved if criteria for RE were no longer met after treatment. 254 patients were identified. 137 were men and 117 were women. Median age at diagnosis was 54 years (19–88). 130 had grade 4 astrocytoma, 49 grade 3 astrocytoma, 16 grade 2 astrocytoma, 46 grade 3/4 oligodendroglioma or oligoastrocytoma, 8 grade 1/2 oligodendroglioma, 2 pleomorphic xanthoastrocytoma, and 1 each of ganglioglioma, anaplastic mixed astrocytoma-ependymoma and gliomatosis cerebri. 121 had seizures at some point during their clinical course, and seizure was the presenting symptom in 98. Eight patients were identified with RE. Of these, 5 had grade 1/2 oligodendroglioma, 2 had grade 3 astrocytomas, and one had grade 4 astrocytoma. Of patients with grade 1/2 oligodendroglioma, 3 had clini-cally and radiographically stable disease and had no treatment other than subtotal resection. All 3 were treated with temozolomide chemotherapy with improvement in seizures and required stable or decreasing doses of AEDs. Two had progressive disease. Of these, 1 had no prior treatment and received radiation therapy with improvement. The other had recurrent dis-ease after treatment with radiation therapy and temozolomide and did not improve. All patients with grade 3 and grade 4 astrocytoma had progressive disease. Patients with grade 1/2 oligodendroglioma are frequently observed following initial resection because of the indolent nature of these tumors. However, they are at risk for developing RE, even without radiographic or clinical progression. Early identification of these patients and treatment with radiation therapy or temozolomide chemotherapy may effectively con-trol seizures. Further investigations regarding the risk factors for develop-ment of RE and mechanisms of multidrug resistance are warranted.

QL-14. EFFICACY OF DRONABINOL FOR NAUSEA, VOMITING, AND APPETITE DISTURBANCES IN ADULT PATIENTS WITH PRIMARY MALIGNANT GLIOMASDeborah Allen, Karen Carter, Cindy Bohlin, Karen Bronson, Lee Jones, Stephen Keir, Bebe Guill, Renee Raynor, Bart Bridigi, Mary Lou Affronti, and James Vredenburgh; Preston Robert Tisch Brain Tumor Center at Duke, Duke University, Durham, NC, USA

Nausea and vomiting (NV) and appetite disturbances (AD) are com-mon adverse effects from tumor placement or treatment for patients with primary brain tumors. Although most treatments administered in this popu-lation are not highly emetogenic or cause anorexia, up to 50% of patients report common gastrointestinal disturbances. Treatments for these side effects may not work or may further alter mental status. Dronabinol may provide a safe alternative option without compromise of neurological sta-tus. This exploratory study seeks to describe toxicities (TOX) associated

with dronabinol administration in patients with WHO Grade III/IV pri-mary gliomas on adjuvant chemotherapy. A secondary aim is to describe the impact that dronabinol has on their quality of life (QOL). Power analysis determined a sample size of 34 subjects for 0.80 effect size. Enrollment is ongoing, with 22/34 patients accrued. Patients take dronabinol 5 mg BID 24 hours prior to, during, and 48 hours after completion of oral/IV chemo-therapy. Patients continue their established antiemetic regimen. Between chemotherapy doses, dronabinol is reduced to 2.5 mg daily. Intolerance of dronabinol is defined as 2 or more Grade 3 or greater non-hematologic TOX according to the CTC v.3 definition handbook. Modified Functional Living Index-Emesis (FLIE), Functional Assessment of Cancer Therapy-Brain Tumor (FACT-Br), Mini Mental Status Exam (MMSE), and NV and appetite visual analog scales are collected at specific points for 2 cycles for TOX and QOL data. Subjects are predominately male (n 5 12, 55%); mean age of 46.0 years (range 26–68 years). A total of six subjects (27%) have withdrawn from the study due to Grade 3 neuro-cognitive changes (n 5 2) despite decreasing dosages; Grade 3 persistent NV (n 5 4) despite increasing dronabinol dosage. Increased dosages were required in 5 additional subjects to better manage CINV or AD without TOX. Two required decreased doses for Grade 2 neuro-cognitive TOX, maintaining the new dosage throughout the remainder of their treatment. Most subjects (n 5 18) reported stable or improved QOL by FACT-Br and FLIE. Dronabinol is safe and effective for administration as an adjunct to standard therapy for NV or AD during treatment for primary glioma. The dose may be titrated with minimal toxici-ties and improvement in QOL.

QL-15. OPHTHALMOPLEGIC MIGRAINE AS A SYMPTOM OF CARCINOMATOUS MENINGITIS FROM ESOPHAGEAL ADENOCARCINOMAHongmei Liang,1 Angela Lu,2 Barry Lembersky,3 and Frank Lieberman4; 1Division of Hematology/Oncology, University of Pittsburgh, Pittsburgh, PA, USA; 2Department of Neurology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 3Division of Hematology/Oncology, Pittsburgh, PA, USA; 4University of Pittsburgh, Pittsburgh, PA, USA

Leptomeningeal carcinomatosis (LC) is an increasingly common meta-static complication of solid tumors, but rarely occurs in esophageal cancer. Although headache is a common presentation of LC, migranous syndromes have been infrequently reported. Here we report a case of LC presenting with opthalmoplegic migraine. A 50-year old man presented with dys-phagia, nausea, and dull holocranial headache. Endoscopy revealed signet-ring adenocarcinoma of the gastroesophageal junction, and laparoscopic esophagectomy was performed. In the post-operative period, he developed episodic right retro-orbital lancinating headache superimposed on a more diffuse headache, associated with lightheadedness and vomiting. MRI showed patchy leptomeningeal enhancement with normal ventricular size; CSF cytology was positive. The patient began treatment with whole brain radiation, dexamethasone, and an Ommaya reservoir was placed. The epi-sodic retro-orbital headache worsened, and when it crescendoed in severity, was associated with episodes of poor responsiveness. The episodes were not associated with sitting or standing, occurring when recumbent as well. During an episode witnessed in the hospital, complete right 3rd nerve palsy with pupillary dilatation was observed. There was papilledema on fundo-scopic exam. CT scan of the head showed no acute changes. Drainage of CSF transiently relieved the holocranial and lancinating retro-orbital com-ponents of the headache. One gram of sodium valproate was administered intravenously along with dexamethasone. The headache, ophthalmoplegia and pupillary dilatation resolved. Gabapentin was begun and valproate and steroids continued. Intermittent recurrent retro-orbital headache with oph-thalmoplegia and pupillary dilatation occurred despite ongoing CSF drain-age, though headaches were decreased in intensity. The patient died four weeks from the diagnosis of LC after colonic perforation related to meta-static esophageal cancer. A syndrome resembling ophthalmoplegic migraine may be associated with LC. LC is an uncommon complication of esophageal carcinoma but needs to be considered in the differential diagnosis of head-ache in this patient population. We review the features of LC complicating esophageal cancer.

QL-16. PREVALENCE OF DIABETES MELLITUS IN ADULT PATIENTS WITH PRIMARY GLIOMAS ON STEROID THERAPYDeborah Allen,1 Susanne Jackson,2 Karen Ziegler,1 Steve Silverman,1 Kara Penne,2 and Mary Lou Affronti2; 1Preston Robert Tisch Brain Tumor Center at Duke, Duke University, Durham, NC, USA; 2Duke University, Durham, NC, USA

It is well known that steroid therapy may induce or escalate problems with diabetes mellitus (DM). While steroids are used in many oncology treatment regimes, steroid therapy in neuro-oncology is utilized to manage cerebral edema and neurological dysfunction. Complications of DM may



worsen neurological functioning if undiagnosed and untreated, and may even be confused with primary brain tumor progression. It is important to recognize the onset of DM in order to diminish quality of life issues that may occur. It was hypothesized that 50% of adult neuro-oncology patients undergoing treatment at a large international NCI-designated outpatient clinic are on steroid therapy. This study seeks to determine the use of steroid therapy in the neuro-oncology population and the incidence of DM while on steroids. Further, guidelines for tapering steroids were developed and longitudinal evaluations collected. This exploratory study was performed with a convenient sample of 277 adult patients presenting to clinic in January 2005. Data collection included steroid use, dosage, chemotherapy regime, diagnosis and onset, tumor status, and medical/family history of DM. Patients on steroid therapy were evaluated for initiation of a taper with patient education instructions provided. Blood glucose, steroid administra-tion, and health status were monitored longitudinally for six months. Chi-square analysis and t-tests were performed. Logistic regression will be used to assess the joint effect of these factors. Of the 277 patients evaluated, 144 (41%) were on steroid therapy. Fifteen patients had elevated blood glucoses, with 11 initiated on DM appropriate interventions. During the six months, a total of 25 patients had increased blood glucoses with 18 initiated on therapeutic interventions. Thirty-three patients on steroid therapy were lost to follow-up due to neurologic deterioration and death. As hypothesized, a large percentage of neuro-oncology patients are on steroid therapy. While patients may not suffer from DM early in their disease course, the majority of patients (p , 0.05) who developed DM had tumor progression nearing their end-of-life. Vigilance to prevent DM complications is necessary when administering steroids in all patients, especially those at the end-of-life. Further study is warranted for comorbidities affecting quality of life in this vulnerable population.

QL-17. FROM BRAIN TO BEHAVIOR: NEUROPSYCHOLOGICAL PROFILES OF GLIOMASChristina Blodgett Dycus, Margaret Primeau, Henry Brown, and Vikram Prabhu; Loyola University Medical Center, Maywood, IL, USA

Malignant brain tumors can present with a variety of behavioral mani-festations. Subtyping cognitive and emotional profiles allows for further understanding of gliomas and brain functioning, which can help direct treatment decisions and improve patient quality of life. Currently, most behavioral data correlated to this type of brain dysregulation has focused on neurological examination. Neuropsychological evaluation can allow for detection of subtle cognitive and emotional symptoms which may not be evident on neurological examination. New research on behavioral (cognitive and emotional) profiles related to particular brain tumors and their loca-tions will be presented. A sample of patients with gliomas and subsequent neurosurgical treatment underwent neuropsychological investigations pre and post operatively. A database was created, which included pathology of tumor, location of tumor, neuropsychological results for specific cognitive domains and emotional functioning pre- and postsurgery, and postsurgical treatments. Analyses of the presurgical data found that all patients (n 5 14) evidenced some cognitive dysregulation, although not necessarily to the point of impairment. A significant relationship was found between pathol-ogy of the tumor and language functioning for both confrontation nam-ing (p 5 0.05) and verbal fluency (p 5 0.01) regardless of the hemisphere of involvement (left 5 57%). Specifically, the more infiltrative the tumor, then a greater degree of dysregulation was noted on these particular tasks, with verbal fluency being the more sensitive measure. These results will be presented and compared to the postsurgical data to investigate intrasubject changes in functioning. Additional neuropsychological profiles will be pre-sented. The goal is to generalize these findings to include as part of the cost/benefit analysis of the intervention for the clinical patient.

QL-18. WERNICKE’S ENCEPHALOPATHY IN CANCER PATIENTS: A CASE SERIES FROM M.D. ANDERSON CANCER CENTERSheng-Han Kuo,1 Gregory Fuller,2 J. Matthew Debnam,2 and John De Groot2; 1Baylor College of Medicine, Houston, TX, USA; 2University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Wernicke’s encephalopathy (WE) is a neurological emergency. A high index of suspicion for this acute encephalopathy in cancer patients is essen-tial because (1) cancer patients are at high risk of this complication due to chronic malnutrition, chemotherapy and rapidly growing tumor consump-tion of thiamine, (2) critically ill patients may not present with the classic triad of symptoms, (3) early empiric treatment with thiamine is potentially life-saving and has no adverse effects. Here we describe the clinical his-tory, predisposing risk factors, and imaging characteristics of 4 cases of WE recently identified at M.D. Anderson Cancer Center. All of our patients had rapid growing cancers and were undergoing treatment. None of the patients were alcoholic. All of them had poor nutritional status, in part due

to chronic nausea from chemotherapy administration. Acute confusion was the most common symptom. Magnetic resonance imaging (MRI) studies of the brain revealed classic findings, including restricted diffusion and T2/FLAIR hyperintensity in medial thalami and periaqueductal gray. In addi-tion to classic imaging findings, one patient had more atypical changes of T2/FLAIR hyperintenisty in the left caudate head, left lentiform nucleus, and superior vermis. In only half of the cases was WE considered antemor-tem, and only one was empirically treated with thiamine, which rapidly reversed the imaging findings within 7 days and led to clinical improve-ment. Other clinical and radiologic diagnoses under consideration included viral encephalopathy, paraneoplastic encephalitis, non-specific inflamma-tory process, and vascular ischemia. In conclusion, it is crucial to consider WE in the differential diagnosis for all cancer patients with altered mental status. Chronically ill cancer patients with malnutrition are at a high risk of developing WE and should be empirically treated with thiamine.

QL-19. COGNITIVE REHABILITATION IN PATIENTS WITH PRIMARY MALIGNANT BRAIN TUMORS: A CASE STUDY SERIES USING AN ACCEPTANCE-BASED COGNITIVE TRAINING APPROACH (N 5 10)Bart Brigidi,1 Henry Friedman,1 and Renee Raynor2; 1Duke University, Durham, NC, USA; 2Preston Robert Tisch Brain Tumor Center at Duke, Duke University, Durham, NC, USA

Existing medications that are used to treat neurocognitive dysfunction in primary malignant brain tumor patients often are limited by their long-term efficacy, potential for interaction with adjuvant therapies, and side effects. There are no published studies on non-medication approaches for treating neurocognitive dysfunction in adult brain tumor patients. Traditional cogni-tive training has fallen short of being able to transfer gains made on specific tasks or tests in artificial environments to functional improvements in every-day activities. Present in both training and “real-life” experience is, at least at some level, the mild-moderate cognitively-impaired brain tumor patient’s perception of their own impairment and the manner in which they deal with perceived changes from premorbid functioning. Incorporating techniques of acceptance-based behavior therapy into cognitive training provides a frame-work for individuals with mild to moderate neurocognitive impairment to effectively cope with making mistakes and maintain focus, first in artifi-cial training situations, and, then later, outside of this context. In this way, acceptance-based cognitive training may bridge a gap between performance on specific training tasks and functioning in everyday activities. As such, we conducted a case study series on acceptance-based cognitive training. Ten adult primary malignant brain tumor patients referred for treatment at the neuropsychology clinic at The Preston Robert Tisch Brain Tumor Center at Duke completed 1 1/2–hour training sessions on 3 out 7 days per week for 6 weeks either at their home or in a sound-attenuated room in an outpatient clinic setting. Each session consisted of a 1/2 hour of mindfulness and accep-tance training followed by 1 hour of computer-based cognitive training. The research design was an A-B-A design. Participants completed a brief neuropsychological/quality of life test battery consisting of verbal memory, verbal fluency, attention, executive functioning, perceived cognitive ability, acceptance, and quality of life measures at baseline, post-intervention, and 3-months post-intervention. Pre- and post-intervention data for 10 adult pri-mary CNS tumor patients with stable disease will be presented. Preliminary analyses show modest improvement on computer training tasks related to verbal attention and working memory; however, over the course of the train-ing and follow-up periods, ratings of acceptance of making mistakes due to cognitive dysfunction showed marked improvement. These results reveal the possibility that adult survivors of brain cancer may benefit from cognitive training with a structured, coping skills component. This approach should be tested in a more rigorous randomized, controlled design and in combi-nation with medication used to treat neurocognitive deficits. If successful, perhaps this method of changing acceptance of cognitive deficits may be used as a template for changing acceptance of other aspects of the disease that impact quality of life.

QL-20. CORRELATES OF PERCEIVED SOCIAL COMPETENCY IN PEDIATRIC BRAIN TUMOR PATIENTSKevin Wong,1 Kristina Hardy,2 Victoria Willard,1 Melanie Bonner,2 and Sridharan Gururangan3; 1Psychology, Duke University, Durham, NC, USA; 2Duke University, Durham, NC, USA; 3Duke University, NC, USA

Survivors of childhood brain tumors consistently report difficulties with social functioning, including isolation and few friends. Consequences of these struggles extend into adulthood as survivors are less likely to achieve the normal social milestones of adult life, including marriage, stable employ-ment and living independently. However, despite reports of social function-ing deficits, many aspects of social functioning have yet to be studied with child and adolescent cancer survivors. In particular, since most studies have examined patients’ social functioning from the perspective of parents or



teachers, very little is known about pediatric brain tumor patients’ own perceptions of their social competence. The primary objectives of the current study are to: (1) assess differences in social competence between patients at baseline and survivors and (2) to determine what risk factors (medical, functional, nonverbal cognitive impairments) are associated with social functioning. From a larger longitudinal study examining the cognitive and psychosocial functioning of pediatric brain tumor patients, 31 partici-pants (17 females and 14 males) ranging in age from 7–16 (M 5 11.8, SD 5 2.44) were selected for participation in the current study. Participants completed a comprehensive neuropsychological assessment which included intellectual (WISC-III), academic (WJ-R), visual-motor (VMI), and psycho-logical screening measures (Perceived Competency Scale, Revised Children’s Manifest Anxiety Scale). Moreover, participants’ parents reported on fam-ily functioning (Family Environment Scale), their own psychological symp-toms (Brief Symptom Inventory), and their children’s emotion and behavior using established questionnaire measures. Fifteen (48.4%) participants were assessed at baseline (i.e., within 60 days of diagnosis) and 16 (51.6%) were assessed at least six months off treatment. Diagnoses were varied, with 7 (22.6%) patients presenting with a history of medulloblastoma, 6 (19.4%) with pilocytic astrocytoma, 4 (12.9%) with ependymoma, and 14 (45.1%) with other tumor types. Age at diagnosis ranged from 1–16 (M 5 9.5, SD 5 3.58). Time since diagnosis ranged from 0 to 165 months at the time of the study. Compared to children assessed at baseline, survivors reported signifi-cantly lower perceived social acceptance, physical appearance, and global self-worth, and a trend for lower perceived athletic competence. Parents of children at baseline rated their children as significantly less aggressive than survivors, but there were no differences between groups in parents’ perception of their children’s social skills. However, as children’s perceived social acceptance decreased, parents’ viewed their children as significantly more depressed and prone to somatic complaints. Finally, several medical and parent psychosocial variables correlated with patients’ perceived social competence. For example, parents with greater hostility had children who reported significantly less social competence and acceptance. Moreover, children diagnosed at a younger age reported significantly worse general social competence. Findings from this study indicate that pediatric brain tumor survivors may experience concerns about their social competence compared to children at baseline, though these differences are not reflected in parents’ observations of their children’s sociability. The study highlights the need for more detailed and specific research on the social functioning of pediatric brain tumor survivors, particularly from the perspective of the survivors themselves.

QL-21. CHARACTERIZING NEUROBEHAVIORAL AND SOCIAL CAUSES OF SCHOOL PROBLEMS IN SURVIVORS OF PEDIATRIC BRAIN TUMORS: THE SURVIVOR EDUCATION AND REINTEGRATION SUPPORT PROGRAMBeverly Barkon,1 Sherri Scheboth,2 and Regina Jakacki3; 1Education, Carlow University, Pittsburgh, PA, USA; 2Child Psychiatry, Carlow University, Pittsburgh, PA, USA; 3Hematology/Oncology, Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA

Survivors of pediatric brain tumors are known to have a constellation of neuropsychological, social and cognitive late effects from tumor and treat-ment. Schools and teachers have insufficient knowledge of and experience with the unique sets of school-related challenges faced by these children. Children and parents need support in negotiating the school terrain in order to get appropriate education services for these students. The SERSP is a multidisciplinary support program that provides in-service training for school administrators and teachers, diagnostic and educational remedia-tion, and psychosocial support for survivors and their families. Prospective studies of survivors were conducted over the course of 1–4 years. Serial evaluations over time included: Woodcock-Johnson III Tests of Cognitive Ability, Woodcock-Johnson III: Tests of Academic Achievement, Barkon School Experience Questionnaire, and structured interviews with parents and students. The case studies focused on the needs of each family and child and included educational evaluation, advocacy, social skills program-ming, transition, and academic support. Children continued to experience a range of school related difficulties up to six years post-treatment. The difficulties students demonstrated included problems of working memory, cognitive efficiency, academic fluency, and social skills. Executive processing problems were common, particularly organizational, and planning issues. Students had difficulty completing schoolwork in a timely fashion and did not remember directions, especially involving newly learned information. It took them longer to learn new information and skills than would be expected of students of their ages and they appeared more confused about how to accomplish designated tasks. Difficulties that students experienced early in the post-treatment period evolve over time, but remain traceable to tumor and treatment effects. Because the time period between treatment and school difficulties may be protracted, schools did not always credit the origin of the difficulty or assumed the student should have already compen-sated for that problem when they had not or could not do so. Schools were not effectively dealing with the problems and had disconnected the student’s current academic and social challenges from the treatment origins. Schools

did not effectively address the ongoing difficulties presented. Families and schools require ongoing support and education to adequately address the needs of survivors. Ongoing advice and oversight by individuals familiar with the school-related issues of survivors is required until the needs of sur-vivors become an integrated part of training for school professionals.

QL-22. NEUROPSYCHOLOGICAL FOLLOW-UP OF HEAD START II SURVIVORSStephen Sands,1 Jennifer Oberg,2 and Jonathan Finlay3; 1Pediatrics and Psychiatry, Columbia University Medical Center, New York, NY, USA; 2New York, NY, USA; 3University of Southern California, Los Angeles, CA, USA

Our objective was to evaluate the neuropsychological late effects arising from having been treated on the Head Start II protocol between 1997—2002. Patients underwent baseline neuropsychological assessment prior to autologous stem cell transplantation and bi-annually thereafter. Patients received induction chemotherapy (five cycles of vincristine, cispla-tin, cyclophosphamide, and etoposide) before undergoing myeloablative consolidation using carboplatin, thiotepa, and etoposide. Irradiation was used to treat either residual disease at completion of induction or relapse upon follow-up. Baseline neuropsychological assessments were obtained on 48 of 73 (66%) patients (mean age 40.76 months; SD 5 24). Twenty-nine males and 19 females were diagnosed with either medulloblastoma/PNET 5 29, ependymoma 5 5, pineoblastoma 5 4, ATRT 5 2, GBM 5 2, or other56. At baseline testing, patients functioned within the lower limits of the average range of overall intelligence compared with their age peers (Full Scale IQ 5 89.84, SD 5 18.29), as well as verbal and non-verbal intelligence (Verbal IQ 5 95.86, SD 5 17.28; Performance IQ 5 95.38, SD 5 16.90, respectively). Additionally, their expressive language skills were within the average range (PPVT 5 101.69, SD 5 18.11); whereas their visual-motor integration abilities were within the low average range (VMI 5 87.50, SD 5 9.76). Parents reported their children displayed social-emotional and behav-ioral functioning well within normal limits (CBCL 5 45.82; SD 5 7.77). Of the 48 patients assessed before undergoing transplantation, 18 (38%) passed away prior to being re-evaluated. Follow-up neuropsychological testing was obtained on 23 of 30 (77%) survivors (mean age 79.3 months; SD 5 24.58). Independent samples t-test comparing baseline functioning between the 25 for whom baseline-only testing was obtained and the 23 at baseline (for whom follow-up testing was subsequently obtained) did not reveal signifi-cant differences between the two groups on any of the neuropsychological measures. A t-test analysis of these 23 survivors for whom both baseline and follow-up neuropsychological assessments were obtained indicated that their overall, verbal and non-verbal intellectual functioning, did not decline from baseline to follow-up. Additional analyses did not reveal significant correlations between worsening functioning and either young age at diagno-sis or length of follow-up. Descriptive analyses of a subset of survivors at fol-low-up indicated average verbal and visual memory abilities, while academic achievement skills (reading, spelling, and math) were within the lower limits of the average range. Results from this study indicate that induction and myeloablative consolidation chemotherapy followed by transplantation may avoid or delay cranio-spinal irradiation while also preserving intellectual functioning. Furthermore, those who were younger at diagnosis were not at increased risk for neuropsychological deficits, nor did a significant degree of deficits emerge over time. These three findings are in contradistinction to expectations based upon published literature and may be influenced by the fact that 17 (74%) of these 23 survivors were never irradiated. Continued follow-up assessments of these survivors is warranted to continue monitor-ing their neuropsychological functioning to determine if these encouraging findings persist beyond the 3 year follow-up in this current study.

QL-23. CHANGES IN FUNCTIONAL CONNECTIVITY AFTER TUMOR RESECTION IN BRAIN TUMOR PATIENTS: A MAGNETOENCEPHALOGRAPHY STUDYLinda Douw,1 Hans Baayen,1 Jaap Reijneveld,1 Ingeborg Bosma,2 Kees Stam,1 Pieter Vandertop,1 Jan Heimans,1 Jan De Munck,1 and Martin Klein1; 1VU University Medical Centre, Amsterdam, Netherlands; 2University of Amsterdam, Amsterdam, Netherlands

Higher cognitive functions require the integrated action of many, sometimes widely distributed specialized brain areas relying on functional interactions; a concept known as functional connectivity. In brain tumor patients, we have previously observed resting state differences in functional connectivity relative to healthy controls, which were not confined to regions close to the tumor. More importantly, we found impaired cognition to be significantly associated with functional connectivity in low-grade glioma patients. However, it is still unknown how brain tumors cause these changes in functional connectivity and cognition. The present study investigates whether tumor resection affects and possibly restores functional connec-tivity patterns in brain tumor patients. We recruited 15 patients with his-



topathologically confirmed brain tumors (8 astrocytomas [4 WHO grade II, 4 grade III], 3 oligodendrogliomas [2 grade B, 1 grade C], 2 glioblas-tomas, and 2 WHO grade I meningiomas; 9 left-sided tumors; 7 males; mean age 39, range 27–66), which have been described elsewhere. Patients were newly diagnosed and all presented with epileptic seizures, for which neurosurgery was warranted. Patients underwent (partial) tumor resection aiming to relieve epileptic seizures. They underwent magnetoencephalog-raphy (MEG) recordings two weeks prior to and six months following surgery. Resting state magnetic fields were recorded using a 151-channel whole-head MEG system. Functional connectivity was assessed by using the phase lag index (PLI), which measures the asymmetry of the distribu-tion of phase differences between two MEG signals and is not sensitive to volume conduction effects. Computation of the PLI was based on 4 carefully selected artifact free epochs of 6.5 seconds (4096 samples). PLI scores were calculated between all channels and averaged to a number of short-distance (within MEG brain regions) and long-distance (between MEG brain regions) scores in 7 frequency bands. Wilcoxon signed-rank tests were performed to investigate whether postoperative PLIs differed sig-nificantly from preoperative PLIs. We found several significant PLI changes after tumor resection, most of which involved long-distance connections. Separate analyses were conducted based on tumor lateralization. In patients with tumors in the dominant hemisphere, surgery induced a decrease of delta (0.5–4 Hz), theta (4–8 Hz), and lower alpha band (8–10 Hz) PLIs, and an increase in the upper alpha (10–13 Hz) and gamma band (30–80 Hz). Patients with right-sided tumors showed significant increases and decreases of PLI in various frequency bands throughout the brain. Interestingly, the majority of PLI changes occurred within the left hemisphere or between hemispheres, regardless of tumor lateralization. Although we cannot con-clude that tumor resection has a beneficial effect on functional connectivity, these results indicate that (1) alterations in functional connectivity occur in brain tumor patients after tumor resection, (2) different patterns of changes are present in left and right hemisphere tumor patients, and (3) changes in functional connectivity are not only seen around the resection site. Future research efforts will focus on further elucidating the effect of tumor resec-tion on functional connectivity. Furthermore, we will investigate changes in functional connectivity and cognition as a function of tumor treatment, including radiation therapy, chemotherapy, and antiepileptic drug use.

QL-24. LONG-TERM SAFETY AND STEROID-SPARING POTENTIAL OF CORTICORELIN ACETATE INJECTION FOR TREATMENT OF PERITUMORAL BRAIN EDEMA: THIRD INTERIM REPORT OF AN OPEN-LABEL STUDY AS PART OF A PHASE III PROGRAMLawrence Recht,1 Lazslo Mechtler,2 John Alksne,3 Edward Arenson,4 Nina Paleologos,5 Nicholas Avgeropoulos,6 Adilia Hormigo,7 James Perry,8 Jeffrey Raizer,9 H.Ian Robins,10 Eric Wong,11 Michael Shulman,12 and Lisa Carr13; 1Stanford University, Stanford, CA, USA; 2Dent Neurologic Institute, Amherst, NY, USA; 3Thornton Hospital, University of California, San Diego, La Jolla, CA, USA; 4Center for Brain & Spinal Tumors, Colorado Neurological Institute, Englewood, CO, USA; 5Neuro-Oncology Program, Evanston Northwestern Healthcare, Evanston, IL, USA; 6Florida Hospital Cancer Institute, Orlando, FL, USA; 7Memorial Sloan Kettering Cancer Center, New York, NY, USA; 8Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada; 9Northwestern University, Chicago, IL, USA; 10University of Wisconsin Hospital, Madison, WI, USA; 11Beth Israel Deaconess Medical Center, Boston, MA, USA; 12Celtic Pharma Development, San Francisco, CA, USA; 13Neurobiological Technologies, Inc., Emeryville, CA, USA

Corticorelin acetate injection is a synthetic peptide with an amino acid sequence identical to human corticotropin-releasing factor (hCRF). Experimental and early clinical studies indicate corticorelin acetate may have steroid-sparing potential for the treatment of peritumoral brain edema (PBE). Preclinical toxicology indicates corticorelin acetate may be safe and devoid of side effects associated with dexamethasone. Initial positive clinical data from earlier studies need to be confirmed in ongoing Phase III controlled clinical trials. To evaluate long-term safety, tolerability, and steroid-sparing potential of corticorelin acetate in patients with primary or metastatic brain tumors (BT) and PBE. Following participation in one of 2 randomized, double-blind Phase III trials comparing corticorelin acetate to dexamethasone or placebo, patients with primary or metastatic BTs, PBE, and Karnofsky Performance Scores (KPS) > 50 took SC injections bid of 1.0 mg corticorelin acetate in an ongoing open-label study. We tapered dex-amethasone maximally as tolerated. Patients were assessed every 4 weeks for dexamethasone dose, steroid and other side effects, and every 12 weeks for KPS, 10-Item Neurologic Exam Score, and serum cortisol. Net cumula-tive change in dexamethasone dose (NCD) was calculated by integrating changes from baseline for each 4-week visit. Twenty patients (13 men, 7 women; age 32 to 69, median 52) completed 20–64 weeks of treatment (median 36 weeks). Typically refractory patients who had NCD reduced or took no dexamethasone included 4/5 (80%) . 55, 6/11(55%) with recurrent BT, and 16/19(84%) who had failed prior attempts. In 7 of 12 patients with

NCD reduction, plus one who took no dexamethasone for 24 weeks, 20 instances of baseline steroid side effects resolved or improved. Six patients with increased NCD experienced worsening of 5, onset of 4, and resolution of 3 steroid AEs. KPS and 10-Item Neurologic Exam Scores were stable or improved in most patients with NCD reduction at 12, 24, 36, and 48 weeks. Serum cortisol levels were within or below the reference range (5–25 mg/dL) except one, which was moderately elevated at 39 mg/dL and then normalized at 25 mg/dL off dexamethasone for 16 weeks. Corticorelin acetate appears safe and well tolerated. There were 4 deaths in NCD increased patients and 1 death in the NCD reduced, group—none related to study drug. There were no SAEs related to study drug. This third interim report provides further evidence of the long-term safety and steroid-sparing potential of corticorelin acetate in patients with primary or metastatic brain tumors and PBE. In the first 20 patients to complete at least 20 weeks of treatment, corticorelin acetate did not induce hypercortisolism, and was associated with reduced exposure to dexamethasone, resolution or improvement of steroid side effects plus neurologic improvement or stability.

QL-25. THE IMPACT OF DIAGNOSIS AND TREATMENT ON THE HIGH-GRADE BRAIN TUMOR FAMILYMichele Lucas,1 and Peter Maramaldi2; 1Neuro-Oncology, Massachusetts General Hospital, Boston, MA, USA; 2Social Service, Massachusetts General Hospital, Boston, MA, USA

This paper reports a pilot study currently being implemented in a large research hospital in the Northeast. It represents an innovative attempt to collect empirical data to describe the impact of brain tumors and their treatment on patients, with an important caveat, the inclusion of patients’ families. The impact of high-grade glioma on the family creates power shifts, reallocation of responsibilities, and may have significant financial consequences long after the patient’s death. The unit of analysis will be the high-grade brain tumor family (HGBTF), defined as the patient and her/his identified family. Although family members are often involved in diagnosis and treatment, little empirical evidence is available to describe HGBTF’s quality of life or responses to symptom management. The high morbidity rate associated with the diagnosis of a high-grade glioma compounds the stress and anxiety on the patient and their family. Patients’ neurocognitive impairments often necessitate the involvement of HGBTF in clinical deci-sion making and data collection on quality of life. Literature indicates that current measures of patients’ functional status fail to capture the subtlety of the cognitive and behavioral changes related to diagnosis and treatment. Participation in survey research is often unfeasible for patients given the debilitating nature of a high-grade glioma. Additionally, elevated stress and anxiety may contribute to difficulty in responding to direct questions from providers. In our clinical experience, we find that patients are fre-quently unable to read, comprehend, and/or remain focused, and suffer from extreme chronic fatigue as a side effect of treatment. The purpose of this study is to test the feasibility of collecting data on HGBTF in a minimally intrusive manner to provide empirical evidence describing the impact of brain tumors and their treatments on the HGBTF. Findings will be used to develop adjuvant psychosocial interventions that will contribute to gains in quality of life and favorable outcomes in symptom management. In the absence of a brain tumor model, we followed the recommendations of the 2005 NCI BTPRG report and looked to the literature on traumatic brain injury (TBI). The conceptual model for this study, the McMaster Model of Family Functioning (MMFF) has been used with TBI populations. The MMFF describes multiple aspects of family functioning, including problem solving, communication, roles, affective responsiveness, affective involve-ment, and behavior control. The study design uses two concurrent focus groups to explore patient and family members’ experiences related to the diagnosis and treatment of the high-grade glioma. One group consists of 10–12 patients with histologically confirmed WHO grade III/IV and IV/IV tumors and the other of 10–12 matched family representatives. We will conduct the groups simultaneously to lessen the burden on the HGBTF. If we interviewed only patients, someone would likely have to assist in trans-porting them. If we conducted groups with family members, caregiving responsibilities or respite would have to be arranged, which would further complicate attendance for participants. This proposed pilot study will sup-port our grant request (pending approval) of the Kenneth B. Schwartz Cen-ter to conduct additional focus groups.



QL-26. COMPARING RBANS NEUROPSYCHOLOGICAL TASKS TO OPTIMIZE BRIEF COGNITIVE BATTERIES FOR CLINICAL TRIALSSarah K. Lageman,1 Jane H. Cerhan,1 Paul D. Brown,2 S. Keith Anderson,3 Wenting Wu,3 and Dona E.C. Locke4; 1Psychiatry and Psychology, Mayo Clinic, Rochester, MN, USA; 2Mayo Foundation for Medical Education and Research, Rochester, MN, USA; 3Cancer Center Statistics, Mayo Clinic, Rochester, MN, USA; 4Psychology, Mayo Clinic, Scottsdale, AZ, USA

Neuropsychological tests are being used increasingly as outcome mea-sures in clinical trials of brain tumor therapies. Cognitive change has been shown to be an early indicator of tumor progression, sometimes before it is detectable on imaging studies. Cognitive functioning is also a strong pre-dictor of quality of life in patients with brain tumors. Selecting specific cognitive tasks to use in clinical trials can be challenging; pitting the sensi-tivity of sampling multiple domains against the need for brevity in research batteries. To inform development of economical batteries for clinical trials, we compared brain tumor patients’ performances across twelve cognitive tasks from the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). This is a retrospective study of brain tumor patients who were receiving radiation therapy at Mayo Clinic Rochester and completed the RBANS as part of a neuropsychological consultation. The RBANS is a well standardized battery sampling multiple cognitive domains using twelve subtests with widely-used task formats. The twelve subtests are List Learn-ing, Story Memory, Figure Copy, Line Orientation, Picture Naming, Seman-tic Fluency, Digit Span, Coding, List Recall, List Recognition, Story Recall, and Figure Recall. A conservative threshold of two standard deviations below published means was used to indicate impairment. A total of 68 brain tumor patients were studied (57% high-grade glioma, 43% other). Forty patients (58.8%) were impaired on at least one subtest. Additional analy-ses were conducted in this subgroup of forty impaired patients. The Cod-ing, Story Recall, and List Recognition subtests were the most frequently impaired at 52.5%, 52.5%, and 45% of the impaired subgroup respectively. A combination of four subtests (Figure Copy, Coding, List Recognition, and Story Recall) captured 90% (36/40) of the impaired subgroup, while the best two-subtest combination (Figure Copy and Story Recall) identified 70% (28/40). A subset of four of twelve cognitive tasks from the RBANS captured 90% of brain tumor patients identified as impaired with the larger, complete RBANS battery. These results suggest visuo-construction, process-ing speed, and verbal memory measures may be the most important domains to assess when evaluating cognitive change in this population. Analyses such as this help inform the design of sensitive but brief cognitive batteries for use in clinical trials.

QL-27. ALL PEDIATRIC POSTERIOR FOSSA TUMOR PATIENTS SUFFER FROM LONG-TERM NEUROCOGNITIVE DEFICITSFriederike Blankenburg, Ines Wuithschick, Hermann Baqué, Guenter Henze, and Pablo Hernaiz Driever; Department of Pediatric Oncology/Hematology, Charité- Universitaetsmedizin Berlin, Virchow Hospital, Berlin, Germany

Long-term survivors of pediatric posterior fossa tumors often present with neurological deficits and a cerebellar cognitive affective syndrome. We hypothesized that patients who had not received chemo- and radiotherapy have a better outcome concerning neurocognitive abilities and health related quality of life (HrQL). Seventeen patients with medulloblastoma (MB) and 17 patients with low grade glioma (LGG) in the posterior fossa were evalu-ated. Mean follow-up time was 5.6 yrs in MB patients and 6.4 yrs in LGG patients. Tumors were resected in all patients but only MB patients received chemotherapy and craniospinal irradiation. Neuropsychological tests included the assessment of attention with a non-verbal computer test (TAP), the German version of the Auditory Verbal Learning Test (VLMT), the Beery Developmental Test of Visual-Motor Integration (VMI), and the Ger-man version of the Wechsler Intelligence Scale for Children (HAWIK-III). Patients were evaluated clinically and responded to a validated question-naire about health related quality of life (KINDL-R questionnaire). Results: (1) Tonic alertness was significantly reduced in both groups. Phasic alertness (the ability to increase attention in expectance of an important stimulus) is conserved in both groups. Vigilance is reduced in both groups, but more pronounced in MB patients. (2) In three of six subscale tests for verbal learn-ing and memory (VLMT) MB patients had significantly worse results than LGG patients. (3) Deficits in visual motor integration (VMI) were found in both patient groups. However, deficits reached significance only in MB patients. (4) In all HAWIK subscale tests MB patients scored at least one SD below age range and were classified as having a learning disability. LGG patients scored low in performance IQ, the other subscales were within normal range. Differences in overall and verbal IQ were significant between MB and LGG patients. (5) The KINDL-R questionnaire showed a tendency towards reduced HrQL in MB and slightly reduced HrQL in LGG patients, but all results were still within normal range. An increased number of medi-

cal problems was associated with a significant decrease in emotional well-being in both patient groups. Several years after diagnosis all patients who suffered from a posterior fossa tumor present with significantly reduced attention, deficits in visual motor integration, auditory learning, memory and reduced IQ. Deficits are significantly more apparent in patients who received chemo- and radiotherapy. Health related quality of life is slightly, but not significantly reduced in all patients. We suggest that not MB patients only, but all posterior fossa tumor patients need long-term specific reha-bilitation programs emphasizing on improvement of attention as well as adapted psychological care.

QL-28. BASELINE COGNITIVE IMPAIRMENT IN PATIENTS WITH SINGLE BRAIN METASTASISMariana Witgert, Frederick Lang, Dima Suki, Eric Chang, Christina Meyers, and Jeffrey Wefel; University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Brain metastasis is the most common intracranial tumor. This investiga-tion aimed to determine the baseline level of impairment in neurocognitive function (NCF) in patients with single brain metastasis prior to undergoing treatment of that metastasis. In addition, relations were determined between NCF and disease variables, such as lesion volume and location and extent of systemic disease. Between 1998 and 2005, seventy-eight patients underwent formal assessment of NCF by a qualified neuropsychologist or trained staff prior to undergoing surgical resection or stereotactic radiosurgery (SRS) for a single brain metastasis. Patients had not received any prior treatment for their brain tumor and had a KPS > 70 and a tumor diameter < 3 cm. Logis-tic regression was used to determine predictors of cognitive impairment. At baseline, 42% of patients exhibited impairment on one or more measures of NCF. The domains most frequently impaired were learning of verbal infor-mation (38.5%), motor dexterity (20%), attention (19.2%), and executive function (15.4%). Cognitive impairment on the most frequently impaired measure (Hopkins Verbal Learning Test Revised: Immediate Recall condi-tion) was not related to the location of the tumor or to the extent or activ-ity of systemic disease, nor was it related to past treatment of the primary cancer, including chemotherapy. However, it was predicted by lesion volume (odds ratio 5 0.987, p 5 0.044). Lesion volume also predicted impairment on a measure of attention (Trail Making Test Part A: odds ratio 5 0.985, p 5 0.047), but not on measures of motor dexterity (Grooved Pegboard) or executive function (Controlled Oral Word Association, Trail Making Test Part B). In addition, past surgical intervention for the primary cancer predicted impairment on a motor dexterity task (Grooved Pegboard) with the nondominant hand (odds ratio 5 0.250, p 5 0.047). In this highly select group of single brain metastasis patients with generally favorable charac-teristics, over one-third exhibited impairments in NCF at baseline. These results are consistent with previous research on multiple metastases. Future studies will investigate the impact of surgical resection versus SRS on NCF and whether pretreatment NCF correlates with survival time or time to neurologic progression in patients with a single brain metastasis.

QL-29. COMPARISON OF LEVETIRACETAM MONOTHERAPY WITH CONTINUED PHENYTOIN THERAPY AFTER SUPRATENTORIAL CRANIOTOMIES IN PATIENTS WITH A HISTORY OF TUMOR-RELATED SEIZURES: A PHASE II, PILOT STUDYDaniel Lim,1 Phiroz Tarapore,1 Edward F. Chang,2 Burt Marlene,1 Lenna Chakalian,1 Nicholas Barbaro,1 Susan Chang,1 Kathleen Lamborn,1 and Michael McDermott1; 1University of California, San Francisco, San Francisco, CA, USA; 2Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA

Our objective was to evaluate the safety and feasibility of seizure pro-phylaxis with levetiracetam monotherapy in post-operative supratentorial glioma patients taking phenytoin preoperatively for tumor-related seizures. Patients met inclusion criteria if they had seizure history attributable to supratentorial glioma, were taking phenytoin monotherapy for seizure prophylaxis, were to undergo a craniotomy as standard management, had at most one previous resection, and were over 18 years of age. Patients were also free of other comorbidities. Thirty patients were enrolled over a 13-month period. After signing an approved IRB consent form, patients were randomized in a 2:1 ratio to initiate levetiracetam therapy within 24 hours of surgery or to continue phenytoin therapy. Patients were followed for 6 months post-operatively with 2 telephone surveys and mail-in ques-tionnaires conducted at 3 month intervals. The study’s anticipated conclu-sion is October, 2007. Of the 30 patients enrolled, 20 were randomized to levetiracetam and 10 to phenytoin. There was 1 fatality within the leveti-racetam group from complications of the primary pathology. As of May 1, 2007, 25 of 30 patients had completed the follow-up period. In the leveti-racetam group, 2 patients had seizures reported: 1 responded to increased dosage; 1 followed self-discontinuation of therapy and, with resumption



of therapy, seizures did not recur. No seizures were reported in the pheny-toin group. Reported side effects were as follows (% levetiracetam group/% phenytoin group): dizziness (0/29), excessive sleepiness (17/57), difficulty with coordination (0/43), depression (28/43), insomnia (44/43), and mood instability (5/0). No side effects required discontinuation of assigned therapy in either group. The pilot data presented here suggest that levetiracetam is a safe agent for seizure monotherapy following craniotomy for supratentorial glioma. Its side effect profile may also be less morbid than that of phenytoin. A large-scale, double-blinded, randomized control trial of levetiracetam vs. phenytoin should now be conducted to assess equivalence in seizure control and better determine differences in side effect profiles.

QL-30. GENDER DIFFERENCES IN CAREGIVER BURDEN IN FAMILY CAREGIVERS IN NEURO-ONCOLOGYElana Farace, Jonas Sheehan, Zarui Melikyan, and Jeff Hollier; Penn State University, Hershey, PA, USA

Previous research in other diseases has shown a gender difference in caregiving (e.g., Navaie-Waliser, 2002) showing that women are more likely to be the primary caregivers, provide more intensive and complex care, have difficulty with care provision and balancing caregiving with other family and employment responsibilities, suffer from poorer emotional health sec-ondary to caregiving, and cope with caregiving responsibilities by forgo-ing respite participation and engaging in increased religious activities. The present study is the first to examine gender differences in caregiver strain in family caregivers of brain tumor patients. Sixty-two brain tumor patients (26 male), age: 20–84 (Mean 5 57.4; SD 5 16.4). 85% right-handed, 98% Caucasian, education: 6–16 (Mean 5 12; SD 5 2). Patients had primary or metastatic tumors of various localizations and different grades of malig-nancy. Procedures included biopsy, resection, or Gamma Knife; caregiver strain was assessed prior to and six weeks following the procedure. T-test and Pearson’s correlations were performed. Prior to (F 5 4.798, p 5 0.043) and following (F 5 13.417, p 5 0.002) the procedure, male caregivers indi-cated having more strain and their responses showed greater variability (SD 5 3.812 vs. SD 5 1.703 prior, and SD 5 3.492 vs. SD 5 1.265 after the surgery) then females. Perceived caregiver strain in females was shown to be significantly positively correlated with patient’s tumor grade (r 5 0.658, p , 0.05) suggesting that caregiver burden increased with disease severity. Qualitative item analysis showed that males were most likely to report being overwhelmed with adjustments to the whole disease situation. Male caregiv-ers reported significantly higher caregiver strain both before and after the surgery than did female caregivers. Although women’s caregiving burden was positively associated with increased disease burden, male caregiving strain was reported to be significant and overwhelming even in relatively benign disease situations. Suggestions for why caregiving would be more burdensome to men unlike previous reports of chronic illness will be dis-cussed. Present findings indicate the necessity for support and education especially for male caregivers.

QL-31. LONG-TERM MEDICAL, PSYCHOLOGICAL, AND EDUCATIONAL ISSUES IN PEDIATRIC LOW-GRADE GLIOMAS TREATED WITH SURGERY ONLYChristopher Turner,1 Heather Begley,2 Christine Chordas,1 Cori Cieurzo,1 Celiane Rey-Casserly,3 Brian Delaney,1 Leslie Kim Cunningham,1 Scott Pomeroy,4 Nicole Ullrich,4 Liliana Goumnerova,5 R. Michael Scott,5 Karen Marcus,6 William Fletcher,1 Susan Chi,1 and Mark Kieran1; 1Pediatric Oncology, Dana-Farber/Children’s Hospital Cancer Care, Boston, MA, USA; 2Tufts University School of Medicine, Boston, MA, USA; 3Department of Psychiatry, Children’s Hospital of Boston, Boston, MA, USA; 4Department of Neurology, Children’s Hospital of Boston, Boston, MA, USA; 5Department of Neurosurgery, Children’s Hospital of Boston, Boston, MA, USA; 6Joint Center for Radiation Oncology, Dana-Farber/ Children’s Hospital Cancer Care, Boston, MA, USA

Low-grade gliomas (LGG) are the most common group of pediatric brain tumors and comprise the largest survivor cohort. Surgical resec-tion is often the only treatment necessary, and survival rates for children with completely resected LGG approach 90%–100%. Historically, many “surgery-only” LGG patients have not been followed by oncology-based survivorship clinics as many of these clinics focus on survivors of LGG who received radiation therapy and/or chemotherapy. Thus, despite the large percentage of LGG survivors treated with surgery only, there is a paucity of literature regarding the quality of survivorship for this group of survivors. The goal of this study was to characterize the multidimensional experience of survivors of pediatric LGG treated with surgical resection only and followed by the Pediatric Neuro-Oncology Outcomes Program at Dana-Farber/Children’s Hospital Cancer Center (DF/CHCC). A retro-spective medical record review was performed to collect multidimensional data from LGG surgery only patients seen at least once between January 2003 and December 2005 within the DFCI’s Outcomes Program. Eligible

patients were required to have surgical diagnoses of a LGG before 22 years of age, received no additional tumor-directed therapy except surgery, and be at least two-years from diagnosis. Multidimensional data was collected using a standardized abstraction form and analyzed using standard data reduction techniques and descriptive statistics. Medical records of 72 (M 5 34, F 5 38) surgery-only LGG survivors were reviewed. The median patient age at time of abstraction was 15.5 years (range 4 to 33) and the median time since diagnosis was 8.0 years (range 2.8–19.3). Diagnoses included 38 pilocytic astrocytomas, 5 fibrillary astrocytomas, 9 gangliogliomas, 2 oligodendrogliomas, and 18 LGG not otherwise specified. Primary LGG locations were posterior fossa (47%), cortical (33%), diencephalic/ brainstem (14%), and intraventricular (6%). Post-treatment medical issues were experienced by 93% of survivors and included: motor (60%) or gait (35%) dysfunction, visual problems (43%), chronic pain (36%), seizure disorders (32%), and problems with weight (29%). At least one or more significant psychosocial issues were identified in 72% of surgery-only LGG survivors. These issues included depression (35%), anxiety (42%), behav-ioral problems (28%), and social difficulties (24%). There were 8 patients who expressed suicide ideation and 3 patients with actual suicide attempts. Special education services were utilized by at least half of the 72 patients. Results of formal neuropsychological testing were recorded on 51 patients. Of those tested, the mean full scale IQ was 97 (median of 97, range 58–147). However, the number of survivors scoring at 85 or less (1 standard deviation below the mean) was twice the expected number in the normative popu-lation (p 5 0.011). The majority of LGG surgery-only survivors followed within DF/CHCC’s Pediatric Neuro-Oncology Outcomes Clinic experience late effects from their previous tumor and/or its treatment. These problems often necessitated multiple long-term medical, psychological, and educa-tional interventions. We recognize that there may be potential selection bias due to referral of more complex patients, thus a prospective study is being planned to validate these findings.

QL-32. MAJORITY OF BRAIN TUMOR PATIENTS SHOW NEUROCOGNITIVE IMPROVEMENT AFTER INITIAL SURGICAL RESECTIONElana Farace, Zarui Melikyan, and Jonas Sheehan; Penn State University, Hershey, PA, USA

Although there is increasing interest in neuro-oncology on the effect of radiation and chemotherapy on neurocognitive outcomes, the measure-ment of baseline (prior to surgery) neurocognitive impairment and any post- operative changes in neurocognitive function are often not addressed. With-out an understanding of patients’ presenting neurocognitive deficits and their post-operative changes it is difficult to further evaluate the effects of adjuvant treatments. Therefore, we performed a careful prospective analysis of consecutive newly diagnosed brain tumor patients with pre- and post-operative neuropsychological assessment. The patient population consisted of 77 adult brain tumor patients (35 male), with mean age of 58 years at diagnosis (range 20 to 84 years). 77.5% were right-handed, mean education was 13 years (range 8–22 years) with estimated baseline IQ in the average range (mean 104.9, SD 5 8.9, range 86–120). 87% were Caucasian, 53% were married. Tumor types included craniopharyngiomas, meningiomas, primary neuroectodermal tumors, central neurocytomas, and all forms of glioma (grades I–IV) in a wide range of locations (left, right, callosal, skull base, etc.). 78% had resection, 11% had biopsy, 11% also had Gamma Knife radiosurgery. The neuropsychological battery measured language (verbal fluency—COWA), memory (verbal memory—HVLT), executive func-tion (attention and cognitive switching—Trails A&B), motor (fine motor control—Grooved Pegboard), psychomotor speed (Digit Symbol Subtest), depression (Beck Depression Inventory—II), a standard battery currently being used in several cooperative group trials. Standardized scores were analyzed with ANOVA and Pearson’s correlations. 88% of patients showed improvement of at least one standard deviation in at least one cognitive domain. 68% worsened by at least one standard deviation in at least one domain (percentages overlap due to some patients who improved in one domain and worsened in another). Approximately one-quarter of patients were impaired at baseline (e.g., more than two standard deviations below expectation) in executive function, 43% were impaired in language, 50% in memory, and 50% in psychomotor speed at baseline. Regardless of base-line performance, language significantly improved in 53% of patients, was stable in 39% of patients, worsening in only 7%. Attention was significantly improved in 46%, stable in 35%, and declined in 19%. Executive function improved in 58% of patients, was stable in 25%, and declined in 17%. Memory significantly improved in 47%, was stable in 40%, and declined in 13%. Psychomotor speed improved in 67%, was stable in 19%, and declined in 14%. Memory, when impaired at baseline, was most likely to improve (88% of patients). Improvements were not correlated with predictors such as presence of hydrocephalus, location of tumor, or other demographics (age, education, race, handedness, marital status). Neurocognitive impairment at presentation, before any surgical intervention was very high in this popula-tion. Neurocognition improved or stayed stable in 901% of the patients in each domain. Although some of the improvement may have been a practice effect, alternative forms were used and certainly not all patients benefited



from practice. Although surgery has the potential to induce deficits through focal damage to surrounding tissue, increased risk of hemorrhage, etc., it may also improve function in some areas, perhaps through resolution of mass effect, relief of intracranial pressure, or even a benefit of debulking. A better understanding of the neurocognitive effects of surgical resection is important as patients face decisions regarding their resection, and to aid researchers in delineating neurocognitive effects of treatments.

QL-33. MALIGNANT GLIOMA PATIENTS WITH SEIZURES TREATED WITH LEVITIRACETAMJohn De Groot,1 Terri Armstrong,1 Kenneth Aldape,2 Melissa Smith,1 Elizabeth Vera,1 and Mark Gilbert3; 1M.D. Anderson Cancer Center, Houston, TX, USA; 2University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3Houston, TX, USA

Seizures are the presenting sign of a brain tumor in 20 to 30 percent of patients and up to 80% of patients will have a seizure at some time during the course of their illness. Tumor-related epilepsy is aggressively treated with anti-epileptic drugs (AED) because of the potential morbidity asso-ciated with seizures in this fragile patient population. Interestingly, many clinical studies have demonstrated that seizures at the time of diagnosis are a positive prognostic finding. This has been attributed to an earlier diagnosis, but the true reason remains uncertain. Laboratory investigations are now actively pursuing the concept that excitatory neurotransmitters and other modulators of seizures may be involved in the biologic behavior of malig-nant brain tumors. Levetiracetam (LEV) has been shown to be a very potent anticonvulsant in animal models of chronic epilepsy and is increasingly used in the primary brain tumor population because of its efficacy, low side effect profile and metabolism. Recently, synaptic vesicle protein SV2A was discov-ered to be the binding site of LEV and the drugs mechanism of action has been linked to the association of LEV with SV2A. This study was initiated to examine the impact of LEV on seizure control in a cohort of patients with glioblastoma being treated concurrently with chemotherapy and to correlate seizure control on LEV with the expression of SV proteins in tumor tissue. Retrospective analysis was performed on a cohort of 265 adult patients with glioblastoma who were treated on chemotherapy protocols at M.D. Ander-son Cancer Center. A total of 38 patients were treated with LEV and were analyzed for the number, types of seizure (focal or generalized) and concur-rent anticonvulsant use before and after treatment with LEV. In addition to analysis of demographic and seizure frequency data, we obtained tumor tissue on 34 patients which is being stained for SV2A and SV2B, inhibi-tion of which is responsible for the anticonvulsant action of LEV. Eighty percent of patients in this cohort had at least one seizure during the course of the protocol treatment. A total of 69 patients (26%) were on non-enzyme inducing anticonvulsants (NEIAED) while 74% were on EIAED. 52% of patients on NEIAED (including LEV) had seizures compared to 22% in the NEIAED group. In the LEV only cohort, 92% of patients had seizures of which 79% occurred while not on LEV. The number of patients with no seizures once LEV was added dropped to 13%. Tissue staining for SV proteins is ongoing and will be correlated with seizure control in response to LEV therapy. LEV has demonstrated anticonvulsant activity in patients with primary malignant gliomas as both monotherapy and add-on therapy. The association between LEV responsiveness and expression of SV2A and SV2B will be reported at the time of presentation. If an association is found, further prospective studies will be performed to assess for association with biologic activity of the tumor. Supported in part by a Young Investigator Research Program for Keppra Initated Studies, UCB Pharma, Inc.

RADIOLOGY

RA-01. QUANTITATIVE OPTICAL SPECTROSCOPIC IMAGING OF BRAIN TUMORSMarlon Mathews,1 Christopher Owen,1 Jae Kim,2 David Abookasis,2 Yuanjie Hu,1 Henry Hirschberg,2 Devin Binder,1 Bruce Tromberg,2 Yi-Hong Zhou,1 and Mark Linskey1; 1Neurological Surgery, University of California, Irvine, Orange, CA, USA; 2Beckman Laser Institute and Medical Clinic, University of California, Irvine, CA, USA

Spatially modulated imaging utilizes the projection of NIR light modu-lated spatially into a sine wave configuration of intensity. Reflected light is captured on a CCD camera and digitally demodulated. The demodulated images contain information about the pathophysiologic state of tissue. Spatial modulation allows separation of light absorption from scattering, yielding absolute concentrations of tissue chromophores from individual molar extinction coefficients (based on the Beer-Lambert Law). The authors report the results of utilizing spatially-modulated near infrared (NIR) light in rodent brain tumor imaging. U251HF glioma cells were implanted ste-reotactically in the parietal lobes of adolescent immunodeficient mice. Four

weeks after tumor implantation, skin overlying the skull was opened and modulated imaging was carried out through the skull. In addition, BT4C tumor cells in BDIX rat brains were imaged ex vivo to map optical absorp-tion and scattering properties of gray matter, white matter and brain tumor. Tissue chromophore maps were generated successfully, demonstrating the spatial distribution of oxy-, deoxy-, total hemoglobin, and oxygen satura-tion. Tumor regions were found to be relatively hypoxic compared to normal brain tissue, despite higher blood flow. Scattering of NIR light was found to be significantly different in tumor and white matter (p 5 1.5 3 10–24; ANOVA). Spatial modulation of NIR light is useful in imaging and quanti-tatively mapping the distribution of tissue chromophores in brain tumors. This technology has great potential in clinical and research areas of neuro-oncology, particularly in understanding and monitoring disease and phar-macology related to angiogenesis.

RA-02. IN VIVO ANALYSIS OF EGFRvIII RAT GLIOMA GROWTH AND INVASION BY MAGNETIC RESONANCE IMAGING (MRI)Gurpreet Kapoor,1 Harish Poptani,2 Oliver Hsu,2 Sungheon Kim,2 and Donald O’Rourke3; 1University of Pennsylvania, Philadelphia, PA, USA; 2Radiology, University of Pennsylvania, Philadelphia, PA, USA; 3Neurosurgery, University of Pennsylvania, Philadelphia, PA, USA

EGFRvIII is a constitutively active truncated mutant of epidermal growth factor receptor (EGFR), which has been shown to increase neoplastic transformation and tumorigenicity. Previous reports indicate that EGFRvIII is expressed in a high proportion of malignant gliomas, and appears to be more expressed in primary or de novo GBMs. EGFRvIII positive tumors have been associated with poor prognosis, shorter life expectancies and fre-quent therapeutic resistance when compared to EGFRvIII-negative tumors, demonstrating the refractory nature of EGFRvIII tumors. While much is known about the constitutively active intrinsic tyrosine kinase properties of EGFRvIII, the effect of EGFRvIII expression on tumor physiology is not well characterized. In the present study, we utilized non-invasive MRI to assess tumor physiology in a 9L/EGFRvIII rat orthotopic brain tumor model. Fischer rats with 9L/EV (Empty Vector) and 9L/EGFRvIII intrac-ranial tumors were serially echo imaged using 4.7–T small-animal MRI scanner. Tumor volumes were estimated by adding all regions of interest (ROI) of the tumor and correlated to postmortem histological findings to understand the molecular mechanism of tumor development in vivo. Over-expression of EGFRvIII significantly enhanced infiltrative tumor volume (S ROI 5 3757) in the rats bearing 9L/EGFRvIII glioma tumors when com-pared to rats with 9L/EV gliomas (S ROI 5 506), suggesting that EGFRvIII overexpression may enhance the infiltrative potential of 9L/EGFRvIII tumors. Immunohistochemical analyses showed increased VEGF expres-sion in invasive EGFRvIII tumors when compared to empty vector controls. Furthermore, 9L/EGFRvIII tumors showed increased phospho-PLC-g, suggesting a possible involvement of VEGF and PLC-g in tumor invasion. Interestingly, there was also an increase in SHP-2 PTPase phosphorylation at Tyr542 and Tyr580 in 9L/EGFRvIII tumors, suggesting that activation of the EGFRvIII-coupled SHP-2 PTPase via phosphorylation may be required for increased tumorigenesis in EGFRvIII-bearing tumor cells. Further inves-tigation is warranted to understand the mechanistic implication of SHP-2 phosphorylation in EGFRvIII-induced tumorigenesis. Collectively, our data indicate that EGFRvIII overexpression enhances the tumorigenic potential of 9L glioma cells as assessed by MRI and that this may occur via SHP-2, PLC-g, and VEGF. MR imaging will allow for the non-invasive quantifi-cation and characterization of physiologic variables of intracranial glioma growth and invasion. Monitoring glioma tumor biology by MRI will also permit longitudinal assessment of therapeutic interventions in vivo.

RA-03. EVALUATION OF TUMOR RESPONSE BY DYNAMIC CONTRAST-ENHANCED MAGNETIC RESONANCE IMAGING IN GLIOBLASTOMA (GBM) PATIENTS TREATED WITH BEVACIZUMAB (BEV) AND IRINOTECAN (CPT-11)Annick Desjardins, Daniel Barboriak, James E. Herndon II, David Reardon, Jennifer Quinn, Jeremy Rich, Sith Sathornsumetee, Henry Friedman, and James Vredenburgh; Duke University, Durham, NC, USA

Impressive response rates have been observed in malignant glioma patients treated with bevacizumab (BEV) and irinotecan (CPT-11). Unfor-tunately, the treatment is not without toxicity and for this reason, ways to predict patients who will benefit from the treatment are necessary. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can be used to evaluate the microvasculature within tumors. The vascular permeability of the tumor can be evaluated with DCE-MRI using Ktrans, a volume trans-fer constant of contrast agent between blood plasma and the extravascular extracellular space. A 50% reduction in Ktrans is demonstrated clinically meaningful. We present the updated results of a phase II trial to determine the correlation between vascular permeability and radiographic response



in glioblastoma multiforme (GBM) patients treated with the combination. Eligibility included patients with recurrent GBM. Both agents were given every 14 days. All patients received BEV at 10 mg/kg IV. CPT-11 was dosed at 340 mg/ m2 for patients on enzyme inducing antiepileptic drugs (EIAED) and 125 mg/m2 for patients not on EIAED. Radiographic responses were assessed every 6 weeks. DCE-MRIs were performed before administration of chemotherapy, one day after treatment and after the first cycle. The pri-mary endpoint was to examine the effect of BEV and CPT-11 treatment on vascular permeability as measured by percent change from baseline in Ktrans. Twenty patients were enrolled, with a median age of 49.5 years. All patients are assessable for response. Best responses include eleven patients with partial response (response rate 5 55%) and nine patients with stable disease. Median time to progression was 31.9 weeks and 6–month progres-sion-free survival was 70%. Ktrans values are available for all 20 patients. A reduction in Ktrans by 50% was observed in 10 patients one day after treat-ment and in 19 patients at the end of cycle 1. Changes in Ktrans value and percentage decrease in enhancing tumor volume after one cycle of therapy showed a statistically significant correlation (Spearman’s rank correlation coefficient 5 0.47, p 5 0.04). Seven patients are still on study. In conclusion, the utilization of DCE-MRI to determine a reduction in vascular permeabil-ity following a combination of BEV and CPT-11 is feasible, and does cor-relate significantly with the change in cross-sectional tumor measurement at six weeks. Analysis of changes in DCE-MRI relative to progression-free survival and overall survival are ongoing.

RA-04. EVALUATION OF PRE-TREATMENT MR MARKERS THAT PREDICT SURVIVAL IN PATIENTS WITH NEWLY DIAGNOSED GBMSuja Saraswathy,1 Forrest Crawford,2 Kathleen Lamborn,1 Andrea Pirzkall,1 Susan Chang,1 Soonmee Cha,1 and Sarah Nelson1; 1University of California, San Francisco, San Francisco, CA, USA; 2CA, USA

The goal of this study was to test the predictive value of MR parameters in relation to the survival of patients with newly diagnosed GBM who had surgical resection and were scanned prior to receiving adjuvant radiation and chemotherapy. The study population comprised 70 patients. The patient age ranged from 27 to 78 (median 5 53) years. Out of 70 patients 27 had gross total resection (GTR), 33 had subtotal resection (STR) and 11 had biopsy. MR data were acquired using either 1.5 T (36 out of 70) or 3T (34 out of 70) MR scanners (GE Healthcare) using commercially available head coils. Each study included anatomical MRI, diffusion and perfusion weighted imaging and 1H MRSI. MR data were acquired approximately 3–5 weeks after surgery and 1 week before treatment with radiation therapy. Median parameter values within every region were adjusted for age and subjected to proportional hazards analysis. The patients with larger contrast enhancement and larger overall regions of T2 hyper-intensity tended to have a higher risk for poor outcome, which suggests that the residual tumor bur-den is an important factor in determining outcome. The volume of CNI2 (Number of pixels with CNI . 2), CBV3 (Number of pixels with CBV . 3), and nADC1.5 (number of pixels with normalized ADC ,1.5), found to be strongly associated with decreased survival. High lipid, lactate, and lipid/lactate values in lesion regions relate to poor patient survival. Low recovery values in all lesion regions and high values of LF in all lesion values were associated with worse survival. Discussion: The high CNI value observed in tumors reflects both loss of NAA due to the absence of functioning neurons and increased choline due to increased cell density and/or more extensive proliferation/higher cell density. As the latter characteristics are both asso-ciated with malignancy, it is not surprising that a high CNI volume was correlated with shorter survival. High CBV indicates that the lesion is more vascular than normal tissue and is a characteristic used to define malignant glioma. Low ADC is thought to be indicative of higher cellularity and hence larger tumor burden. Increased lactate may occur when the cellular capacity for exporting lactate to the blood stream is impaired. This would indicate tumor metabolism, infiltration and growth. Lipid is present when cell mem-branes become disrupted. Low recovery and high leakage factor indicator rupture of blood brain barrier and hence points to malignant tumor. The survival for patients with glioma can depend on both the malignancy of the tumor and its response to treatment. Since both tumor progression and response to radiation or cytoxic drugs are intrinsic properties of tumor cells, the goal of the present study was to examine if there is a relationship between the pre treatment MR parameters and survival. Our study shows that several of the pre-treatment MR parameters are predictive of survival. This information may be important for assigning patients to specific treat-ment protocols and in planning focal therapy.

RA-05. THE ROLE OF 11C METHIONINE POSITRON EMISSION TOMOGRAPHY IN ASSESSING TREATMENT RESPONSE FOLLOWING CONCURRENT TEMOZOLOMIDE/RT IN PRIMARY GLIOBLASTOMA MULTIFORMEChristina Tsien,1 Yue Cao,2 Theodore Lawrence,1 Larry Junck,1 Lisa Rogers,3 Randall Ten Haken,1 and Morand Piert2; 1University of Michigan, Ann Arbor, MI, USA; 2University of Michigan, MI, USA; 3University of Michigan, Cleveland, OH, USA

Combined chemo-RT may increase the likelihood of early delayed radia-tion effects. The use of metabolic imaging may improve our ability to dif-ferentiate between tumor progression and post-treatment effect. The aim of the study was to determine the value of post-treatment 11C Methionine PET in predicting patient outcome. Nineteen patients with primary glioblastoma multiforme (GBM) were treated with concurrent temozolomide 75 mg/m2 and dose escalation radiation on a clinical prospective study (median RT dose 75 Gy). All patients had 11C Methionine performed prior to treatment and 3 months following completion of chemo-RT. PET and MR images were co-registered to initial treatment planning CT scan. PET_GTV was defined quantitatively as the region of greater than or equal to 1.5× uptake of the normal cerebellum. Pre- and post-treatment PET were analyzed for comparison regarding extent of tumor volume and mean uptake. Response was defined as progression free survival (PFS) of greater than 6 months, based on MRI. Clinical parameters and PET parameters including residual PET volume, percent change in PET-GTV at 3 months, change in mean PET-GTV uptake, and percent change in mean GTV uptake at 3 months were analyzed using Wilcox rank-sum test. Eleven patients were respond-ers and 8 patients, non-responders. Seven cases had no appreciable uptake at the 3-month follow-up PET; all were responders. Clinical parameters were not significantly different in responders versus non-responders. How-ever, quantitative PET analysis demonstrated that the percent change in PET-GTV (p , 0.01), change in mean PET-GTV uptake (p , 0.02), and percent change in mean PET-GTV uptake at 3 months (p , 0.01) were asso-ciated with improved progression-free survival. In 5 cases, percent change in PET-GTV 3 months post-treatment was less than 50%. In four of these five cases, the initial PET-GTV was not completely encompassed by the 95% isodose in the RT plan (range: 40%–83%). Our findings suggest that post-treatment functional PET imaging may be useful in predicting patient outcome in patients with glioblastoma multiforme treated with concurrent chemo-RT. Our data from non-responders suggest that an additional RT boost to the 11C Methionine positive tumor tissue may be beneficial and might improve patient outcome.

RA-06. IMPROVED COVERAGE OF BRAIN TUMORS WITH MAGNETIC RESONANCE SPECTROSCOPIC IMAGING FOR RADIATION TREATMENT PLANNINGJoseph Osorio,1 Andrea Pirzkall,1 Soonmee Cha,2 Susan Chang,1 and Sarah Nelson1; 1University of California, San Francisco, San Francisco, CA, USA; 2CA, USA

Although 3D PRESS Magentic Resonance Spectroscopic Imaging (MRSI) is able to provide excellent quality metabolic data for patients with brain tumors and has been shown to be important for defining tumor bur-den, the method is currently limited by how much of the anatomic lesion can be covered within a single examination. One reason is that PRESS selection is rectangular, whereas the head is more elliptical in shape. The goal of the current study is to combine PRESS localization with improved outer volume suppression (OVS) pulses in order to obtain larger coverage for 1H MRSI of the human brain. This has been achieved by integrating fixed cosine modulated, and graphically placed non-cosine modulated, very selective suppression (VSS) pulse scheme that are optimized for high field strengths and provide improved coverage for supratentorial brain tissue from various head sizes, shapes, and treated brain tumor patients. Two symmetric sup-pression bands were produced using a single RF pulse by modulating the RF pulse by a cosine function. Each cosine modulated VSS pulse that was gener-ated in real time produced two suppression bands that were spatially placed based on the desired prescription of the localization volume. This design of the cosine modulated VSS pulse allowed for 12 fixed-saturation bands in addition to 6 graphically prescribed bands. Eight of the 12 fixed cosine modulated saturation bands were used to generate an octagonal selection region. The six additional graphically prescribed bands uniquely conformed the selected region to the shape of the individual. Volunteer and patient studies were performed on a GE 3T MR scanner using body coil excitation and reception with an 8-channel phased array head coil. The pulse was implemented using 3D MRSI with PRESS volume localization in 4 min-utes; TR/TE 5 1100/144ms for a single slice. These acquisitions employed spectral arrays with a nominal spatial resolution of 1 cc. Ten brain tumor patients were scanned and compared to the conventional MRSI sequence. Twenty-eight patient scans were retrospectively investigated for tumor cov-erage with PRESS 1H MRSI, which used a conventional VSS pulse scheme. The mean coverage by MRSI per slice of brain tissue was 55 cc, with 63% mean coverage of the region of T2 hyperintensity. Coverage was improved by a factor of 2.5 in ten patients using the new OVS scheme, with the mean



volume of brain tissue covered in patients being 133.3 cc per MRSI slice. The efficiency of saturation of water from the VSS pulse was 91%. The selection was conformed to an octagon shaped region. Comparison of the PRESS MRSI selected volume using both methods was demonstrated show-ing a significant improvement of tumor coverage using the improved OVS technique. This study demonstrated improved coverage of the PRESS region prescribed by using improved OVS pulses for maximum lipid suppression in volunteers and patients by employing cosine modulated VSS pulse scheme. Tumor coverage was significantly improved. This is likely to be particularly important for using MRSI in radiation treatment planning and evaluating treatment response.

RA-07. COMBINING MATHEMATICAL MODELING WITH SERIAL MR IMAGING TO QUANTIFY AND PREDICT RESPONSE TO RADIATION THERAPY IN INDIVIDUAL GLIOMA PATIENTSKristin Swanson, Russ Rockne, Jason Rockhill, and Ellsworth Alvord; University of Washington, Seattle, WA, USA

Malignant gliomas are uniformly fatal brain tumors with extensive peripheral invasion evident at all stages of growth. After surgical resection, malignant gliomas generally receive radiotherapy which has been classically considered to double the length of survival. However, clinical quantification of glioma response to treatment has relied primarily on observed changes in gross tumor volume as measured on MRI. Mathematical modeling of glioma growth and invasion has already proven useful in predicting the behavior of untreated gliomas (Swanson, 1999; Swanson and Alvord, 2002; Harpold et al., 2007; Mandonnet et al., 2003). Using the classic linear-quadratic model for radiation efficacy, we extend Swanson’s primary model to simulate, quantify and assess response to radiation therapy. This can be done within an individual patient in vivo and in real time requiring as few as two pre-treatment and one post radiation therapy observations on each of T1-weighted gadolinium-enhanced and T2-weighted MRIs. As previously described by us (Swanson and Alvord, 2002), the segmented pre-treatment MRIs are fit to the pre-treatment mathematical model to quantify the net rates of invasion (D) and net proliferation (r) for each patient’s glioma. Finally, our spatio-temporal radiation model is then fit to the remaining post-treatment MR observation yielding linear-quadratic measures of radiotherapy effectiveness (a and b) for that patient. Initial results show good agreement between the mathematical model expected behavior and that observed in individual patients. Further, even with a small number of patients, a significant correlation (p , 0.01 with r 5 0.98) is found between the net proliferation rate (r) of the glioma cells before the initiation of treat-ment and the radiation effectiveness (a). Although more patient studies are necessary to see if this relationship persists, these initial data suggest that individual response to radiation therapy may be predictable from the pre-treatment model-defined net proliferation rate (r) in individual patients. References: (1) K.R. Swanson. “Mathematical Modeling of the Growth and Control of Tumors” (PhD dissertation, University of Washington, 1999); (2) K.R. Swanson and E.C. Alvord Jr., The Concept of Gliomas as a Traveling Wave: The Application of a Mathematical Model to High- and Low-Grade Gliomas. Canadian Journal of Neurological Sciences 2002;29:395; (3) H.L.P. Harpold, E.C. Alvord Jr., K.R. Swanson, Visualizing Beyond the Tip of the Iceberg: The Evolution of Mathematical Modeling of Glioma Growth and Invasion. Journal of Neuropathology and Experimental Neurology 2007;66:1–9; (4) E. Mandonnet, J-Y Delattre, M-L Tanguy, K.R. Swanson, A.F. Carpentier, H. Duffau, P. Cornu, R. Van Effenterre, E.C. Alvord Jr., and L. Capelle, Continuous growth of mean diameter in a subset of WHO grade II gliomas. Annals of Neurology 2003;53:524–528.

RA-08. RADIOGRAPHIC EVALUATION OF RECURRENT GLIOBLASTOMA MULTIFORME (GBM) IN PATIENTS TREATED WITH BEVACIZUMAB AND IRINOTECANJalal Andre,1 Kenneth Spearman,1 Stanley Lu,1 and Sumul Raval2; 1Diagnostic Radiology, Monmouth Medical Center, Long Branch, NJ, USA; 2The David S. Zocchi Brain Tumor Center, Monmouth Medical Center, Long Branch, NJ, USA

Recurrent GBM carries a poor prognosis after first-line therapies have been exhausted, even in the setting of gross total resection. Bevacizumab and Irinotecan have shown promising results in patients with recurrent GBM. We sought to retrospectively document the short-term effects of this chemotherapeutic regimen on recurrent GBM, as evidenced by comparative MRI brain scans obtained prior to, and one-month following initiation of treatment. We collected brain MRI data from August 2005 to December 2006, in which post-contrast spin-echo T1-weighted images demonstrated measurable enhancement and/or GBM tumor mass. Having failed temo-zolomide and radiation therapy, 15 consecutive patients’ MRI scans were available for review at this institution by a neuro-radiologist, in which both pre- and post-treatment hard and/or soft copy MR images were available

for direct measurement. Each was treated with Bevacizumab 5 mg/kg IV and Irinotecan 125 mg/m2 IV infusion every 2 weeks until disease progres-sion, or development of unacceptable toxicity. We measured pre- and post- treatment recurrent GBM bulk tumor in anteroposterior, transverse, and cranio-caudad dimensions, and calculated volumetric data, assuming an ellipsoid tumor configuration. Pre- and post-treatment peritumoral T2 sig-nal abnormality was measured in this same manner on FLAIR images (avail-able for 10 of these patients). Pre-treatment MRI scans were performed 2 weeks prior to initiation of therapy (median: 10 days). Post-treatment scans were performed at approximately one month following initial treatment (median: 28 days). All patients witnessed significant decrease in tumor bulk volume ranging from 15.3% to 96.7%, having received an average of two cycles of chemotherapy. We observed a mean decrease in tumor volume of 50.0% (SD 5 25.3%; SEM 5 6.5%; 95% CI 5 35.9%–64.0 % decrease). FLAIR images demonstrated a mean reduction in peritumoral T2 abnormal-ity of 44.3%. We report 50.0% mean reduction in recurrent GBM tumor bulk at an average of 1-month post-treatment in patients treated with an average of two cycles of Bevacizumab and Irinotecan therapy. These prom-ising initial results necessitate further long-term prospective evaluation of this chemotherapy.

RA-09. DUAL-AGENT DYNAMIC MAGNETIC RESONANCE IMAGING IN THE EVALUATION OF ANTITUMOR EFFECT OF ANTIANGIOGENIC DRUGS: PRECLINICAL IMAGING DATA ON A 12-TESLA SCANNERCsanad Varallyay,1 Leslie Muldoon,1 Seth Lewin,1 Kristoph Jahnke,2 James Goodman,3 Jeffrey Wu,1 and Edward Neuwelt1; 1Neurology, Oregon Health & Science University, Portland, OR, USA; 2Neurology and Medicine, Oregon Health & Science University, Portland, OR, USA; 3Advanced Imaging Research Center, Oregon Health & Science University, Portland, OR, USA

The aim of this study was to evaluate the potential of dual agent dynamic magnetic resonance imaging (MRI). During antiangiogenic treatment of a human glioma model, the iron compound ferumoxytol was used for cerebral perfusion, and the gadolinium contrast agent Omniscan was used for vascu-lar permeability measurement. Male athymic nude rats received intracerebral injection of U87 human glioma cells and were scanned 3–4 weeks later on a 12 tesla MRI scanner. Imaging consisted of high resolution T2-weighted anatomical scans, T2*-weighted perfusion measurement using the blood pool agent ferumoxytol and T1 weighted permeability measurement using gadolinium. Rats were injected with 45mg/kg of intravenous bevacizumab (an antibody against vascular endothelial growth factor) and scanned with the same MRI protocol 24 and 48 hours after treatment. The cerebral blood volume perfusion parameter of the tumor dropped dramatically and reached the level of the normal appearing brain tissue in 48 hours. The delayed peak of gadolinium enhancement and the decreased signal intensity sug-gested lower vascular permeability as a result of antiangiogenic effect of bevacizumab. In a single imaging session, it is feasible to use the blood pool agent ferumoxytol for T2*-weighted blood perfusion measurements, and the extravasation of gadolinium for T1-weighted vascular permeability mea-surements (in this scanning order). This imaging protocol could be used in the evaluation of early therapeutic effect in antitumor therapy.

RA-10. IN VIVO MR SPECTROSCOPY AND DIFFUSION MR IMAGING OF THE DESMOPLASTIC SUBTYPE OF MEDULLOBLASTOMAAshok Panigrahy, Jonathan Finlay, Marvin Nelson, Floyd Gilles, Ignacio Gonzalez-Gomez, Mark Krieger, and Stefan Bluml; Childrens Hospital Los Angeles, Los Angeles, CA, USA

Elevation of taurine, measured with quantitative in vivo MR spec-troscopy (MRS) has been observed in medulloblastoma by several groups independently. The goals of this study were to (i) evaluate whether taurine levels correlate with the degree of differentiation (i.e., it is hypothesized that taurine concentrations are higher in regular medulloblastoma than in the desmoplastic subtype) and (ii) to determine whether metabolite levels cor-relate with tumor cellularity assessed by quantitative diffusion imaging. MR spectroscopy and diffusion data obtained from 27 patients at initial diagno-sis were reviewed. This included 18 classic medulloblastoma (MB), five des-moplastic medulloblastoma (DMB), one atypical rhabdoid tumor (ATRT), and three supratentorial primitive neuroectodermal tumors (ST-PNET). MRS and MRI of two patients with recurrent MBL (RMB) were also reviewed. All data were obtained on a 1.5 T clinical MR scanner (Signa LX, GE Healthcare, Milwaukee, WI, USA). A single-voxel PRESS sequnce was used for spectroscopy acquisitions and data were processed and quantified using fully automated commercially available software (LCModel, Stephen Provencher Inc., Ontario, Canada). Mean apparent diffusion coefficients (ADC) were determined for tumor tissue and white matter and cerebrospi-



nal fluid for reference. Tau was significantly higher in the classical histologi-cal subtype of medulloblastoma compared to the desmoplastic subtype of medulloblastoma (p , 0.0001, unpaired t-test). There were no apparent dif-ferences in levels of other metabolites such as total choline (tCho), creatine (Cr), or myo-inositol (mI) in MB and DMB. High levels of taurine were also noticed in the supratentorial PNET and recurrent medulloblastoma when compared to the desmoplastic medulloblastoma group. There was no differ-ence between ADC values between the desmoplastic group, classic subtype of medulloblastoma, supratentorial PNET and recurrent PNET case. There was no statistically significant correlation between the ADC values and tau-rine level. The desmosplastic subtype of medulloblastoma demonstrated low taurine levels when compared with other histological subtypes of medullo-blastoma (classic, supratentorial and recurrent). Elevated taurine did not correlate with increased cellularity (as measured by diffusion weighted MR). These findings suggest that elevated taurine may be a specific marker for the “classic” histological subtype of medulloblastoma and support the role of proton MRS in predicting histological subtypes.

RA-11. IN VIVO PROTON MR SPECTROSCOPY OF INTRACRANIAL GERM CELL TUMORSAshok Panigrahy, Marvin Nelson, Jonathan Finlay, Mark Krieger, Ignacio Gonzalez-Gomez, Floyd Gilles, and Stefan Bluml; Childrens Hospital Los Angeles, Los Angeles, CA, USA.

Objectives: (a) To characterize the metabolic fingerprint of intracranial germ cell tumors in pediatric patients and (b) to compare spectra of pure germinoma with mixed germ cell tumors. MR spectra from 20 pediatric patients with pathology-proven intracranial germ cell tumors (13 pure and 7 mixed) were retrospectively reviewed. Metabolic features were compared with other common pediatric brain tumors such as medulloblastoma (n 5 14), ependymoma (n 5 8), astrocytoma (n 5 8), and pilocytic astrocytoma (n 5 14) that have been previously studied at this institution. All spec-tra were acquired with a short echo time (TE 5 35ms) PRESS sequence. Spectra were processed with fully automated LCModel software (Ste-phen Provencher Inc., Ontario, Canada) and metabolite concentrations of N-acetyl-aspartate (NAA), creatine (Cr), choline (tCho), myo-inositol (mI), taurine (Tau), and lipid intensities were determined. Also reviewed were fea-tures of spectra such as the full width at have maximum (FWHM), a mea-sure of homogeneity of the magnetic field in the region of interest. Serum and cerebrospinal fluid (CSF) levels of AFP and b-HCG were obtained from the clinical records. MR spectra of pure pineal germinoma showed features generally also found in other tumor such as prominent lipids, low NAA, elevated tCho and reduced Cr. In addition, Tau (3.6 61.3 mmol/kg, n 5 8) was consistently observed. There was no evidence for taurine in mixed pineal tumors (0.0 60.0, n 5 3, p , 0.05). But it needs to be acknowledged that the quantitation of taurine was compromised by the larger linewidth of peaks in mixed tumor spectra (13 66 vs. 7 62 hertz (Hz), n.s.). Also, lipids appeared to be more prominent in pure germinoma than in mixed but significance was not reached due to the small number subjects studied. Pure and mixed germinoma showed a large linewidth (11 63 Hz) which was significantly higher than the linewidth observed in other common pediatric brain tumors (5 61, p , 0.001). There was no difference in metabolite or lipid levels between mixed and pure germinoma that occurred outside the pineal region. There was no correlation between serum and CSF tumor markers and MR spectroscopy data. This study demonstrated two major MR spectroscopy features of germ cell tumors. (1) Pure pineal germinoma present with prominent taurine. (2) Non-pineal germ cell tumors (pure 1 mixed) demonstrate a high linewidth which likely reflects the heterogeneity of the lesions. These characteristics may help to (a) differentiate germ cell tumors which cannot be separated by serum and CSF tumor markers and (b) may be useful to distinguish germ cell tumors from other tumors.

RA-12. MR IMAGING CHARACTERISTICS OF OLIGODENDROGLIOMAS WITH THE 1P/19Q DELETIONJonathan Sherman,1 Daniel Prevedello,2 Nader Pouratian,1 Lubdha Shah,1 Prashant Raghavan,1 Mark Shaffrey,1 and David Schiff1; 1University of Virginia, Charlottesville, VA, USA; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA

Patients with oligodendrogliomas with allelic loss of chromosomal arm 1p and 19q have been shown, especially with anaplastic oligodendrogliomas, to have both a better initial and long term response to chemotherapy as well as an improved overall survival. Effective treatment of patients with these tumors requires accurate diagnostic techniques. MR imaging can be used to help differentiate between low- and high-grade tumors. We hypothesize that certain MR imaging characteristics can be used to differentiate between patients with and without the 1p/19q deletion. Using our clinical database from 1989 until 2007, we collected adult patients with oligodedrogliomas who underwent treatment at the University of Virginia. Age at diagnosis, gender, tumor grade, tumor deletion status, time of follow-up, and MR

imaging characteristics were analyzed; the latter by two blinded neuroradi-ologists. The imaging characteristics included cerebral hemisphere and lobe involvement, T1- and T2-signal intensity, tumor margin character, presence of enhancement, degree of heterogeneity, presence of restricted diffusion, and tumor cross-sectional area. Between 1989 and 2007, 113 patients were identified. There were 67 men and 46 women. Mean age at diagnosis was 42.7 years (range 16 to 82). Pathologic examination of patient tumor speci-mens displayed 49 patients with the 1p/19q deletion and 63 patients without this deletion. Thirty-seven patients (75.5%) remain alive with tumors that contain the deletion with a mean time of follow-up of 44.4 months (range 3.2 to 166.4). In comparison, 40 patients (63.5 %) with tumors that do not contain this deletion remain alive with a mean time of follow-up of 41.6 months (range , 1 to 214.2). After analyzing all pertinent MR imaging characteristics, two features displayed statistical significance. The greatest cross-sectional area (mean) of the tumor (p 5 0.008) measured at 47.7 cm2 for the patients with the deletion and 64.1 cm2 for the patients without the deletion. In addition, patients with the deletion displayed tumors more often confined to a single lobe as compared to those patients without the deletion (p 5 0.019). Six tumors (12.2%) of the former group spread to mul-tiple lobes as compared to 18 tumors (28.1%) of the latter group. MRI is a valuable imaging modality for differentiating between oligodendrogliomas with or without the 1p/19q deletion. Tumors without the deletion are both larger in size and more often involve multiple lobes than those with the dele-tion. Analyzing MR imaging for these characteristics can assist clinicians in assessing patients prior to surgical and medical intervention.

RA-13. CLINICAL, RADIOLOGIC, AND PATHOLOGIC CHARACTERISTICS OF PSEUDOPROGRESSION IN MALIGNANT GLIOMASElham Jafarimojarrad and Alexis Demopoulos; Neurology, North Shore-LIJ Health System, Manhasset, NY, USA

Evaluating therapeutic response in malignant brain tumor patients typi-cally includes periodic neuroimaging. Magnetic resonance imaging (MRI) with intravenous contrast material is the preferred modality. Tumor pro-gression is more likely when an increase in hyperintensity on T2/FLAIR weighted sequences or new/progressive contrast enhancement is observed more than three months from radiation therapy. Occasionally, however, such radiographic changes do not represent worsening of disease. Indeed, such changes may reflect treatment effects, termed “pseudoprogression.” (M.C. De Wit et al., 2004) Pseudoprogression has important implications for clinical trial design because many patients may either be inappropriately characterized as failing first line therapy or falsely recorded as responding to second line therapy. In clinical practice, changes on imaging need to be carefully weighed with other clinical information when deciding whether to continue current therapy or initiate new treatments. Unfortunately, true progression of disease immediately following radiotherapy carries a worse prognosis and is important to exclude. (L.E. Gaspar et al., 1993) In gliomas, the combination chemoradiation protocols may result in a greater incidence pseudoprogression. We describe the clinical characteristics, MR findings, pathology (when available), and clinical outcomes of glioma patients in order to better understand the phenomenon. We conducted a single insti-tution retrospective review of sixty five patients with malignant gliomas treated with chemoradiation (R. Stupp et al., 2005) between 2005 and 2007. Twenty five patients underwent a second resection. Two patients were offered surgery and declined. Pathology revealed aggressive neoplasm (n 5 21) or treatment effect plus no (3) or a minority of (1) persistent neoplasm. In four nonsurgical patients, progressive disease seen on imaging gradually resolved without change in therapy, suggesting pseudoprogression without pathologic correlates. Detailed clinical characteristics are presented.

RA-14. ELLIPSOIDAL SENSE FOR FAST PROTON MAGNETIC RESONANCE SPECTROSCOPIC IMAGING OF GLIOMAS AT 3TEsin Ozturk-Isik, Susan Chang, and Sarah Nelson; University of California, San Francisco, San Francisco, CA, USA

Addition of 3D MRSI to the anatomical MR imaging for patients diag-nosed with gliomas has been shown to provide better brain tumor localiza-tion, treatment planning, and assessment of treatment response. Although 3D MRSI provides a vast array of molecular information, conventional spectroscopic imaging results in long MRSI data acquisition times ranging up to 301 minutes. In this study, we have implemented a new combined ellipsoidal SENSE data acquisition strategy with an effective nine fold scan time reduction that results in 4:36 min scan time for a 16 3 16 3 8 spectral array. We have shown its application for imaging glioma patients at 3T, and quantitatively compared it to the current clinical ellipsoidal MRSI protocol in tumor and normal regions. Three volunteers and six glioma patients (4 grade IV, 2 grade III, 1 female, 5 male, mean age 5 53) were scanned on a 3 T GE MR scanner with an eight channel RF coil. The imaging proto-



col included the acquisition of axial T1-weighted SPGR, axial T2-weighted FLAIR, and proton-density weighted coil sensitivity images. FLAIR abnor-mality (FL) regions excluding cavity areas were segmented using an in-house region growing algorithm for the patients. Ellipsoidal SENSE spectral data acquisition was implemented in a custom PRESS MRSI sequence (TR/TE 5 1.1s/144 ms). 16 3 16 3 8 ellipsoidal SENSE (4:36 min) and 16 3 16 3 8 original ellipsoidal (17:32 min) data were acquired from volunteers. The original ellipsoidal spectral array size was reduced to 12 3 12 3 8 (9:28 min) for patients due to scan time limitations. Data reconstruction for ellip-soidal SENSE spectra was implemented using Matlab 7.0. The ellipsoidal and ellipsoidal SENSE spectra were quantified using in-house software. Spearman rank correlation coefficients were computed to assess the simi-larity of Cho/NAA ratio between the ellipsoidal and the ellipsoidal SENSE spectra. A Mann-Whitney rank sum test was utilized to assess if the tumor regions had significantly different Cho/NAA values than normal regions for ellipsoidal SENSE or ellipsoidal spectra for the patients. Ellipsoidal SENSE k-space sampling reduced the scan time from 37:42 min to 4:36 min for a 16 3 16 3 8 array. Cho/NAA ratios were significantly (p , 0.001) corre-lated for all subjects between the two methods. The median Cho/NAA ratio calculated from the ellipsoidal spectra for patients were (0.86, 0.54, 0.75, 0.78, 0.67, and 0.63) in normal regions and (1.06, 0.66, 0.95, 1.61, 0.74, and 0.91) in tumor regions. The median Cho/NAA ratio calculated from the ellipsoidal SENSE spectra for patients were (0.82, 0.49, 0.67, 0.7, 0.64, and 0.59) in normal regions, and (1.01, 0.66, 0.92, 1.02, 0.63, and 0.83) in tumor regions. Tumor regions had significantly higher (p , 0.05) Cho/NAA for both techniques for the second, third, fourth, and sixth patients. The Cho/NAA ratio was not significantly different for both techniques in the first and fifth patients. A new technique has been implemented for imag-ing gliomas at high field with an effective nine fold scan time reduction to 4:36 min. The results of ellipsoidal SENSE spectra correlated well with the clinically accepted ellipsoidal spectra. Ellipsoidal SENSE spectra also had the ability to distinguish tumor regions like the ellipsoidal spectra.

RA-15. NEW DOUBLE B-VALUE DIFFUSION IMAGING SEQUENCE TO BETTER DISTINGUISH TUMOR FROM EDEMA FOR PATIENTS WITH HIGH-GRADE GLIOMASInas Khayal,1 Soonmee Cha,2 Susan Chang,1 and Sarah Nelson1; 1University of California, San Francisco, San Francisco, CA, USA; 2CA, USA

Development of an imaging biomarker that is capable of distinguishing tumor from non-tumor regions within the anatomic lesion could provide an important opportunity for guiding surgery and distinguishing recurrent tumor from treatment effect for patients with brain tumors. Multiple high b-value diffusion-weighted imaging has been suggested for differentiating between edema and tumor, but requires a long imaging time that is typically not clinically feasible. The goal of this study was to develop and validate a two b-value sequence that provides a differential signal (dS) capable of differentiating tumor (enhancing lesion, CEL) from edema (non-enhancing lesion, NEL) than the conventional apparent diffusion coefficient (ADC) and fractional anisotropy (FA) maps for patients with high grade gliomas. The double b-value sequence employed 6 directional diffusion tensor imag-ing (DTI) with low (b 5 1000 s/mm2) and high (b 5 2250s/m m2) b-values. Eight patients with grade IV gliomas were scanned on a 3T using this tech-nique along with postGd T1- and T2-weighted images. ADC, FA, and dS (median of the difference of the expected and measured high b-value signal) were processed using in-house software and normalized by normal appear-ing white matter to generate nADC, nFA, and ndS. The largest percent difference between CEL and NEL is seen for ndS (25%) as compared to nADC (8%), and nFA (6%). The percent differences are significantly larger in ndS compared to nADC and nFA (p 5 0.0078) using a Wilcoxon signed rank test. This technique shows the capability of this approach for sepa-rating enhancing vs. non-enhancing lesions as an estimate of tumor and non-tumor. This technique may also be utilized to distinguish progressive tumor from treatment response within the contrast enhancing lesion for recurrent patients.

RA-16. IMAGING TREATMENT EFFECTS IN BRAIN TUMORS USING SWIJanine Lupo,1 Kathryn Hammond,2 Susan Chang,1 and Sarah Nelson1; 1University of California, San Francisco, San Francisco, CA, USA; 2CA, USA

Susceptibility-weighted imaging (SWI) is a powerful tool for high resolu-tion imaging of the vasculature, aiding in the diagnosis of many pathologic conditions at 1.5T, including brain neoplasms, neurological trauma, vascu-lar malformations, and a variety of cerebrovascular and neurodegenerative diseases. With this technique, the post-processing of the phase information is used to amplify contrast between tissues that have different susceptibili-ties in the magnitude image. SWI is expected to be important for measuring

blood volume at higher field strengths because the heightened susceptibil-ity effect magnifies phase variations and does not suffer from the severe spatial distortions due to off-resonance effects associated with traditional sequences. At 7T, the enhanced sensitivity to microvasculature due to higher susceptibility effects can provide a major advantage by allowing the detec-tion of vessels as small as 100–200 m. High resolution T2*-weighted brain MR imaging was performed on 20 treated brain tumor patients using either a 3T GE Signa system (10 patients), with 8-channel phased array receive coil (Medical Devices) and body coil excitation, or 7T whole body GE scanner (10 patients), using uniform excitation by a volume transmitter and recep-tion by an 8-channel head coil (Nova Medical). The SWI employed a 3D flow compensated, GRE (3T) or SPGR (7T) sequence with TE 5 28/16 ms (3T/7T) TR 5 46/80 ms (3T/7T), flip angle 5 20o, BW 5 62.5 kHz, and 24 3 24 cm3 FOV. GRAPPA with R 5 2 was acquired using a 512 3 144 image matrix with 16 ACS lines and reconstructed with a GRAPPA-based technique developed by our group. The coverage in z was varied to image the extent of the entire tumor at 1mm resolution. Phase masks were constructed from the raw complex data of each individual coil element through complex division by a low-pass filtered image and scaling the resulting negative phase values between zero and one. The phase masks were then multiplied into the magnitude image from each coil m times and the resulting susceptibility-weighted images were combined by the traditional square root of sum of squares method, low pass filter corrected, and projected through 4–15 mm slabs depending on the tumors. Both small and large vessels were easily depicted, with a 1.5-fold increase in vessel contrast observed at 7T com-pared to 3T. SWI provided additional specificity to the contrast-enhancing portion of the lesion, aiding in the visualization of vessels, blood products, and radiation effects in the tumor region. High field SWI is a promising technique, feasible for use in patient studies. The implementation of parallel imaging with a GRAPPA-based reconstruction allowed a 2-fold reduction in scan time without compromising the contrast between veins and sur-rounding brain tissue. Large brain tumor lesions can be covered in less than seven minutes at 7T and under 5 minutes at 3T, facilitating the incorpora-tion of this technique into routine patient studies. This could be especially advantageous for monitoring the effectiveness of new anti-angiogenic and radiation therapies.

RA-17. PERFUSION-WEIGHTED IMAGING IDENTIFIES MR SURROGATES OF MALIGNANT GLIOMA MOLECULAR SUBTYPESRobert Whitmore,1 Casey Halpern,1 Gurpreet Kapoor,2 Ronald Wolf,2 John Woo,2 Jennifer Baccon,2 Sanjeev Chawla,2 Laura Oleage,2 Joanna Lopinto,2 Justin Plaum,2 Elias Melhem,2 and Donald O’Rourke1; 1University of Pennsylvania, Philadelphia, PA, USA; 2PA, USA

Gliomas are increasingly being characterized by specific molecular sub-types that predict response to therapeutics and prognosis. Chemosensitivity of oligodendrogliomas and prolonged patient survival are predicted by loss of heterozygosity (LOH) of chromosome 1p and 19q. A frequent genetic alteration in glioblastoma multiforme (GBM) is amplification of epidermal growth factor receptor (EGFR), implicated in tumor angiogenesis. Coex-pression of EGFRvIII mutation and PTEN by GBMs is associated with therapeutic response to EGFR kinase inhibitors. We have utilized MR per-fusion-weighted imaging to predict and identify MR surrogates of 1p/19q deleted oligodendrogliomas and EGFRvIII amplified GBMs. 30 patients with oligodendroglial tumors and 24 patients with primary GBMs were retrospectively reviewed for preoperative MR perfusion imaging. Tumors were resected and subjected to histopathologic and cytogenetic analysis. Oligodendroglial tumors were stratified into two cytogenetic groups: 1p or 1p/19q loss of heterozygosity (LOH) (group 1; n 5 11), and 19q LOH or intact alleles (group 2; n 5 19). Primary GBM tumors were grouped as EGFR-negative (n 5 14) and EGFR-positive (n 5 10) tumors. The EGFRvIII mutation was present in three EGFR-positive GBMs. The relative tumor blood volumes (rTBV) were calculated in relation to contralateral white mat-ter (WM) and we converted maximum rTBV values to z-scores based on WM blood volume measurements. In WHO grade II neoplasms, the rTBV was significantly greater (p , 0.03) in group 1 (n 5 7; mean 2.63; range 0.96–3.28) compared to group 2 (n 5 7; 1.71; range 1.27–2.23). In grade III neoplasms, the differences between group 1 (n 5 4; 2.83; range 1.59–6.26) and group 2 (n 5 12; 2.88; range 1.81–3.76) were not significant. The rTBV was significantly greater (p , 0.01) in grade III neoplasms (n 5 16; 2.88; range 1.59–6.26) compared to grade II neoplasms (n 5 14; 1.99; range 0.96–3.28). Predictive models integrating rTBV with cytogenetic profile and grade showed accuracies of 68% and 73%, respectively. Notably, there was increased expression of VEGF and VEGF-induced angiogenic proteins in 1p/19q-deleted tumors, suggesting that the genes on chromosomes 1p and 19q may include negative regulators of angiogenesis and tumor vascular invasion. EGFR-positive GBMs had higher rTBV z scores (4.59; range 2.97–8.57) than EGFR-negative tumors (3.95; range 1.55–6.64), and between-group comparisons of z-scores revealed a significant difference (p , 0.01). EGFRvIII GBMs had higher rTBV relative to EGFR-negative GBMs, and z-score analyses were consistent with this finding (p , 0.0020. There was a trend of higher rTBV in EGFRvIII-GBMs relative to EGFR-positive



tumors without this mutation. 1p/19q deletions in oligodendrogliomas and EGFRvIII mutation in GBMs are associated with elevated rTBV. These find-ings may reflect increased angiogenesis in these glial subtypes, but further investigation is warranted. We have developed predictive algorithms from our results to improve the accuracy of glioma grading, provide prognostic information, and potentially influence treatment decisions. Collectively, our data demonstrate the utility of advanced imaging modalities to identify MR surrogates of molecular subtypes of human glial brain tumors.

RA-18. PROTON MR SPECTROSCOPY IDENTIFIES SURROGATES OF MALIGNANT GLIOMA MOLECULAR SUBTYPESRobert Whitmore,1 Sanjeev Chawla,2 Casey Halpern,2 Gurpreet Kapoor,2 Ronald Wolf,3 John Woo,4 Laura Oleaga,4 Joanna Lopinto,4 Justin Plaum,4 Elias Melhem,4 and Donald O’Rourke5; 1Neurosurgery, Hospital of the University of Pennsylvania, Philadelphia, PA, USA; 2University of Pennsylvania, Philadelphia, PA, USA; 3Wynnewood, PA, USA; 4PA, USA; 5Philadelphia, PA, USA

Primary glioblastoma multiforme (GBM) frequently overexpresses epidermal growth factor receptor (EGFR), a major genetic alteration com-monly associated with tumor angiogenesis. We recently demonstrated the utility of magnetic resonance (MR) perfusion-weighted advanced MR imaging in identifying molecular subtypes and angiogenic profiles of glial tumors. We observed elevated relative tumor blood volume in EGFR-posi-tive GBMs using MR perfusion-weighted imaging. In the present study, we correlate the proton MR spectroscopy (1H MRS) profiles of EGFR-positive GBMs with the molecular subtype of EGFR-negative GBMs. Preoperative 1H MRS of 12 patients with primary GBMs were retrospectively reviewed. Tumors were resected and subjected to histopathologic analysis. Primary GBMs were grouped as EGFR-positive (n 5 10) and EGFR-negative (n 5 2) tumors based upon immunohistochemistry. 1H MRS was performed using a 3 Tesla scanner. Volumes of interest (VOI) were selected in a standard fashion, and spectroscopy data (0.2 and 4.0 ppm) were analyzed from only those voxels with good spectral resolution and signal-to-noise ratio that encompassed contrast-enhanced regions on T1-weighted images. Concen-trations of lipid 1 lactate (Lip1Lac), as well as choline, myo-inositol, and glutamate 1 glutamine were measured and normalized with respect to cre-atine (Cr). Between-group comparisons of concentration ratios (metabolite/Cr) were compared using t-tests, and ratios were converted to z-scores based on the entire patient sample for individual analyses. Lip1Lac levels were found to be higher in EGFR-negative GBMs (mean 6SD: 34.42 616.57) than EGFR-positive GBMs (17.58 613.13), however between-group com-parisons revealed only a trend. Individual analyses using a Chi-Square test demonstrated that both of the EGFR-negative GBMs had elevated levels of Lip1Lac compared to the entire patient sample, however, seven of 10 (70%) EGFR-positive GBMs had relatively low levels of Lip1Lac (p , 0.06). No differences were found for other metabolites. 1H MRS does appear to have potential in identifying surrogates of GBM subtypes. High lipid and lactate content, as seen in the EGFR-negative GBMs included in this study, has been well-documented and ascribed to a greater extent of necrosis in GBMs rela-tive to lower grade astrocytic tumors. The lower levels of these metabolites in EGFR-positive GBMs is consistent with increased angiogenesis in these glial subtypes which may be associated with less necrosis, however, further investigation is warranted. These data provide further evidence for the util-ity of advanced imaging modalities to identify MR surrogates of molecular subtypes of human glial brain tumors based on their metabolic content.

RA-19. DEXAMETHASONE AND ENHANCING GLIOMAS: ALTERATIONS IN PERFUSION AND BLOOD-TUMOR BARRIER KINETICS SHOWN BY MAGNETIC RESONACE IMAGINGDavid Jellinek,1 Nigel Hoggard,2 Iain Wilkinson,3 David Levy,4 F. Giesel,3 Charles Romanowski,3 Barbara Miller,3 and Paul Griffiths3; 1University of Sheffield, Sheffield, United Kingdom; 2Department of Radiology, University of Sheffield, Sheffield, United Kingdom; 3University of Sheffield, United Kingdom; 4Oncology, Weston Park Hospital, Sheffield, United Kingdom

Dexamethasone is routinely used in the management of gliomas, reduc-ing peri-tumoral oedema, it changes the blood brain barrier (BBB) permea-bility but there are conflicting reports about its effects on cerebral perfusion. However, radiological grading of gliomas is based on tumor enhancement determined by BBB permeability and MR measures of cerebral perfusion. The objective of this study was to study blood-lesion barrier (BLB), BBB and cerebral perfusion of glioma patients treated with dexamethasone using magnetic resonance imaging. Thirteen patients with biopsy proven gliomas underwent MR imaging before and after 3 days of dexamethasone therapy. Dynamic T1-weighted uptake imaging was performed to assess permeabil-ity and dynamic T2*-weighted imaging was used to assess cerebral perfu-

sion. Analysis was performed on both the enhancing lesion and contralateral normal-appearing white matter (CNAWM). Eleven grade IV gliomas and 2 grade III oligodendrogliomas were imaged. There was a 30.2% reduction in lesion T1-uptake after treatment (p , 0.0005). There was no significant dif-ference between T1-uptake in CNAWM. The mean lesional reduction rCBV was –25% (p , 0.05). A strong trend to reduced rCBV was demonstrated in CNAWM (–16.6%, p 5 0.06). These data suggest that dexamethasone ther-apy not only affects the permeability of the BLB but may also reduce blood flow. In this data there is a strong trend to reduced flow in the CNAWM and in a larger study group CNAWM was significantly reduced (20% reduction, p , 0.0010). There is a need to realize that that dexamethasone therapy may affect all of the factors used in so-called radiological grading of gliomas.

RA-20. A NEW MEASUREMENT CRITERION FOR VOLUMETRIC EVALUATION OF METASTATIC BRAIN TUMORSDimitri Sigounas, Paul Stewart, Samer Elbabaa, Elizabeth Bullitt, Michaux Kilpatrick, and Matthew Ewend; University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Manual segmentation is the gold standard (3D) for volumetric evalu-ation of metastatic brain tumors on MRI; however, this method is time-consuming and often impractical. Therefore, lesion volume is typically estimated by uni-dimensional (1D) or bi-dimensional (2D) measurements—the respective bases for the Response Evaluation Criteria in Solid Tumor (RECIST) and WHO guidelines for assessing metastatic disease—both of which demonstrate limited accuracy and precision. Our purpose is to propose and evaluate a novel, rapid hybrid metric (UNC) and compare it to the 1D, 2D, and 3D methods to define the optimal radiological metric for accurately assessing metastatic tumor volume and determining clinical response. To assess the agreement of each linear metric (1D, 2D, UNC) with the manual segmentation metric (3D), a single observer performed all four measurements for each of 658 metastatic brain lesion image sets obtained via T1-weighted axial MRI studies (longitudinal imaging sets of 133 hetero-geneous lesions from 94 patients treated at UNC Hospitals, 2001–2007). Per lesion, measurements consisted of the greatest diameter on any single axial slice (1D), the greatest diameter and orthogonal length (2D), and circum-scribing the lesion on every slice (3D, calculated with institutional software). The UNC metric was computed using the greatest diameter and orthogo-nal lengths, and product of axial slice number and collimation thickness. For these data, the first step in the statistical analyses of agreement was transformation of the four measures to log10 scale. Components of variance and corresponding correlations among measurements were then estimated via linear mixed-effects models. Agreement with the 3D metric was evalu-ated for each metric using the Bland-Altman approach generalized to this partly hierarchical, partly factorial repeated-measures study design. In a 4-component-of-variance model for each of 3 volumetric measures (UNC, 2D, 1D) conditional on 3D as a covariate, the estimate of total variance was 0.007, 0.018, and 0.028, respectively. Approximate 95% confidence intervals for these variance estimates and a resampling test procedure of size 5 0.05 indicated that there is a statistically significant difference between the 3 variances. The differences among these variances indicate that UNC is the metric which deviates the least from any given 3D value. This result is visually evident in bivariate graphs. Most importantly, the Bland-Altman plots clearly suggest that the UNC metric is superior to 2D and 1D in terms of agreement with the 3D gold standard. Increasing the sophistication of a linear metric criterion (1D to 2D to UNC) appears to decrease total vari-ance when conditioning on given values of segmentation analysis (3D) and improve agreement with the 3D metric. These results suggest that the UNC hybrid criterion has accuracy and precision similar to the 3D gold standard. Furthermore, the UNC method has a speed of computation similar to 1D and 2D methods. These characteristics of the UNC metric suggest that it may be better-suited for volumetric evaluation of metastatic brain tumors than RECIST and WHO guidelines for routine patient care and clinical tri-als. PDF figure files available upon request.

RA-21. EVALUATION OF MR SPECTROSCOPY IN DIAGNOSIS OF PERIVENTRICULAR ENHANCING BRAIN LESIONS: PRIMARY CNS LYMPHOMA VERSUS HIGH-GRADE GLIOMASamer K Elbabaa, Dimitri Sigounas, Lester Kwock, J. Keith Smith, Sharon Cush, and Matthew G. Ewend; University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Both primary CNS lymphoma (PCNSL) and high grade glioma (GBM) can present as periventricular enhancing brain lesions; differentiation with-out biopsy may be challenging due to the diffusely infiltrative growth pat-tern. MR spectroscopy (MRS) allows to non-invasively determine metabolic changes in vivo. This is a second unique attempt to evaluate the efficacy of pre-biopsy MRS for diagnosing periventricular enhancing brain lesions. We performed a retrospective evaluation of all patients with periventricular



PCNSL or GBM having undergone pre-operative MRS at our institution from 2000 to 2006. Medical records and radiological studies were reviewed. All patients underwent MRS on a 1.5–T imaging unit. Six PCNSL and 10 GBM patients (mean age 56.1 years, 9 males, 7 females) were identified. Sta-tistical analysis to assess diagnostic validity of metabolite level on MRS was performed. Lactate, choline, lipid, N-acetyl aspartate and myo-inositol lev-els were qualitatively categorized as decreased, absent, present, or elevated. Lipids were present in 80% of GBM (8/10) and 33% of PCNSL (2/6); if lipids were detected in a periventricular lesion by MRS, there was an 80% probability of the lesion being a GBM. Furthermore, the presence of lipids and lactate (without marked elevation) was highly predictive of GBM (PPV 5 100%). Conversely, lymphomas had a 90% probability of demonstrating very elevated lactate levels and absent lipids. Choline levels were elevated and myo-inositol was absent in virtually all GBM and PCNSL—limiting diagnostic significance for either metabolite. In our series, MRS detection of lipids and lactate without substantial elevation constitutes a useful diag-nostic parameter for identifying periventricular GBM versus PCNSL. Like-wise, absence of lipids and very elevated lactate (approximately two-fold the average level in GBM) suggests PCNSL rather than GBM. Increased lipid levels represent greater central necrosis observed in GBM, whereas elevated lactate levels reflect larger lymphocytic populations in PCNSL. Assessment of a larger number of lesions and quantitative analysis of metabolites may better clarify the role of MRS in differentiating periventricular enhancing lesions.

RA-22. TUMOR REGROWTH BETWEEN SURGERY AND INITIATION OF RADIATION THERAPY IN PATIENTS WITH NEWLY DIAGNOSED GBMAndrea Pirzkall, Colleen McGue, Suja Saraswathy, Soonmee Cha, Raymond Liu, Kathleen Lamborn, Mitchel S. Berger, Susan Chang, and Sarah Nelson; University of California, San Francisco, San Francisco, CA, USA

To assess incidence and degree of tumor regrowth in patients s/p surgi-cal resection of a GBM relative to the time between surgery and begin of RT; and to correlate tumor regrowth with survival and quantitative imag-ing parameters acquired prior to surgery. Thirty-two patients with newly diagnosed GBM accrued through a SPORE protocol underwent advanced MR imaging [MRI, 3D MR Spectroscopy (MRSI), perfusion (PWI), diffu-sion weighted imaging (DWI)] at multiple time points including immedi-ately post-surgery and prior to initiation of adjuvant treatment (conformal RT and Temodar-based chemotherapy). Regions of contrast enhancement (CE) on pre-RT MRI were outlined and compared visually to the extent of reduced diffusion seen on postsurgical DWI to differentiate tumor growth from postsurgical injury. Quantitative MR parameters were calculated and included: from MRSI, peak heights for Choline (Cho), Creatine (Cr), N-actetylasparatate (NAA), Lactate, Lipid; from PWI, the height of the peak in the concentration time curve during delivery of the bolus (PH) and percent recovery to baseline levels (Rec); from DWI, apparent diffusion coef-ficient (ADC) and fractional anisotropy (FA). Statistical analyses included rank sum, log rank and Wilcoxon tests. Postsurgical MRI indicated that 18 patients had undergone gross total resections, and 14 patients subtotal resections. RT was initiated 14 to 46 days after surgery (median 32.5) with pre-RT imaging acquired 5–21 days prior to RT (median 6). Regions of reduced diffusion were seen in 21 patients post surgery and 25 patients showed new/increased CE pre-RT. In 8 patients (25%) the new CE was spatially confined/related only to areas of postsurgical reduced diffusion. In 17 patients (53%) at least some portion of new CE was spatially unre-lated to postsurgically reduced diffusion and was considered indicative of tumor growth (6, 19%) or a combination of tumor growth and surgical injury (11, 34%). MR imaging parameters obtained within the CE prior to RT demonstrated higher Cho, Cho-to-NAA and Cho-to-Cr indices with a trend toward reduced ADC in patients with presumed tumor growth vs. patients with postsurgical injury, suggesting active metabolism and tumor cell proliferation. Similarly, MR data acquired prior to surgery indicated that higher perfusion and creatine values within the non-enhancing por-tion of the tumor predict subsequent tumor growth in respective patients. Twenty-one (66%) patients have died; median survival was 14.6 months in patients with interim tumor growth vs. 24 months in patients without. The Wilcoxon test which emphasizes differences between groups in early deaths was statistically significant (p 5 0.02) while the log-rank test which empha-sizes later events was not (p 5 0.1). Increases in CE that are interpreted as corresponding to tumor growth occur in a considerable portion of patients (53%) during the interval between surgery and RT, with a negative impact on survival. Our data support maximal safe resection in newly diagnosed GBM and encourage considering an earlier start of adjuvant therapy. These data also support the value of pre-RT imaging in conjunction with metabolic/ physiologic imaging and the acquisition of postsurgical DWI in order to refine interpretation of contrast enhancement and, ultimately, to improve patient selection and management as well as RT treatment planning.

RA-23. PREOPERATIVE MRI CHARACTERISTICS OF GLIOBLASTOMA MULTIFORME: IMPLICATIONS FOR UNDERSTANDING GLIOMA ONTOGENYLeif-Erik Bohman,1 Christopher Mandigo,2 Todd Hankinson,2 Marcela Assanah,2 Kristin Swanson,3 Peter Canoll,4 and Jeffrey Bruce2; 1Columbia University, New York, NY, USA; 2Neurological Surgery, Columbia University Medical Center, New York, NY, USA; 3Pathology, University of Washington, Seattle, WA, USA; 4Neuropathology, Columbia University Medical Center, New York, NY, USA

Glioblastoma multiforme (GBM) is one of the most common intra-cranial neoplasms in adults yet its ontogeny remains unclear. Recent inves-tigation has focused on the neural stem cells of the sub-ventricular zone (SVZ) as candidate “cancer stem cells” (Jackson et al., 2006). However, the largest population of cycling cells in the adult CNS are multi-potent glial progenitors distributed throughout the subcortical white matter (Roy et al., 1999; Dawson et al., 2003). An animal model has also been developed which induces glioma-like tumors by infecting these progenitors via growth factor overexpression (Assanah et al., 2006). Recent work in our lab has shown human gliomas contain large numbers of cells bearing white matter progenitor markers (Ogden et al., submitted). In this context, we sought to investigate whether a pattern would emerge from an analysis of the pre-operative radiographic appearance of GBMs which suggested an predomi-nant origin in a particular brain region. Sixty-three consecutive patients pathologically diagnosed with glioblastoma multiforme with preoperative contrast-enhanced MRIs were included. Tumors were assessed with fine-cut images in 3 planes and their proximity of both the contrast-enhancing rim and the estimated center point of the tumor to the ventricle was mea-sured. Tumor volume was estimated by measuring average tumor radii in 3 planes and treating the tumor as an sphere. Comparisons of means were performed with Mann-Whitney U-tests and correlation was assessed using Spearman’s Rank Correlation Coefficient. The average volume of tumors was 29.6 cm3. 46% of tumors abutted the ventricle. Of those that did not abut the ventricle, 76% had Edge-to-Ventricle Lengths (EVLs) of at least 5 mm, and 20.6% were .10mm from the ventricle. Tumors which abutted the ventricle were significantly larger, with mean volumes of 36.4 cm3 com-pared to 23.7 cm3 for tumors not abutting the ventricles (p 5 0.0076). EVL showed a significant negative correlation with tumor volume (r 5 –0.4886, p , 0.0001) whereas Center-Point-to-Ventricle-Length (CPVL) showed no significant correlation with volume (r 5 0.2212, p 5 0.08). Our results sup-port that most gliomas are found in the subcortical white matter rather than the SVZ. More than half of the tumors are radiographically distinct from the ventricles. Increasing tumor size correlated with increased proximity to the ventricles, suggesting that ventricular contact may be more a function of a growing mass in a limited intracranial space rather than an indicator that the tumors necessarily originate in the SVZ. These results demonstrate that for a majority of gliomas an origin in the SVZ is unlikely. Instead gliomas may originate from neuroprogenitors in the subcortical white matter. We are currently using computerized models of glioma growth to further explore these patterns.

RA-24. MRI IMAGING OF THE EFFECTS OF CEREPRO IN HIGH-GRADE GLIOMAAnu-Maaria Sandmair,1 Yla-Herttuala Seppo,2 Gillian Langford,3 and Neil Murray3; 1Neurochirurgische Klinik, Klinikum Ingolstadt GmbH, Ingolstadt, Finland; 2Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland; 3Ark Therapeutics Ltd, London, United Kingdom

A randomized, controlled study involving 36 patients with operable primary or recurrent glioma showed that CereproTM plus standard care was associated with an 81% mean increase in survival vs. standard care alone. To illustrate typical outcomes as seen on MRI imaging following use of CereproTM in patients with operable high-grade glioma, highlight issues that neurosurgeons should be aware of when using this therapeu-tic agent, and evaluate clinical benefit of CereproTM using MRI imaging. Seventeen patients were randomized to CereproTM (3 3 1010 pfu in 10 ml) by 30–70 injections to a depth of 10 mm into the wound bed following tumour resection; or control (resection and no injections) (n 5 19). All patients received radiotherapy. CereproTM patients received GCV (5 mg/kg IV b.d.) on days 5–19. MRI was conducted within 14 days prior to surgery; on Day 1 or 2 post-operation; after GCV treatment (Day 19) and every 3 months thereafter. MRI images will be presented for a sample of patients at all available time-points pre- and post-operation. Most patients showed no marked oedema following administration of CereproTM in Day 1 images compared with pre-operative images. Some oedema is visible in the Day 1 post-operative images; however, this could be due to the surgical procedure or the tumor as well as a result of CereproTM administration. It is relatively easy to distinguish between residual tumor and oedema due to the contrast enhancement. In addition, a bright, thin layer can be seen around the tumor cavity on the MRI post-operative images, probably due to oxygenized cel-lulose. In many Day 19 images, i.e., after GCV-treatment, the tumor cavity appears bright; this could be an effect of the CereproTM administration. In this study, CereproTM provided a significant survival benefit over standard



care (Immonen 2004;967–72). MRI provides a useful tool for assessing the effectiveness of surgery and responses to CereproTM therapy. Understand-ing and accurate interpretation of these images will enable more effective treatment of patients with high-grade glioma with surgery, radiation plus CereproTM.

RA-25. CO-REGISTRATION OF FUNCTIONAL MRI DATA AND DIFFUSION TENSOR TRACTOGRAPHY WITH INTRAOPERATIVE NEURONAVIGATION: METHODS AND APPLICATIONSBharat Guthikonda,1 James Leach,2 Ronald Warnick,1 Christopher McPherson,1 Philip Theodosopoulos,1 and John Tew Jr1; 1Department of Neurosurgery, University of Cincinnati, Cincinnati, OH, USA; 2Department of Radiology, University of Cincinnati, Cincinnati, OH, USA

Intra-operative neuronavigation (IGS) during brain tumor surgery is a technique that has become more sophisticated over the last decade. Simultaneously, neuroimaging advances in tractography and functional MRI have made glioma surgery much safer. The combined use of these two modalities is an even more recent advance. We present our experience of co-registration of functional MRI and tractography data in an attempt to increase the safety of intra-operative neuronavigation in eloquent regions. We also show its utility in pre-operative decision making regarding totality of planned resection, need for awake craniotomy and decision to perform biopsy rather than a more aggressive resection. Furthermore, we confirmed the accuracy of the imaging data using direct awake cortical stimulation in a single case study. Pre-operative tractography and functional MRI scanning (3T MRI) for motor and/or language function were performed in 14 patients with intraaxial brain tumors. Areas localized by these studies included Bro-ca’s area, Wernicke’s area, primary motor cortex, the corticospinal tract and the arcuate fasciculus. Tractography and functional MRI results were co-registered onto a standard anatomical MRI scan using BrainLab soft-ware for the purpose of pre-operative decision making and intra-operative neuronavigation. In a single case, standard awake craniotomy techniques were performed in a patient with a left insular glioma. Using direct cor-tical stimulation, the face motor cortex and areas causing speech arrest, naming difficulty and paraphasias were identified and correlated with IGS predictions. Co-registration of tractography and functional MRI was suc-cessfully achieved in all patients. The representation of crucial language and motor regions by preoperative tractography and functional MRI was confirmed to be highly accurate (86%) based on intraoperative awake testing. In one patient with a left opercular tumor, the operative plan was modified to perform a stereotactic biopsy rather than a resection because of the high risk to language function predicted by the pre-operative imaging. The corticospinal tract could be accurately delineated at least as caudal as the ponto-mesencephalic junction, as demonstrated in a patient with a tectal mass. Safe corridors of approach to these intraaxial lesions were planned pre-operatively based upon the advanced imaging data. We describe our experience of 14 cases of co-registration of tractography and functional MRI data for the purpose of pre-operative planning and intra-operative neuronavigation. Co-registration was feasible in all cases and helped guide the optimal surgical treatment of gliomas in eloquent regions of the brain. In one case, the neuroimaging information was confirmed, by direct awake cortical stimulation, to be highly accurate.

RA-26. REAL-TIME INTRAOPERATIVE BRAIN IMAGING WITH 3-DIMENSIONAL ULTRASOUND: THE UK EXPERIENCE IN RESECTION OF GLIOMASDavid Jellinek and Charles Romanowski; University of Sheffield, Sheffield, United Kingdom

Intraoperative real time 3D ultrasound imaging has been available in Scandinavia and Germany since the beginning of this decade. It has reported to provide a practical alternative to intraoperative MRI scanning as like intraoperative MRI it overcomes the problems associated with preoperative neuronavigation datasets. Its value as a surgical tool was recently reviewed by Unsgaard (Acta Neurochir 2006;148:235–253. Sheffield is the only unit in the UK to have access to intraoperative 3D ultrasound imaging. 3D real time intraoperative ultrasound has been routinely employed during glioma resection since October 2006. Objective: To assess the accuracy of intra-operative 3D ultrasound images in the determination of glioma resection. Patients undergoing planned radical resection of their glioma are imaged intraoperatively with a 3D ultrasound system—“Sonowand.” At the end of the procedure a final image data set is recorded to establish the apparent vol-ume of residual disease. This intraoperative imaging is then compared with MRI imaging performed within 24 hours of surgery to establish the veracity of the ultrasound intraoperative 3D imaging. The average additional operat-ing time is under 10 minutes for this step. Assessment of post operative MRI

imaging has confirmed the accuracy of intraoperative 3D ultrasound as a reliable tool in determining extent of tumor resection. The learning curve in interpretation and acquisition of intraoperative 3D ultrasound is very steep. The system is easy to use and rapidly produces high quality intraoperative imaging. It appears to be of the greatest value in aiding resection of low-grade tumors, where published intraoperative MRI data suggest there is maximal survival advantage from a radical surgical resection.

RA-27. INTRAOPERATIVE MRI AT 3 TESLA: FEASIBILITY, SAFETY, AND PRELIMINARY RESULTSChristian Raftopoulos, Aleksandar Jankovski, Geraldo Vaz, Edward Fomekong, Thierry Duprez, Marie-Agnes Docquier, Michel Van Boven, and Guy Cosnard; Université Catholique de Louvain, Brussels, Belgium

We developed a twin neurosurgical-MR suite with a 3T intraoperative MRI (iMRI) available for the neurosurgeons, but also for independent use by the radiologists. Our aim is to report on the feasibility of such a suite. Our new suite has two areas, one dedicated to neurosurgery, the other to the iMRI. The operating table is completely motorized enabling easy transfer of the patient into the iMRI. These two areas can be completely independent allowing the iMRI to be used also for non-surgical cases. We report our findings from the first 50 patients to have been planed for surgery with iMRI in this suite (average age: 47.8 yrs; weight: 5–110 kg; intracra-nial tumor 37; epilepsy surgery 5; tumor 1 epilepsy surgery 2; and 6 were planed but not performed). Fifty-one iMRI examinations were performed: 6 immediately pre-surgery, 16 during surgery (skull not definitely closed), and 29 immediately post-surgery. Minor technical dysfunctions lengthened 20 iMRI scans. No serious iMRI related incident occurred. Thirty-two iMRI examinations took an average of 83 mins (min 58, max 129 min) to per-form. Seven patients benefited from a second surgical look after their iMRI examination to remove a residue. Complete tumor resection was achieved in 27 cases. The interpretations of 5 of the iMRI scans (total resections) were modified within a few hours after surgery (not total resection). Control MRI (2 months later) of these 5 cases demonstrated that 3 of them had, in fact, been total resections. In 2 cases the radiologist could not ascertain whether the removal was total or near total. Our new complex allows the radiologists to use the 3T iMRI at all times except when required by the neurosurgeons. Three patients benefited from the iMRI examination to achieve total resec-tion. However, the scans in 2 patients were wrongly interpreted as total resection. Although time consuming, 3T iMRI appears to be a safe tool.

RADIATION THERAPY

RT-01. INITIAL EXPERIENCE WITH BEVACIZUMAB TREATMENT FOR BIOPSY-CONFIRMED CEREBRAL RADIATION NECROSISRoy Torcuator,1 Y.S. Mohan,2 Ian Lee,2 Jack Rock,3 Thomas Doyle,2 Joseph Anderson,2 Samuel Ryu,2 Jorge Gutierrez,2 Rajan Jain,4 Mark Rosenblum,2 and Tom Mikkelsen3; 1Department of Neurosurgery, Henry Ford Hospital, Detroit, MI, USA; 2MI, USA; 3Detroit, MI, USA; 4Henry Ford Hospital, Detroit, MI, USA

Cerebral radiation necrosis is a recognized potential treatment compli-cation among patients with brain tumor that develops 2 to 3 years after radi-ation. Clinical manifestations produced by localized brain necrosis depend upon the location resulting to focal neurological deficits or more general-ized signs and symptoms of increased intracranial pressure. It is caused by vascular endothelial damage resulting to fibrinoid necrosis of small arte-rial vessels. The occlusion of vessels results in focal coagulative necrosis and demyelination of overlying brain parenchyma. Surgical decompres-sion of any radionecrosis-related mass effect can provide helpful palliative effect. Corticosteroids usually produce prompt symptomatic improvement. Therapeutic anticoagulation and hyperbaric oxygen therapy have also been reported to provide benefit, but their efficacy is far from established. Beva-cizumab is a humanized monoclonal antibody against vascular endothelial growth factor (VEGF, also known as vascular permeability factor). Since blockage of VEGF from reaching its capillary targets is a logical treatment strategy for radiation necrosis, bevacizumab might be an effective treat-ment option. Four patients with glioblastoma multiforme previously treated with radiation were biopsied for imaging progression suggestive of radiation necrosis. In addition to MRI, perfusion CT scans were performed as part of the pre-operative studies. Biopsy sites were correlated with areas of low blood volume and permeability on CT perfusion. Multiple biopsy sites were sampled, and 3 out of 4 patients showed pure radionecrosis with 1 patient revealing mixed tumor and necrosis. The patients were then treated with bevacizumab (2/4) alone (10 mg/kg) or combined with Irinotecan (2/4), both given as infusion every 2 weeks. MRI studies were then obtained at 6 week



intervals to determine response to treatment. Bi-dimensional measurements of both Gadolinium enhanced and FLAIR sequences of the largest radia-tion necrosis lesions were performed. The difference in the measurements was expressed as a percentage change from the pretreatment MRI images. The post-treatment MRI for all 4 subjects performed at 6 weeks after the start of bevacizumab therapy showed a reduction in both the FLAIR and T1-weighted post-Gadolinium contrast abnormalities. The average area change in the post-Gad studies was 77.5% and 44.25% for the FLAIR images. The significant imaging changes seen in the follow-up studies of these 4 patients with biopsy-confirmed radiation necrosis are probably related to the relative normalization of the blood brain barrier secondary to decreased levels of VEGF brought about by bevacizumab. Gonzales et al. first reported the effect of bevacizumab in 8 patients with radiographic evidence of radiation necrosis. In this case series, we report our experience with the use of bevacizumab for biopsy-confirmed cerebral radiation necro-sis. Bevacizumab is a potential treatment option for patients with cerebral radionecrosis. Further studies are needed to establish the role of this agent in the treatment of radionecrosis.

RT-02. CLINICAL EXPERIENCE WITH RADIATION THERAPY IN THE MANAGEMENT OF NEUROFIBROMATOSIS-ASSOCIATED CENTRAL NERVOUS SYSTEM TUMORSStacy Wentworth,1 Thomas Ellis,2 Steven Glazier,1 Kevin McMullen,1 Volker Stieber,1 Stephen Tatter,1 and Edward Shaw1; 1Wake Forest University, Winston-Salem, NC, USA; 2Neurosurgery, Wake Forest University, Winston-Salem, NC, USA

Patients with neurofibromatosis (NF) frequently develop tumors of the central nervous system (CNS). Radiation therapy (RT) is sometimes used in treating these lesions. To better define the efficacy of RT in controlling NF-associated CNS tumors, we reviewed our 20 year experience. Seventy-four lesions in 16 patients with NF were treated with RT from 1986 to 2006. One third of patients had NF1, two thirds NF2. Median follow-up was 36 months. Progression was defined as tumor growth or recurrence in an irradiated lesion on serial imaging. Progression-free survival was measured from date of treatment to date of last imaging follow-up. The actuarial rates of progression-free survival were calculated according to the Kaplan-Meier method. On average, 5 lesions were treated per patient. The most common indication for treatment was growth on serial imaging. Median age at time of treatment was 24.1 years (range 4.3–57.1). The treated lesions included acoustic neuromas (9%), ependymomas (7%), low-grade gliomas (12%), meningiomas (62%), and non-acoustic schwanommas/neurofibromas (8%). Most patients received stereotactic radiosurgery. The others received frac-tionated external beam RT. Overall survival at 5 years for all patients was 94%. Five-year progression-free survival was 100% (acoustic neuromas), 75% (ependymomas), 100% (low-grade gliomas), 86% (meningiomas), and 100% (non-acoustic schwannomas). This is the largest published experience on the results of RT in NF patients with CNS tumors. The progression-free survival rates herein are similar or superior to those published for non-NF patients treated with RT. RT should be considered in NF patients with CNS tumors.

RT-03. DELAY IN RADIATION LEADS TO SHORTER LENGTH OF SURVIVAL IN GBM: ANALYSIS OF THE GLIOMA OUTCOMES DATAJonas Sheehan,1 Stuart Burri,2 Edward R. Laws,3 and Elana Farace1; 1Penn State College of Medicine, M.S. Hershey Medical Center, Hershey, PA, USA; 2Carolinas Medical Center, Charlotte, NC, USA; 3Stanford University, Palo Alto, CA, USA

Although there is a common belief that a delay in the start of radiation following diagnosis of a glioblastoma should have a negative effect, there is surprisingly little research to this area. Burnet (2006), in a mathematical model of 129 GBM patient outcomes, found that the mean time to tumor doubling was 24 days, suggesting a theoretical adverse effect to a delay in radiation therapy following diagnosis. Do (2000) showed shorter length of survival in patients with prolonged waiting time in 182 patients with malig-nant glioma equating to a 2% increased risk of dying per day of waiting for radiotherapy, and found that patients who started radiotherapy immediately as opposed to within 4 weeks of surgery lived on average 6.4 weeks longer. The purpose of this study is to evaluate the relationship between timing of radiation and survival outcomes in the Glioma Outcomes dataset. The Glioma Outcomes Project is a database of 788 patients which was a natural history database of all surgical glioma patients at 52 institutions in North America between December 1997 and December 2001. For this analysis, a multivariate Cox proportional hazards analysis was performed with the established predictors of length of survival (baseline KPS, age at diagnosis) as covariates. Patients were included if first pathology was GBM, and if in the perioperative period radiotherapy was planned. Length of survival was

compared between those who did and did not receive radiotherapy by three months post-diagnosis. Data are only reported from those patients who lived to the first outcome assessment at three months post-diagnosis. Out-comes from 380 patients were therefore available for analysis with complete data for all elements in the multivariate model. The patient population was 60% male, 89% righthanded, 86% white, 3% black, 1% Asian, 8% other. The median age at diagnosis was 58 years (range 18–87). Of the patients with a perioperative plan to receive radiation, 90% did so within the first three months. The overall model was significant (p , 0.001) as expected. The covariates were also significant predictors of outcome (age at diagno-sis p 5 0.006; baseline KPS p 5 0.009). Receipt of radiotherapy by three months following surgery trended toward significance (p 5 0.09) with sharp differentiation in survival curves in the multivariate model; in the univari-ate model Kaplan-Meier curve also trended toward significance (p 5 0.08). Differences in median survival were 47 weeks in the patients who received radiotherapy versus 41 weeks in those who did not. Radiation delay appears to have a negative impact on length of survival, both in this dataset and in one previous report, to a surprisingly large extent. The length of survival extended by radiating a patient earlier rather than later is suggested to have an effect similar to the extended length of survival of approved chemothera-peutic treatment for GBMs including carmustine wafers and temozolomide. This finding bears further analysis in other datasets.

RT-04. SPINAL CORD ASTROCYTOMA: A SURVIVAL BENEFIT FOR POST-OPERATIVE RADIOTHERAPYPaul Brown,1 Kiernan Minehan,1 Bernd Scheithauer,1 Michael Wright,2 and William Krauss1; 1Mayo Foundation for Medical Education and Research, Rochester, MN, USA; 2University of Washington, WA, USA

The objective of this study was to identify prognostic factors and deter-mine the impact of surgery and radiotherapy on outcome of spinal cord astrocytomas. This study consists of 136 consecutive patients with a spinal cord astrocytoma treated at a single institution between 1962 and 2005. Extent of surgery included incisional biopsy (59%), subtotal resection (25%), and gross total resection (16%). Post-operative radiotherapy was delivered to 75% of patients. There were 69 pilocytic and 67 non-pilocytic astrocytomas with a median follow-up of 8.2 years. Patients with pilocytic tumors had significantly better median overall survival (MOS) than those with non-pilocytic astrocytomas (1.85 vs. 39.9 years; p , 0.0001). For both pilocytic and non-pilocytic astrocytomas, patients had a worse outcome with more extensive resections (pilocytic 39.7 vs. 18.1 years; p 5 0.066; non-pilocytic 42 vs. 25 months; p 5 0.137 MOS). Post-operative radio-therapy did not result in a significant survival benefit for pilocytic tumors but did for the non-pilocytic astrocytomas (pilocytic 39.8 vs. 18.1 years; p 5 0.329; non-pilocytic 24 vs. 3 months; p 5 0.006 MOS). Multivariate analy-ses found pilocytic histology, diagnosis in the era of MRI, longer symptom duration, younger age, minimal extent of surgery, post-operative radiother-apy all significantly predictive of a better survival. Histology is the most important prognostic variable for spinal cord astrocytomas. Extensive sur-gical resection greater than a biopsy produces a less favorable survival and remains an unproven treatment. Post-operative radiation therapy improves survival significantly for the diffuse fibrillary astrocytomas subgroup but not for pilocytic tumors.

RT-05. RE-IRRADIATION USING HIGH-PRECISION RADIOTHERAPY AND CONCOMITANT TEMOZOLOMIDE IN PATIENTS WITH RECURRENT GLIOMAStephanie E. Combs, Johanna Wagner, Daniela Schulz-Ertner, Marc Bischof, and Juergen Debus; Radiation Oncology, Neuro-Radiation Oncology Research Group, Heidelberg, Germany

This study was aimed to assess the toxicity and efficacy of radiochemo-therapy (RCHT) with temozolomide performed as re-irradiation in patients with recurrent glioma. We treated 17 patients with RCHT for recurrent glioma between July 2005 and December 2006. Nine patients were female, 8 were male. Pathology evaluation revealed WHO Grade II Astrocytoma in 5, Anaplastic Astrocytoma in 4, and Glioblastoma multiforme (GBM) in 7 patients. One patient suffered from Gliosarcoma. Median age at primary diagnosis was 33 years (range 6–60 years). Surgery had been performed as total resection in 4 patients, as subtotal resection in 9 patients, and as a biopsy in 4 patients. Median doses of radiotherapy (RT) of 60 Gy had been applied. Prior treatment with temozolomide either as RCHT or as che-motherapy alone for tumor progression had been performed in 8 patients. The median time interval between primary RT and re-irradiation was 29.5 months (range 5–180 months). Re-irradiation was performed as high- precision radiotherapy using fractionated stereotactic radiotherapy (FSRT) in 16 patients with a median total dose of 36 Gy (range 28–36 Gy); in 1 patient stereotactic radiosurgery (SRS) was applied for a very small lesion with a single dose of 18 Gy/80% isodose. The target volume was defined as the area of contrast enhancement on T1-weighted MRI-scans including



a safety margin of 0.5 to 1 cm. Chemotherapy was applied concomitantly in a median dose of 50 mg/m2 per day (median of 100 mg TMZ qd); in 14 patients RCHT was followed by adjuvant cycles of temozolomide. Treat-ment could be completed in all patients without interruptions due to side effects; no severe acute or long-term toxicities were observed. One patient with recurrent GBM showed tumor progression during therapy and treat-ment was stopped at 32 Gy out of 36 Gy. Progression-free survival was 56% at 6 months after FSRT; survival after FSRT was 75% at 6 months. Median overall survival was 39 months, with 92% at 1 and 2 years. High-precision RT and concomitant temozolomide is safe and effective when performed as re-irradiation in recurrent glioma. The data demonstrate that re-challenge with temozolomide justifies further evaluation.

RT-06. RADIOCHEMOTHERAPY WITH TEMOZOLOMIDE FOR PRIMARY GLIOBLASTOMA MULTIFORME COMPARING TWO DOSE REGIMENS: 50 VERSUS 75?Stephanie Combs, Johanna Wagner, Marc Bischof, Juergen Debus, and Daniela Schulz-Ertner; Radiation Oncology, Neuro-Radiation Oncology Research Group, Heidelberg, Germany

Standard treatment for patients with glioblastoma multiforme (GBM) is currently considered to be post-operative radiochemotherapy (RCHT) with temozolomide (TMZ) with 75 mg/m2. Previously, we had performed a phase I/II study with a dose of 50 mg/m2 yielding comparable survival data in our instituton. In the present analysis, we evaluated toxicity and outcome in 160 patients with GBM treated with post-operative RCHT comparing these two dose regimens. One hundred sixty patients with histologically confirmed GBM were treated with post-operative RCHT with TMZ; 66 patients were female, 94 were male. Median age was 60 years (range 19–76 years). After primary diagnosis, a biopsy had been perfomed in 42 patients; subtotal and total resections were conducted in 66 and 52 patients, respectively. Patient characteristics were equally distributed in both groups, including age, extent of neuorosurgical resection, as well as performance status. In all patients post-operative radiotherapy was with a median dose of 60 Gy, applied in a median fractionation of 5 3 2 Gy/week. Concomitant TMZ was applied at 50 mg/m2 in 123 patients (group A) and 75 mg/m2 in 37 patients (group B). Adjuvant cycles of TMZ were prescribed in 13 patients in group A and in 21 patients in group B. Patients were followed in 3-months intervals after RCHT, the median follow-up time was 13 months (range 1–75 months). Median overall survival (OS) in group A and in group B was 67% versus 79% at 1 year and 43% versus 49% at 2 years, respectively (p 5 0.65). Progression-free survival (PFS) was 49% versus 54% at 1 year and 22% versus 29% at 2 years (p 5 0.46). Patients receiving adjuvant cycles of TMZ showed OS of 93% versus 65% at 1 year and 63% versus 40% at 2 years (p 5 0.03); PFS in patients receiving adjuvant TMZ was 71% as compared to 45% in the RCTH-only group (p 5 0.008). Hematological toxicity was not statistically significant over the 6-week treatment course except for a significant decrease in platelets in week 6 (p 5 0.01) in group B. Comparing these two dose regimens, overall survival seems to be comparable in both groups; however, there is a longer follow-up and a larger group of patients is unaltered in both treatment groups.

RT-07. HYPOFRACTIONATED RADIOTHERAPY (XRT) PLUS CONCURRENT AND ADJUVANT VERSUS SALVAGE TEMOZOLOMIDE (TMZ) IN ELDERLY PATIENTS WITH GLIOBLASTOMA MULTIFORME: A RETROSPECTIVE REVIEWJeffrey Cao,1 Glenn Bauman,1 Barbara Fisher,1 David Macdonald,2 Joseph Megyesi,3 and Chris Watling3; 1Radiation Oncology, London Regional Cancer Program, London, Ontario, Canada; 2Oncology, London Regional Cancer Program, London, Ontario, Canada; 3Clinical Neurological Sciences, London Health Sciences Centre, London, Ontario, Canada

Glioblastoma multiforme (GBM) is the most frequently diagnosed and most aggressive primary malignant brain tumor in adults. A recent land-mark study (Stupp et al., NEJM 2005) demonstrated a survival advantage for concomitant and adjuvant TMZ chemotherapy added to a standard course of XRT (60 Gy in 30 fractions over 6 weeks) compared to XRT alone. The overall survival benefit of this aggressive treatment, however, was attenuated in older or poor performance status patients. In a study reported by Roa et al. (J Clin Oncol 2004), elderly GBM patients treated with a shorter hypofractionated course of XRT (40 Gy in 15 fractions over 3 weeks) demonstrated similar survival and palliative benefit compared to a stan-dard 6-week course (overall survival 5.6 vs. 5.1 months, p 5 0.57). Taken together, these trials suggest that hypofractionated XRT with concurrent and adjuvant TMZ may be a reasonable palliative option. To evaluate the effectiveness of this strategy, we conducted a retrospective review of elderly patients diagnosed with GBM between 2000 and 2006 who were treated in this fashion at our institution. We identified 34 patients who received

hypofractionated XRT plus concurrent and adjuvant TMZ. Over the same time period, we also identified 20 elderly patients with GBM who received hypofractionated XRT alone with TMZ delivered as salvage treatment at the time of clinical or radiographic progression. Among the concurrent XRT 1 TMZ patients, mean age was 69.1 years (range 59–81, SD 5.9), median KPS was 80 (range 30–100), and 18/34 (53%) had received prior debulk-ing surgery. Median overall survival among the XRT 1 TMZ patients was 5.2 months (95% CI, 4.5–8.6), similar to that reported by Roa et al. for hypofractionated XRT alone. The sequentially treated patients were simi-lar in demographics (mean age 67.3 years [range 60–81, SD 5.1], median KPS 80 [range 30–90]), 14/20 (70%) had received debulking surgery, and median overall survival was greater at 13.4 months (95% CI, 10.0–19.3). Our results demonstrate that concurrent TMZ chemotherapy does not con-fer an additional survival benefit in this elderly population. Interestingly, our data suggest that a sequential approach (XRT initially with TMZ for sal-vage) may be a more effective strategy in this elderly patient population. This could be because it allows better selection of patients with sensitive disease (i.e., responding to initial XRT alone) who may benefit from more intensive therapy. A randomized phase III study comparing TMZ and short-course XRT versus short-course XRT alone for elderly GBM patients has recently been opened (NCIC CE.6/EORTC 26062-22061) and will help answer this question in a definitive fashion.

RT-08. USE OF FOCAL RADIATION FOR NEWLY DIAGNOSED HIGH-GRADE GLIOMAAllen Sills,1 Allen Redmond,2 Mary Mynatt,1 and Nilesh Dubal3; 1Semmes Murphey Clinic, Memphis, TN, USA; 2Memphis Regional Brain Tumor Center, TN, USA; 3Methodist University Hospital, TN, USA

The use of radiotherapy as an adjunct in the treatment of newly diag-nosed high-grade glioma is well established. We are exploring the delivery of radiation by a balloon brachytherapy catheter implanted at the time of surgical debulking of high-grade gliomas. We have used focal radiation as the initial treatment regimen, with deferment of external beam radiotherapy until disease progression. A total of 14 patients with high-grade glioma have been treated with surgical debulking and implantation of the brachytherapy catheter. Median age is 57.5 years (range 26–84). Within 21 days after sur-gery, a 125I solution has been instilled into the brachytherapy catheter to deliver focal radiation therapy. Concomitant temozolomide has been given during brachytherapy treatment in 8 patients. Adjuvant temozolomide in monthly cycles was continued after brachytherapy in 11 patients, including those patients who received concomitant temozolomide. All patients have tolerated brachytherapy well. Subsequent external beam radiotherapy has been given in 10 patients at an average of 6.3 months after initial surgery (range 1–12 months). Follow-up has ranged from 2 to 17 months. In those patients who have been followed for at least 6 months (n 5 12), median sur-vival is 14 months. Radiation necrosis has been noted to date in 2 patients who also received subsequent external beam radiotherapy. Focal radiation with and without concomitant temozolomide may be given to newly diag-nosed high-grade glioma patients with reasonable safety. This strategy may offer an alternative for patients who wish to defer or who decline conven-tional radiotherapy.

RT-09. A PHASE II STUDY UTILIZING FOCAL RADIATION IN PATIENTS WITH 1–3 BRAIN METASTASESAllen Sills,1 Stephen Tatter,2 Robert Fraser,3 Anthony Asher,3 Michael Vogelbaum,4 Kevin Judy,5 Gregory Canute,6 Randy Jensen,7 and Allen Redmond8; 1Semmes Murphey Clinic, Memphis, TN, USA; 2Wake Forest University, Winston-Salem, NC, USA; 3Carolinas Medical Center, Charlotte, NC, USA; 4Cleveland Clinic Foundation, Cleveland, OH, USA; 5University of Pennsylvania, Philadelphia, PA, USA; 6SUNY Upstate Medical University, Syracuse, NY, USA; 7University of Utah, Salt Lake City, UT, USA; 8Memphis Regional Brain Tumor Center, Memphis, TN, USA

Addition of radiation following brain metastases resection has shown reduction in local failure rates. Deferring whole brain radiation may reduce the risk of neurological deficits related to radiation toxicity. This study eval-uates brachytherapy in combination with stereotactic radiosurgery (SRS) in patients with 1–3 brain metastases. The balloon brachytherapy catheter is implanted at surgery and filled within 21 days with a 125I solution. A total dose of 60 Gy to 5 mm is delivered to the tumor bed. Remaining lesions may be treated with SRS. Primary objectives include 6-month and 1-year local control. Forty-eight patients have been treated. Median age is 62 years (range 37–79). Pathology includes: lung (26), breast (10), melanoma (5), unknown (2), renal (2), uterine (1), ovarian (1), and liver (1). Local tumor recurrence is documented in 1 case at 3 months and 1 case at 9 months post-brachytherapy. Distant recurrence is documented in 5 cases at 1 month, 9 cases at 3 months, 3 cases at 6 months, 2 cases at 9 months, and 1 case at 12 months. Local and distant recurrence are documented in 1 case at



3 months. One case of biopsy-proven radiation necrosis is documented at 12 months, and 1 case of PET positive radiation necrosis is documented at 15 months. Quality-of-life measures remain stable in patients reaching 12-month follow-up. Grade 3 or greater toxicity attributed to treatment includes: radiation necrosis (2), cerebral abscess (1), tumor cavity hemor-rhage (1), seizure (1), and wound infection (1). Enrollment will continue for a total of 50 patients. Preliminary results seem encouraging regarding safety and efficacy.

RT-10. A DOSIMETRIC COMPARISON OF FIVE BRAIN TUMOR TREATMENT PLANS FOR TREATMENT WITH HELICAL TOMOTHERAPY AND GAMMA KNIFELinda Grossheim1 and Paul Jursinic2; 1Society for Neuro-Oncology, Milwaukee, WI, USA; 2Radiation Oncology, West Michigan Cancer Center, MI, USA

Our objective was to determine whether treatment plans developed for helical Tomotherapy IMRT delivery are acceptable compared to Gamma Knife plans for brain tumors less than 3 cm in size. Gamma Knife treatment plans were developed with the Leksell Gamma-plan software (Elekta Instru-ments, Norcross, GA). The 5 cases that were studied included 2 acoustic neuromas, 2 meningiomas, and 1 glomus jugularis, all with tumor diam-eters less than 3 cm. These cases were planned and treated with Gamma Knife in our clinic. Helical Tomotherapy treatment plans were developed with the treatment-planning platform of the HiART system (TomoTherapy, Inc., Madison, WI). The HiART system can deliver radiation with a 6 MV X-ray beam that is intensity modulated and moves in a helical arc around the target volume of a patient. Tomotherapy is now used at MCW for IMRT treatments of extra-cranial tumors. The original CT and MR images used for these cases were imported into the Tomotherapy planning system and were used for developing treatment plans. Tomotherapy plans had a single isocenter and were prescribed as in the Gamma Knife plans for a single delivery fraction with the 50% isodose line coincident with the outer edge of the target volume. The plans were compared for the dose drop-off and dose gradient in directions from the center of the tumor to the patient’s left, to the anterior, and to the superior. DVHs for the tumor and critical struc-tures and treatment times were also analyzed and compared. Tomotherapy is not capable of producing as rapid a fall-off in dose or as high a dose gradient as Gamma Knife, especially in the helical-axis direction (Fig. 1), which is superior/inferior of the patient. Dose gradients for Gamma Knife are as large as 18%/mm, while for tomotherapy they are 12%/mm. Even with the use of the smallest Tomotherapy jaw width, 1 cm, and a small pitch size, tomotherapy still cannot match the dose gradients of Gamma Knife plans. Tomotherapy produces a more uniform dose distribution in the tumor and does not generate the huge hot spot in the center of the tumor that is produced by Gamma Knife. Tomotherapy plans had tumor coverage by the 50% isodose line as conformal as, and sometimes more conformal than, the Gamma Knife plans. In addition, the tomotherapy plans were able to spare the critical structures as well as the Gamma Knife plans in most cases. For these 5 cases, Gamma Knife plans, for a 5-year-old source, were delivered in 36 to 88 min, while tomotherapy plans could be delivered in 13 to 17 min. Tomotherapy plans are suitable for treating these types of intracranial neoplasms. Dose coverage of the target is very conformal, and the fall-off of dose toward a critical structure is rapid enough to provide adequate sparing. Tomotherapy treatment times are significantly shorter than those of Gamma Knife. Tomotherapy is not suitable for targets less than 1 cm in diameter, due to the smallest jaw width being 1 cm. Use of Tomotherapy for stereotactic type treatments will require the development of adequate immobilization and localization methods that are compatible with Tomotherapy hardware.

RT-11. HYPOFRACTIONATED INTENSITY-MODULATED RADIATION THERAPY AND TEMOZOLOMIDE FOR PATIENTS WITH GLIOBLASTOMALuis Souhami, David Roberge, Petr Kavan, Thierry Muanza, Christine Lambert, Marie Christine Guiot, Rolando Del Maestro, Richard Leblanc, and George Shenouda; McGill University, Montreal, Quebec, Canada

Despite multimodality treatments, the outcome of patients with glio-blastoma multiforme (GBM) remains poor. In an attempt to improve results, we have begun a program of accelerated hypofractionated IMRT with concomitant and adjuvant temozolomide. We report our initial expe-rience with this novel approach. Between March 2004 and June 2006, 35 unselected patients (median age 63 years), with histologically proven GBM, were treated. A total of 26 patients (74.3%) had RTOG RPA class 5 or 6. Nine patients underwent biopsies only, while 26 patients had partial or com-plete resections. The methylation status of the MGMT promoter was known in the pathological specimens of 30 patients. Of these, 20 patients had a methylated-MGMT promoter, while the other 10 had an unmethylated pat-tern. IMRT was delivered by either a forward or an inverse planning tech-

nique. Gross tumor volume (GTV) consisted of the contrast-enhanced area and surgical cavity on the post-operative CT/MRI images. Planning target volume (PTV) was the GTV 1 15 mm. Patients received 60 Gy in 20 frac-tions (3 Gy per fraction) over 4 weeks, prescribed to the edge of the GTV, while 40 Gy was delivered to the periphery of the PTV. Temozolomide was administered according to the Stupp regimen. The median follow-up was 12.6 months (range 3.2–26.5). Twenty-nine patients (82.8%) completed the combined modality treatment, and 25 of them (71.4%) received adjuvant temozolomide, with a median number of 4 cycles. Median survival was 14.2 months, and median disease-free survival was 6.1 months. Median survival differed significantly between patients who underwent a biopsy versus a partial or total resection (8.6 vs. 16.8 months, p , 0.01). Median survival was also significantly different between patients with methylated versus unmethylated MGMT promoters (17.23 vs. 7.083 months, p , 0.049). The pattern of failure was mainly central, within the initial GTV. Grade III–IV toxicity was limited to 1 patient with nausea and emesis during adjuvant temozolomide administration. Seven patients who relapsed received further treatment: 5 received temozolomide and procarbazine second-line chemo-therapy, 3 underwent further debulking surgery, 1 underwent stereotactic radiosurgery, and 1 started metronomic temozolomide. Accelerated hypof-ractionated IMRT with concomitant and adjuvant temozolomide is well tolerated with a useful two-week shortening of radiotherapy. Despite a high number of patients with poor prognostic features (74.3% RPA class 5–6), median survival was comparable to standard radiotherapy fractionation schedules plus temozolomide.

RT-12. ANALYSIS OF CLINICAL SYMPTOMS AND PROGNOSTIC FACTORS IN BREAST CANCER-RELATED MENINGEAL METASTASESEtsuyo Ogo,1 Toshi Abe,1 Mizuhiko Terasaki,2 Gen Suzuki,1 Hidehiro Etou,1 Hiroaki Suefuji,1 Chiyoko Tsuji,1 and Naofumi Hayabuchi1; 1Radiology, Kurume University School of Medicine, Kurume, Fukuoka, Japan; 2Neurosurgery, Kurume University School of Medicine, Kurume, Fukuoka, Japan

Meningitis carcinomatosa is an uncommon but aggressive complication of advanced breast cancer. It occurs in 1 to 5% of patients with breast can-cer. The prognosis in meningitis carcinomatosa is extremely poor. Materi-als and methods: We reviewed 6 cases of meningitis carcinomatosa caused by breast cancer at our hospital from June 2002 to May 2007. The mean age at the time of diagnosis was 44 years (range 33–56), and the median Karnofsky status was 50% (range 30–80). The neurological symptoms, pre-treatment characteristics, prognosis, and treatment methods were analyzed. Results: The mean interval from breast cancer diagnosis was 25 months (range 3–72 months). All of the cases were previously treated with systemic chemotherapy as well as metastatic setting. One of them was Her2-positive. One of them was ER-positive. Other metastatic sites were associated with meningitis carcinomatosa in all patients. The clinical symptoms at the time of diagnosis were headache, nausea/vomiting, confusion, hyperexcitability, cerebellar signs, paresis, and pain in the thoraco-lumbal region. All patients were diagnosed with meningitis carcinomatosa by MRI. Whole brain irra-diation was performed in all patients. The treatment intrathecal injection of methotrexate was administrated with radiation in 1 patient. The median duration of survival was 3.6 months. One patient survived beyond 6 months (only those who received methotrexate and temosoromide). Conclusion: Meningitis carcinomatosa is a rare and poor prognosis. We suggest that the prognostic factors are systemic chemotherapy, Karnofsky status at the time of the diagnosis of meningitis carcinomatosa, and the clinical response of chemotherapy. The number of analyzed patients was low, and we did not observe statistical relevance in multivariate analysis.

RT-13. APPLICATION OF DEFORMABLE IMAGE REGISTRATION TO HIPPOCAMPAL DOSES AND NEUROCOGNITIVE OUTCOMESAnita Mahajan,1 Lei Dong,2 Sujit S. Prabhu,3 Catherine Wang,2 Zhang Yongbin,2 Lifei Zhang,2 Christina Meyers,4 and Shiao Woo1; 1Radiation Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Radiation Physics, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3Neurosurgery, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 4Neuro-Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Deformable image registration (DIR) is a technique to facilitate efficient region of interest delineation for radiotherapy (XRT) planning. We applied a new method of DIR to a cohort of patients using a reference template patient and determined the relationship of hippocampal dose to neurocogni-tive outcomes. Forty-nine patients treated at our institution with primary low-grade or anaplastic brain tumors were identified. The DIR protocol was applied to their planning CT and MRI scans for 33 intracranial structures. The dose volume histograms (DVH) of the right and left hippocampi were



evaluated of 18 righthanded patients who had full neurocognitive evalua-tions before and after XRT, with a mean interval of post-XRT testing of 14 months. The relationships between laterality, mean and maximum dose radiotherapy dose and neurocognitive changes were evaluated. In this group of 18 patients (11 male, 7 female), 16 had anaplastic tumors and 2 had low-grade tumors. Median age at diagnosis was 42 years (range 21–76) with a median follow-up from diagnosis of 29 months (range 4–58 months). Presenting symptoms were seizures, speech difficulties, and headaches in 11, 3, and 2 patients, respectively. Two patients had tumors found inciden-tally. Ten patients had left-sided tumors (4 frontal, 4 temporal, 2 parietal); 8 patients had right-sided tumors (2 frontal, 5 temporal, 1 thalamic). Median prescribed dose was 57 Gy in 30 fractions. Median maximum dose to the left and right hippocampus was 53.3 Gy and 30.4 Gy, respectively (range: left 17.2–61.2 Gy; right 3.4–61.4 Gy), with median mean dose to the left and right hippocampus of 45.5 Gy and 25.8 Gy, respectively. A correlation between the maximum dose to the left hippocampus and a decline in learn-ing (p 5 0.014) and delayed recall (p 5 0.01) was identified. There was no relationship noted between the dose delivered to the right hippocampus, the volume of the hippocampus treated, or the mean dose to the hippocampus on either side. In this study we identified an objective relationship between the radiation dose to the left hippocampus and neurocognitive function with these organs delineated by automated DIR. This method may be useful to apply prospectively in brain XRT planning to assess doses and potential morbidities to normal tissues.

RT-14. LOCAL CONTROL OF VESTIBULAR SCHWANNOMAS UTILIZING FRACTIONATED LINAC STEREOTACTIC RADIOSURGERYFrancis Cardinale,1 Isaac Goodrich,2 Vanna Dest,1 Jonathan Haas,3 Robert Sinha,4 and Justin Barry-Jerome5; 1Radiation Oncology, Hospital of Saint Raphael, New Haven, CT, USA; 2Neurosurgery, Hospital of Saint Raphael, New Haven, CT, USA; 3Winthrop Hospital, NY, USA; 4Mountain View, CA, USA; 5GA, USA

Vestibular schwannomas are the most common primary cerebellopon-tine angle tumor occurring in the United States. Between 2,500 and 3,000 new cases are diagnosed each year. A variety of therapies have been uti-lized in the past for this tumor, including observation, surgery (suboccipital craniectomy, translabyrinthine, middle fossa, combined approaches), and stereotactic radiosurgery/radiotherapy (appropriate for lesions 2.5 centime-ters or less in diameter). In 2004, we reported on our initial experience of results with fractionated LINAC stereotactic radiotherapy for vestibu-lar schwannomas at the American Society of Therapeutic Radiology and Oncology (ASTRO). The purpose of this paper is to provide a follow-up of previously reported cases, as well as additional cases compiled since then. Retrospective review of all patients who underwent fractionated stereotactic radiotherapy for vestibular schwannomas at the Hospital of Saint Raphael (Saint Raphael Healthcare System) in New Haven, Connecticut, between 1998 and 2006 was accomplished. A total of 60 patients were identified. Patient age ranged from 26 to 81 (median 55). Forty-one (68%) patients had useful hearing prior to treatment. Therapy was accomplished with a Varian® 600C linear accelerator with an attached BrainLab® micro-multileaf col-limator. The total dose of radiation varied from 46.8 Gy to 54 Gy (median 50.4 Gy). Daily fractions of 1.8 Gy prescribed to the 90th percentile isodose line were utilized. Static beams were utilized for all patients with 7 to 16 (median 16) per treatment plan. Target volumes ranged from 0.17 cm3 to 21.34 cm3 (median 3.75 cm3). In all cases, the percent isodose volume/target volume ranged from 1.49 to 2.76 (median 1.55). The median follow-up was 37 months (range 4.7 to 76.4 months). Local control was achieved in 58 of 60 patients (96.7%). Twenty (33.3%) patients had a decrease in size of their tumor, and 18 (30%) patients exhibited evidence of central tumor necrosis on follow-up MRI scans. Thirty-five (85%) patients retained useful hearing. Sixteen (26.7%) patients with tinnitus prior to treatment reported resolution after therapy completed. Eight percent of patients (5/60) reported transient worsening of tinnitus, and 15% (9/60) reported no change in tinnitus. Nine-teen patients (32%) reported improved tinnitus with radiotherapy. Long-term side effects are infrequent in this population. However, 2 patients (3%) developed cranial nerve V and VII dysfunction. Ten percent of patients have noted decreased to loss of previous useful hearing. Fractionated LINAC stereotactic radiotherapy utilizing a micro-multileaf collimator is a useful treatment option for patients with appropriately sized (,2.5 cm in diameter) vestibular schwannomas that is very well tolerated. Over a median period of 37 months, useful hearing can be preserved in the vast majority of patients with functional hearing. Local control rates with other cranial nerve pres-ervation thus far are excellent. Still further follow-up will be required to ascertain the durability of these results.

RT-15. SINGLE-FRACTION, SINGLE-ISOCENTER INTENSITY-MODULATED RADIOSURGERY (IMRS) FOR MULTIPLE BRAIN METASTASES: DOSIMETRIC AND EARLY CLINICAL EXPERIENCELauren VanderSpek,1 Jia-Zhu Wang,1 John Alksne,2 and Kevin Murphy1; 1Radiation Oncology, Moores Cancer Center, San Diego, CA, USA; 2Neurosurgery, Moores Cancer Center, San Diego, CA, USA

Our objective is to present our early clinical experience using inten-sity-modulated radiosurgery (IMRS) for the treatment of multiple (> 3) brain metastases using a single-fraction, single-isocenter technique. Ten patients with multiple brain metastases were treated between March 2006 and February 2007 on a Varian Trilogy linear accelerator equipped with a 120 leaf multi-leaf collimator. The median patient age was 59 years (range 36–82). The primary cancers were lung (5), breast (3), and melanoma (2). The median KPS was 75 (40–100). The median number of metastases was 6 (range 3–12). Three patients received prior whole brain irradiation. Treatment was delivered using a frameless optically guided immobilization approach in 9 patients and a traditional frame-based approach in 1 patient. The median number of fields utilized was 9 (range 8–14). Acute radiation toxicities were analyzed. The median total planning target volume (PTV) was 35.0 cm3, and the median prescribed dose was 16 Gy (range 14–18). The median integral dose to normal brain was 6.2 Gy-kg with a SD of 1.6 Gy-kg. The median dose to normal brain was 3.5 Gy with a SD of 1.3 Gy. The median maximum and minimum PTV coverage was 107% and 87%, respectively. Doses to normal structures were minimal. One patient had an acute, medically reversible, grade 3 toxicity of confusion. One patient had grade 2 headache and left lateral visual changes that improved with steroid administration. One patient had grade 3 arm weakness prior to IMRS that had worsened at first follow-up. At the time of last follow-up, 8 patients either had an improvement in their symptoms or reported no complications with IMRS. For a comparison analysis, a patient with 6 lesions was re-planned using a 5-isocenter stereotactic radiosurgery (SRS) technique using 20 arcs and multiple circular collimators. The prescribed dose was 18 Gy. The integral dose to normal brain was 1.6 Gy-kg and the median normal brain dose was 1.0 Gy, compared to 4.5 Gy-kg and 3.2 Gy, respectively, for the IMRS plan. Doses to critical structures were decreased compared to IMRS except for dose to the eyes. Single-fraction, single-isocenter IMRS is a feasible and well-tolerated treatment of select patients with multiple brain metastases. The SRS multiple isocenter plan had improved dosimetric parameters; however, the differences may not be clinically significant. We are currently accruing patients to assess late toxicity and tumor control using this approach.

RT-16. ULTRAFRACTIONATED RADIOTHERAPY (THREE DOSES PER DAY) FOR INOPERABLE GLIOBLASTOMAS: A PHASE II STUDYPatrick Beauchesne,1 Luc Taillandier,2 Valerie Bernier,3 Mohamed Djabri,4 Xavier Michel,5 Jean-Philippe Maire,6 Laurent Martin,7 Pierre Verrelle,8 and Remy Pedeux9; 1Society for Neuro-Oncology, Nancy, France; 2Neuro-Oncology, CHU de Nancy, Nancy, France; 3Radiotherapy, Centre A Vautrin, Nancy, France; 4Radiotherapy, CHG de Thionville, Thionville, France; 5Radiotherapy, CHG de Metz, Metz, France; 6Radiotherapy, CHU de Bordeaux, Bordeaux, France; 7Radiotherapy, Centre G Le Conquerant, Le Havre, France; 8Radiotherapy, Centre J Perrin, Clermont-Ferrand, France; 9Unite INSERM U578, Institut A Bonniot, Grenoble, France

Ultrafractionated radiotherapy consists in irradiating cells or tumors sev-eral times daily, delivering low doses at which hyperradiosensitivity occur. We recently reported the high efficiency of ultrafractionated radiotherapy in glioma cell lines and xenografts (Beauchesne et al. 2003) and are now con-ducting a phase II clinical trial to determine the effect of an ultrafraction-ated regimen for glioblastoma patients. A prospective, multicenter, phase II study opened for accrual in September 2003. Patients over 18 years of age who are able to give informed consent and have histologically proven, newly diagnosed and unresectable, supratentorial glioblastoma (WHO grade IV) are eligible. Three doses of 0.75 Gy spaced by at least 4 hours are delivered daily, 5 days a week for 6 consecutive weeks for a total of 67.5 Gy. Confor-mal irradiation includes the tumor bulk including a margin of 2.5 cm. Toler-ance and toxicity are the primary endpoints; survival and progression-free survival are secondary endpoints. To date, 31 patients have been enrolled in this study (25 were evaluable): 16 men and 15 women, median age 58 (range 37–76), median Karnofsky performance status 80 (range 70–100). The median time between histological diagnosis and the start of treatment is 7 weeks. The ultrafractionated radiotherapy has been well tolerated; no acute grade 3 and/or 4 CNS toxicity has been observed. Minor responses at the end of irradiation were seen in 5 patients. Median survival from initial diagnosis was 10 months; 8 patients remain alive. Ultrafractionated radio-therapy is safe and well tolerated. No acute CNS toxicity has been observed. Overall survival of over 10 months for patients without prior debulking surgery compares favorably with other reports.



RT-17. EDEMA AND RADIATION NECROSIS FOLLOWING GAMMA KNIFE RADIOSURGERY FOR BRAIN METASTASES FROM NON-SMALL CELL LUNG CARCINOMAMarlon Mathews,1 Franklin Westhout,2 Andrew Kim,3 Kathleen Thomas,4 Mark Linskey,1 and Christopher Duma4; 1Neurological Surgery, University of California, Irvine, Orange, CA, USA; 2Surgery, Cedar Sinai Medical Center, Los Angeles, CA, USA; 3University of California, Irvine, Orange, CA, USA; 4Hoag Memorial Presbyterian Hospital, Newport Beach, CA, USA

Our objective was to examine the incidence of edema and radiation necrosis (RN) following Gamma Knife stereotactic radiosurgery (GKSR) for non-small cell lung cancer (NSCLC) brain metastases. From 1997 to 2005 (100 months), 340 cases (302 patients) underwent GKSR for NSCLC metastatic brain tumors. Patients were followed prospectively, including regular neuroimaging. Median age was 64.5 years (range 34–87). Median pretreatment KPS was 90 (range 30–100; 9.3% KPS < 60). One hundred forty-one cases (130 patients) were treated for single tumors and 199 cases (172 patients) for multiple tumors (median 3 tumors; range 1–10). Eighty patients (26.5%) received whole brain radiation therapy (WBRT) before, and 36 (11.9%) after, GKSR (38.4% WBRT). Forty patients (13.2%) received additional GKSR for new lesions or for local control failure. Median follow-up was 6 months (range 0–80). Twenty-three cases (6.8%) developed post-GKSR edema, defined as increased signal on FLAIR or T2 (11 in the single-metastasis group and 12 in the multiple-metastases group). Eleven patients (3.2%) were found to have RN (8 patients in the single-metastasis group and 3 in the multiple-metastases group) at a median follow-up of 13 months (range 5–39). Five of the 11 RN patients received WBRT prior to their GKSR (62.5%), while none received post-GKSR WBRT. The risk of RN was 4.3% with, versus 1.6% without, WBRT. Median margin and maximal treatment doses to the RN cases were 50% isodose and 17 Gy, respectively (ranges 50–80% isodose and 13–20 Gy). Median volume of the RN tumors was 4.2 cm3 (range 0.52 ¨C 33.49 cm3). None of the repeat GKSR cases developed RN. Four patients required surgical resection from edema/radiation necrosis. The occurrence of edema and RN is infrequent following GKSR for single metastatic brain tumors, but it does increase with the addition of pre-GKSR WBRT.

RT-18. INCIDENCE OF EDEMA AND RADIATION NECROSIS FOLLOWING GAMMA KNIFE RADIOSURGERY FOR SINGLE METASTATIC BRAIN TUMORSMarlon Mathews,1 Franklin Westhout,2 Andrew Kim,3 Kathleen Thomas,4 Mark Linskey,1 and Christopher Duma4; 1Neurological Surgery, University of California, Irvine, Orange, CA, USA; 2Surgery, Cedar Sinai Medical Center, Los Angeles, CA, USA; 3University of California, Irvine, Orange, CA, USA; 4Hoag Memorial Presbyterian Hospital, Newport Beach, CA, USA

Our objective was to examine the incidence of edema and radiation necrosis (RN) following Gamma Knife stereotactic radiosurgery (GKSR) for single brain metastasis. From 1997 to 2005 (110 months), 292 consecutive patients received GKSR for a single metastatic brain tumor at one center. All were followed prospectively. Primary histologies included non-small cell lung (NSCLC) (n 5 130), breast (n 5 47), melanoma (n 5 46), colon (n 5 21), and other (n 5 45). Median age was 61.5 years (range 30–94; mean 61.2 years; SD 12.8). Median Karnofsky performance score (KPS) was 90 (range 40–100; n 5 18 [5.6%] with KPS ,70). Twenty-two percent of cases (n 5 71) received whole brain radiation therapy (WBRT) before, and 8% (n 5 26) after, GKSR (30% received WBRT). Forty-two patients (13.8%) received additional GKSR for local failure (n 5 7) or newly developing lesions (n 5 34). Median follow-up was 6 months (range 0–80). Increased edema following GKSR defined by new or increased signal on T2 or FLAIR MRI developed in 24 patients (8.2%). Sixteen patients developed RN (5.5%). Six of the 16 RN cases received WBRT prior to their GKSR (37.5%), while 1 received post-GKSR WBRT. Incidence of RN was 7.2% with WBRT, versus 4.6% without. Median margin isodose and maximal treatment dose to the RN cases were 50% and 18 Gy, respectively (ranges 45–80% and 13–20 Gy). Median volume of the RN tumors was 4 cm3 (range 0.12–33.49 cm3). One patient with repeat GKSR developed RN. Two patients required surgical resection for edema/radiation necrosis. The occurrence of edema and RN is infrequent following GKSR for single brain metastatic tumors, but it does increase with the addition of WBRT.

RT-19. STEREOTACTIC RADIOSURGERY AS THE PRIMARY TREATMENT FOR PATIENTS WITH MELANOMA AND RENAL CELL CARCINOMA BRAIN METASTASES: SURVIVAL, LOCAL CONTROL, AND NEED FOR WHOLE BRAIN RADIOTHERAPY SALVAGERandy Jensen1 and Shrieve Dennis2; 1Neurosurgery, Huntsman Cancer Institute, Salt Lake City, UT, USA; 2Huntsman Cancer Institute, UT, USA

We hypothesized that early diagnosis and aggressive treatment of patients with “radioresistant” brain metastases such as melanoma and renal cell carcinoma with SRS would allow delivery of early systemic therapy, pos-sibly spare many patients whole brain radiotherapy (WBRT), and ultimately lead to improvement of survival. Over a 6-year period, 156 radiosurgery (SRS) procedures were performed on 115 patients with brain metastases from melanoma or renal cell carcinoma. Of these, 95 patients with 252 newly diagnosed metastases were treated with radiosurgery either alone (81) or combined with WBRT. Local control follow-up was available for 202 lesions with 46 local failures. The median time to failure was 14.5 months, compared to 7.1 months for 72 lesions treated after previous radiotherapy failed (p , 0.05). Overall median survival for these 95 patients was 8.9 months following diagnosis of brain metastases with no significant differ-ence between patients with MEL or RC. Survival was similar regardless of whether WBRT was used as part of initial therapy. Overall survival by RPA class was apparently improved compared to published results for patients treated with WBRT: RPA I median survival (MS) not reached; RPA II MS 5 8.9 months; RPA III MS 5 4.7 months. These results are most similar to published data for patients treated with surgery and WBRT for single brain metastases. Sixty-nine of 81 (77%) patients initially treated without WBRT had never received WBRT by last follow-up. WBRT-free survival was 55% at 2 years following diagnosis. We have found that aggressive screening for brain metastases in patients with high-risk melanoma or renal cell carci-noma followed by SRS alone for patients found to have <5 brain metastases is well tolerated, allows patients to proceed to systemic therapy in a rapid fashion, leads to excellent local control while sparing a majority of patients WBRT, and results in excellent overall survival compared to other series.

RT-20. DIFFUSION TENSOR IMAGING AS A BIOMARKER FOR EARLY PREDICTION OF THE DELAYED RADIATION-INDUCED NEUROCOGNITIVE CHANGES IN CEREBRAL TUMOR PATIENTSVijaya Nagesh, Christina Tsien, Henry Buchtel, Pia Sundgren, Thomas Chenevert, Larry Junck, Lisa Rogers, Brian Ross, Theodore Lawrence, and Yue Cao; University of Michigan, Ann Arbor, MI, USA

Cranial radiation therapy (RT) is known to affect the central nervous system, resulting in delayed neurological complications and deficits in long-term survivors. We hypothesize that structural degradation in cerebral tis-sue after RT will evoke deficits in neurocognitive functions involving a large neural network. Twenty-five patients with low- and high-grade gliomas or benign tumors treated with RT participated in an IRB-approved MRI study. The biologically corrected doses ranged from 50 to 81 Gy. The MRI proto-col included T1, T2, and diffusion tensor imaging (DTI). Temporal changes in normal-appearing tissue were assessed pre-, during, and up to 18 months post-RT. Four DTI indices were calculated: (1) mean diffusivity of water, D; (2) fractional anisotropy of diffusion, FA, an indicator of white matter integrity; (3) diffusivity perpendicular () to the fiber long axis, a measure of myelination; and (4) diffusivity parallel (||) to the long axis, a measure of axonal injury. Neuropsychological tests included the Trail Making Test and the Folstein Mini-Mental State Exam (MMSE). In normal-appearing gray and white matter tissue of the ipsilateral and contralateral hemispheres of the tumor, and || increased by 15% and 10%, respectively, by 3 months post-RT, signifying global and diffuse effects of RT and suggest-ing dose dependency. Further analysis of radiation-induced injury to large white matter fibers of the corpus callosum indicated demyelination evident from the substantial increase in (22%) and only 5% in || 3 months post-RT. Demyelination progressed up to 9 months post-RT. In addition to global gray matter degradation, specific structural injury was seen in the hippocampus, which is implicated in learning and memory. Even in the contralateral hippocampus remote from the tumor and remote from high radiation dose up to an 18% increase in was noted 1 month post-RT, suggesting that radiation can affect distant structures. Neurocognitive mea-sures were obtained up to 18 months for 5 patients; the percentage changes in FA and at week 3 during RT were significantly correlated with the percentage change in (1) memory component of the MMSE at 18 months (Pearson correlation coefficient, r 5 0.92; p 5 0.03) and (2) Trail Mak-ing Test B performance at 18 months (Pearson correlation coefficient, r 5 0.90; p 5 0.04), indicating sensitivity of DTI to subtle structural changes, which in turn affect the neural networks with subsequent neurocognitive decline. The correlation between changes in DTI indices and neurocognitive function reinforce our central hypothesis that early neurostructural changes in functional pathways could predict delayed radiation-induced neurobe-havioral changes. Our data suggest that early detection of neurostructural changes in normal tissue offers a window of opportunity for interventional



therapy to minimize neurotoxicity and neurological deficit. This work is supported by NIH grants 3 PO1 CA59827 and PO1 CA85878.

RT-21. DOSIMETRIC ANALYSES OF REPEAT GAMMA KNIFE RADIOSURGERY CASESYoichi Watanabe,1 Chung Lee,1 Stephen Haines,2 and Paul W. Sperduto3; 1Therapeutic Radiology, University of Minnesota, Minneapolis, MN, USA; 2Neurosurgery, University of Minnesota, Minneapolis, MN, USA; 3Minnesota Radiation Oncology, Inc., Minneapolis, MN, USA

Our objective was to investigate whether the failure of local control was due to dosimetric deficit of the initial treatment for patients with repeat Leksell Gamma Knife (LKG) radiosurgery. We analyzed a total of 11 cases (10 patients): 3 recurrent GBM, 7 metastases (2 renal cell, 2 non-small cell lung, 3 breast), and 1 meningioma, which were treated from July 2005 to April 2007. The average length between the first and the second treatments was 214 days (126 to 399 days). The lesions were treated twice because of increase in size for 6 lesions and because of persistent appearance of con-trast-enhanced lesions or evidence of growth to the periphery of the initial target in post-treatment MRI for 5 lesions. The prescription dose ranged from 13 Gy to 18 Gy at 45%, 50%, or 90% isodose surface. The MRI data for the second treatment (MRI-2) was co-registered with those for the first treatment using the MuliView software (Elekta, Sweden), which utilizes the normalized mutual information algorithm. The failed lesion was corre-lated with the dose given to the first treatment by visually examining MRI-2 superimposed with the first planning isodose lines. Four cases failed inside the target of the first treatment (intra-target failure), and 7 lesions failed at the periphery of the target of the first treatment (marginal failure). All intra-target failures occurred in regions exhibiting ring-like enhancement on T1-weighted MRI images. All recurrent GBM cases showed heterogeneous or ring-like enhancement and had marginal failures. One recurrent GBM case clearly showed that the tumor grew toward a lower dose region (less than 19 Gy) outside the periphery of the initial target volume. One metas-tasis case (from breast cancer) failed in the non-enhancing lesion inside the initial target volume, although the dose to the failed lesion was greater than 27 Gy. One lesion with a prescription of 18 Gy to 90% isodose surface (or the maximum dose of 20 Gy) failed. The current post-LGK radiosurgery analysis was performed on the failed cases for which treatment was repeated at the same lesion. This allowed us to use the same high-resolution MRI images for comparison. We have observed some correlation of local failure with the low dose at the periphery of the target and the type of contrast enhancement. More systematic analysis of patients treated at our center, which treats about 250 patients per year, is planned and the results will be presented.

RT-22. INTRACRANIAL LOW-GRADE GLIOMAS: LONG-TERM FOLLOW-UP OF THE MAYO CLINIC EXPERIENCEDavid Schomas, Nadia Laack, Ravi Rao, Fredric Meyer, Brian O’Neill, and Paul Brown; Mayo Clinic, Rochester, MN, USA

Low-grade gliomas (LGG) are uncommon tumors, and the long-term clinical behavior and impact of various prognostic factors on outcomes remains to be defined. The records of 314 adult (>18 years old) patients with grade 1 or 2 intracranial, non-pilocytic LGGs diagnosed between 1960 and 1992 at the Mayo Clinic, Rochester, Minnesota, were retrospectively reviewed. Lesions of the optic tract or brain stem were excluded. Data of clinical outcomes, therapy received, and pertinent prognostic factors were extracted for analysis. The Kaplan-Meier method was used to estimate pro-gression-free survival (PFS) and overall survival (OS) as well as to determine the impact of well-defined prognostic factors on outcomes. The chi-square test was used to analyze prognostic factors with the likelihood of receiving post-operative RT. The median age at diagnosis was 36 years. The median follow-up was 13.6 years. Operative-pathology revealed pure astrocytoma (58%), mixed oligoastrocytoma (31%), and oligodendroglioma (11%). The median OS was 6.9 years (range 1 month to 38.5 years). The extent of surgi-cal resection was assessed by operative report, neurosurgeon’s impression, and post-operative imaging when available. Gross total resection (GTR) was achieved in 13%, radical subtotal resection in 11%, subtotal resection in 41%, and biopsy only in 35% of patients. Post-operative radiation ther-apy (RT) and chemotherapy (CT) were given to 73% and 6% of patients, respectively. The 10- and 15-year OS rates were 36% and 23%, respectively. Adverse prognostic factors for OS identified by univariate analysis included age >40 (p 5 0.008), size >5 cm (p , 0.0001), pure astrocytoma histol-ogy (p 5 0.0025), undergoing less than a radical-subtotal resection (p 5 0.0002), grade 2 disease (p 5 0.0045), presentation with motor-sensory symptoms (p 5 0.0006), and supratentorial location (p 5 0.0072). Neither post-operative RT or CT had an impact on OS (p 5 0.955 and p 5 0.946, respectively). On multivariate analysis, size >5 cm, pure astrocytoma histol-ogy, grade 2 disease, and undergoing less than a radical-subtotal resection remained statistically significant. A separate analysis of PFS was accom-

plished. Adverse prognostic factors for PFS identified by univariate analysis included presentation with motor-sensory symptoms (p 5 0.0073), size >5 cm (p 5 0.0005), grade 2 disease (p 5 0.05), and undergoing less than a radical-subtotal resection (p 5 0.0004). Neither post-operative RT or CT had an impact on PFS (p 5 0.259 and p 5 0.74, respectively). On multivari-ate analysis, only size >5 cm and undergoing less than a radical-subtotal resection remained statistically significant. The following adverse prognos-tic factors were significantly associated with the use of RT: pure astrocy-toma histology (p 5 0.0085), extent of surgical resection (p , 0.001), and grade 2 disease (p 5 0.0051). This large, single-institution retrospective series of patients with intracranial LGGs provides evidence that a substan-tial proportion of patients have good long-term prognosis and remain alive with stable disease up to and beyond 15 years after diagnosis. Post-operative RT or CT did not impact OS or PFS, though patients with negative prognos-tic factors were preferentially treated with such modalities. The prognostic value of tumor size, histology, grade, and extent of resection is confirmed in our analysis.

RT-23. THE USE OF GLIA-SITE IN THE PRIMARY MANAGEMENT OF BRAIN METASTASES RESULTS IN A HIGH RATE OF RADIATION NECROSIS AND RE-OPERATIONStuart Burri, Anthony Asher, Meghan Mueller, and Robert Fraser; Carolinas Medical Center, Charlotte, NC, USA

Balloon catheter based brachytherapy (Glia-site) is an innovative way of delivering radiation to resection cavities in the brain. It has been used for both primary and metastatic lesions. The published risk of radiation necrosis has been noted to be fairly low. In our experience, patients with longer follow-up frequently develop imaging changes and symptoms from treatment effect that require further intervention. We maintain a pro-spective database at our institution of all patients treated with Glia-site brachytherapy. Beginning in June 2001, 37 patients have been treated with Glia-site at our institution. A total of 20 patients had Glia-site balloons implanted for brain metastases as initial primary therapy without external beam radiation. Of the 20 patients with brain metastases, the 7 patients who required re-operation for symptomatic progressive imaging changes are the focus of this abstract. Patients were managed conservatively with other measures, primarily high-dose steroids, and only went to re-operation when they subsequently developed progressive symptoms. The median age of the patients requiring resection was 63, all of whom had a KPS >90 following the resection. Five patients were treated with the 2 cm balloon (volume 4–5 cm3), and 2 with the 3 cm (15 cm3) balloon. All of the patients were treated to 60 Gy, with the first 3 to a depth of 1 cm and the last 4 to a depth of 5 mm. The median time to re-operation was 17 months. All 7 of the patients who underwent re-operation for brain metastases had .90% radiation necrosis without any clearly viable tumor cells. Thus a crude re-operation rate of 35% was noted for patients treated with Glia-site as primary therapy for brain metastases. None of the 20 patients treated suffered biopsy-proven recurrent malignancy. The actuarial risk of re-operation for symptomatic biopsy-proven radiation necrosis was 7% at 6 months, 26% at 12 months, 40% at 18 months, 68% at 20 months, and 84% at 24 months. In long-term survivors, the risk of symptomatic radiation necrosis with Glia-site therapy for brain metastases is very high. The fact that radiation necrosis is a late effect with a median time of 17 months to re-operation likely understates the actual risk in a population of patients with brain metastases. In long-term survivors of brain metastases treated with Glia-site, imaging and symp-tomatic changes should be presumed to be radiation necrosis rather than recurrent tumor. Glia-site appears to be effective at controlling metastatic disease in the doses used in this series, but it results in far too much radia-tion necrosis. Continued study of dose reduction lower than 60 Gy at 5 mm needs to be undertaken if Glia-site is to be used in the initial management of brain metastases.

RT-24. SALVAGE STEREOTACTIC RADIOSURGERY FOR CENTRAL NEUROCYTOMAJames L. Leenstra,1 Paul D. Brown,1 Scott L. Stafford,1 and Bruce E. Pollock2; 1Radiation Oncology, Mayo Clinic, Rochester, MN, USA; 2Neurosurgery and Radiation Oncology, Mayo Clinic, Rochester, MN, USA

Central neurocytomas (CN) are rare tumors of young adults, typically located in a deep midline position near the foramen of Monro. Gross total resection (GTR) is the mainstay of treatment. When only subtotal resec-tion (STR) is feasible, post-operative radiotherapy (RT) is often utilized but remains controversial. Observation for progression is an alternative strategy. Local failure predominates with only very rare distant metastases. Salvage therapeutic options include re-operation, fractionated radiotherapy, and ste-reotactic radiosurgery (SRS). Five patients—2 males and 3 females—with histologically proven CN treated with SRS at time of recurrence or progres-



sion were evaluated retrospectively. Two patients were treated at our insti-tution with Gamma Knife SRS (Leksell Instruments, Norcross, GA), and 3 were seen in consultation prior to SRS performed elsewhere. Age at initial presentation ranged from 23–33 years (mean 29 years). All had undergone prior surgery, GTR in 2 and STR in 3. After surgery, median time to progres-sion/recurrence seen on neuroimaging was 3.0 years (range 2 months to 10.5 years). All five patients were asymptomatic at the time of SRS. Treatment doses ranged from 14–20 Gy at the margin and a maximum of 28–40 Gy. Average tumor size was 7.3 cm3 (range 2.7–20.6 cm3). One patient was lost to follow-up immediately after SRS. Treatment resulted in decreased tumor size on subsequent imaging for 2 patients and stable disease for another. These 3 patients are asymptomatic without further progression at 2.6–6.5 years after SRS. The fifth patient (treated at an outside facility) underwent a subsequent GTR 16 months after SRS for radiation-induced tumor necrosis with symptomatic cerebral edema. He was alive, asymptomatic, and with-out evidence of progression 9.7 years after reoperation. Including these 5 patients, a total of 17 cases of salvage SRS for CN have been reported in the literature. Radiosurgery offers a number of therapeutic advantages over fractionated RT in this setting. CN recurrences are often small, focal, and well-circumscribed, so inclusion of the tumor visible to magnetic resonance imaging within the dose plan allows for precise, complete tumor coverage. Because of the steep decrease in dose gradient at the margin, SRS delivers a high dose of radiation to the tumor with less radiation exposure to adjacent brain tissue than conventional RT. The intraventricular growth pattern of CN enhances this benefit because much of the tumor is surrounded by cere-brospinal fluid and only a minority is in contact with brain parenchyma. Thus the risk of late radiation toxicity and iatrogenic tumor development is minimized. Finally, with SRS, the treatment dose can be delivered in a single treatment session. The more potent cytotoxic effect of a much higher single dose may account for the marked tumor regression seen in several reported cases. Given these benefits, SRS should be considered for salvage therapy when tumor recurrence is small. The patient from our series that developed radiation necrosis after SRS suffered the only adverse outcome of those reported in the literature. Further long-term follow-up and experience with salvage SRS for CN are needed.

RT-25. RESULTS OF STEREOTACTIC RADIOSURGERY FOR NF2 PATIENTS WITH ACOUSTIC NEUROMAMelva Pinn,1 J. Daniel Bourland,1 Thomas Ellis,2 Allan Deguzman,1 Kenneth Ekstrand,1 Kevin McMullen,1 Steven Glazier,2 Michael Munley,1 Doug Powell,3 Volker Stieber,4 Peter Rossi,5 Stephen Tatter,2 Stacy Wentworth,1 and Edward Shaw1; 1Radiation Oncology, Wake Forest University School of Medicine, Winston-Salem, NC, USA; 2Neurosurgery, Wake Forest University School of Medicine, Winston-Salem, NC, USA; 3Internal Medicine, Madigan Army Medical Center, Tacoma, WA, USA; 4Wake Forest University School of Medicine, Winston-Salem, NC, USA; 5Radiation Oncology, Emory Clinic, Atlanta, GA, USA

This retrospective study included 10 patients with Neurofibromatosis Type 2 (NF2) who had 20 acoustic neuromas. There were 9 females (includ-ing 1 mother and daughter) and 1 male who ranged in age from 14 to 66 years (median 35 years). The average time from the diagnosis of NF2 to the treatment of the first acoustic neuroma was 3.2 years. Management of the 20 acoustic neuromas was observation in 6 (1 of whom had surgery at progression, 1 fractionated external beam radiation at progression), stereot-actic radiosurgery (SRS) in 10, and surgery in 4 (2 with subsequent GK SRS at recurrence). Thus 12 acoustic neuromas had SRS and form the basis of this report. The mean maximum diameter of the 12 acoustic neuromas was 2.47 cm (range 1.41–3.52 cm) with a mean volume 6.4 cm3 (range 1.0–20 cm3). Eleven received Gamma Knife (GK) SRS, 1 linear accelerator SRS. The mean margin dose (usually the 50% isodose line) was 12.1 Gy (range 10–14 Gy, 11–14 Gy in 10 of 12 acoustic neuromas). Prior to SRS, useful hearing was present in 6 of the 12 acoustic neuromas. After SRS, useful hearing was preserved in 3 acoustic neuromas (2 in the same patient who received 12 Gy to each tumor; the other patient’s tumor received 10 Gy) and lost in the other 3 acoustic neuromas (tumor dose 12 Gy in all 3). Facial weakness was present prior to SRS in 1 patient (post-op complication). Four of 12 acoustic neuromas (33%) developed facial weakness following SRS. Local control was achieved in all 12 acoustic neuromas (100%). In conclusion, compared to non-NF2 patients treated with SRS for acoustic neuroma, NF2 patients have larger tumors, a lower likelihood of useful hearing pre- and post-SRS, a higher incidence of facial weakness, and a high degree of local control. These data suggest that earlier treatment with lower dose SRS (e.g., 10Gy) when the acoustic neuromas are smaller and when useful hearing is present may reduce the toxicity of treatment.

RT-26. SURVIVAL FOLLOWING STEREOTACTIC RADIOSURGERY FOR RECURRENT SMALL CELL LUNG CANCER BRAIN METASTASES AFTER WHOLE BRAIN RADIOTHERAPYCarl R. Peterson,1 Sam Chao,2 Gene H. Barnett,3 Michael A. Vogelbaum,3 Paul Elson,4 Gregory M.M. Videtic,2 and John Suh2; 1Cleveland Clinic Foundation, Cleveland, OH, USA; 2Radiation Oncology, Cleveland Clinic Foundation, Cleveland, OH, USA; 3Brain Tumor and Neuro-Oncology Center, Cleveland Clinic Foundation, Cleveland, OH, USA; 4Cleveland Clinic Foundation, Cleveland, OH, USA

Our objective was to determine the survival of patients with recur-rent small cell lung cancer (SCLC) brain metastases after previous whole brain radiotherapy (WBRT) who were treated with salvage stereotactic radiosurgery (SRS) and to analyze for prognostic variables. A retrospective chart review of the records of 18 patients treated at the Cleveland Clinic between September 28, 1993, and December 11, 2003, who received SRS for brain metastasis following recurrence after WBRT were analyzed for survival outcomes. Patients were classified according to the RTOG brain metastases criteria. Factors evaluated with univariate analysis for potential impact on survival included gender, age at SRS, KPS at SRS, number of lesions at SRS, location (supra-tentorial vs. infra-tentorial vs. both), pres-ence of systemic metastasis at SRS, if the primary disease was controlled at the time of SRS treatment, and extent of disease. All patients had died at the time of analysis. Median WBRT dose delivered was 3,000 cGy in 10 fractions (range 2,700–5,600 cGy in 9–28 fractions); 2 patients received WBRT boost (1,250 and 1,000 cGy in 5 fractions). Seven patients (39%) had extensive disease, and 11 (61%) had limited disease at diagnosis of brain metastasis. The median interval between completion of WBRT and salvage SRS was 5.1 months (range 2.7–16.1 months). At SRS (median dose 1,800 cGy per lesion), median age was 62 (range 42–47). Karnofsky Performance Scores were 90 5 33%, 80 5 28%, 70 or less 5 39%. Eleven percent had infra-tentorial disease; 72% had supra-tentorial disease; 17% of the patients had both. Patient ranking per the RPA criteria was class I 5 33% (n 5 6), class II 5 50% (n 5 9), class III 5 17% (n 5 3). Thirty-nine lesions were treated overall, with 45% of patients having a single brain metastasis, 33% 2 metastases, and 22% 3 or more metastases at SRS. Eleven of the 18 patients (61%) underwent SRS for local relapse only, 2 (11%) had distant relapse, and 3 (17%) had relapses both distantly and locally (type of relapse unknown for 2 patients). Seventy-two percent of patients had no evidence of systemic disease at the time of SRS. Sixty-seven percent of patients had local control of the primary SCLC. Median survival from SRS following WBRT failure was 3.3 months (range 0.2–22.6 months); two patients lived more than 1 year, with one surviving 22 months. Variables significantly associated with survival included gender (p 5 0.04) and extent of disease (p 5 0.03). The median survival of males (n 5 6, 33%) was 2.4 months and for females (n 5 12, 67%), 4.9 months. Median survival for limited disease was 2.8 months, compared to 5.7 months for extensive disease. Although this series is small, the results suggest a possible role for SRS as a salvage therapy for selected patients with recurrent SCLC brain metastasis after WBRT in selected patients. Prospective evaluation of the role of SRS in this setting is warranted.

RT-27. STEREOTAXIC GAMMA KNIFE SURGERY IN TREATMENT OF CRITICALLY LOCATED RECURRENT CYSTIC PILOCYTIC ASTROCYTOMARaef Hafez; Neurosurgery and Gamma Knife Center, International Medical Center, Cairo, Egypt

Low-grade gliomas are uncommon primary brain tumors, located more often in the posterior fossa, optic pathway, and brain stem and less commonly in the cerebral hemispheres. Case presentations: 2 patients with diagnosed recurrent cystic pilocytic astrocytoma critically located within the brain (thalamic and brain stem) were treated with Gamma Knife sur-gery. Gamma Knife surgery (GKS) very much improved the patient’s clini-cal condition, which remained stable later on. Progressive reduction on the magnetic resonance imaging (MRI) studies of the solid part of the tumor and near disappearance of the cystic component were achieved within the follow-up period of 36 months in the first case (with the thalamic-located lesion) and 22 months in the second case (with the brain stem–located lesion). Gamma Knife surgery represents an alternate tool in the treatment of recurrent and/or small post-operative residual pilocytic astrocytoma, especially if they are critically located.



RT-28. BRAIN DISEASE IS RARELY THE CAUSE OF DEATH IN NON-SMALL CELL LUNG CANCER PATIENTS: PROPHYLACTIC CRANIAL RADIATION MAY NOT BE WARRANTEDJonas Sheehan,1 Logan Douds,1 Robert Cavaliere,2 and Elana Farace1; 1Penn State University, Hershey, PA, USA; 2Ohio State University, Columbus, OH, USA

Lung cancer is the most common cause of cancer death and is the most common source of brain metastatic disease overall (first in men, second in women). Thirty-three percent of patients with lung cancer are found to have brain metastatic disease at some time in their disease course. Previous stud-ies have found that nearly two thirds of brain metastases are symptomatic, but with more frequent neuroimaging, the number of asymptomatic brain metastases cases reported is increasing. Nearly all patients with stage IV lung cancer will die from their disease within 5 years, despite the avail-able treatment modalities (whole brain radiotherapy, targeted radiosurgery, chemotherapy, and surgery). The RTOG 0214 study was opened in 2002 to give prophylactic cranial irradiation to non-small cell lung cancer patients, justified on the basis of high CNS failure rates and the fact that the brain is frequently the first site of recurrence. However, an analysis of the relation-ship of brain disease to actual cause of death in non-small cell lung cancer patients has not yet been reported. An analysis of 321 consecutive patients with non-small cell lung cancer seen at this institution from 2000 to 2005 was obtained from the Penn State Cancer Institute Registry. Only patients with a histological confirmation of non-small cell lung cancer were included. All available data concerning age, sex, date of diagnosis, date of death, neuroimaging, clinic and ER visits with documented physical examination, discharge and death summaries, and previous therapeutic intervention were recorded. The time from last known study to time of death averaged 6.5 months, with a median of 3 months and a mode of 1 month. Cause of death was assigned in the following ways: (1) negative brain imaging within 6 months of death was assigned to non-brain causes, (2) neurologi-cally intact within 3 months of death with either no imaging study or nega-tive study outside of 6 months was assigned to non-brain causes, (3) known stable brain metastases or lack of imaging studies with progressive lung or systemic disease was assigned to non-brain causes, (4) progressive brain metastatic disease or worsening neurological exam was assigned to brain causes, (5) lack of sufficient records or confounding factors was assigned to unknown cause of death, (6) patient still alive. Of the 321 patients, 241 were deceased. Out of the 241 deceased patients, 161 (66.8%) were defini-tively assigned to a non-brain cause of death (category 1, 2, or 3), while an additional 70 patients (29%) had no known cause of death (category 5) but also no evidence of any brain image or reported neurological worsening and can be presumed to have had non-brain related deaths. Only 10 out of 241 (4.1%) were assigned to the brain disease cause of death group (category 4). The overwhelming cause of death in patients with advanced non-small cell lung cancer appears to be their lung or systemic cancer burden and not metastatic brain disease. There is a limitation that patients may have had asymptomatic lesions that went undetected, but if there was no documenta-tion of a neurological image or a neurological change, it is unlikely that the patient died of neurological causes. Although brain disease and recurrence are common in non-small cell lung cancer, it does not appear that brain disease has a significant effect on length of survival. Therefore the quality-of-life trade-offs with the effects of prophylactic cranial irradiation should be seriously considered.

RT-29. ADJUVANT RADIOTHERAPY AFTER SURGICAL RESECTION OF SINGLE BRAIN METASTASES: AN ANALYSIS OF 358 PATIENTS BY PRIMARY TUMOR SITEFrederick Lang,1 Christopher McPherson,2 Dima Suki,1 Eric Chang,3 Moshe Maor,3 Raymond Sawaya,1 and Anita Mahajan3; 1Department of Neurosurgery, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Department of Neurosurgery, University of Cincinnati, Cincinnati, OH, USA; 3Department of Radiation Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

The use of adjuvant whole brain radiation therapy (WBRT) after resec-tion of single brain metastases remains highly controversial. Despite class 1 evidence to the contrary, many clinicians still withhold WBRT after resec-tion of a single brain metastasis based in part on the argument that WBRT is ineffective against many tumor types because of their relative radioresis-tance. Therefore we undertook an observational study in order to determine the extent to which the type of primary cancer influenced the effectiveness of adjuvant WBRT after surgical resection of single brain metastases. We reviewed prospectively collected data from 358 adult patients with newly diagnosed, single brain metastases who underwent surgical resection. The median duration of follow-up was 60.1 months (range 0.6–156 months). Of these patients, 142 (40%) received adjuvant WBRT (WBRT group), and 216 (60%) did not (observation group). There were 105 patients with lung can-cer, 75 with melanoma, 71 with renal cell carcinoma, 39 with breast cancer, 20 with gastrointestinal cancer, 12 with sarcomas, 12 with gynecological cancer, and 24 with other cancer types. Based on these sites, 197 (55%)

tumors were considered “radiosensitive,” and 161 (45%) were considered “radioresistant.” Adjuvant WBRT was not a predictor of overall survival regardless of tumor type. Even when tumors were grouped as radiosensitive or radioresistant, there was no difference in overall survival of each group, whether the patients received adjuvant WBRT or not. However, adjuvant WBRT had a significant effect on recurrence. In the 358 patients, there were 67 (19%) local recurrences (at site of surgery), 21 (15%) in the WBRT group and 46 (21%) in the observation group (hazard ratio [HR] 5 0.58; 95% confidence interval [CI], 0.35–0.98; p 5 0.04). In addition, there were 156 (44%) distant recurrences (in brain locations away from the site of sur-gery), 42 (30%) in the WBRT group and 114 (53%) in the observation group (HR 5 0.43; 95% CI, 0.30–0.61; p , 0.001). Multivariate analyses demon-strated that withholding WBRT was an independent predictor of local and distant recurrence, but only when interactions with other variables were considered. For local recurrence, there was an interaction between WBRT and tumor size; i.e., patients whose tumors were large (.3 cm) and who did not receive adjuvant WBRT had an increased risk of recurring locally (HR 5 3.14; CI 1.02–9.69; p 5 0.05). For distant recurrence, there was an inter-action between WBRT and the status of the systemic cancer; i.e., patients whose primary disease was progressing and who did not receive WBRT had an increased risk of distant recurrence (HR 5 2.16; CI 1.01–4.66; p 5 0.05). Importantly, radioresistant tumors, such as melanoma and sar-comas, benefited from WBRT to a similar extent, as did the radiosensi-tive tumors, such as breast and lung cancer. This study, which includes the largest number of patients reported to date with surgically resected, single brain metastases treated with or without adjuvant WBRT, indicates that adjuvant WBRT significantly reduces local and distant recurrence in subsets of patients, regardless of the type of primary tumor. We recommend post-operative WBRT for patients with single metastases .3 cm or for patients with active systemic disease.

RT-30. NEUROPSYCHOLOGICAL PROFILE IN CHILDREN WITH LOW-GRADE BRAIN TUMORS BEFORE STARTING TREATMENT WITH FOCAL RADIOTHERAPY: BASELINE DATA FROM AN ONGOING RANDOMIZED TRIALRakesh Jalali,1 Debnarayan Dutta,1 Savita Goswami,2 Rashmi Kamble,3 Rajiv Sarin,1 and Ketayun Dinshaw1; 1Radiation Oncology, Tata Memorial Hospital Parel, Mumbai, India; 2Clinical Psychology, Tata Memorial Hospital Parel, Mumbai, India; 3Brain Tumour Foundation of India, Tata Memorial Hospital Parel, Mumbai, India

The objective was to report neuropsychological profile and activities of daily living in the pediatric patient population with residual/progressive low-grade brain tumors before starting focal radiotherapy (RT). The aim is to report the baseline pre-RT data collected in an ongoing prospective ran-domized trial comparing stereotactic conformal RT versus conventional RT in these patients to generate level 1 evidence for the efficacy of high-precision RT techniques. Between April 2001 and April 2007, out of 93 children and young adults (5–25 years) accrued so far, the present analysis focuses on 60 pediatric patients (46 males, 14 females) in the age group of 5–16 years. Prior surgery was in the form of partial excision in 29 patients, subtotal excision in 16 patients, and biopsy in 15 patients. Tumor subtypes included 18 chiasmatic-hypothalamic gliomas, 17 craniopharygiomas, 14 cerebellar astrocytomas, 8 supratentorial low-grade gliomas, and 3 ependymomas. Patients underwent a detailed baseline neuropsychological evaluation as per designed protocol. Neuropsychological function was assessed by Wechsler Intelligence Score Chart (WISC), which included measurement of age- adjusted intelligence quotients (verbal quotient, performance quotient, full-scale intelligence quotient [IQ]). A Lowenstein Occupational Therapy Cog-nitive Assessment (LOTCA) battery, which measured cognitive parameters of visual, spatial, visuomotor, and attention, was performed in 41 suitable patients. Anxiety was measured by State-Trait Anxiety Inventory for Chil-dren (STAIC) (C1—state and C2—trait) and activities of daily living by the modified Barthel’s index. Binomial distribution analyses were performed to assess whether the proportion of patients with impaired performance on each measure exceeded normal expectations. As many as 32 (60.4%) of the 53 evaluable patients had full-scale IQ values below normal expected levels at baseline before starting RT, although the overall mean verbal IQ (87.9), performance IQ (85.33), and full-scale IQ (84.6) were only slightly less than the expected values. Proportion of patients with less-than-expected scores was seen significantly more (p 5 0.003) in the performance IQ domain (34 patients, 64%) than in the verbal IQ domain (24 patients, 45%). A signifi-cantly higher number of patients showed severe anxiety (score more than 30) in the state C1 form (19 out of 41 evaluable patients patients, 46%) than in the trait C2 form (14 patients, 34%) (p 5 0.008). In the LOTCA battery, the proportion of patients with less-than-expected scores was seen significantly more in the visual (p 5 0.007), orientation (p , 0.001), and spatial perception (p , 0.001) than visuomotor, motor praxia, thinking, and attention domains. The mean score of Barthel’s index was 18.21 (range 10–20). Our prospective study in children with residual/progressive benign and low-grade brain tumors after prior surgery demonstrates a considerable pre-RT cognitive and neuropsychological morbidity. These suggest that fac-tors other than RT, such as tumor and surgery, could have a possible impact



on neuropsychological parameters. Performance IQ rather than verbal IQ was seen to be impaired in a greater number of patients. Cognition was poorer in some specific domains than others. We are collecting prospective follow-up data to show the further impact of various RT techniques on these parameters.

RT-31. CEREBRO-SPINAL IRRADIATION IN PEDIATRIC POSTERIOR FOSSA TUMOR PATIENTS CAUSES PERMANENT ALTERATION OF NEURAL METABOLITE CONCENTRATIONS AS DETECTED BY SINGLE-VOXEL MR PROTON SPECTROSCOPYStefan Rueckriegel,1 Friederike Blankenburg,1 Günter Henze,1 Harald Bruhn,2 and Pablo Hernaiz Driever1; 1Department of Pediatric Oncology and Hematology, Charite-Universitätsmedizin Berlin, Berlin, Germany; 2Department of Radiology, Charite-Universitätsmedizin Berlin, Berlin, Germany

Neurotoxicity of posterior fossa tumors, chemotherapy, and cerebro-spinal irradiation (CSI) in children leads to neurological and neurocognitive long-term deficits. Survivors’ sequelae are more pronounced in high-grade tumor patients being treated with chemotherapy and CSI in comparison to patients who underwent surgery only. By comparing these two otherwise similar patient groups, we tried to determine the critical neurotoxic effect of CSI. Metabolite concentrations in white matter (WM) and in gray mat-ter (GM) as determined by quantitative single-voxel proton MR spectros-copy (MRS) served as a measure of neurotoxicity of CSI and chemotherapy. Twenty-three medulloblastoma survivors and 1 ependymoma (WHO III) survivor (age 6SD: 13.0 65.2 y; interval therapy to examination 6SD: 46 631.7 m) constituted the first group that had received chemotherapy and CSI including a boost on the posterior fossa. Nineteen pilocytic astrocytoma (WHO I) survivors and 1 ependymoma (WHO II) survivor were included in the second group (age 6SD: 11.9 64.7 y; interval 6SD: 30.4 624 m). All patients were subjected to MR imaging and MRS on a 3T-MR system equipped with an 8-channel headcoil (3T Signa Excite, GE Healthcare) after ethically approved written informed consent had been obtained. MRS was performed in parietal WM (4 ml) and parietal midline GM (8 ml) using the PRESS technique (TR/TE/NEX 5 6,000ms/30ms/64). Quantification of the resonances of creatine and phosphocreatine (Cre), N-acetylaspartate and N-acetylaspartylglutamate (tNAA), choline-containing compounds (Cho), glutamate (Glu), and myo-inositol (mI) was done with the operator-independent LCModel method. In the high-grade tumor group concentra-tions of Cho (p 5.004, Mann-Whitney-U-test), tNAA (p 5 0.029), and Glu (p 5 0.045) were significantly decreased in WM, while mI (p 5 0.035) was increased in GM. Throughout measurements, water linewidth was at FWHM (full width at half maximum) 5–7 Hz. The decrease of metabolite concentrations in irradiated patients allows us to suggest that craniospi-nal irradiation has a long-lasting effect on the metabolism of brain tissue. Predominantly in WM, decreases of Cho, a marker of membrane turnover in astrocytes and oligodendrocytes, and tNAA, an indicator of neuronal integrity and myelination, were noted. The physiological concentration of tNAA increases in childhood up to an age of 10 years. As the mean age of the patients under 10 years is similar in both groups (7.2 y), the influ-ence of age should not have confounded results for tNAA. MRS in vivo provides valuable data on the molecular level for a better understanding of the mechanisms and extent of irradiation-induced neurotoxicity. More extended longitudinal follow-up studies are warranted to evaluate the degree of metabolism alteration and correlate the predictive value of metabolite quantification versus neurocognitive outcome. A thesis scholarship from the Kind-Philipp Foundation to S.R. is gratefully acknowledged.

RT-32. LONG-TERM NEUROTOXICITY OF THERAPY IN PEDIATRIC POSTERIOR FOSSA TUMOR SURVIVORS DETECTED BY DIFFUSION TENSOR IMAGING: A COMPARISON OF ANALYTIC APPROACHES BASED ON REGIONS OF INTEREST AND VOXEL-BASED MORPHOMETRYStefan Rueckriegel,1 Friederike Blankenburg,1 Günter Henze,1 Harald Bruhn,2 and Pablo Hernaiz Driever1; 1Department of Pediatric Oncology and Hematology, Charite-Universitätsmedizin Berlin, Berlin, Germany; 2Department of Radiology, Charite-Universitätsmedizin Berlin, Berlin, Germany

Posterior fossa tumors per se, chemotherapy (CT) and craniospinal irradiation (CSI) cause neurological and neurocognitive long-term deficits in survivors. Sequelae are more pronounced in high-grade tumor (HGT) patients due to CT and CSI than in low-grade tumor (LGT) patients that underwent surgery only. Damage of fiber tract integrity is a critical mor-phological marker of neurotoxicity. By comparing the fractional anisotropy (FA) data obtained by diffusion tensor imaging (DTI) between both patient groups, we tried to distinguish the neurotoxic effects of CSI and CT on pre-

specified and skeletonized fiber tracts. We compared results achieved when analyzing specific regions of interest or voxel-based morphometry. Twenty-four medulloblastoma (WHO IV) survivors and 1 ependymoma (WHO III) survivor (HGT) constitute the first group of patients having received CSI, including a boost on the posterior fossa, and CT (age 6SD: 13.0 64.8 y; interval therapy to examination 6SD: 49 633.5 m). Twenty-one pilocytic astrocytoma (WHO I) and 1 ependymoma (WHO II) survivors (LGT) were included in the second group (age 6SD: 12.1 65.1 yrs; interval 6SD: 33 626.7 m). All patients were subjected to MR imaging and DTI on a 3T-MR system equipped with an 8-channel headcoil (3T Signa Excite, GE Healthcare) after ethically approved written informed consent had been obtained. For a region of interest (ROI) analysis, FA maps were generated by Functool. Seventeen ROIs were set on different anatomical landmarks, including the cerebellar peduncles, crura cerebri, internal capsules, and the corona radiata. Voxelwise statistical analysis of FA data was carried out using Tract-Based Spatial Statistics (TBSS) (Smith, 2006) as part of the FSL (FMRIB Software Library, Oxford). TBSS projects all subjects’ FA data onto a mean FA tract skeleton before applying voxelwise cross-subject sta-tistics. No significant difference of FA in the 17 ROIs between both patient groups was observed. When applying TBSS, two clusters of voxels appeared to be significantly decreased in the HGT group (Monte Carlo permutation t-test). The first cluster (p , 0.002) was located in the cerebellar vermis, the second cluster (p , 0.05) in the right parahippocampal gyrus. Discus-sion: So far, the ROI-approach did not yield statistical differences of FA contrasting previous results between medulloblastoma patients and healthy controls (Khong, 2003). Compared to TBSS, the ROI approach is in risk of a higher variability due to subjectivity of manual ROI placing. Sensitivity may be lower because of the reduction of tract-analysis on the ROIs. On the other hand, determination of the exact anatomical localization of the voxel compared is more limited in TBSS due to the brain normalization on standard space. Of note, both TBSS and the ROI approach did not show significant FA decreases in the main fiber tracts. Our results allow us to assume that cerebellar vermis and temporal structures are more sensitive to radiation toxicity and/or have a worse capacity of regeneration. Tract-based spatial statistics on FA maps proved to be a useful tool for spatially resolved, more objective measure of neurotoxicity on fiber tracts in children. More extended follow-up studies are warranted to discriminate the neurotoxic effects of the tumor itself, hydrocephalus, resection, CSI, and CT. A thesis scholarship from the Kind-Philipp Foundation to S.R. is gratefully acknowl-edged. References: Smith SM, NeuroImage 2006;31:1487–1505. Khong P, AJNR 2003;24:734–40.

RT-33. LITHIUM: PROTECTION FROM NEURONAL HIPPOCAMPAL CELL DEATH AND PROMOTION OF DNA REPAIR DURING THERAPEUTIC RADIATIONHong Wang1 and Fen Xia2; 1Radiation Oncology, Vanderbilt University Medical Center, Nashville, TN, USA; 2Academy of Clinical Laboratory Physicians and Scientists, Nashville, TN, USA

The clinical use of cranial radiation for the treatment of brain cancer is known to sometimes result in irreversible cognitive defects in patients. Evi-dence suggests that this phenomenon is caused by radiation-induced damage to proliferating neuronal progenitor cells in the hippocampus. Previously we showed that lithium, a known neuroprotective agent against a wide variety of insults, attenuates radiation-induced apoptosis in hippocampal neurons in vivo and improves learning and memory in cranially irradiated mice. To better elucidate the mechanism of lithium’s radioprotection of hippocampal neurons, we studied the effects of lithium on chromosomal double-strand breaks (DSBs), one of the most toxic radiation-induced lesions in proliferat-ing cells. Additionally, we examined the effects of lithium on the functional regulation of MRE11, a critical protein involved in multiple aspects of cellu-lar response to chromosomal DSBs. Murine primary hippocampal neuronal cells and malignant glioma cells were cultured either with or without 3 mM lithium chloride for 7 days prior to being subjected to 3 Gy irradia-tion. Chromosomal DSBs were subsequently quantified by measuring the -H2AX nuclear foci that are the in situ marker of DSBs. -H2AX foci were measured at 0, 0.5, 1, and 4 hours after radiation by immunostain-ing of cells with anti–-H2AX antibody. Intranuclear function of Mre1 1 was analyzed in each of the cell populations by means of western blot and ionizing radiation-induced nuclear foci (IRIF). Apoptosis in each of the cell populations was directly determined by morphological change and tunnel staining. Hippocampal cells that were pretreated with lithium demonstrated a relative 90% reduction in radiation-induced DSBs at 1 hour post-radiation and 75% reduction at 4 hours post-radiation, as compared to cells that were not pretreated with lithium. Correspondingly, apoptosis was significantly reduced in the hippocampal cells pretreated with lithium. Second, in the malignant glioma cells, lithium pretreatment resulted in no difference in radiation-induced DSBs or apoptosis. Finally, pretreatment with lithium also resulted in a 2–3-fold increase of Mre11 protein expression with a simultaneous paradoxical 2–3-fold decrease of Mre11 IRIF. These results suggest that lithium protects hippocampal neurons from death by means of up-regulation of Mre11, thereby promoting the efficiency of DNA repair. The protective effect of lithium appears to apply only to hippocampal cells



and not to malignant glioma cells. The mechanisms by which lithium pro-vides neuroprotection for hippocampal cells are important in optimizing the concomitant use of lithium with radiation. Furthermore, these molecular targets provide a basis for future clinical strategies in the safe and effective administration of radiation for brain cancer.

RT-34. A LARGE-SCALE siRNA SCREEN REVEALED THAT PARP1 AND CDK7 INHIBITION SELECTIVELY SENSITIZED EGFR AND RAS OVER-EXPRESSING GLIOMA CELL LINES TO IONIZING RADIATIONClark Chen1 and Alan D’Andrea2; 1Harvard University, Boston, MA, USA; 2Radiation Oncology, Dana Farber Cancer Institute, Boston, MA, USA

While the incorporation of ionizing radiation (IR) into the treatment of high-grade gliomas has led to improvement in patient survival, the therapeu-tic efficacy of IR is largely limited by the intrinsic resistance of gliomas. The therapeutic gain of many radio-sensitizing agents developed to overcome this resistance, however, has been disappointing due to comparable sensi-tization of both the normal and the tumor cells. To address this issue, we carried out a large-scale siRNA screen with the intent of identifying gene targets that when inactivated caused selective IR sensitization of gliomas transformed by the over-expression oncogenes. To minimize the likelihood of cell line–specific artifacts, two isogenic pairs were used in this study: the U87 and U87 1 EGFRvIII pair as well as the THV and THV 1 H-RasV12G pair. A siRNA library (Qiagen) consisting of 712 siRNAs directed against 356 genes implicated in the major DNA repair/damage response pathways was screened. We first identified targets that sensitized both the U87 1 EGFRvIII and the THV 1 H-RasV12G cell lines to IR. Candidate genes were divided into 5 groups based on their known functions. Representa-tive candidate genes from each group (ADPRT, CDK7, RPA3, FANCF, or HDAC2) were subsequently characterized as to whether they selectively sen-sitized gliomas harboring EGFRvIII or H-RasV12G relative to their isogenic counterparts. We found that siRNA silencing of ADPRT and CDK7, but not RPA3, FANCF, or HDAC2, preferentially sensitized the U87 1 EGFRvIII cells to IR relative to the U87 cells. Similarly, silencing of ADPRT and CDK7 caused selective IR sensitization of THV 1 H-RasV12G cells relative to the THV cells. To exclude the possibility that this selective sensitization was related to siRNA “off-target effect,” we showed that pharmacologic PARP inhibition using INO1001 (Inotek) caused similar selective IR sensitization of U87 1 EGFRvIII relative to the U87 cell line. These results suggest that PARP inhibition caused preferential IR sensitization of glioma cells harbor-ing aberrant EGFR and RAS activation and provide a basis for the design/analysis of clinical trials involving PARP inhibition.

RT-35. PHASE 3 ENRICH (RT-016) COMPARATIVE STUDY OF EFAPROXIRAL ADMINISTERED CONCURRENT WITH WHOLE BRAIN RADIATION THERAPY (WBRT) IN WOMEN WITH BRAIN METASTASES FROM BREAST CANCERJohn Suh,1 Baldassarre Stea,2 K. Tankel,3 H. Marsiglia,4 Y. Belkacemi,5 H. Gomez,6 S. Falcone-Lizaraso,7 J. May,8 J. Hackman,8 and M. Saunders8; 1Cleveland Clinic Foundation, Cleveland, OH, USA; 2University of Arizona, Oro Valley, AZ, USA; 3Cross Cancer Institute, Edmonton, Alberta, Canada; 4Institut Gustave-Roussy, Villejuif, France; 5Centre Oscar Lambert, Lille, France; 6Instituto de Enfermedades Neoplasicas, Lima, Peru; 7Hospital Edgardo Rebagliati, Lima, Peru; 8Allos Therapeutics, Inc., Westminster, CO, USA

Study RT-016 (ENRICH) was a Phase 3, randomized, open-label comparative study of standard whole brain radiation therapy (WBRT) with supplemental oxygen, with or without concurrent efaproxiral, in women with brain metastases from breast cancer to determine if adding efaproxiral to WBRT prolongs survival and increases tumor response. Key eligibility criteria included confirmed breast cancer with brain metastases, Karnofsky performance status (KPS) > 70, and no other active concurrent malignancy. All patients received a standard 30 Gray (Gy) course of WBRT (over 2 weeks) at 3.0 Gy per fraction. Patients were randomized 1:1 to the Efaproxiral Arm or Control Arm. Patients randomized to the Efaproxiral Arm received efaproxiral and supplemental oxygen within 30 minutes prior to daily WBRT. Patients randomized to the Control Arm received supple-mental oxygen and WBRT. The planned sample size was 360 patients (180 patients/Arm). Patients were stratified for KPS (70–80 and 90–100) and known liver metastases (presence or absence), for a total of 4 strata. The primary endpoint was overall survival. The final analysis occurred at 281 deaths. From February 2004 to September 2006, 368 patients were random-ized at 78 sites in 15 countries. Efficacy analyses were conducted on 365 eligible patients (n 5 182 Efaproxiral Arm, n 5 183 Control Arm). No sta-tistically significant difference in survival was detected between the 2 arms using the stratified log-rank test (hazard ratio [HR] 5 0.87, p 5 0.233). The median survival time (MST) was 8.5 months in the Efaproxiral Arm,

compared to 7.5 months in the Control Arm. Secondary efficacy endpoints of response rate in the brain at 3 months (confirmed complete response [CR] plus partial response [PR] 5 31% in the Efaproxiral Arm and 27% in the Control Arm), KPS, and neurological signs and symptoms improvement, also failed to achieve statistical significance. Nausea and headache were the most frequent adverse events (AE). In this randomized trial, the addition of efaproxiral to WBRT failed to improve overall survival in women with brain metastases originating from breast cancer. No impact was observed in any of the pre-specified secondary endpoints.

STEM CELLS

SC-01. COMPARISON BETWEEN microRNA SIGNATURE OF GLIAL TUMORS, NEURONAL PROGENITOR CELLS, AND NORMAL ADULT BRAINIris Lavon,1 Daniel Zrihan,1 Malkiel A. Cohen,2 Benjamin Reubinoff,2 Ofira Einstein,3 Tamir Ben-Hur,3 Yoav Smith,4 and Tali Siegal1; 1Gaffin Center for Neuro-Oncology, Hebrew University–Hadassah Medical School, Jerusalem, Israel; 2Human Embryonic Stem Cell Research Centre, Hebrew University–Hadassah Medical School, Jerusalem, Israel; 3Department of Neurology, Hebrew University–Hadassah Medical School, Jerusalem, Israel; 4Genomic Data Analysis Unit, Hebrew University–Hadassah Medical School, Jerusalem, Israel

microRNA (miRNA) is a family of small nucleotides that regulates gene expression. The miRNA family contains 300–400 members in human, and each miRNA can inhibit up to 200 different genes. Therefore miRNA fam-ily has the potential to regulate almost all of the genetic and physiological pathways of the cell, including development, metabolism, proliferation, dif-ferentiation, and programmed cell death. Impaired miRNA expression is related to different diseases, including malignancy, which is characterized by uncontrolled cell renewal. Stem cells (SC) also have self-renewal ability, suggesting a strong relationship between SC and cancer. Our objective was to compare the profile of miRNA expression in glial tumors, embryonic SC (ESC), neural progenitor cells (NPC), and normal adult brain. The identified deviations in miRNA expression in these tissues may facilitate our under-standing of glioma genesis. Expression of most of the validated (186) human miRNAs was evaluated in tissue RNA samples of 7 low- and high-grade gliomas, ESC, NPC, and commercially available normal adult brain, using stem-loop primers for reverse transcription (RT) followed by real-time PCR. In gliomas, 58 miRNAs out of the 186 tested demonstrated distinct expres-sion when compared with normal adult brain. Strikingly, a similar expres-sion pattern of these 58 miRNAs was present in NPC or/and ESC. However, in SC 15 other miRNAs had a unique signature not observed in normal brain or in gliomas. No significant difference was noted between miRNA expression profile of low- and high-grade gliomas. Substantial similarity was found between miRNA signature of gliomas and NPC and/or ESC. Additional studies are needed to determine whether the origin of gliomas is from adult SC or from de-differentiated glia cells that acquired “stem-like” property. Careful study of the 15 miRNAs that differed between SC and gliomas may enhance our understanding of gliomagenesis. Furthermore, our finding that miRNA signature of low- and high-grade gliomas is similar may imply that low-grade gliomas already carry the potential to become highly aggressive tumors.

SC-02. HYALURONAN ANTAGONISM IN GLIOMA PROGENITOR TUMOR SPHERESAnne Gilg, Mark Slomiany, Sandra Tye, Bryan Toole, and Bernard L. Maria; Medical University of South Carolina, Charleston, SC, USA

Hyaluronan (HA), a large polysaccharide constitutively expressed in the brain extracellular matrix, constitutively regulates key receptor tyrosine kinases (RTKs), drug efflux transporter expression and localization, and cell survival activities, including the PI3K/Akt, Ras, Erk, and FAK pathways in malignant cancer cells. HA oligomers (o-HA) and other hyaluronan antag-onists inhibit endogenous interactions between hyaluronan and CD44, a key receptor for hyaluronan, resulting in suppression of these activities and inhibition of anchorage-independent growth, invasiveness, and resistance to therapy. The purpose of this study was to target hyaluronan/CD44 interac-tions in treatment-resistant glioma progenitor cells. These cells were isolated using two methods. First, a side population (SP) of C6 glioma progenitor cells was isolated by FACS analysis based on their efflux of Hoechst dye. These cells also have higher expression of ABCG2 (breast cancer resistance protein, BCRP) and CD133, a marker of glioma progenitor cells. Second, using neurosphere isolation techniques, BCRP1/CD1331 populations were enriched from several human glioma cells lines and primary human glioma biopsy samples. By immunocytochemistry, we demonstrated the presence of an HA-rich extracellular coat on live neurospheres. Short-term treatment



of these spheres with o-HA disrupted the HA-enriched coat and caused the rapid disaggregation of spheres. This is the first demonstration of dis-solution of the HA pericellular coat in drug- and radiation-resistant cancer progenitor cells. Inhibition of HA/CD44 interactions with o-HA in these glioma progenitor cells decreased phosphorylation of EGFR, c-MET, and Akt and decreased BCRP production. Based on these findings, we propose that hyaluronan oligomers decrease survival of glioma progenitor cells and that the mechanism is mediated by suppressing EGFR and c-MET activity and Akt-mediated BCRP function.

SC-03. HUMAN CYTOMEGALOVIRUS IE1 GENE IS EXPRESSED IN GLIOMA STEM-LIKE CELLS IN SITU AND PROMOTES SELF-RENEWAL OF NEURAL PRECURSOR CELLS IN VITROLiliana Soroceanu,1 Matthias Kraus,2 Lualhati Harkins,2 and Charles Cobbs1; 1California Pacific Medical Center Research Institute, San Francisco, CA, USA; 2University of Alabama at Birmingham, AL, USA

We have previously identified human cytomegalovirus (HCMV) gene products in over 90% of malignant gliomas in vivo. We now have evidence that the essential HCMV IE1 gene product is preferentially expressed in the CD1331 “stem-like” glioma cells in situ. Double labeling immunofluores-cence of glioblastoma tissues and CD1331 sorted primary glioblastoma cells revealed that 40–60% of the CD1331 cells are also expressing IE1. These findings prompted our investigation into the potential role of HCMV in gliomagenesis. We subjected normal human astrocytes (NHA), primary adult neural precursor cells (NPC), and glioma cell lines to HCMV infection or sustained IE1 gene expression and monitored the status of key signaling pathways regulating cell survival, proliferation, and self-renewal. HCMV infection of primary adult NPCs and glioma cells induced activation of Bmi-1, a critical positive modulator of neural tumor stem cell self-renewal. BrdU incorporation and MTT survival assays revealed that sustained IE1 expression induced proliferation of some glioma cell lines, while it did not significantly alter the growth rate of NHA, suggesting that IE1-induced growth modulation is dependent on the differentiation status of the cell. IE1-induced glioma cell proliferation was paralleled by down-regulation of the p53 family of tumor suppressors and a reduction in the cyclin-dependent kinase inhibitors, such as p27Kip. Stable IE1 expression induced sustained AKT activation and MAPK signaling in both malignant glioma cells and NHA. We are currently investigating the effects of stable IE1 expression on the self-renewal, Bmi1 levels, and differentiation potential of adult human NPCs. Given the frequent association of HCMV with glioma stem-like cells and its role in modulating key oncogenic pathways, reactivation of latent HCMV in a susceptible neural precursor cell population may underlie early events in gliomagenesis.

SC-04. ELEVATED SOX-11 EXPRESSION IS ASSOCIATED WITH PROLIFERATION, MIGRATION, AND POOR OVERALL SURVIVAL IN GLIOBLASTOMATimothy Van Meter,1 Catherine Dumur,2 Gary Tye,2 and William Broaddus2; 1Virginia Commonwealth University, Richmond, VA, USA; 2Richmond, VA, USA

Glioblastoma (GBM) is a notoriously heterogeneous cancer of the cen-tral nervous system which rarely metastasizes but has a high propensity for extensive local diffuse invasion of the adjacent brain. Recently, our research team has prospectively collected regional biopsies from tumor periphery and core of GBM and other tumors, with the intent to study the regional differences in gene expression which might arise from different microen-vironments within the tumor. In a study comparing invasive rim versus perinecrotic core samples of 12 untreated glioblastomas, we identified the neural stem cell marker Sox-11 as one of a suite of CNS stem cell-related genes up-regulated in the tumor rim using Affymetrix microarrays, quan-titative PCR, immunostaining, and western blotting. Importantly, Sox-11 expression was tightly correlated with expression of the cell cycle markers including TOPO2A, SKIP1, Ki67, CD133, and Nestin expression levels in the Affymetrix datasets and by qPCR. Sox-11 expression predicted poor overall survival in an independent cohort of 35 GBM specimens. Because Sox-11 was reported to be expressed in proliferating neural progenitor cells during development of the neocortex and had been reported to potently activate Nestin transcription, we sought to investigate whether a functional relationship between Sox-11 and Nestin existed in glioblastoma. Sox-11 binding of the enhancer region of the human Nestin gene was examined using a quantitative PCR-based chromatin immuno-precipitation assay in U251MG cells and in Sox-11 positive GBM biopsy cultures. Finally, we used an siRNA silencing approach to suppress Sox-11 expression and examined the effects on cell cycle, motility, and transcriptional activation in GBM cultures. RNA silencing experiments demonstrated that Sox-11 suppression significantly attenuated GBM cell growth and diminished Nestin expres-

sion and cell migration in vitro. Sox-11 expression in primary GBM cul-tures co-labeled with CD133 and mitotic markers by confocal microscopy. These data strongly implicate Sox-11 as a cell cycle associated gene related to a highly migratory and proliferative neural stem cell-like population in GBM.

SC-05. HUMAN BONE MARROW–DERIVED MESENCHYMAL STEM CELLS AS DELIVERY VEHICLES FOR ONCOLYTIC ADENOVIRUS DELTA-24–RGD IN THE TREATMENT OF MALIGNANT GLIOMARaymund L. Yong,1 Juan Fueyo,2 Frank Marini,3 Nobuhiro Hata,1 Joy Gumin,1 Michael Andreeff,4 and Frederick F. Lang1; 1Department of Neurosurgery, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Department of Neuro-Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3Department of Blood and Marrow Transplant, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 4University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Delta-24–RGD is a tumor-selective, infectivity-augmented, replication-competent oncolytic virus with significant antiglioma effects. Although direct intratumoral delivery of Delta-24–RGD may be effective, the devel-opment of less invasive systemic delivery methods remains a major goal of viral therapy. Recent evidence from our laboratory has demonstrated that human bone marrow–derived mesenchymal stem cells (hMSCs) are useful delivery vehicles for brain tumor therapy (Nakamizo et al., Cancer Res 2005;65:3307–3318). Therefore we tested in vivo the hypothesis that packaging Delta-24–RGD within hMSCs allows for systemic delivery of Delta-24–RGD. To demonstrate that hMSCs infected with Delta-24–RGD are capable of delivering Delta-24–RGD to gliomas in vivo, Delta-24–RGD-infected hMSCs (hMSCs-Delta-24, 106 cells), PBS-treated hMSCs (hMSCs), or “naked” Delta-24–RGD (108 pfu) were injected into the carotid artery of nude mice bearing intracranial U87 glioma xenografts (n 5 3/group). After 7 days, animals were sacrificed, their brains were removed, and immunostaining using anti-viral-E1A or anti-viral-hexon protein antibod-ies was undertaken. Whereas no staining was seen after delivery of empty hMSCs or “naked” Delta-24–RGD, positive immunostaining for both viral E1A and hexon protein was seen widely throughout the tumors treated with hMSCs-Delta-24. To specifically document at the cellular level that hMSCs-Delta-24 localize to human gliomas and release Delta-24–RGD into the tumor, hMSCs were transduced with an adenovirus containing green fluorescent protein (Ad-gfp). These gfp-infected hMSCs were subse-quently infected with Delta-24–RGD (MOI 10) and injected into the carotid artery of nude mice bearing U87 glioma xenografts in their frontal lobes. At increasing times after the intra-carotid injection, animals (n 5 3 per time point) were sacrificed, their brains were removed, and paraffin sections were analyzed by immunofluorescence using fluorescent antibodies against gfp (green fluorescent secondary antibody to track hMSCs) and viral E1A or hexon protein (Texas Red secondary antibodies to track Delta-24–RGD). By 4 days after hMSC delivery, gfp-positive cells (i.e., hMSCs) were seen exclusively throughout the gliomas (average density 5 16 1 0.7 cells per mm2). Red fluorescent cells (viral E1A-positive cells) were also identified. Merging of the images indicated that E1A was localized within the hMSCs (yellow cells), indicating that replicating Delta-24–RGD was located within the hMSCs. At day 7 after injection, hMSC density within the tumors decreased slightly to 14 1 0.1 cells per mm2, while both E1A and hexon expression increased. Merged images revealed that E1A and hexon proteins were present within hMSCs (yellow cells). In addition, cells containing only red fluorescence (E1A protein) were also identified, indicating that the virus had spread to U87 tumor cells (gfp-negative cells). By 11 days, gfp-express-ing hMSCs were rare (average density of 2 1 0.04 cells per mm2), whereas E1A and hexon staining had increased significantly. Merged images showed that tumors were dominated by E1A-expressing or hexon-expressing U87 cells (red cells). Taken together, these studies are the first to demonstrate in vivo that hMSCs are capable of delivering Delta-24–RGD to intracranial human gliomas after systemic (intra-carotid) injection. Specifically, Delta-24–RGD-carrying hMSCs (but not naked Delta-24–RGD) selectively local-ize to gliomas, support the replication and release of virus into the tumor, and produce a propagating viral infection within the tumor.



SC-06. PDGF-BETA MEDIATES THE TROPISM OF HUMAN BONE MARROW–DERIVED MESENCHYMAL STEM CELLS FOR MALIGNANT GLIOMASNobuhiro Hata,1 Raymund L. Yong,1 Joy Gumin,1 Frank Marini,2 Rui-Yu Wang,2 Michael Andreeff,2 and Frederick Lang1; 1Department of Neurosurgery, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Department of Blood and Marrow Transplantation, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Previous studies have shown that human mesenchymal stem cells (hMSCs), which are derived from the bone marrow, are capable of local-izing to human gliomas after systemic delivery and can be used to delivery therapeutic agents to gliomas (Nakamizo et al., Cancer Res 2005;65:3307–3318). However, the mechanism underlying the tropism of hMSCs for gliomas remains unclear. In vitro studies have suggested that tumor- derived growth factors, particularly platelet-derived growth factor (PDGF-beta), may mediate the tropism between hMSCs and gliomas. We therefore tested the hypothesis that PDGF-beta is causally associated with attracting hMSCs to gliomas. U87-XO cells were transfected with a plasmid encoding human PDGF-beta. The stable transfected clone with the highest secretion of PDGF-beta (U87–PDGF-high, mean PDGF 23 x102 pg/ml by ELISA) and the clone with the lowest secretion (U87–PDGF-low, mean PDGF 0.26 x102 pg/ml) were chosen for further studies. In matrigel in vitro trans-well invasion assays, significantly more hMSCs migrated toward the U87 PDGF-high (86 61 hMSCs/10hpf) compared with U87–PDGF-low (40 12 hMSCs/10hpf) (p , 0.006). The increase in migration toward U87–PDGF-high was abrogated by treatment with neutralizing PDGF-beta antibody. To evaluate hMSC tropism in vivo, sex-mismatched transplant experiments were undertaken. Specifically, xenografts from U87–PDGF-high (n 5 3) or U87–PDGF-low cell lines (n 5 3) (both with XO genotype) were established in the frontal lobes of nude mice, and animals were treated with intraca-rotid injections of hMSCs derived from the bone marrow of a male donor (genotype XY, 106 cells). After 7 days, animals were sacrificed, paraffin sections prepared, and FISH analyses undertaken using probes for human 3 and Y chromosomes. Triple fluorescent microscopy identified populations of human Y-positive cells (hMSCs) only in the U87–PDGF-high xenografts (average 19 12 hMSCs/4hpf for U87–PDGF-high vs. 0 hMSCs/4hpf for U87–PDGF-low; p , 0.005). Importantly, immunohistochemical staining of the tumors for PDGF-beta demonstrated PDGF only in the U87–PDGF-high xenografts and no staining in the U87–PDGF-low xenografts. To con-firm the role of PDGF, in vivo bioluminescence imaging (BLI) of hMSCs was undertaken. Intracranial xenografts of U87–PDGF-high (n 5 4) or U87–PDGF-low (n 5 6) cells were established in the frontal lobes of nude mice. Seven days after tumor inoculation, hMSCs were transduced with an adenovirus vector containing the cDNA of the firefly luciferase gene and injected (106 cells) into the carotid artery of the tumor-bearing mice. Seven days later, animals were treated with Luciferin (150 mg/kg, IP) and imaged with the IVIS Imaging System (Xenogen, CA). The average signal intensity in U87–PDGF-high tumors (614 3 103 646 3 103 photons/sec/cm2) was significantly higher than that of the U87–PDGF-low tumors (87 3 103 613 3 103 photons/sec/cm2; p , 0.0002). Taken together, these studies demon-strate that intratumoral expression of PDGF-beta causally mediates at least in part the attraction of hMSCs for gliomas. This finding can be exploited for improving the specificity of hMSCs for gliomas, or for selecting proper patients for successful hMSCs treatment.

SC-07. HUMAN BONE MARROW–DERIVED MESENCHYMAL STEM CELLS AS DELIVERY VEHICLES FOR GLIOMA THERAPY: IN VIVO FUNCTIONAL ANALYSES USING BIOLUMINESCENCE IMAGINGNobuhiro Hata,1 Raymund Yong,1 Joy Gumin,1 Frank Marini,2 Raymond Sawaya,1 Michael Andreeff,2 and Frederick Lang1; 1Department of Neurosurgery, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Department of Blood and Marrow Transplantation, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

In our continuing effort to improve delivery of anticancer agents to malignant gliomas, we have shown previously that bone marrow–derived human mesenchymal stem cells (hMSCs) selectively localize to human gliomas after systemic delivery and can be exploited as delivery vehicles for brain tumor therapy (Nakamizo et al., Cancer Res 2005;65:3307–3318). Although the hMSCs appear to be capable of integrating into gliomas, their selectivity for tumors and their functional capacity once within a tumor remain unclear. Consequently, in vivo bioluminescence imaging (BLI) was undertaken to track hMSCs after systemic delivery. Importantly, BLI iden-tifies functionally intact (living) hMSCs, because light production requires the intracellular translation of a catalytically active protein (luciferase). Intracranial xenografts of U87 cells were established in the frontal lobes of nude mice. Seven days after tumor inoculation, hMSCs were transduced with an adenovirus vector containing the cDNA of the firefly luciferase gene (Ad-Luc, 1000 vp/cell), and these hMSC-Luc (106 cells) were injected into the carotid artery of the tumor-bearing mice (experimental group, n 5 10). As a control for nonspecific localization, hMSCs-Luc were injected into

mice in which PBS, rather than tumor cells, was inoculated into the brain (sham-tumor control, n 5 10). As another control, mice with U87 gliomas were injected with PBS rather than hMSCs (PBS control, n 5 10). Seven days after hMSC injection, animals were treated with Luciferin (150 mg/kg, IP) and imaged with the IVIS Imaging System (Xenogen, CA). BLI color images were overlaid on grayscale photographic images of the mice to local-ize the light source within the animals using the LIVINGIMAGE V. 2.11 software overlay (Xenogen, CA) and IGOR image analysis software (V. 4.02 A, WaveMetrics, OR). Of the 10 mice with intracranial xenografts who received hMSCs-Luc (experimental group), 100% (10/10) had high signal detected in the tumor, and the average signal intensity over the right frontal tumor (ROI-right frontal) was 147.1 3 102 620.4 3 102 photons/sec/cm2. Of the 10 mice without intracranial tumors that received hMSCs-Luc (sham-tumor control), 20% (2/10) had low signal detected in the right fron-tal region, and the mean ROI-right frontal was only 3.1 3 102 60.3 3 102 photons/sec/cm2 (p , 0.04, compared with experimental group). No signal was detected in the brains of tumor-bearing mice that received intracarotid PBS (PBS control), and the mean ROI-right frontal was 0.9 3 102 60.01 3 102 photons/sec/cm2, indicating that signal detected in the experimental group was specific for hMSCs-Luc. Taken together, these studies demon-strate that hMSCs localize selectively to human gliomas after systemic deliv-ery and that the cells within the tumor are functionally intact (capable of translating a catalytically active protein). Moreover, nonspecific localization at sites without tumor is rare.

SC-08. HETEROGENEITY OF CD111 (NECTIN-1) EXPRESSION GOVERNS INFECTION OF HUMAN GLIOMA TUMOR OR PROGENITOR CELLS BY GENETICALLY ENGINEERED HERPES SIMPLEX VIRUS (HSV)Gregory Friedman, Suman Bharara, Catherine Langford, Jennifer Coleman, and Yancey Gillespie; University of Alabama at Birmingham, Birmingham, AL, USA

Wild-type HSV and genetically engineered HSV have been shown to infect and kill human glioblastoma multiforme cells; however, the extent of cell killing has been variable from tumor to tumor. Expression of CD111 (nectin-1), a cell surface adhesion molecule that serves as an important entry receptor for HSV, was assessed by FACS analysis to determine whether this could predict the degree of HSV-mediated oncolysis. Three human glio-blastomas, GBM12, D456MG, and UAB1066, grown in the flanks of nude mice, were disaggregated and grown in Neurobasal-A serum free tissue cul-ture medium with B27 supplement, basic FGF, and EGF. Cell suspensions were treated with fluorochrome-labeled antibodies to CD111 and CD133, a transmembrane cell surface protein thought to be a marker for neural precursor cells and glioma stem cells in gliomas. CD133 expression levels were highest immediately after solid tumor disaggregation averaging 54% in GBM12, 25% in D456MG, and 50% in UAB1066. These proportions of positive cells dropped to 29% in GBM12, 18% in D456MG, and 35% in UAB1066 after being maintained for 7–14 days in tissue culture. In double-label analyses, CD111 expression varied from 82% in D456MG to 55% in GBM12 to 13% in UAB1066 cells. Importantly, both CD1331 and CD133– populations of glioma cells expressed very similar relative proportions of CD111 for all three xenografts at all time points studied. Cultured cells were next separated into CD1331 and CD133– fractions (.90% purity) via a magnetic bead separating kit (MACS, Miltenyi) and immediately tested for relative sensitivity to killing by wild-type HSV-1(F) using the in vitro ala-marBlue assay. Calculated PFU-LD50s (number of PFU/cell required to kill 50% of the cells in a 3-day period) were lowest for D456MG, the xenograft with highest nectin-1 expression (0.3 pfu/cell for CD133– cells; 1.2 pfu/cell for CD1331 cells). PFU-LD50 values for GBM12 were 11.5 pfu/cell for CD133– cells and 4.7 pfu/cell for CD1331 cells. PFU-LD50 values for UAB1066 cells were not attainable as the cells were relatively insensitive even at 100 pfu/cell. Similar results were obtained using genetically engineered herpes viruses G207 (currently in clinical trials) and M002 (IL-12 express-ing). These findings suggest that nectin-1 expression may predict sensitivity to herpes virus-mediated oncolytic therapy irrespective of the proportions of glioma progenitor cells comprising the tumor. Supported in part by Dixon Fellowship (GKF) and NCI grants CA71933 and CA097247.



SC-09. TUMOR STEM CELL ENRICHMENT CULTURE SELECTS IN FAVOR OF CHROMOSOME 10–INTACT CELLS FROM A FRESHLY DISSOCIATED GBMYi-Hong Zhou,1 Yuanjie Hu,1 Marlon Mathews,1 Chung-Ho Sun,2 Henry Hirschberg,2 Charles Limoli,3 and Mark Linskey1; 1Neurological Surgery, University of California, Irvine, Orange, CA, USA; 2Beckman Laser Institute and Medical Clinic, University of California, Irvine, Orange, CA, USA; 3Radiation Oncology, University of California, Irvine, Orange, CA, USA

GBM is characterized by histological heterogeneity, suggesting powerful selection pressures favoring genetic changes in critical target cells which may trigger GBM onset and drive persistence and progression. Recent reports characterize a small sub-population of radio-resistant cells in GBM that exhibit multiple “stem cell–like” properties. A population of tumor stem cells (TSCs) relatively refractory to standard treatments could complicate efforts to sterilize the tumor while promoting tumor recurrence in the treated tumor bed. Tumorigenesis may also be driven by multiple cellular interactions that define regional tumor microenvironments. The microenvi-ronmental niche likely promotes neovascularization that favors the delivery of many survival and growth factors. Our study is focused on differential genetic changes within TSCs versus stromal tumor cells (STCs) and the relative contribution/importance of each in the progression of GBM. Loss of heterozygosity (LOH) of chromosome 10 is the most common genomic alteration distinguishing GBM from other lower grades of gliomas. We thus hypothesize that loss of chromosome 10 occurs in the dominant GBM cell population (STCs), which provide a tumor microenvironment that favors the TSC survival and expansion. In contrast, persisting TSCs may maintain genomic stability, as genomically instability leads to transition from TSC to STC phenotype when it occurs. From single cells dissociated from freshly dissected GBM, we used non-adherent culture conditions favoring TSC growth to enrich the rare TSC population from the tumor mass containing both normal cells and STCs. Using normal adherent culture condition, STCs were enriched over cell passages. To analyze the TSCs and STCs enriched by different in vitro culture system, we used real-time quantitative reverse tran-scription (QRT-) PCR method to compare the expression of genes encoding stem cell markers (CD133 and ABCG2) and the makers for GBM microenvi-ronmental compartment (VEGF). We developed a novel real-time compara-tive quantitative (CQ-) PCR to quantify the degree of chromosome 10 loss. Three genes in chromosome 10 were subjected to quantification (PTEN at 10q23.3, ZNF22 at 10q11, and MBL2 at 10q11.2–q21) and normalized to 2 reference genes (SPAG16 at 2q34, ERC2 at 3p14). Measured against three housekeeping genes (ENO1, ACTB, and GAPDH), the results show that both PROM1 and ABCG2 expression were dramatically increased in multi-cellular spheroids (MCSs) following an initial increase of VEGF expression during TSC-enrichment culture. Interestingly, VEGF expression in MSCs dropped significantly by week 3 in culture and was nearly abolished by week 4. This later trend was also evident in the expression of both PROM1 and ABCG2, which decreased over the duration of cell culture, suggesting a dependence of TSC on VEGF for longer-term growth. In contrast, VEGF expression in the adherent cells, isolated from the same GBM, showed an increase over 5 cell passages. Quantification of chromosome 10 copy num-bers showed 50–70% loss of loci in chromosome 10 in adherent cultures over 4 passages, while decreasing level of chromosome 10 loss in MCSs subjected to TSC-enrichment culture, e.g., MCSs at third week of culture with peak level of PROM1 and ABCG2 has nearly balanced chromosome 10 copy number. Our data demonstrate that primary cultures of GBM can be manipulated to select for cells with varying anchorage dependence and growth factor requirements. Monolayer culture of primary GBM selects in favor of STCs with an increased expression of VEGF and loss of chro-mosome 10, while non-adherent culture conditions select in favor of TSCs which appeared not to express VEGF and maintain chromosome 10 integ-rity. Our data also suggest that survival of TSC appears to be dependent on the microenvironment provided by STCs. Collectively, these data support the idea that genetic heterogeneity within tumors is under selection pressure for different cell populations that interact to promote persistence and expan-sion of the tumor mass, and that may be a source of therapeutic resistance.

SC-10. ANTITUMOR ACTIVITY OF HISTONE DEACETYLASE INHIBITORS AGAINST GBM STEM-LIKE CELLSPeng Sun,1 Shuli Xia,1 Bachchu Lal,1 Sara M. Piccirillo,2 Angelo L. Vescovi,2 and Laterra John1; 1Department of Neurology, Kennedy Krieger Institute, Baltimore, MD, USA; 2Department of Biotechnology and Biosciences, University of Milan–Bicocca, Milan, Italy

Brain tumor stem-like cells possess a capacity to proliferate, self-renew, differentiate, and function as tumor-initiating cells in animal models. His-tone deacetylases (HDAC) modulate gene expression patterns via chromatin modifications in ways that may influence tumor-initiating mechanisms. We investigated the effects of the HDAC inhibitors trichostatin A (TSA) and MS-275 on gene expression, in vitro growth characteristics, and the tumor-initiating properties of three human GBM stem-like neurosphere forming cell lines. Both TSA (200 nM) and MS-275 (4 m) had antiproliferative effects

on the three cell lines examined. Histone H4 acetylation was increased ~350%, and expression of the putative brain cancer stem cell maker CD133 was decreased ~75% in response to HDAC inhibition. Neurosphere cell pro-liferation as determined by MTS assay and cell counting was inhibited by ~70% in all three cell lines. Both TSA and MS-275 reduced neurosphere size by ~75% and inhibited neurosphere clonogenicity by 70–80%. Pretreating neurosphere cultures with TSA in vitro for 72 hours inhibited their ability to form subcutaneous tumor xenografts by ~57%. When animals bearing pre-established neurosphere-derived xenografts (9 weeks post-implantation) were treated with TSA (500 g/kg/day i.p.), tumors completely regressed within 10 days of therapy. Thirty percent of tumors recurred within 4 weeks of discontinuing TSA. The number of primary neurospheres that could be cultured from these recurrent xenografts decreased by 50–60% compared to the controls, consistent with TSA-mediated depletion of stem-like neurosphere-forming cells. Microarray-based gene expression analysis was used to identify gene expression changes induced by TSA in 2 of the GBM neurosphere forming cell lines derived from a single patient. Thirty-six genes including VEGF changed significantly (3-fold change), and a sub-set of these are currently being confirmed by qRT-PCR and immunoblot analyses. For example, VEGF mRNA decreased 4.6-fold and VEGF protein decreased 2.3-fold in response to TSA. These results indicated that HDAC inhibitors have the potential to target GBM stem-like, tumor-initiating cells. Candidate, TSA-sensitive genes of mechanistic interest have been iden-tified and are currently being evaluated. This project is funded by Brain Tumor Founder’s Collaborative.

SC-11. ISOLATION OF BRAIN CANCER STEM CELLS USING SIGNALING PATHWAY REPORTERSLaurie Ailles,1 Samuel Cheshier,2 Tal Raveh,2 Dominique Higgins,2 Griffith Harsh,2 Michael Edwards,3 and Irving Weissman4; 1Stanford Institute for Stem Cell Biology, Palo Alto, CA, USA; 2Stanford University, Stanford, CA, USA; 3Department of Neurosurgery and Pediatrics, Lucile Packard Children’s Hospital, Stanford, CA, USA; 4Pathology, Stanford University, Stanford, CA, USA

A major challenge in neuro-oncology is the identification of brain tumor–initiating cells. Within each tumor, only a subset of the cells, the “cancer stem cells,” is capable of re-creating the disease. This study is designed to improve the definition and functional characterization of brain tumor stem cells (BTSC). In various cancers, stem cells have been distin-guished by specific cell surface antigens. In some brain tumors, the presence of the normal neural stem cell–associated antigen, CD133, could predict the tumorigenic properties of the cells. However, experiments in our lab have shown that CD133 may not be a universal marker for brain tumor stem cells, and alternative experimental strategies are required. Our work-ing hypothesis is that the activity level of signaling pathways may also be a distinctive hallmark of BTSC. Our methodology involves the fractionation of tumor cells based on biochemical activities associated with self-renewal and thus provides a functional method that complements the phenotypic method used for cancer stem cell isolation. Our laboratory has developed a paradigm for the identification of cancer stem cells in human medulloblas-toma (MB) and glioblastoma multiforme (GBM): First, a xenograft model for these human brain tumors was established using newborn immunocom-promised mice. Injection of fresh, non-fractionated human MB or GBM cells into the cerebellum or lateral ventricles, respectively, has resulted in successful recapitulation of the original tumors. This xenograft model pro-vides the platform by which the tumorigenicity of select tumor cell popula-tions is assessed. Fractionation of fresh tumor cells from MB and GBM is based on the activity of two signaling pathways associated with self-re-newal: the Wnt and Hedgehog (Hh) pathways. The important roles of these pathways in normal progenitor self-renewal, and mutations found in CNS malignancies, suggest that their deregulation may contribute to MB and GBM tumorigenesis. To assess the state of intracellular Hh or Wnt pathway stimulation, specific intracellular reporters were generated, in which GFP reporter gene expression is dependent on pathway-activated transcription. Conserved elements responsive to either Gli or beta-catenin were placed upstream of a GFP gene to report the activity level of the Hh or Wnt path-ways, respectively. These cassettes were inserted into Lentiviral vectors, with which we have achieved high efficiency of transduction into primary human tumor cells. These specific intracellular reporters for the Wnt and Hh pathways were used to isolate primary human medulloblastoma cell populations based on the level of activity. The relevance of these signaling pathways to tumor initiation was then tested in in vitro clonogenic assays as well as in vivo tumor transplantation experiments. Future experiments will assess the direct effect of specific pathway inhibitors on the growth of brain tumor stem cells. The isolation of an enriched population of BTSC based on signaling pathways will advance our understanding of brain tumors. The purification of BTSC will be followed by the characterization of mutations, chromosomal aberrations, active signaling pathways, and unique surface antigens. This knowledge will guide the development of highly specific and more effective anti–brain cancer therapies.



SC-12. NOTCH PATHWAY BLOCKADE INHIBITS GLIOBLASTOMA GROWTH BY DEPLETING CD133-POSITIVE CANCER STEM CELLSXing Fan,1 Leila Khaki,1 Cheryl Koh,1 Jiangyang Zhang,2 Yue-Ming Li,3 Francesco DiMeco,4 Angelo Vescovi,5 and Charles Eberhart1; 1Department of Pathology, Johns Hopkins University, Baltimore, MD, USA; 2Department of Radiology, Johns Hopkins University, Baltimore, MD, USA; 3Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA; 4Department of Neurosurgery, National Neurological Institute “C. Besta,” Milan, Italy; 5Department of Biotechnology and Biosciences, University of Milan–Bicocca, Milan, Italy

Cancer stem cells (CSCs) are thought to be critical for propagating many tumors, including glioblastoma (GBM), but are spared by traditional chemo- and radiation therapies. Finding new treatments that can target CSCs may therefore be critical for improving the survival of GBM patients. It has been shown that the Notch signaling pathway regulates normal stem cells in the central neural system (CNS) and that GBMs contain CSCs with higher Notch activity (Cancer Research 2004;64:7011–7021; Cancer Cell 2006;9:391–403). However, it is not known if Notch pathway blockade can target CSCs in GBM. We have previously demonstrated that Notch pathway inhibition depletes CSCs and blocks engraftment in medulloblas-tomas (Cancer Research 2006;66:7445–7452). In the present study, we used GBM-derived neurospheres to examine the requirement for the Notch pathway in malignant glioma CSC. We found that Notch pathway blockade by 2 mM Gamma-Secretase Inhibitor GSI-18 reduces expression of CD133 and inhibits tumor growth in vitro. Furthermore, over-expression of an active form of Notch2 (NICD2) increases tumor growth in vitro. When equal numbers of viable GSI-18 or vehicle (DMSO) treated GBM neuro-sphere cells were injected subcutaneously into nude mice, DMSO treated cells always formed tumors, whereas GSI-18 treated cells did not. Moreover, mice injected intracranially with GSI-18 treated GBM neurosphere cultures have significantly longer survival compare with mice injected with DSMO treated cells. Most importantly, in vivo delivery of GSI-18 by implantation of drug-impregnated polymer beads also effectively blocks tumor growth and significantly prolongs survival. While investigating the mechanism of CSC loss, we found that Notch pathway inhibition appears to deplete CSCs through reduced proliferation and Akt-mediated apoptosis. In summary, we demonstrate that Notch pathway blockade depletes stem-like cells in GBMs, suggesting that gamma-secretase inhibitors can be used as chemotherapeutic reagents to target CSCs in malignant gliomas.

SC-13. PROSPECTIVE IDENTIFICATION OF A CD133-NEGATIVE, PUTATIVE CANCER STEM CELL POPULATION IN ADULT HUMAN GLIOBLASTOMASSara G.M. Piccirillo,1 Annunziato Mangiola,2 Laura Cajola,3 Giulio Maira,4 Giuseppe Lamorte,3 Giovanni Broggi,5 Francesco DiMeco,5 and Angelo L. Vescovi1; 1Department of Biotechnology and Biosciences, University of Milan–Bicocca, Milan, Italy; 2Department of Neurosurgery, Catholic University, Rome, Italy; 3Stemgen s.p.a., Milan, Italy; 4Catholic University, Rome, Italy; 5National Neurological Institute “C. Besta,” Milan, Italy

The expression of CD133—a 120 kDa 5-transmembrane cell surface protein found in hematopoietic and neural stem cells—has recently been shown to identify a small population of stem-like, cancer-initiating cells in medulloblastomas, glioblastomas (GBMs), and ependymomas. Notably, it has been proposed that the CD133– cells from these tumors do not bear cancer stem cell features in vitro and cancer-initiating ability in vivo. Here we report that in 2 cases out of 6 samples a CD133– population can be iso-lated from acutely dissociated and cultured, adult human GBM cells that, similar to its CD1331 counterpart from the same specimens, is endowed with clonogenic capacity, multipotentiality, aberrant proliferation, and dif-ferentiation, as well as extensive self-renewal in culture. This might suggest that in rare cases adult human GBMs contain putative cancer stem-like cells that do not express CD133. In order to determine if this is the case, we set out to assess if the clonogenic, self-renewing CD133– cells also bear cancer-initiating ability. We have now transplanted from 10,000 to 300,000 CD133– cells into the lateral striatum of immunodeficient SCID mice and are comparing their cancer-initiating capacity to that of the CD1331 popu-lation from the same specimens.

SC-14. CYCLOPAMINE-MEDIATED HEDGEHOG PATHWAY INHIBITION DEPLETES STEM-LIKE CANCER CELLS IN GLIOBLASTOMAEli E. Bar,1 Aneeka Chaudhry,1 Alex Lin,1 Karisa Schreck,2 William Matsui,3 Xing Fan,4 Angelo L. Vescovi,5 Francesco DiMeco,6 Alessandro Olivi,4 and Charles G. Eberhart4; 1Pathology, Johns Hopkins University, Baltimore, MD, USA; 2Neurosurgery, Johns Hopkins University, Baltimore, MD, USA; 3Oncology, Johns Hopkins University, Baltimore, MD, USA; 4Johns Hopkins University, Baltimore, MD, USA; 5Department of Biotechnology and Biosciences, University of Milan–Bicocca, Milan, Italy; 6National Neurological Institute “C. Besta,” Milan, Italy

Brain tumors can arise following deregulation of signaling pathways normally activated during brain development and may derive from neural stem cells. Given the requirement for Hedgehog (Hh) in non-neoplastic stem cells, we investigated if Hh blockade could target the stem-like population in glioblastoma (GBM). We found that Gli1, a key Hh pathway target, was highly expressed in 5 of 19 primary GBMs and in 4 of 7 GBM cell lines. Shh ligand was expressed in some primary tumors and in GBM-derived neurospheres, suggesting a potential mechanism for pathway activation. Hh pathway blockade by cyclopamine caused a 40–60% reduction in growth of glioma lines highly expressing Gli1, but not in those lacking evidence of pathway activity. When GBM-derived neuropheres were treated with cyclo-pamine, then dissociated and seeded in media lacking the inhibitor, no new neurospheres formed, suggesting that the clonogenic cancer stem cells had been depleted. Serum-induced differentiation of GBM neurospheres also resulted in a reduction in markers of stem/progenitor cells and Hedgehog activity. Consistent with our hypothesis that Hh is required in less-differen-tiated cancer cells, the stem-like fraction in gliomas marked by both alde-hyde dehydrogenase activity and Hoechst dye excretion (side population) was significantly reduced or eliminated by cyclopamine. In contrast, we found that radiation treatment alone of our GBM neurospheres resulted in increased percentages of these stem-like cells and rapid recurrence of the tumors, indicating that this standard therapy may target only better-differentiated neoplastic cells. Most importantly, viable GBM cells injected intracranially following Hh blockade were no longer able to form tumors in athymic mice, indicating that a cancer stem cell population critical for ongoing growth had been removed.

SC-15. MIR-124A AND MIR-137 INDUCE DIFFERENTIATION OF POSTNATAL NEURAL PROGENITOR CELLS AND BRAIN TUMOR STEM CELLSJoachim Silber,1 Daniel Lim,1 Claudia Petrisch,1 Anders Persson,2 Alika Maunekea,1 Scott VandenBerg,3 David James,1 Joseph F. Costello,1 William Weiss,2 Gabriele Bergers,1 Arturo Alvarez-Bullya,1 and Graeme Hodgson1; 1Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA; 2Neurology and Pediatrics, University of California, San Francisco, San Francisco, CA, USA; 3Pathology, University of California, San Francisco, San Francisco, CA, USA

MicroRNAs (miRs) are emerging as important regulators of develop-ment, stem cell division, and cancer. However, the detailed mechanisms by which miRs are regulated in tumor cells and the mechanisms by which miRs regulate tumorigenesis remain largely unknown. To assess the role of microRNAs in differentiation of normal adult neural stem cells (NSCs) and brain tumor stem cells (TSCs), we investigated miR expression and function in adult human astrocytomas, NSCs, and TSCs. Quantitative RT-PCR analyses of miR expression in non-neoplastic brain tissues (glio-ses), anaplastic astrocytomas, and glioblastoma multiforme (GBM) revealed 6 miRs, miR-7, -124a, -129, -137, -139, and -218, that were significantly down-regulated in both primary tumor classes compared to the glioses (p , 0.01). Of these 6 miRs, only miR-124a and miR-137 showed increased expression in differentiating adult mouse NSCs following withdrawal of EGF, FGF, and FCS from the culture medium. Transfection of either miR-124a or miR-137 oligonucleotides induced morphological changes consis-tent with neuronal differentiation and significantly increased staining of the neuronal marker Tuj-1 in NSCs, and in TSCs derived from S100â-v-erbB mouse oligodendrogliomas. Transfection of miR-124a or miR-137 also induced morphological changes consistent with increased differentiation in magnetic bead-sorted fractions of both CD1331 and CD133– human primary GBM cells. We also determined that GBM lines U87 and U251 treated with the DNA demethylating agent 5–aza-dC increased miR-137 expression up to 12-fold, suggesting that its transcription is epigenetically suppressed in GBMs. Collectively, our results suggest that suppression of miR-124a/137 expression by growth factor signaling and/or DNA methyla-tion promotes gliomagenesis by impairing neuronal differentiation of adult NSCs, and suggest that miR-124a/137 agonists may be useful agents for treatment of GBMs and oligodendrogliomas.



SC-16. INHIBITORS OF STAT3 PREVENT PROLIFERATION OF GLIOBLASTOMA STEM CELLSMaureen Sherry,1 Andrew Reeves,1 Maciej Kotecki,1 Julian Wu,2 and Brent Cochran1; 1Department of Physiology, Tufts University, Boston, MA, USA; 2Department of Neurosurgery, Tufts–New England Medical Center, Boston, MA, USA

Signal transducer and activator of transcription–3 (STAT3) is an impor-tant mediator of growth factor and cytokine transcriptional responses. It has been shown to be a critical mediator of growth and survival of a number of human tumor cell lines. We have previously found that knockdown of STAT3 by RNAi in several serum-derived glioblastoma cell lines induces apoptosis. We have also found that STAT3 regulates the expression of the catalytic subunit of telomerase (hTERT) in glioblastoma cells. In murine embryonic stem cells as well as in neural stem cells, STAT3 is necessary for the maintenance of pluripotency. Therefore we have examined the role of STAT3 in putative tumor stem cells isolated from human glioblastomas. These cells form neurospheres when cultured in serum-free medium in the presence of EGF and FGF and can form tumors in mouse brains from the injection of as few as 1,000 cells. In culture, these cells express STAT3 that is phosphorylated on both serine and tyrosine residues, indicating that it is both present and active. STA-21 is a small molecule inhibitor of STAT3 that functions by binding to the STAT3 SH2 domain. STA-21 strongly inhibits the formation of neurospheres by glioblastoma stem cells and inhibits pro-liferation of these cells as judged by BrdU incorporation. However, these cells did not undergo apoptosis as judged by annexin V staining. Treat-ment with STA-21 strongly inhibits the expression of the STAT3 target gene c-myc in these same cells. Preliminary results suggest that dominant negative STAT3 and STAT3 RNAi have a similar effect on the proliferation of the glioblastoma stem cells. Moreover, inhibitors of the STAT3 upstream kinase Jak2 also inhibit the proliferation of the putative tumor stem cells. These data suggest that inhibitors of STAT3 could be effective in tumor stem cell–directed therapy of glioblastoma. This work was supported by a grant from the Brain Tumor Society.

SC-17. STUDYING PREMALIGNANT STEM CELLS IN OLIGODENDROGLIOMAClaudia Petritsch,1 Markus Waldhuber,2 Anders Persson,3 William Weiss,4 and Gabriele Bergers4; 1Neurological Surgery, Brain Tumor Research Center and Comprehensive Cancer Research Center, University of California, San Francisco, San Francisco, CA, USA; 2Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA; 3Neurological Surgery and Neurology, University of California, San Francisco, San Francisco, CA, USA; 4University of California, San Francisco, San Francisco, CA, USA

The “cancer stem cells hypothesis” claims that in pathological adult stem cells, the cancer stem cells give rise to a heterogeneous tumor and maintain it by aberrant differentiation and proliferation (Vescovi et al., 2006). It has not been determined yet how cancer stem cells are generated. We utilized transgenic mice heterozygous for p53 and with activated EGFR signaling in the postnatal stem cell lineage by expressing the v-erb oncogene via the S100b-promotor (Weiss et al., 2003). High-grade oligodendroglioma develop in these S100b-verbB, p53 6mice at a significant latency, provid-ing a unique opportunity to study the potential early changes in the stem cell population inflicted by ectopic EGFR signaling. We discovered for the first time that adult neural stem cells residing in the subventricular zone in transgenic mice already showed differentiation and self-renewal abnormali-ties before the actual tumor occurrence due to ectopic EGFR activation; we therefore referred to them as “premalignant stem cells.” The identification of premalignant stem cells is unique and prompted us to investigate further how they are being generated. We propose the hypothesis that initiating mutations such as EGFR amplification in normal stem cells generate prema-lignant stem cells by disrupting either their asymmetrical cell division, their proliferation rate, or their survival abilities. Studies to identify the mecha-nism for generation of premalignant stem cells and markers for their identifi-cation will be presented. The discovery of premalignant changes in the adult neural stem cells is of high relevance for human patients, because they set a foundation to identify markers of premalignant stem cells; these markers may allow identification of early or residual lesions in human brain tumor patients even before macroscopic tumor occurrence. References: Vescovi, AL, Galli, R, and Reynolds, BA. Brain tumor stem cells. Nat Rev Cancer 2006;6:425–436. Weiss, WA et al. Genetic determinants of malignancy in a mouse model for oligodendroglioma. Cancer Res 2003;63:1589–1595.

SC-18. REGULATION OF GLIOBLASTOMA STEM CELL SELF-RENEWAL BY STAT3Sonya Popoff,1 Kenneth Aldape,2 Shuzhen Wang,1 Charles Conrad,1 Frederick Lang,3 Ke Sai,1 Alfred Yung,1 Waldemar Priebe,4 and Howard Colman1; 1Neuro-Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Neuropathology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3Neurosurgery, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 4Experimental Therapeutics, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

The majority of glioblastomas (GBM) are resistant to standard ther-apies. Growing evidence indicates that tumor stem cells, defined in part by the property of self-renewal and expression of the cell-surface marker CD133, may underlie tumor initiation and treatment resistance in GBM. In order to identify mechanisms regulating GBM stem cell maintenance and tumorigenicity, we have isolated and expanded 12 GBM stem cell lines from human tumors that display the ability to self-renew in vitro (months to years) when maintained as floating neurosphere cultures. These stem cell lines also initiate tumors with histopathology closely resembling the parent tumor after intracranial injection into the brain of immune deficient mice. STAT3 is a pleiotropic signaling molecule and transcription factor activated in response to a large number of cytokines, growth factors, and hormones. Immunohistochemical analysis demonstrates that STAT3 is activated (phos-phorylated) in the majority of high-grade gliomas. STAT3 also is critical in development (STAT3-deficient mice die during early embryogenesis) and plays an important role in maintaining self-renewal of a number of stem cell types. We thus set out to determine the extent to which STAT3 regulates glioblastoma stem cell maintenance and self-renewal. Western blot analy-sis demonstrated high levels of phosphorylated STAT3 at both the Ser727 and the Tyr705 sites in a high percentage (10/12, 83%) of the GBM stem cell lines examined. STAT3 activation was reduced using two specific small molecule inhibitors of STAT3 (WP1066 and WP1193) as well as transient STAT3 shRNA transfection. Inhibition of STAT3 activation by WP1066 and WP1193 resulted in a dose-dependent decrease in levels of both phos-pho-STAT3 (Ser727 and Tyr705 sites) and total STAT3. Treatment with WP1066 and WP1193 (0–10 mM, 4–10 days) also demonstrated a dramatic dose-dependent inhibition of the ability of GBM stem cells to reform neu-rospheres from single cell suspensions in the presence of serum-free media supplemented with EGF and FGF. These effects on neurosphere formation by WP1066 and WP1193 were mimicked by partial knockdown of STAT3 RNA using transient shRNA transfection, suggesting a specific role for STAT3 in this process. Because neurosphere formation has been used as a functional assay for neural stem cell self-renewal, these findings suggested that inhibition of STAT3 activation reduces GBM stem cell self-renewal. To determine the effects of STAT3 inhibition by WP1066 and WP1193 on puta-tive stem cell markers in GBM cells, we performed PCR for selected genes relative to GAPDH in treated cells versus control cultures. We observed a dose-dependent decrease in expression of multiple stem cell markers includ-ing CD133, Bmi-1, and Notch 1 after treatment with STAT3 inhibitors for as little as 4 days. In the case of CD133, quantitative RT-PCR demonstrated the average decrease in expression was 9-fold for 5 mM and more than 100-fold for 10 mM treatment at 5 days. Together, these results indicate a specific role for STAT3 activation in the maintenance and self-renewal of GBM stem cells and suggest a potential central role for STAT3 in the regulation of GBM tumorigenicity and treatment resistance.

SC-19. UMBILICAL CORD BLOOD STEM CELLS SHOW TROPISM TO GLIOMA CELLS IN VITRO AND IN VIVO AND INDUCE APOPTOSIS BY FASL ACTIVATION IN SNB19 AND U87 HUMAN GLIOMA CELLS OR RELEASE CYTOCHROME C IN 5310 HUMAN GLIOMA EGFR–OVER-EXPRESSING XENOGRAFT CELLSChristopher Gondi,1 Venkata Ramesh Dasari,2 Peggy Mankin,3 Neelima Kandhukuri,4 Krishna Veeravalli,4 Kay Saving,3 Dzung Dinh,5 Meena Gujrati,6 and Jasti Rao2; 1University of Illinois at Peoria, Peoria, IL, USA; 2Cancer Biology and Pharmacology, University of Illinois College of Medicine–Peoria, Peoria, IL, USA; 3University of Illinois College of Medicine–Peoria, Peoria, IL, USA; 4IL, USA; 5Peoria, IL, USA; 6Pathology, University of Illinois College of Medicine–Peoria, Peoria, IL, USA

The use of stem cells for the treatment of various diseases is gaining considerable popularity. Here we studied stem cells isolated from umbilical cord blood and their interaction with glioma cells. We have previously dem-onstrated the multipotent nature of stem cells derived from human umbili-cal cord blood. In this study, we show the utility of human umbilical cord blood stem cells in glioma therapy. From our matrigel invasion and spher-oid invasion assays, we demonstrate that human umbilical cord blood stem cells show tropism toward glioma cells. Human umbilical cord blood stem cells failed to invade human astrocyte spheroids, indicating their specificity toward glioma cells. To further evaluate the migration of human umbilical cord blood stem cells to glioma cells in vivo, nude mice were implanted



intracranially with GFP-expressing human glioma cells SNB19 in one hemi-sphere and human umbilical cord blood stem cells labeled with cell tracker red dye in the opposite hemisphere 7 days after glioma cell implantation. It was observed that, 3 days after stem cell implantation, migration of stem cells toward glioma cells had been initiated, and migration was complete in 5 days, indicating that stem cell migration was toward glioma cells in vivo. In contrast, controls did not show any in vivo migration. To determine whether receptor ligand interaction is involved in stem cell migration, we assessed the levels of VEGF, VEGFR, and PDGF in glioma cells alone or in stem cell:glioma cell co-cultures. We observed that VEGF and VEGFR were over-expressed in co-cultures as determined by western blot analysis and immunohistochemistry. We also observed PDGF accumulation at the con-tact points between stem cells and glioma cell contact regions. To determine the mode of apoptosis induction, we used glioma cells singly or in co-cul-tures and determined the levels of cleaved FasL, caspase 8, and cytochrome c release into the surrounding media by western blot analysis of conditioned media. We observed that, in stem cell co-cultures with glioma cells, cleavage of FasL was enhanced except in the case of 5310 human glioma xenograft cells. Cleavage of caspase 8 was also enhanced except in the case of 5310 human glioma xenograft cells. Human glioma xenograft cells co-cultured with human umbilical cord blood stem cells showed release of cytochrome c in the conditioned media, indicating an alternate method of apoptosis induction. This study shows that stem cells derived from umbilical cord blood induce apoptosis in human glioma cells by multiple pathways and show promise as potential therapy for treatment of gliomas.

SC-20. BONE MARROW–DERIVED MESENCHYMAL STEM CELLS ARE RECRUITED TO AND ALTER THE GROWTH OF HUMAN GLIOMASFrederick Lang,1 Toshiyuki Amano,1 Nobuhiro Hata,1 Joy Gumin,1 Kenneth Aldape,2 and Howard Colman3; 1Department of Neurosurgery, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Department of Pathology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3Department of Neuro-Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Bone marrow–derived human mesenchymal stem cells (BM-hMSCs) are stem/progenitor cells that give rise to bone marrow stromal elements (e.g., fat, bone cartilage, pericytes) and provide the microenvironment for marrow homeostasis. BM-hMSCs can be isolated and cultured as adher-ent, spindle cells and are CD34–, CD38–, CD45–, CD133–, CD1051, CD441, CD1661, CD291. We have shown that BM-hMSCs localize to human gliomas after intravascular delivery (Nakamizo et al., Cancer Res 2005;65:3307–3318), raising the possibility that circulating BM-hMSCs may be recruited into gliomas during tumorigenesis. Thus we tested the hypothesis that BM-hMSC–like cells can be isolated from human brain tumors and can alter the growth of human gliomas. To determine whether hMSC-like cells can be isolated from gliomas, surgical specimens from 41 patients with malignant gliomas were cultured in “BM-hMSC media.” In 29 cases (71%) adherent spindle cells morphologically identical to BM-hMSCs were isolated and serially passaged. Flow cytometric analyses indicated that the surface antigen profile of these tumor-derived cells was consistent with BM-hMSCs (i.e., CD34–, CD38–, CD45–, CD1051, CD441, CD1661, CD291). In 12 cases the percentage of CD1051 cells in the culture was low (1.1–5.6%), whereas in 17 cases the percentage was high (32.5–89.7%). All cultures were CD133–, indicating that they were not neural-like stem cells. Importantly, single clones of the tumor-derived BM-hMSC–like cells were capable of differentiating into all three mesenchymal elements (osteocytes, chondrocytes, adipocytes), indicating that these cells had differentiation potentials similar to BM-hMSCs. In order to demonstrate that these tumor-derived BM-hMSC–like cells were potentially recruited from the circula-tion, we analyzed the percentage of CD1051 cells in the blood of 19 cases. Whereas the median percentage of CD1051 cells in normal volunteers (n 5 5) was 19.6% (range 11.1–23.0), the percentage of circulating CD1051 cells in patients with gliomas was significantly higher (median 34.9%, range 17.2–69.8%; p , 0.05), suggesting that tumor-derived BM-hMSCs–like cells may be recruited from the bloodstream. Interestingly, when tumor-derived BM-hMSC–like cells (106) from each case were injected into the frontal lobes of SCID mice, 98% of samples did not form tumors after 6 months’ observation, indicating that the cells were not tumorigenic. How-ever, BM-hMSCs appeared to alter the growth of gliomas cells. Specifically, BM-hMSCs, fibroblasts, or normal human astrocytes (NHA) were mixed with U87 glioma cells (1:5 ratio of hMSC: U87) and implanted into the fron-tal lobes of SCID mice. Whereas the median survival of mice with mixtures of NHA/U87 or fibroblasts/U87 was no different from unmixed controls (U87 alone), the survival of mice with hMSC/U87 was significantly shorter (55 days vs. 65 days for control; p , 0.05). We conclude that cells with fea-tures similar to BM-hMSCs can be isolated from human gliomas and that these cells may be recruited from the circulation. Although not themselves tumorigenic, the tumor-derived BM-hMSCs appear to be capable of alter-ing the growth of gliomas. Thus recruited BM-hMSCs may be important elements of the glioma microenvironment and may contribute to the growth of gliomas and to the resistance of gliomas to many therapies.

SC-21. GLYCOGENE PROFILING OF GLIOMA STEM CELLSRoger Kroes,1 Charles Conrad,2 Frederick Lang,2 Howard Colman,2 Waldemar Priebe,2 Mark Emmett,3 Carol Nilsson,4 and Joseph Moskal1; 1Northwestern University, Evanston, IL, USA; 2University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL, USA

The aberrant cell surface glycosylation patterns present on virtually all tumors have been linked to altered cellular morphology, oncogenic transfor-mation, tumor progression, metastasis, and invasivity. We have previously characterized altered cell surface glycosylation patterns present on glioblas-tomas using an oligonucleotide microarray platform representing all of the cloned human glycogenes (i.e., glycosidases, glycosyltransferases, polysac-charide lyases, carbohydrate esterases, and carbohydrate-binding proteins). In the present study, we compared glycogene expression profiles of cultured NS11 cells with NS11 cells treated for 24 hours with either 10% FBS or 5 mM WP1193. Of the 359 glycogenes evaluated, 33 genes exhibited statistically significant expression differences between the FBS-treated and control cul-tures, and 22 genes exhibited statistically significant differences between the WP1193-treated and control cultures. Significant alterations in glycolipid met-abolic and catabolic pathways were observed following treatment with either agent. Specifically, altered expression of ST6–(alpha N-acetyl-neuraminyl- 2,3–beta -galactosyl-1,3)-N-acetylgalactosaminide alpha 2,6–sialyltrans-ferase 6 (ST6GALNAC6), phosphatidylinositol glycan (PIGA), and hexo-saminidase A (HEXA) following FBS treatment and beta 1–galactosidase (GLB1), and sialidase 1 (NEU1) following WP1193 treatment were identified. Among the unique changes in glycogene expression observed following serum-induced differentiation were increases in transcripts related to proteoglycan deposition and ECM remodeling, including beta 1,3–glucuronyltransferase 1 (B3GAT1), beta 1–galactosidase (GLB1), and UDP-glucose ceramide gluco-syltransferase-like 2 (UGCGL2). Altered glucose ceramide glucosyltransferase expression has been previously observed in multidrug and radiation resistant malignancies. Conversely, WP1193-mediated inhibition of STAT3 had little effect on matrix-related genes, and the predominant effect was increased expression of genes responsible for protein N- and O-linked glycosylation; genes were not altered following serum stimulation. Among the key genes identified were UDP-N-acetyl-alpha -D-galactosamine:polypeptide N-acetyl-galactosaminyltransferase 7 (GALNAC-T7), core 1 synthase, glycoprotein-N-acetylgalactosamine 3–beta -galactosyltransferase 1 (C1GALT1), protein-O-mannosyltransferase 2 (POMT2), mannosyl (alpha 1,6–)-glycoprotein beta 1,2–N-acetylglucosaminyltransferase (MGAT2), mannosyl (alpha 1,3–)-gly-coprotein beta 1,4–N-acetylglucosaminyltransferase, isozyme b (MGAT4B), dolichyl-phosphate mannosyltransferase polypeptide 1 (DPM1), UDP-Gl-cNAc: beta gal beta 1,3–N-acetylglucosaminyltransferase 1 (B3GNT1), and UDP glucuronosyltransferase A1 (UGT1A1). Overall, these results demon-strate that differentiation of glioma stem cells results in significant alterations in discrete biochemical pathways that modulate the expression of cell surface glycoconjugates. Moreover, these changes provide the foundation for studies that will define the molecular basis for the utilization of alternative differen-tiation pathways during glioma formation.

SC-22. OPTIMIZATION OF GROWTH CONDITIONS FOR NORMAL NEURAL AND TUMOR-DERIVED PROGENITOR CELLSPaul Clark,1 Haviryaji Kalluri,1 Daniel Treisman,1 Frank Hospod,1 Gordana Raca,2 and John S. Kuo1; 1Neurological Surgery, University of Wisconsin–Madison, Madison, WI, USA; 2University of Wisconsin–Madison, Madison, WI, USA

Recent evidence from many tumor types reveals that a small fraction of tumor cells possess “stem-like” properties (self-renewal, multipotency), are likely critical for tumor initiation and propagation, and maintain geno-typic similarity to the parental tumor over long-term cell culture. Therefore tumor progenitor cells (TPC) rather than traditional tumor cell lines are hypothesized as a more clinically relevant model for testing new therapies. This study aimed to optimize growth conditions for TPC isolation and expansion from glioblastoma multiforme (GBM). We compared GBM TPC and neural progenitor cell (NPC) proliferation as free-floating cell aggre-gates (neurospheres) in various media formulations and mitogenic growth factor concentrations (epidermal growth factor, EGF, and basic fibroblast growth factor [bFGF]). NPC proliferation significantly increased in one medium as compared to other formulations: rat NPC in NBE medium (neu-robasal medium, 0.5 3 N2 supplement, 0.5 3 B27) supplemented with 50 ng/ml EGF and bFGF, and human NPC in “passaging medium” (PM: 70% DMEM/30% F12, 1 3 B27) with either 20 or 50 ng/ml EGF and bFGF. TPC proliferated similarly in all media formulations and growth factor concen-trations, with PM and 50 ng/ml EGF and bFGF only slightly (10%) better than other media formulations. Additionally, we showed that NPC required at least one mitogenic factor (EGF or bFGF) for neurosphere formation, whereas TPC formed neurospheres even in the absence of EGF and bFGF. TPC proliferation increased in the presence of mitogenic factors at 20 ng/ml: 20% for EGF and 30% for bFGF in MTS metabolic assay. No synergistic effects were observed: adding both EGF and bFGF was equivalent to bFGF



alone. Therefore TPC growth conditions were observed to be less stringent than required by NPC. These optimized conditions are now being applied in establishing additional GBM TPC lines for molecular and cellular analyses and for use in identifying and developing novel therapeutic approaches to the treatment of brain cancer.

SC-23. PERSISTENCE OF CD1331 CELLS IN HUMAN AND MOUSE GLIOMA CELL LINES: DETAILED CHARACTERIZATION OF GL261 GLIOMA CELLS WITH CANCER STEM CELL–LIKE PROPERTIESAnhua Wu,1 Walter Low,1 Walter Hall,2 and John Ohlfest1; 1University of Minnesota, Minneapolis, MN, USA; 2Upstate Medical University, Syracuse, NY, USA

The concept of cancer stem cells suggests that there are malignant stem-like cells within a tumor that are responsible for tumor renewal and resistance to cytotoxic therapies. A CD1331 stem cell–like subpopulation has been isolated from malignant human glioma that is enriched for tumor-initiating cells. However, preclinical trials that target human glioma stem cells can be conducted only in immunodeficient xenograft models, which ignore adaptive immunity. Identification of the tumor-initiating cell in murine glioma would enable cancer stem cell–targeted therapy to be tested in immune competent animals preclinically. Studies have identified tumor stem-like cells that extrude Hoechst 33342 dye, representing a double nega-tive “side population” (SP) thought to be selectively resistant to drug ther-apy. The glioma-initiating population has not been clearly identified in any species other than humans, but an SP containing stem-like cells has been reported in murine glioma cells lines. We investigated whether CD1331 and SP cells existed in the GL261 cell line, a syngeneic C57BL/6 mouse glioma model. Intracerebral injection of fewer than 50 CD1331 GL261 cells formed tumors, whereas up to 1,000 CD133– cells did not. CD1331 GL261 cells expressed stem cell–associated genes such as nestin, formed tumor spheres, and differentiated into glial and neuronal-like cells. Similar to GL261, all six human glioma cell lines analyzed also contained a rare (,0.5%) CD1331 population. Surprisingly, we found that CD1331 GL261 cells did not reside in the SP, nor did the majority (~94%) of CD1331 human glioma cells. These results demonstrate that CD133 is a marker for glioma-initiating cells that is conserved from mice to humans and identify non-SP CD1331 GL261 cells as a rational target for preclinical immunotherapy. The lack of overlap in the expression of CD 133 and SP staining also sug-gests that subpopulations of glioma stem-like cells exist in cell lines. Further study will be required to determine the differences (or similarities) between CD1331 cells and cells within the SP.

SC-24. MOLECULAR, CELLULAR, AND PATHOLOGICAL PROPERTIES OF GENETICALLY MODIFIED SYNTHETIC ASTROCYTES DIFFERENTIATED FROM EMBRYONIC STEM (ES) CELLS IN VITRODeepak Kamnasaran,1 Cynthia Hawkins,1 and Abhijit Guha2; 1Arthur and Sonia LaBatts Brain Tumor Centre, Hospital for Sick Children, Toronto, Ontario, Canada; 2Neurosurgery and Cell Biology, Toronto Western Hospital and Hospital for Sick Children Research Institute, Toronto, Ontario, Canada

ES cells have the ability to differentiate into derivatives of all three germ layers. We obtained “synthetic astrocytes,” differentiated from ES cells under artificial culture conditions, toward an alternative source for high-throughput modeling of gliomas using known candidate astrocytoma genes. ES cells harboring wild-type (wt) and p53 (6) genetic backgrounds were dif-ferentiated to produce “synthetic astrocytes” with high efficiency (.90%). These cells expressed several structural and biochemical markers of somatic astrocytes and were most similar to primitive/early progenitor astrocytes of E13.5–cortex and P4–hippocampus, with the expression of stem cell mark-ers (CD133, NESTIN, BMI, MUSHASHI1). These cells do not demonstrate any unusual apoptotic index; however, proliferation was enhanced. While the wild-type and p53 6ES cells transformed in soft agarose and developed intracranial teratomas in Nod-SCID mice (100%—3 weeks), the synthetic astrocytes of either genetic background overall did not. Genetic modifica-tions of the wild-type plus p53 6synthetic astrocytes with over-expression of candidate MDM2, myr-AKT, and V12H-RAS brain tumor oncogenes induced high-grade intracranial tumors within a 3–4 month period in Nod-SCID mice. Over-expression of the same cocktail of oncogenes in terminally differentiated somatic astrocytes of either a wild-type or p53 6host genetic backgrounds did not induce any intracranial tumors. We have developed an effective high-throughput assay for the analysis of the in vitro and in vivo transformation potentials of candidate genes for astrocytomas, using syn-thetic astrocytes. Our findings demonstrate that these synthetic astrocytes are very similar to primitive/early differentiated somatic astrocytes and when genetically modified can potentiate the development of intracranial tumor lesions, unlike terminally differentiated astrocytes.

SC-25. IDENTIFICATION OF A2B51/CD133– TUMOR-INITIATING CELLS IN ADULT HUMAN GLIOMASPeter Canoll,1 Alfred Ogden,2 Allen Waziri,3 Kim Lopez,3 David Fusco,4 Marcela Assanah,3 and Jeffrey Bruce4; 1Pathology, Columbia University, New York, NY, USA; 2Neurological Surgery, Columbia University, New York, NY, USA; 3Columbia University, New York, NY, USA; 4New York, NY, USA

Human gliomas contain a small population of cells with stem cell–like features, and it has been proposed that these “cancer stem cells” may be uniquely responsible for glioma formation and recurrence. However, human gliomas also contain an abundance of cells that resemble glial pro-genitors, and animal model studies have shown that these cells also possess the capacity to form malignant gliomas. To further investigate the contri-butions of stem-like and progenitor-like cells in human gliomas, we have used flow cytometry to characterize the expression of a cancer stem cell marker (CD133) and a glial progenitor marker (A2B5). We have also used these markers to isolate subpopulations of cells from glioblastomas and test their capacity to form tumors when injected into nude rats. Human tumor samples were mechanically dissociated after enzymatic digestion, and cells were isolated by centrifugation through a 30% sucrose gradient. Cells were immunostained with fluorescent conjugated antibodies; CD133, A2B5, and CD45 (microglial marker). Flow cytometry and FACS were performed using a FACSCalibur flow cytometer and analyzed using Flow-Jo software. Three subpopulations of cells were collected (A2B51/CD1331, A2B51/CD133–, A2B5–/CD133–) and stereotactically injected into the right striatum of adult nude rats. Animals were sacrificed at the first signs of tumor-induced morbidity. Histological analysis was performed on all brains. Twenty-seven adult gliomas, grades II–IV, were assayed by flow cytometry. A2B5 was expressed in every glioma assayed ranging from 21% to 90% (mean 5 61.7% 63.8% SEM) of the CD45 negative fraction. CD133 expression was absent in most (10/11) grade II–III gliomas but was relatively abun-dant in most (11/16) glioblastomas assayed, comprising 14–60% of CD45– cells in those tumors. When present, virtually all CD1331 cells were also A2B51. Thus most glioblastomas contained 3 distinct populations (A2B51/CD1331, A2B51/CD133–, A2B5–/CD133–). To test their tumorigenic potential, these populations were isolated by FACS from 6 gliomas (5 glio-blastomas and 1 anaplastic oligodendroglioma) and transplanted into nude rats. Of the 6 human gliomas tested, 4 contained a population of A2B51/CD133– cells that formed tumors when transplanted into nude rats, 3 con-tained a population of A2B51/CD1331 cells that formed tumors, and only 1 contained a population of A2B5–/CD133– cells with the capacity to form tumors. Most of the xenograft tumors showed similar histological features characterized by diffuse infiltration with little or no vascular proliferation and no necrosis. However, in 1 glioblastoma the A2B51/CD1331 cells formed a well-circumscribed tumor with marked vascular proliferation and pseudopalisading necrosis, whereas rats injected with A2B51/CD133– cells from the same tumor formed highly infiltrative tumors with little or no vas-cular proliferation and no necrosis. Contrary to previous reports, we found that CD133 is not an obligate marker of tumor-initiating cells in malignant gliomas. Furthermore, we have identified a population of tumor-initiating cells that are CD133– but express the progenitor cell marker A2B5. Our previous animal studies have shown that A2B51 glial progenitors in adult rat white matter can form malignant gliomas. These results suggest that human A2B51 progenitors can also give rise to gliomas.

SC-26. ONCOLYTIC HERPES SIMPLEX VIRUS VECTORS EFFECTIVELY KILL HUMAN GLIOBLASTOMA STEM-LIKE CELLSHiroaki Wakimoto, Christopher Farrell, Manish Aghi, Robert Martuza, and Samuel Rabkin; Massachusetts General Hospital, Boston, MA, USA

Accumulating evidence indicates that certain types of brain tumors, including glioblastoma, are initiated and maintained by a small fraction of cells that have characteristics of neural stem cells: self-renewal and multi-potency. These cells, often named brain tumor stem cells, are regarded as an important target of therapeutics because they may be responsible for tumor recurrence. We have established glioblastoma stem-like cell cultures from human glioblastoma specimens and examined their susceptibility to oncolytic herpes simplex virus (HSV) vectors. Using neural stem cell culture conditions, we achieved long-term growth of nestin-positive tumor spheres from 4 surgical specimens of glioblastoma. Two cultures enriched for stem cell marker CD133–positive cells exhibited efficient tumor-initiating capa-bility, forming fatal malignant gliomas upon intracerebral transplantation of fewer than 50,000 cells into immunodeficient mice. In vitro infection studies using oncolytic HSV vectors expressing GFP revealed that all 4 glioblastoma stem-like cell cultures were highly infectable. Furthermore, G47delta-BAC virus, which has deletions of the ICP6 and gamma 34.5 genes, showed potent killing of the cells with infection at a multiplicity of infection (MOI) of 0.2, producing a greater than 85% reduction in surviv-ing cells in 7 days. Importantly, after infection with G47delta-BAC virus, secondary tumor sphere formation from remaining aggregates/neurospheres was markedly impaired. These results suggest that oncolytic HSV vectors



may be efficacious in killing glioblastoma stem-like cells or blocking their self-renewal. Orthotopic tumor sphere-derived xenografts will provide a useful model for validating this hypothesis.

SC-27. CANCER STEM CELLS AVOID CHEMOTOXICITY BY ENTERING A REVERSIBLY QUIESCENT STATE IN RESPONSE TO LOW DOSES OF BCNU AND TEMOZOLOMIDEAlicia Mihaliak, Li Li, and Alonzo Ross; Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA

Tumors frequently regrow following treatment, suggesting that they contain a population of cells resistant to current therapies. Stem-like cells in brain cancer are likely candidates for tumor regrowth because they can self-renew and remain in a quiescent state for long periods of time. Using both functional assays and the stem cell marker CD133, we are determining whether stem cells are more resistant to chemotherapy than transit ampli-fying cells, and whether glioma stem cells are more resistant than wild-type (WT) neural stem cells. Experiments using the chemotherapy drugs BCNU and temozolomide clearly show that stem cell self-renewal measured by neurosphere formation is inhibited at a concentration 4–10-fold lower for BCNU and temozolomide, respectively, than that required to inhibit growth of transit amplifying cells measured by MTT assay. The concen-tration of drug required to kill 90% of glioma transit amplifying cells is much higher (10–20-fold) than that required to inhibit cell growth. Con-sistent with decreased neurosphere formation, FACS analysis shows that the percentage of CD1331 cells also decreases on treatment with a low concentration of drug. To determine if stem cells were reversibly quiescent, we assayed neurosphere formation following removal of 50 mM BCNU or 200 mM temozolomide, cultures in which minimal sphere formation was observed. Only glioma stem cells recover from 50 mM BCNU, whereas both WT and tumor stem cells recover from 200 mM temozolomide treatment. We therefore conclude that BCNU is more toxic to WT than to glioma stem cells. Glioma cells, but not WT, undergo G2 cell cycle arrest at these high concentrations of chemotherapy drugs. Cell cycle arrest and inhibition of neurosphere formation in response to these drugs may be a protective mechanism for cancer stem cells, facilitating repair of DNA damage and resistance to chemotherapy.

SC-28. EMBRYONIC STEM CELL (ESC)–DERIVED ASTROCYTES EXPRESSING MELANOMA DIFFERENTIATION ASSOCIATED GENE-7 (MDA-7/IL-24) SELECTIVELY INDUCE GROWTH SUPPRESSION, APOPTOSIS, AND RADIO-SENSITIZATION IN MALIGNANT GLIOMASMahmud Uzzaman,1 Gordon Keller,2 and Isabelle Germano1; 1Neurosurgery, Mount Sinai School of Medicine, New York, NY, USA; 2Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY, USA

Malignant gliomas are aggressive tumors currently lacking effective treatment modalities. Adenovirus-mediated gene transfer shows a prom-ising approach to deliver new genes. However, gene transfer is halted by lack of tissue penetration. We have published (JNS 2005;103:115–123 and 2006;105:88–95) a new method of gene delivery using ESC-derived astrocytes to deliver gene therapy. The aim of this study is to assess the pro-apoptotic effects of ESC-derived transgenic mda-7/IL-24 astrocytes on malignant gliomas in vitro. ESC-derived astrocytes were engineered to express tet-inducible mda-7/IL-24 (the gene sequence was gifted by Paul Fisher, Ph.D., Columbia University, NY). Malignant glioma U87 cells were used for transwell experiments in standard condition in the bottom well, while ESC-derived astrocytes conditionally expressing mda-7/IL-24 were used in the top well. Doxycycline (Dox) was used for gene induction. In another set of experiments, U87 glioma cells were exposed to ionizing radiation (IR 4Gy) immediately before transwell plating. Cell viability and apoptosis was detected by flow cytometry (FACS analysis). RT PCR was used to quantify mda-7/IL-24 expression. A significant increase in apop-totic rate was found in U87 glioma cell 24 hours after Dox induction (11% from baseline). Concomitantly, a significant decrease in cell viability was found reaching its peak at 72 hours (80%). This was not observed when Dox was omitted. Pretreatment with IR 4Gy increased the apoptotic rate to 40% compared to controls. Our data suggest that ESC-derived astrocytes conditionally expressing mda-7/IL-24 can be used as vectors to deliver gene therapy for malignant gliomas. The observed increased apoptotic rate is secondary to the radio-sensitizing effects of mda-7/IL-24. These preclinical experiments hint that ESC-derived astrocytes could be a promising tool for adjuvant treatment of malignant gliomas.

SURGICAL THERAPIES

ST-01. ADJUVANT CEREPRO TREATMENT OF PATIENTS WITH OPERABLE MALIGNANT GLIOMA—A POOLED ANALYSIS OF TWO CLINICAL STUDIESAnu-Maaria Sandmair,1 S. Yla-Herttuala,2 Gillian Langford,3 and Neil Murray3; 1Neurochirurgische Klinik, Klinikum Ingolstadt GmbH, Ingolstadt, Germany; 2Kuopio University, Kuopio, Finland; 3Ark Therapeutics Ltd., London, United Kingdom

Intracerebral administration of CereproTM is an innovative technology that transfects the herpes simplex thymidine kinase gene into cells via an adenoviral vector, which, in combination with ganciclovir (GCV), induces cell death in proliferating cells. The aim of this post hoc pooled analysis was to assess the efficacy of CereproTM in prolonging time to death or re-opera-tion among patients with operable primary or recurrent malignant glioma. In two clinical studies, patients received CereproTM (3 3 1,010 pfu in 10 ml; n 5 24) by 30–70 injections into the wound bed following tumor resection to a depth of 10 mm; or control (radical excision and no injections) (n 5 19). All patients received radiotherapy. CereproTM patients received GCV (5 mg/kg IV b.d.) on days 5–19. Median time to death or re-operation of patients treated with adjuvant CereproTM was 62.4 weeks versus 37.7 weeks in the control patients (p 5 0.002, log rank analysis). Analysis with a Cox model including prognostic covariates gave a hazard ratio of 3.31 (95% CI: 1.37; 8.02) (p 5 0.008), representing a significant risk reduction. CereproTM was well tolerated in both studies. CereproTM prolongs the time to death or re-operation in patients with operable malignant glioma when combined with surgery and radiotherapy. CereproTM is a promising potential new treatment in this devastating condition.

ST-02. A CONTROLLED, RANDOMIZED, PARALLEL-GROUP, MULTICENTER STUDY OF THE EFFICACY AND SAFETY OF HERPES SIMPLEX VIRUS–THYMIDINE KINASE GENE THERAPY (CEREPRO), WITH SUBSEQUENT GANCICLOVIR, FOR THE TREATMENT OF PATIENTS WITH OPERABLE HIGH-GRADE GLIOMAM. Westphal,1 P. Menei,2 Zvi Ram,3 D. Eckland,4 Gillian Langford,4 Neil Murray,4 and S. Yla-Herttuala5; 1Universitätsklinikum Hamburg–Eppendorf Klinik und Poliklinik für Neurochirurgie, Hamburg, Germany; 2CHU d’Angers, Angers, France; 3Tel Aviv Medical Center, Tel Aviv, Israel; 4Ark Therapeutics Ltd., London, United Kingdom; 5Kuopio University, Kuopio, Finland

There are 35,500 cases of high-grade glioma per year in Europe, of which 17,700 are operable (calculated from accurate 2003 European popu-lation figures [incidence rate of 1.3 per 10,000 pop.]). Patients with operable high-grade glioma are susceptible to early death due to recurrence of the tumor. In a phase II clinical study CereproTM, an adenoviral vector contain-ing the herpes simplex virus–thymidine kinase gene in conjunction with ganciclovir (GCV), has been shown to almost double survival time, provid-ing on average an extra 7 months of life with an acceptable safety profile that meets this high clinical need. CereproTM is administered by multiple injections into the normal brain tissue in the wound bed following resec-tion of the tumor. Infected cells subsequently express the herpes simplex thymidine kinase transgene. This enzyme phosphorylates GCV, resulting in the production of a cytotoxic nucleotide that induces cell death in dividing cells. Normal neurones surrounding the tumor are non-proliferative and are therefore not affected by CereproTM. The therapeutic effect of CereproTM/GCV is further enhanced by the “bystander effect,” in which the cytotoxic nucleotide analogue spreads to the neighboring non-infected cells and induces apoptosis. This communication describes the design of a confirma-tory phase III, multicenter, controlled, randomized, parallel group study to determine if CereproTM/GCV is superior to standard care for the treatment of operable primary glioblastoma. The study was designed to randomize 250 patients in a 1:1 ratio to either the active group, receiving CereproTM, followed by intravenous GCV as an adjunct to standard therapy, or the con-trol group, which will receive standard therapy alone. The primary efficacy endpoint of this study is time to death or re-intervention, and the principal secondary efficacy endpoint is all-cause mortality. Additional secondary objectives are tumor progression by MRI, safety, and quality-of-life assess-ments. Each patient will be followed until death or the end of the study. As of April 2007, recruitment is complete and 250 patients have been random-ized into the study. The safety data from Study 904 has been reviewed twice by the Data Safety Monitoring Board (DSMB), most recently on December 13, 2006. On each occasion the DSMB has recommended that recruitment into the study should continue with no protocol amendments. The next DSMB meeting will be held in July 2007. The status of the trial as well as preliminary data about the safety of CereproTM and the biodistribution of the viral vector will be presented.



ST-03. HIGH-FREQUENCY, LOW-TEMPERATURE RADIOSURGERY IN NEUROSURGERYAlfred Bowles; Neurosurgery, Conemaugh Health Initiatives—John P. Murtha Neuroscience and Pain Institute, Johnstown, PA, USA

High-frequency, low-temperature radiosurgery is gaining popularity with many surgical subspecialties, including neurosurgery. With radio wave surgery, high-frequency radio waves are directed to tissue through electrode tips. Impedance to passage of radio waves through tissue generates heat within the cells, resulting in volatilization with specific and precise cutting or coagulation. The radio frequency electrode does not provide resistance and therefore remains cold, limiting collateral damage. Paucity of heat gen-erated to these surgical sites allows the surgeon to work in direct proximity to functional neural elements. Over a 12-month period, 220 operations were performed utilizing the Ellman Radio Frequency Generator or radio wave surgery. Forty-eight patients ranging from 19 to 81 years of age were treated for symptomatic brain tumors defined in radiographic and/or clinical pro-gressions. Tumors consisted of cerebral metastases, primary malignant brain tumors, and also biologically malignant yet histologically benign tumors. Radical resections were achieved in the majority of procedures, confirmed operatively and by post-operative MRI. Lateral thermal tissue destruction was minimized, and no new neurological deficits were recorded. Variable characteristics of radio wave surgery include tissue contact time, power intensity, and waveform with a variety of electrodes used. Optimization of the above characterization achieved safe, precise and complete resections in 48 brain tumor cases. Collateral damage was minimized with work in direct proximity with delicate structures with controlled hemostasis, dissection, and debulking. Using high-frequency, low-temperature radiosurgery can be a valuable adjunct for brain tumor surgery, especially when precision and total resection are mandatory. The positive heat generated with minimal lateral tissue injury allows the surgeon to work in proximity to delicate neural structures. With high-frequency radiosurgery, greater resections and low morbidity can be obtained in brain tumor surgery.

ST-04. SURVIVAL IS INCREASING AFTER CRANIOTOMY FOR PATIENTS WITH GLIOBLASTOMA MULTIFORMEMichael Chicoine,1 Michael Zahner,2 Ricky Kalra,3 Feng Gao,4 Lisa Nicoletti,3 Keith Rich,3 Robert Grubb,3 Gregory Zipfel,3 Ralph Dacey,3 Gerald Linette,3 Joshua Dowling,1 Neill Wright,3 Muhammad Ali,3 Shabbir Safdar,3 and Joseph Simpson3; 1Neurosurgery, Washington University in St. Louis, St. Louis, MO, USA; 2MO, USA; 3Washington University in St. Louis, St. Louis, MO, USA; 4Biostatistics, Washington University in St. Louis, St. Louis, MO, USA

Recent advances in neuro-oncology are leading to more intensive treat-ments for patients with glioblastoma multiforme (GBM), including implant-able carmustine impregnated chemotherapy wafers, temozolomide chemo-therapy, and other novel strategies. We assessed whether patients with GBM treated in the past 6 years are surviving longer than patients treated prior to this time period. Retrospective and prospective analysis of survival for 161 patients who underwent craniotomy for GBM at the time of initial diagnosis at Barnes-Jewish Hospital between October 1996 and April 2007. Patients were divided into 2 groups: (1) surgery prior to 2001 or (2) surgery in 2001 or after. Logistic regression of Kaplan-Meier analysis was performed, with comparisons made between the 2 groups. The median ages were equivalent for the 2 groups: 61 years for group 1 and 60 years for group 2. Logistic regression analysis demonstrated that the survival for the 109 patients in group 2 (median 13 months) was longer than the survival for the 52 patients in group 1 (median 10 months, p 5 0.02). The 2-year cumulative survival was 18% for group 1 and 38% for group 2. Survival times after craniotomy for initial resection of GBM are longer for patients who had surgery in the time epoch from 2001 to 2007 than for patients who had surgery in the time epoch from 1996 to 2000. This suggests that survival after craniotomy for GBM may be improving, but long-term prognosis remains poor.

ST-05. FRAME-BASED STEREOTAXY IN A FRAMELESS ERA: CURRENT CAPABILITIES, RELATIVE ROLE, AND THE POSITIVE AND NEGATIVE PREDICATIVE VALUES OF BLOOD THROUGH THE NEEDLEChristopher Owen1 and Mark Linskey2; 1University of California, Irvine, Orange, CA, USA; 2Neurological Surgery, University of California, Irvine, Orange, CA, USA

In the modern era of frameless stereotaxis (FL), the role of frame-based (FB) stereotactic needle biopsy is evolving. A retrospective review was done of prospective database of 106 lesions in 91 consecutive patients undergoing FB stereotactic needle biopsy with a systematic “geologic core” technique by a single surgeon. Diagnostic accuracy was calculated comparing biopsy diagnosis with final pathology in 11 patients who underwent subsequent sur-gical resection. All instances of intraoperative bleeding through the needle

were prospectively noted and compared with post-biopsy CT scan. Lesions were classified as risky for FB technique if they were (1) infratentorial or pineal, (2) within 10 mm of the circle of Willis or root of the Sylvian fis-sure, or (3) within 10 mm of deep cerebral veins. Diagnostic yield was 94%. Diagnostic accuracy was 91%. Of 18 lesions involving the corpus callosum, 13 (72.2%) were GBM, 2 were anaplastic astrocytoma, and 1 each were found to be anaplastic oligodendroglioma, primary central nervous system lymphoma (PCNSL), and tumescent MS. Of 25 multifocal lesions, malig-nant primary brain tumor was diagnosed in 17 (68%) (11 GBM, 3 PCNSL, 2 anaplastic ologodendroglioma, and 1 anaplastic astrocytoma). Mortal-ity was 0%. Three patients developed temporary neurological deficits, and 1 had permanent deficit. Absence of persistent blood through the biopsy needle had a negative predicative value of 98.8% for subsequent neuroim-aging of blood vessels .5 mm diameter. According to our criteria, 80% of patients would have been candidates for FL biopsy. Stereotactic biopsy is an effective, safe, and important technique for histological diagnosis of brain lesions, particularly for multifocal and corpus callosum lesions. Post-biopsy CT can be safely reserved for patients who demonstrate persistent bleeding through the biopsy needle. FB stereotaxy remains an important technique for the 20% with small or deep-seated lesions or when it is advantageous to avoid an incision, a burr hole, or general anesthesia.

ST-06. THE ROLE OF EXTENT OF RESECTION IN THE LONG-TERM OUTCOME OF LOW-GRADE HEMISPHERIC GLIOMASJustin S. Smith,1 Edward F. Chang,1 Kathleen R. Lamborn,1 Susan Chang,2 Michael D. Prados,2 Soonmee Cha,3 Tarik Tihan,4 Scott VandenBerg,4 Michael W. McDermott,1 and Mitchel S. Berger1; 1Department of Neurological Surgery, Brain Tumor Research Center, University of California, San Francisco, San Francisco, CA, USA; 2Department of Neuro-Oncology, University of California, San Francisco, San Francisco, CA, USA; 3Department of Radiology, University of California, San Francisco, San Francisco, CA, USA; 4Department of Pathology, University of California, San Francisco, San Francisco, CA, USA

The prognostic role of extent of resection (EOR) of low-grade gliomas (LGG) is a major controversy. Attempts to address this issue have been lim-ited by a lack of quantitative assessment of tumor resection. We designed a retrospective study using volumetric analysis to assess the influence of EOR on long-term outcomes in adult patients with hemispheric infiltrative LGG. The study population included consecutive adults (>18 years old) undergoing initial resection of a hemispheric infiltrative LGG between 1989 and 2005 at UCSF with pre-operative (pre-op) and post-operative (post-op) MR imaging for review. Patients undergoing biopsy only, with gemistocytic histology or with gliomatosis cerebri, were excluded. Manual segmenta-tion was performed with region-of-interest analysis to measure pre-op and post-op tumor volumes based on FLAIR axial slices (5 mm thickness, no gap). EOR was defined as (pre-op volume – post-op volume)/pre-op volume. Outcome measures included overall survival (OS), progression-free survival (PFS), and malignant progression-free survival (MPFS). Progression was based on unequivocal increase in tumor size and/or malignant progression on follow-up imaging. Malignant progression was based on the appearance of new contrast enhancement on follow-up imaging and/or histology from subsequent surgery. Multivariate assessments were performed with propor-tional hazards modeling The 216 patients studied had a median age of 38 years (range 19–72). All tumors were grade 2 and consisted of 43% astro-cytomas, 42% oligodendrogliomas, and 15% mixed oligoastrocytomas. Median KPS was 90 (range 80–100). Median pre-op and post-op tumor vol-umes and EOR were 36.6 cm3 (range 0.7–246.1), 3.7 cm3 (range 0–197.8), and 88.0% (range 5–100), respectively. There was no operative mortality. New post-op deficits were noted in 36 (17%) patients; however, all but 4 subsequently had complete recovery. There was no significant association between EOR and presence of new post-op deficit (p 5 0.36). There were 34 (16%) deaths, with a median follow-up of 4.4 years (range 0.01–14.7). Progression and malignant progression were identified in 95 (44%) and 44 (20%) cases, respectively. Patients with >90% EOR had 5- and 8-year OS, PFS, and MPFS rates of 97% and 91%, 75% and 43%, and 93% and 76%, respectively, while patients with ,90% EOR had 5- and 8-year OS, PFS, and MPFS rates of 76% and 60%, 40% and 21%, and 72% and 48%, respectively. After adjusting each measure of tumor burden (pre-op volume, post-op volume, EOR) for the effects of patient age, KPS, tumor location, and tumor subtype, OS was predicted by EOR (HR 5 0.972, 95% CI 5 0.960–0.983, p , 0.001) and post-op tumor volume (HR 5 1.010, 95% CI 5 1.001–1.019, p 5 0.03); PFS was predicted by pre-op tumor volume (HR 5 1.009, 95% CI 5 1.004–1.013, p , 0.001) and post-op tumor volume (HR 5 1.007, 95% CI 5 1.001–1.014, p 5 0.035); and MPFS was predicted by EOR (HR 5 0.983, 95% CI 5 0.972–0.995, p 5 0.005) and pre-op volume (HR 5 1.007, 95% CI 5 1.001–1.013, p 5 0.027). OS and MPFS among adult patients with hemispheric LGG are significantly influenced by greater EOR. When used with functional mapping techniques, increased EOR is not associated with additional morbidity. We recommend that, when



feasible, management of adult hemispheric LGG include maximal surgical resection, using mapping techniques as appropriate, to improve long-term patient outcomes.

ST-07. ECONOMIC ANALYSIS OF AWAKE CRANIOTOMY COMPARED TO GENERAL ANESTHESIAMartina Stippler and Arlan Mintz; Neurosurgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA

Awake craniotomy under local anesthesia and monitored conscious sedation is infrequently used in neurosurgery practice. It requires an expe-rienced operating team and poses unique challenges. Most often, awake craniotomy is employed to minimize neurological deficits when operating in eloquent regions. The purpose of this retrospective chart review is to assess the economic outcomes of awake craniotomies used for routine tumor resections compared to similar cases done under general anesthetic. Out-come measures include length of stay, discharge disposition, and associated hospital charges. Method: This is a retrospective cohort study involving 46 patients who had glioma surgery from January 2006 to February 2007. The experimental cohort consisted of 20 consecutive patients who under-went awake craniotomy, compared to the control group composed of 26 patients who underwent craniotomy under general anesthesia during the same period. Total charges, post-operative length of stay, and discharge dis-position were compared between the two groups. Fewer high-grade gliomas (anaplastic astrocytomas and GBMs) were present in the awake craniotomy group (80% vs. 92%). The average post-operative length of stay was signifi-cantly shorter in the awake craniotomy group (1.6 days vs. 3.4 days; p , 0.001). All patients who underwent an awake craniotomy were discharged home post-operatively. Only 68% of the patients who underwent craniotomy under general anesthesia were discharged home (p 5 0.006), while the rest were sent to rehabilitation. The total mean hospital charges per patient were higher in the awake craniotomy group ($123,711 vs. $109,839 in general anesthesia cohort; p 5 0.136). The increased charges reflect the mean opera-tive charges, which were significantly higher in the awake craniotomy group ($64,179 vs. $45,898; p , 0.001). The sum of the non-operative charges was lower in the awake craniotomy group, but the difference was not statisti-cally significant (mean charges $59,531 vs. $63,711; p 5 0.631). The opera-tive charges reflect the supply charges (awake $29,153 vs. general $14,929; p , 0.001) rather than operative time charges, which were similar (awake $12,104 vs. general $10,079). Our analysis showed that awake craniotomy is associated with increased total mean hospital charges compared to resec-tion under general anesthesia. This is due to increased mean operative charges, since the mean non-operative charges were decreased. Overall, awake craniotomies may affect quality of life after tumor resection, since there was a significantly shorter post-operative stay and all patients were discharged home, compared to 32% of patients in the general anesthesia group who were discharged to a rehabilitation facility.

ST-08. ENDOPORT NEUROSURGERY: FULLY ENDOSCOPIC STEREOTACTIC GUIDED RESECTION OF INTRA-AXIAL TUMORSDevin Amin, Amin Kassam, and Arlan Mintz; Neurosurgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA

Endoscopic neurosurgical techniques have been utilized to access the skull base and the ventricles. These techniques have been limited for the access of intra-axial tumors. We present our experience with a novel endo-scopic technique to access intraparenchymal tumors. We will document the clinical outcomes following Endoport neurosurgery in 12 consecutive patients with primary brain tumors (n 5 7) and metastatic lesions (n 5 5). The lesions were supratentorial in 8 cases and infratentorial in 4 cases. An endoscopic approach for tumor resection was developed utilizing frame-less stereotaxis and air medium rod lens endoscopy. A small stereotactic guided craniotomy 15–20 mm in diameter was completed, followed by a 14 mm cortisectomy centered over a gyrus away from cortical veins, and then a tubular port was placed with blunt dilation to create an 11.5 mm transcortical corridor. The transparent tubular retractor or Endoport assists with hemostasis and is able to pivot about two axes to resect lesions much larger than the diameter of the Endoport. Endoscopic removal was per-formed in all 12 patients without the need to convert to open craniotomy in any patient. Gross total resection was achieved in 6 patients, near total resection in 2 patients, and partial resection in 4 patients. Post-operatively, 1 patient developed a wound infection, 1 patient had a posterior fossa CSF leak treated with operative repair, and there was no operative mortality. The length of hospital stay was 3.4 days on average and ranged from 24 hours to 6 days. Endoscopic resection combined with stereotactic placement of an Endoport enables a minimally invasive resection of cortical tumors with only transient morbidity and no operative mortality. This technique is not limited by the nature of the pathology or the size of the lesion based on the conditions treated in this series.

ST-09. THE IMPACT OF MULTIPLE RESECTIONS FOR GLIOBLASTOMA MULTIFORMEDeborah T. Blumenthal, Felix Bokstein, Sigal Freedman, Tal Shachar, Andrew A. Kanner, Razi Sitt, and Zvi Ram; Tel Aviv Medical Center, Tel Aviv, Israel

The role of multiple resections in the management and prognosis of glioblastoma multiforme (GBM) is unclear. The data on re-operation for resection for recurrent GBM are sparse. We retrospectively searched our prospectively collected database for primary (de novo) GBM patients who underwent 31 resections from 1995 to the present by our neurosurgical team. We examined our neurosurgical and neuro-oncology databases from 1995 to the present for diagnoses of GBM patients. Initially 633 patients were identified. We deleted those who had prior history of lower-grade pathology and underwent transformation to GBM as a secondary tumor. We considered only tumor resection; we did not consider diagnostic biopsy, shunting for hydrocephalus, or wound revision as 1 of the 3 or more surger-ies. From the remaining 525 primary GBM patients, we tallied the number of tumor resection surgeries, categorizing them as having undergone biopsy only; 1 surgery; 2 surgeries; or 31 surgeries. Survival was calculated from date of diagnosis to date of death. Performance score was estimated for all patients for whom data were available. For the 31 surgery group, addi-tional information was collected, including gender, laterality and location of tumor, and time intervals between the first and second, second and third, and subsequent surgeries to date of death. Survival was plotted by Kaplan-Meier curves. Cases with insufficient data or uncertain dates of death were removed from the lists. Cases still alive were censored. We identified 27 patients from our database with 31 operations for primary GBM. Of the 27 patients, 4 had diagnosis of gliosarcoma. Nineteen of these patients underwent 3 tumor-resective operations; 7 underwent 4 operations; and 1 underwent 5 operations. Twenty patients were male, and 7 were female. Ages ranged from 25–76, with median age 54. Fifteen tumors were right-sided, and 13 were left-sided (1 patient had bilateral, multifocal disease). The frontal and temporal lobes were most frequently represented, with sev-eral tumors spanning several adjacent lobes. Range of survival from date of diagnosis to date of death was 9 months (a case with 4 resections) to 45 months (also 4 resections). One patient who underwent 3 surgeries is still alive, more than 5 years from his date of diagnosis. Median survival times for biopsy only, 1 surgery, 2 surgeries, and 31 surgeries were, respectively, 101 days, 243 days, 434 days, and 582 days. Multivariate analysis of age, performance score, and number of resections relative to survival time will be presented. Multivariate analysis shows statistical significance for the num-ber of resections as an favorable independent variable for prolonged sur-vival. The results of our retrospective study support the aggressive approach of multiple interventions for a selected group of GBM patients. Increased survival progressively correlated with an increasing number of tumor resec-tions. It is generally accepted that re-operation is indicated for palliation in selected symptomatic cases; our data support the impact of 31 resections as statistically significant in increasing length of survival for a selected group.

ST-10. RESTORING AMBULATORY CAPACITY IN PATIENTS WITH METASTATIC TUMORS OF THE L5 VERTEBRAL BODY: THE MOFFITT CANCER CENTER EXPERIENCESurbhi Jain and Frank Vrionis; NeuroOncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA

Metastatic L5 vertebral body tumor is difficult to approach for pallia-tive gross total resection (GTR) and reconstruction. These patients have restricted quality of life, intractable radicular pain, neural compromise, and mechanical instability. The technical challenge in dissecting the L5 region comes from the unique angulation of the sacral spine, the biomechanical properties of the transitional lumbosacral column, non-resectable large exit-ing nerve roots, and iliac vessels. We report our experience with metastatic L5 tumors treated with GTR and spinal column stabilization using differ-ent surgical schemes without added morbidity or mortality. Between 2002 and 2007, the medical records of 10 patients (mean age 59 years, range 41–80 years; 7 male and 3 female) with metastatic L5 tumor from remote sites treated at the H. Lee Moffitt Cancer Center were reviewed. Patients with at least 6 months of expected survival were considered for surgery. Pre-operative embolization was performed in hypervascular tumors. The operative approach combined radical resection of tumor, L5 vertebrectomy, and vertebral body reconstruction, depending on the degree and stability of the involved segment. Six patients had previous radiation therapy to spine and were nonambulatory. Four patients had cauda equina compression. The following surgical approaches were used: 360-degree single/two stages (3 cases; retro/transperitoneal), transpedicular posterior approach (4 cases), and only anterior retroperitoneal approach (3 cases). For fusion, expand-able cage, stackable cage, or methylmethacrylate were used in 4, 1, and 5 patients, respectively. Mean intraoperative blood loss was 3,660 cm3 (range 500–5,000 cm3). Gross total resection was achieved in 9/10 patients. All patients, including those who were nonambulatory before surgery, regained ambulatory capacity. There was no instrumentation failure. Spinal align-ment was maintained in all. The type of surgical approach used did not



influence post-operative course, survival, and outcome. Complications included dural tear, and superficial wound infection in 2 patients with prior radiation and multiple surgeries. Newer resection and reconstruction tech-niques are making surgery a feasible option in the treatment of L5 tumors. Since L5 is difficult to fuse, it frequently requires the gold standard 360-degree-circumferential approach for effective stabilization. However, in selected cases, either anterior or posterior approach alone equally suffices and provides ambulatory capacity in patients.

ST-11. CIRCUMFERENTIAL DECOMPRESSION AND PLACEMENT OF ANTERIOR LUMBOSACRAL INSTRUMENTATION VIA LATERAL RETROPERITONEAL APPROACH IN LUMBOSACRAL TUMORSSurbhi Jain and Frank Vrionis; NeuroOncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA

The authors describe a technique for inserting a sacral body screw via an anterior retroperitoneal approach while protecting the segmental nerves and major iliac vessels. The advent of sacral instrumentation has allowed the use of a less invasive posterior-lateral approach to the lumbosacral col-umn, especially posterolateral sacral body, thereby decreasing the surgery-induced insult to the patient and minimizing the need to turn the patients into a prone position for posterior screw placement. In an attempt to minimize this trauma, the authors have used an anterolateral position that has allowed them to perform neural decompression, anterior reconstruc-tion with expandable cage, radical resection of tumor, L5 vertebrectomy, and stabilization with anterior lumbosacral screw placement. The authors describe their surgical technique of anterolateral circumferential decompres-sion, which entails the use of a lateral/oblique position of the patient, which could be easily performed in L5 tumors, especially tumors predominantly located to the left, and performing instrumentation with special custom-made tools, thus respecting the segmental nerves of the lumbar spine. One case example treated at the H. Lee Moffitt Cancer Center is provided to illustrate the technique.

ST-12. MYXOPAPILLARY EPENDYMOMA OF THE FILUM TERMINALE: TREATMENT AND OUTCOME IN ELEVEN CASESSameh Badran, Mohamed Elbeltagy, Ahmed Hegazy, and Mohamed Elsisi; Department of Neurosurgery, Cairo University, Cairo, Egypt

Myxopapillary ependymomas are the most common primary tumors of the spinal cord. Recurrence-free survival depends on local control of the tumor. The value of additional radiotherapy is still a matter of debate. The aim of this study was to analyze radiotherapy, surgery, and the pre-operative state with regard to the recurrence rate and post-operative neurological out-come. Eleven patients with filum terminale tumors whose tumors were oper-ated on and whose pathology was quite uniform, of a myxopapillary type, were included in the study. Pre-operative state and neurological outcome were assessed according to the McCormick scale. Total resection of the pri-mary tumor was achieved in 6 of the 11 patients in our series (54.5%), and partial resection was achieved in 5 patients (45.5%). Adjuvant radiotherapy was administered only in 40% of patients with partial tumor resection. No recurrences occurred after macroscopic total resection, and only 1 patient showed tumor recurrence after subtotal resection. Gross total resection of filum teminale ependymomas, whenever feasible, is the modality of choice in treatment of such tumors and a strong predictor in recurrence-free sur-vival. Furthermore, radiotherapy for partially resected myxopapillary filum ependymoma lesions does not appear to have a beneficial effect on recur-rence-free survival and is more controversial. Pre-operative neurological condition is the main prognostic factor for the post-operative neurological outcome and is not different for patients with totally resected tumors as compared to those with partially resected tumors.

ST-13. COMBINED MOTOR AND LANGUAGE DTI FIBER TRACKING AND INTRAOPERATIVE SUBCORTICAL MAPPING FOR SURGICAL REMOVAL OF GLIOMASLorenzo Bello,1 Giorgio Carrabba,1 Francesco Acerbi,1 Anna Gambini,2 Antonella Castellano,2 Enrica Fava,1 Marcello Gadioli,2 Valeria Blasi,2 Alessandra Casarotti,3 Costanza Papagno,3 Sergio Maria Gaini,1 Giuseppe Scotti,2 and Andrea Falini2; 1University of Milan, Milan, Italy; 2Ospedale San Raffaele, Milan, Italy; 3Psicology, Italy

We report the results of the combined use of DTI fiber tracking (DTI-FT) for visualization of language and motor tracts and intraoperative subcor-tical mapping (ISM) in patients with gliomas involving speech or motor areas or pathways. Data are available on 64 patients (23 rolandic tumors,

16 prerolandic, 17 temporal, 6 parietal, and 3 paralimbic): 52 low-grade gliomas, 12 high-grade gliomas. DTI-FT was acquired by a 3T MR scanner with a single-shot echo-planar sequence with gradients applied along 32 non-collinear directions. The cortico-spinal (CST) and the subcortical tracts involved in the phonologic (arcuate fascicle included in superior longitudinal fascicle) or semantic loop (inferior fronto-occipital and uncinate fascicles) were reconstructed using a dedicated software, and data were transferred to the neuronavigational system. ISM was performed during awake or asleep anesthesia, using a Ojemann stimulator. DTI-FT helped pre-operatively in understanding the anatomical relationship between tracts and the tumor mass. DTI-FT reconstructions show a good concordance with ISM find-ings. However, a decrease in FA threshold for start and stop tracking and additional ROIs were needed for a proper reconstruction of the tracts, par-ticularly in low-grade gliomas, where most of the tracts were located inside the tumor mass, and in F3 tumors, for the anterior branch of the superior longitudinalis tract. In high-grade gliomas, DTI-FT depicted the tracts mostly at the tumor periphery, which corresponded to what was identified by subcortical mapping. DTI-FT and subcortical mapping decreased the duration of surgery, patient fatigue, and the occurrence of intraoperative seizures. Combination of DTI-FT and ISM allows accurate identification of fiber tracts associated with motor or language functions and enhances surgical safety maintaining a high rate of functional preservation.

ST-14. CORTICAL AND SUBCORTICAL MAPPING OF MOTOR AREAS AND PATHWAYS WITH POLYGRAPHIC RECORDING OF EMG RESPONSES AND OF CEREBRAL ACTIVITYLorenzo Bello, Francesco Acerbi, Giorgio Carrabba, Carlo Giussani, Enrica Fava, Nino Stocchetti, and Sergio Maria Gaini; University of Milan, Milan, Italy

We describe our experience with parallel recording of multiple motor evoked responses with electromyographic technique and of cerebral activ-ity with electrocorticographic and electroencephalographic techniques as a guide for cortical and subcortical mapping during resection of tumors involv-ing motor areas or pathways. Our data refer to 170 patients (22–68 years of age, median 43) who underwent resection for tumors involving motor areas or pathways during the last 3 years. The procedure was performed under asleep anesthesia when the tumor involved only motor areas or pathways (52 patients), or under awake anesthesia when also language or visuospatial functions were mapped (118 patients). Multichannel polygraphic recording of EMG responses and of cerebral activity (EEG and ECoG) was achieved in all cases during the entire time of the procedure. EEG activity was recorded to monitor brain activity when ECoG was not available, at the opening and closure, and to assess brain activity at distance from the operating field. ECoG was used to define the working current, to monitor for the occur-rence of spikes or electrical seizure during the resection. Multichannel EMG recordings monitored the activity of several muscles from the upper phar-ynx, upper and lower face, neck, arm, forearm, hand, upper leg, and lower leg. The exact montage may vary according to the location of the lesion. EMG recording was associated with clinical evaluation of motor responses. We will present the methodology used and the usefulness and results of the EMG recording during cortical and subcortical motor mapping, detailing the findings registered in asleep or awake patients. We will also discuss the different pattern of EMG response recorded during subcortical mapping, according to the site and depth of stimulation, correlating them with DTI findings. Results of mapping will be correlated with extent of resection and immediate and delayed post-operative morbidity.

ST-15. TREATMENT OF CNS TUMORS BY TARGETED DRUG DELIVERY: INTRATHECAL CHEMOTHERAPY FOR BRAIN TUMORSMichael Vloeberghs,1 Richard Grundy,2 and David Walker2; 1University of Nottingham, Nottingham, United Kingdom; 2University of Nottingham, Turks and Caicos Islands

This paper looks to the future potential of direct intrathecal chemother-apy for CNS tumors in children and combines the results of mathematical modeling of CSF flow and the authors’ clinical experience with intrathe-cal drug delivery. The authors have the largest European intrathecal drug delivery practice in children and run an extensive research program at the University of Nottingham on CSF flow. We believe that direct intrathecal chemotherapy is a valid option for the treatment of CNS tumors. In paral-lel with the use of intrathecal Baclofen for the treatment of spasticity in children, where the intrathecal dose is 1/1,000 times smaller than the orally effective dose, intrathecal chemotherapy for CNS tumors can avoid systemic toxicity and be more effective as the agent is delivered near the target. A 12-year-old girl, previously treated surgically and medically for a posterior fossa medulloblastoma, presented with extensive leptomeningeal spread and spinal metastasis. In joint discussion with the parents and the involved clini-



cians, we agreed to implant a passive drug delivery system consisting of a subcutaneous reservoir and a lumbar intrathecal catheter. The subcutaneous reservoir was injected with Gadolinium a few days after the implantation and MRI studies of the CNS were done. This showed extensive diffusion of the contrast medium throughout the CNS, including the ventricles and the subarachnoid space. This distribution confirmed that all areas of the CNS can be accessed by injecting into the subcutaneous chamber. Intrathecal chemotherapy was then given but failed to control the disease. Despite the failure to control the relapse in this particular case, we are encouraged by the images showing the Gadolinium distribution. Adequate selection of the agent, the development of more target-specific delivery media, e.g., nano-particles and the use of patient-specific MRI data in CSF flow prediction, will allow us to reach and treat tumors in the CSF spaces and potentially avoid the systemic toxicity associated with intravenous chemotherapy.

Neuro-Oncology 9, 467–621, 2007 (Posted to Neuro- Oncology [serial online], September 12, 2007. URL http://neuro-oncology.dukejournals.org; DOI: 10.1215/ 15228517-2007-039)

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