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Pathologic Characteristics of Pediatric Intracranial PilocyticAstrocytomas and Their Impact on Outcome in

3 Countries: A Multi-institutional Study

Tarik Tihan, MD, PhD,* Ayca Ersen, MD,*w Ibrahim Qaddoumi, MD,zMaher A. Sughayer, MD,ySahsine Tolunay, MD,8 Maysa Al-Hussaini, MD,y Joanna Phillips, MD, PhD,*zNalin Gupta, MD, PhD,z Patricia Goldhoff, MD,* and Anu Baneerjee, MD#

Abstract: Pilocytic astrocytoma (PA) is one of the most common

glial neoplasms in the pediatric population, and its gross total

resection can be curative. Treatment of partially resected or

recurrent tumors is challenging, and the factors associated with

increased recurrence risk are not well defined. Identification of

favorable and unfavorable factors can lead to a better under-

standing and management of patients with PA. We studied

the pathologic characteristics of 116 intracranial PAs from

4 institutions representing 3 distinct geographic regions to

identify factors that may be associated with biological behavior.

The study included 65 boys and 51 girls with a median age of

6 years. Median follow-up for all patients was 101 months,

during which time 38 patients had recurrence. Progression-free

and overall survivals were better in patients who underwent

gross total resection. We were not able to identify any

differences in pathologic and molecular markers among the

4 institutions from 3 different countries. However, progression-

free survival varied significantly among institutions. Sox-2 was

the most prevalent stem cell marker in PA, and many tumors

showed synaptophysin positivity. BRAF immunostaining was

not useful in determining BRAF duplication. BRAF duplication

was more typical of posterior fossa tumors. There was a strong

correlation between BRAF duplication and pERK immunos-

taining, suggesting that the RAF/MEK/ERK pathway is active

in these tumors. This finding has significant implications given

its role in oncogen-induced senescence and possible influence on

treatment decisions of subtotally resected tumors.

Key Words: astrocytoma, pilocytic astrocytoma, pediatric

glioma, BRAF, Sox-2

(Am J Surg Pathol 2012;36:43–55)

P ilocytic astrocytoma (PA) is one of the most commoncentral nervous system (CNS) neoplasms in the

pediatric age group, and it usually occurs in the posteriorfossa.40 Recent studies have shown that the overwhelmingmajority of PAs harbor alterations in the BRAF/MEK/ERK signaling pathway in the form of BRAF duplica-tions.2 Whether this pathway alteration is causative or amere reflection of another underlying etiology is not clear.There is a need to correlate these molecular alterations inthe clinical context to determine whether BRAF altera-tions define unique subsets of PAs.

Data on the type and nature of precursor/stem cellsthat may give rise to PAs are also limited.33,42 Further-more, it is not certain whether PAs in different locationsarise from different precursor cells or whether the putativestem cell markers can aid in the diagnosis, classification,or management of these indolent tumors.

There are a number of additional uncertaintiesabout the pathologic evaluation and clinical managementof PAs.7,10,16,19,25,26 Although the extent of resectionhas been shown to affect survival, the influence of otherclinical variables on prognosis is less clear. For instance,it has been suggested that PAs that arise in the settingof neurofibromatosis 1 (NF-1) have a less aggressivecourse11,38; however, the data for this suggestion aresomewhat tenuous. Some PAs with unusual histologicfeatures may show early local recurrence or dissemi-nation,1 necessitating adjuvant therapy.8,22 Occasionally,PAs harbor >1 unusual feature, raising the possibilitythat they may be distinct entities or at least variants.Evidence in favor of this suggestion is the recentreclassification of some PAs as pilomyxoid astro-cytoma, a more aggressive variant that carries the WorldHealth Organization grade II designation.30,44 Thesefindings raise the possibility that tumors classified asPA may represent a more heterogeneous group thanpreviously thought.

From the *Departments of Pathology; zNeurological Surgery; #Divi-sion of Hematology-Oncology, University of California San Fran-cisco, San Francisco, CA; zDepartments of Pediatrics; yPathology,King Hussein Cancer Center, Amman, Jordan; wDepartment ofPathology, Dokuz Eylul University School of Medicine, Izmir,Turkiye; and 8Department of Pathology, Uludag University Schoolof Medicine, Bursa, Turkiye.

Conflicts of Interest and Source of Funding: Dr Ersen’s work in thisproject was supported by a Grant from TUBITAK (The Scientific &Technological Research Council of Turkey; grant code 2214). Forthe remaining authors none were declared.

Correspondence: Tarik Tihan, MD, PhD, UCSF School of MedicineNeuropathology Unit, Room M551, 505 Parnassus Avenue, SanFrancisco, CA 94143-0102 (e-mail: [email protected]).

Copyright r 2011 by Lippincott Williams & Wilkins

ORIGINAL ARTICLE

Am J Surg Pathol � Volume 36, Number 1, January 2012 www.ajsp.com | 43

It is critical to recognize the effect of clinicalvariables such as tumor location and age on prog-nosis.19,20,23,35 PAs in eloquent and deep locations presentgreater challenges in terms of accessibility and totalresection. Some studies suggest that tumor location isindependent of the extent of surgery as a prognosticvariable, and PAs in different locations have differentbiological properties.42 This suggestion, although notconclusively validated, may adversely influence the con-clusion of studies that do not account for tumor location.

One additional factor that may confound clinicalstudies is the possibility that pediatric tumors such as PAsin different geographic regions of the globe may havedifferent properties.3 This issue is often confounded bydifferences in expertise, technological and financialresources, and long-term care in different parts of theworld.5 Nevertheless, it is important to determine whetherPAs from different regions are similar enough to beincluded in a single cohort for clinical trials.

Management of PA after a subtotal resection (STR)is also controversial. Even though patients who undergoSTR can have long progression-free survival (PFS) andoverall survival (OS), many oncologists recommendadjuvant therapy. Some tumors show eventual recur-rence, whereas others almost disappear or show noevidence of recurrence for many years.4,13,39 Given thepossibility of “spontaneous regression,” further manage-ment beyond surgery deserves more consideration.Particularly, the increasing knowledge on the value ofthe BRAF/MEK/ERK pathway may shed more light onthe behavior of PA.9

Our study aimed at finding differences and simila-rities from 3 distinctly different regions of the world. Wealso attempted to provide a more detailed histopathologiccharacterization of PAs using markers in the BRAFpathway and also markers of stem cells.

It is not at all possible to find satisfactory answers toall the complex issues in retrospective or single institu-tional studies of rare tumors such as PA, but the studiesgive us targets to investigate in prospective, consortium-based studies. Multinational, multi-institutional studiescan accrue sufficient number of patients in a reasonablyshort period. Through this effort we hope to emphasizethe necessity of organizing prospective collaborativestudies on a global scale and renew a call for cooperationfor such studies.6

MATERIALS AND METHODS

PatientsWe conducted a retrospective review of the pathol-

ogy archives of the University of California, SanFrancisco (UCSF), the Dokuz Eylul University MedicalCenter (DEUMC), Izmir, Turkey, Uludag UniversityMedical Center (UUMC), Bursa, Turkey, and KingHussein Cancer Center (KHCC), Amman, Jordan, forall patients with a diagnosis of PA diagnosed and/ortreated during a 10-year period. Pathology materials fromall surgical procedures were reviewed to confirm diagnosis

and identify sufficient material. The inclusion criteria wereas follows: patients 16 years or younger with a diagnosis ofPA, intracranial tumor location, availability of initialsurgical pathology material, and sufficient initial clinicalinformation. Adults, spinal tumors as well as pure opticgliomas (tumors limited to the optic nerve), patients withdiagnosis other than PA, the pilomyxoid variant ortumors with a pilomyxoid component, cases withoutinitial pathology material or cases with insufficient clinicalinformation were excluded. Medical records for eachpatient were reviewed to determine the sex, age at initialdiagnosis, localization and lateralization of the tumor,type(s) of surgery, date of first recurrence, type and natureof adjuvant therapy, and date of death or last encounter.For the purposes of this study, recurrence was defined asthe emergence of a new radiologically defined lesion orlesions that required subsequent treatment. The date ofrecurrence was taken as the date of radiologic study thatfirst demonstrated newly defined lesions. Information onthe extent of resection was obtained from operativerecords and the neurosurgical report. Wherever available,we also reviewed postoperative imaging records todetermine the extent of resection. Appropriate permissionwas obtained from the institutional review boards of eachparticipating institution. Tissues used in this study wereconsidered excess material and were not required forclinical purposes.

Pathology Material and Tissue MicroarraysAll pathology specimens were reviewed by 2 of the

authors (T.T. and A.E.) to confirm the diagnosis of PA onthe basis of the current World Health Organizationscheme32 and identify appropriate blocks for immunohis-tochemical studies and tissue microarray (TMA) genera-tion. Autopsy and surgical specimens used as controltissue for TMA included developing fetal brain (cerebralcortex and white matter, cerebellum, and germinal matrixfrom autopsy blocks), adult brain (cerebral cortex, whitematter, and cerebellum from autopsies and non-neoplasticsurgical specimens), fetal heart, adult kidney, liver,placenta, ovary, tonsil, and skeletal muscle. In addition,external control tissues suggested by the manufacturerswere used for each specific antibody.

All TMA and whole slide sections were performed atUCSF Surgical Neuropathology Laboratory. TMAs weregenerated from the available blocks of 54 PA cases, 20normal fetal brain tissues, and 10 normal adult braintissues. For each case, a minimum of 2 representative1-mm cores were obtained from the designated areas ofeach available tumor block. The cores were placed intoa recipient block at 2mm intervals in a 5�10 matrix usinga tissue arrayer (Beecher Instruments, Silver Spring, MD).Each TMA block contained 20 samples in duplicate (40cores) and 10 internal controls. For adult brain TMAs, 1-mm cores from the cerebral cortex (3 cores per block),cerebral white matter (3 cores per block), and cerebellum(2 cores per block) were placed in a 5�10 matrix recipientblock. Fetal TMAs were prepared from 15 cases withgestational ages ranging from 20 to 40 weeks. None of the

Tihan et al Am J Surg Pathol � Volume 36, Number 1, January 2012

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adult or fetal brain specimens used had any significantCNS pathology. Whole blocks and all TMAs were seriallysectioned at 4-micrometer thickness for immunohisto-chemical analysis or special stains. Four-micron-thickwhole sections were used for blocks that were not suitablefor TMA.

ImmunohistochemistryImmunohistochemistry conditions were optimized

for each individual antibody using manufacturers’ re-commendations for concentrations and antigen retrieval/blocking. In addition to the internal controls withinTMAs, each staining batch was run with an externalnegative control (no primary antibody), external positivecontrol (known positive tissue as recommended by themanufacturer), and an external biological negative con-trol (tissue known not to demonstrate any positivestaining). The conditions given below represent the rangesused for each step for specific antibodies. In brief,unstained sections from TMAs were allowed to air dryand were baked at 561C overnight. The sections weredeparaffinized in xylene, followed by immersion indecreasing concentrations of ethanol (100%, 70%, 50%,and 30%). Target retrieval was performed using a RussellHobbs programmable pressure cooker (Guilford, CT) at1251F for 5 to 25 minutes. Blocking for endogenousperoxidase activity was performed by 3% H202 treatment

for 10 to 30 minutes, followed by quenching with 0.5%casein solution. To eliminate nonspecific staining causedby endogenous biotin, specimens were blocked with anavidin-biotin blocking system (DAKO Corp, Carpinteria,CA). The sections were then incubated with species-specific secondary antibody kits, and the signal wasdeveloped with diaminobenzidine (DAKO) for 5 minutes.There were two 1-minute wash cycles between each stepusing the DAKO wash buffer solution. Finally, the slideswere counterstained with dilute hematoxylin solution for1 minute, dehydrated, and coverslipped. The primaryantibodies, resources, and final dilutions have been listedin Tables 1 and 2.

Assessment of Cytoplasmic/Membranous StainingWe scored immunohistochemical staining for cyto-

plasmic and membranous antibodies using a semiquanti-tative scale [epidermal growth factor receptor (EGFR),neurofilament, CD34, BRAF, pERK, bcl-2, synaptophysin,vimentin, glial fibrillary acidic protein (GFAP), CD133,p75NTR, and nucleostemin]. By using the areas onthe glass slide that are appropriately stained withthe antibody, and with the aid of positive and negativecontrol stains, we determined the ratio of tumor cellsstaining positive to those staining negative for theparticular antibody. This interpretation did not considerstaining intensity variations among tumor cells but

TABLE 1. Types, Sources, Dilutions, and Staining Results for Cytoplasmic/Membranous Antibodies

Staining

Antigen Dilution Source N 0 1 2 3 4

Bcl-2 1:500 DAKO 42 36 3 3BRAF 1:100 82 27 13 9 33CD133 1:2000 Abcam 61 60 1CD34 1:100 DAKO 65 65EGFR 1:200 DAKO 68 68GFAP 1:5000 DAKO 94 0 2 2 13 77Neu-N 1:2000 Chemicon 51 49 2Neurofilament* 1:20000 Sigma-Aldrich 60 47 13*Nucleostemin 1:200 ProSci Inc. 29 25 4P75NTR 1:100 Abcam 73 72 1pERK 50 mg/mL Zymed 73 46 4 6 17Synaptophysin 1:150 DAKO 90 48 19 10 7 6Vimentin 1:1200 Zymed 91 11 80

*Neurofilament staining was interpreted within the background of the tumors and all positive cases were related to the staining of the background of tumor cellssuggestive of parenchymal infiltration. None of the tumor cells stained positively with this antibody.

TABLE 2. Types, Sources, Dilutions, and Staining Results for Nuclear Antibodies

Antigen Source Dilution N* Mean LI* Median LI* Range

CC3 Cell signaling 1:50 56 5.3% 0.1% 0%-60%Ki-67(MIB-1) DAKO 1:1000 75 2.4% 2.0% 0%-8%Neu-Nw Chemicon 1:2000 51 N/A N/A N/AOlig-2 DAKO 1:250 57 35% 30% 0%-90%p53 protein Santa Cruz 1:200 97 0 0 N/ASox-2 Santa Cruz 1:5000 47 45% 40% 0%-90%

*The numbers in the N column reflect the number of stainings performed and not the total number of positively stained tumors. Please refer to the Results section forthe number of positively stained cases.

wNeu-N staining reflects the staining of trapped neuronal cells in tumor tissue in 2 cases. None of the tumor cells showed Neu-N positivity.

Am J Surg Pathol � Volume 36, Number 1, January 2012 Pediatric Pilocytic Astrocytomas

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considered any staining similar to the staining intensity incontrol cells as positive. On the basis of this interpretation,the results were categorized into 4 groups: (0) negativestaining=no staining in any of the tumor cells; (1)staining in <1 quarter (<25%) of tumor cells; (2) stainingin more than 1 quarter but less than half(25% to 50%) of the tumor cells; (3) staining in morethan half but less than 3 quarters (50% to 75%) of tumorcells; and (4) staining in more than 3 quarters (>75%) oftumor cells. Visual estimation was facilitated by the use ofa microscopic scale that divided the interpretable area ofthe tissue on the slide into microscopic grids.

Assessment of Nuclear StainingStaining of nuclear antigens using antibodies [Sox-2,

Olig2, p53, MIB-1, Neu-N, cleaved caspase-3 (CC3)] wasscored by counting approximately 1000 tumor cells orcells of interest. Scoring was performed at the region ofmaximum staining to achieve the highest possible index.The quality of staining was evaluated in comparison withthe appropriate control tissues.

Fluorescent In situ Hybridization Analysis

1p/19q codeletionAfter an overnight incubation at 561C, unstained

slides were deparaffinized using xylene 3 times each for10 minutes and using 100% ethanol 3 times each for10 minutes. They were placed in 0.2 N HCL/DiH2Osolution at room temperature for 20 minutes. Thereafter,the slides were washed in running tap water for 5 minutesand in distilled water for 3 minutes. They were transferredto citrate buffer (pH 6.0) that contained heat-inducedretrieval solution, treated in a Hobbs pressure cooker for15 minutes, and then cooled down in citrate buffer pH 6.0for 20 minutes. All slides were then washed with runningtap water for 5 minutes and with distilled water for5 minutes. Pepsin digestion buffer 0.4% solution wasstored at 371C for 30 minutes before use. The slides werethen digested in pepsin digestion buffer 0.4% solution at371C for 30 minutes and washed in running tap water for5 minutes, after which they were transferred to 2X SSC(pH 7.0) at room temperature for 5 minutes and air dried.Probe/Denhyb solution was prepared, and the mixturewas applied on the target area. Slides were coverslippedwith Zymed CISH slip. The slides were codenaturated inslide warmer at 901C for 13 minutes. Thereafter, the slideswere hybridized in a humidified slide moat at 371Covernight. The next day, the slips were removed; the slideswere placed in 50% formamide/1X SSC/H2O solution ona rocker at room temperature in the dark for 5 minutesand transferred to fresh 2X SSC/DiH2O at roomtemperature in the dark for 5 minutes. Afterwards theslides were air dried in the dark. A volume of 10mL ofDAPI II LSI counterstain was applied, and the slideswere coverslipped. A fluorescence microscope equippedwith double-band-pass filters (Chroma Technology,Brattleboro, VT) and a �60, numerical aperture 1.3, oil-immersion objective was used for simultaneous visualiza-

tion of 1p/19q. The slides were scored by counting theabsolute numbers of 1p and 19q signals in 100 cells.

BRAF DuplicationDual-color FISH was performed on TMA slides as

previously described.31 Hybridization was achieved usingthe following probes: RP11-355D18 (KIAA) and RP4-726N20 (BRAF), labeled with fluoroisothiocyanate(green) and rhodamine (red), respectively. MetaphaseFISH was performed to verify correct mapping of theclones, and non-neoplastic samples were used as controls.For each hybridization, a minimum of 100 nonoverlap-ping nuclei were assessed for the presence of fused signals,where a positive result was defined as overlapping red andgreen signals resulting in a yellow signal.

Statistical AnalysisStatistical analyses were performed using SPSS

11.0.1 with an advanced statistical package (SPSS Inc.,Chicago, IL). Descriptive data were presented as meanswith standard deviations for variables in which a normaldistribution was presumed or medians with range forvariables not assumed to have a normal distribution.Cross tables were generated to outline the variablesobtained from each institution. Comparisons amongcategorical variables such as sex, extent of surgery,patients with or without radiotherapy, and patients withor without progression were made with either the w2 orthe Fisher exact test. Comparisons among nominalvariables were made with the t test. Survival analysiswas carried out using the Kaplan-Meier procedure, withthe log-rank method to test the difference betweensurvival curves. Multivariate analysis was carried outusing the Cox proportional hazards model. Correlationsof nominal values were analyzed with the Spearmancorrelation test. Results with a P value <0.05 wereaccepted as statistically significant.

RESULTS

Patient CharacteristicsWe identified 116 patients (51 girls, 65 boys) from 4

institutions on the basis of the inclusion criteria. Therewere 68 patients from UCSF, 18 cases from DEUMC, 18cases from UUMC, and 12 cases from KHCC. Pertinentclinical characteristics at the time of diagnosis are listedin Table 3. There were 41 boys and 27 girls with posteriorfossa tumors and 24 boys and 24 girls with supratentorialtumors limited to the optic chiasm and hypothalamicregion. There were insignificant differences among institu-tions in terms of tumor location. The posterior fossatumors were slightly less than twice the number ofsupratentorial tumors at all centers. There were a total of17 patients (10 boys and 7 girls) with NF-1, and 83 patientshad no evidence or family history. NF-1 was confirmed byNational Institutes of Health (NIH) diagnostic criteria, andthe NF-1 status could not be determined in 16 patients.

As detailed below, there were no significantdifferences among the 4 institutions in terms of clinical



presentation, age, NF-1 status, percentage of adjuvanttreatment, and pathologic features analyzed in this study.

The median age at the time of diagnosis for allpatients was 6 years (71.9mo), with a range of 0.2 to 190months (mean, 83.8±50mo). The median age at diagnosiswas 63 months for UCSF patients, 81 months for DEUMCpatients, 90 for UUMC patients and 39 for KHCCpatients. Despite an apparent difference in median agesamong institutions, the differences did not reach statisticalsignificance (P=0.069). There was no statistically sig-nificant difference between male and female patients as wellas NF-1 and non-NF-1 patients in terms of age atdiagnosis. There was no difference in terms of tumorlocation, sex, or extent of surgery between NF-1-associatedtumors and tumors known not to be associated with NF-1.

The presenting symptoms in all institutions weresimilar. Most patients with posterior fossa tumorssuffered gait disturbance and nausea and vomiting atthe time of presentation. Other presenting symptoms forthe entire group included headache (66%), seizures(21%), visual disturbances, lower or upper extremityweakness, and failure to thrive.

A total of 66 patients (57%) underwent STR, and 50patients had gross total resection (GTR). This impressionwas confirmed radiologically in about half of the patients.

The extent of resection was significantly different in 1 of theinstitutions compared with the other 3 (P=0.013). Theextent of resection in the other 3 institutions was similar.

Subsequent to initial surgery, 15 patients receivedchemotherapy, 17 patients received radiation therapy, and13 patients received both modalities. There was nostatistically significant difference among institutions in termsof percentage of patients undergoing radiation treatment(P=0.146) or chemotherapy (P=0.218). We were not ableto obtain specific details about radiation treatment orchemotherapy; thus, a meaningful calculation of possibledifferences in types of adjuvant therapy across institutionscould not be made. The number of boys and girls whoreceived radiotherapy or chemotherapy was the same.

Median follow-up for the entire cohort was 98months. At the end of the follow-up period, 13 patients(11%) were dead, 19 were alive with stable disease, 26 hadno evidence of tumor, and 58 patients were lost to follow-up.

Clinical OutcomeThirty-six patients experienced recurrence, and 78

had no evidence of disease at the end of the follow-upperiod. There were significant differences in terms ofrecurrence among institutions (P=0.017), and therecurrence rates varied from 5.6% to 42.6%. In addition,there were statistically significant differences amongoverall follow-up times from each institution rangingfrom a median of 10 months to a median of 171 months.The differences in recorded recurrence rates were evenmore pronounced after accounting for the differences infollow-up times (P<0.01). The institution with thehighest recurrence rate had the shortest follow-up period.

There was a significant positive correlation betweenthe number of recurrences at 80 months (median follow-up time) and patients younger than 36 months. Therewere 16 patients younger than 36 months, and 13 of thesetumors were located supratentorially. Fifteen of the 16patients underwent STR, and 12 recurred within the first80 months. A correlation could not be made for posteriorfossa tumors because of sample size. A higher number ofrecurrences was seen in children younger than 36 months,in patients with supratentorial tumors, and after STR. Inmultivariate analysis, age younger than 36 months stillremained an independent prognostic factor.

The number of recurrences also varied by tumorlocation and extent of resection. Among 68 posterior fossatumors, 13 recurred during the entire follow-up period,whereas 25 of 48 supratentorial tumors demonstratedrecurrence. Among 68 tumors in the posterior fossa, 12 of29 tumors with STR demonstrated recurrence, whereasonly 1 of 39 tumors with GTR recurred during the follow-up period. Among 48 supratentorial tumors, 22 of 37 afterSTR and 3 of 11 after GTR showed recurrence. There wasa statistically significant difference in the number ofrecurrences between posterior fossa and supratentorialtumors and between STR and GTR. The difference in thenumber of recurrences between posterior fossa andsupratentorial tumors was significant even after account-ing for the extent of resection and follow-up times.

TABLE 3. Pertinent Clinical Features of 116 Patients with PA

Patient Characteristics No. Cases (percentage)

AgeMedian 71.9moMean 83.8mo

SexMale 65 (56%)Female 51 (44%)

LocationPosterior fossa 68 (59%)Supratentorial 48 (41%)

LateralizationRight 35 (30%)Left 40 (34%)Midline 38 (33%)Bilateral 3 (3%)

NF-1No 83 (71%)Yes 17 (15%)Unknown 16 (14%)

Extent of resectionGross total 50 (43%)Subtotal 66 (57%)

RecurrenceNo 78 (67%)Yes 38 (33%)

RadiotherapyNo 86 (74%)Yes 30 (26%)

ChemotherapyNo 88 (76%)Yes 28 (24%)

OutcomeDied of disease 13 (11%)Alive with tumor 19 (16%)No evidence of disease 26 (23%)Lost to follow-up 58 (50%)



Among 68 patients with posterior fossa tumors,there were 4 deaths and 22 patients without any evidenceof residual disease. For supratentorial tumors, thesenumbers were 9 and 4, respectively. These differences werestatistically significant on univariate analysis (P=0.007).However, on multivariate analysis, the differences inoutcome between posterior fossa and supratentorialtumors became insignificant (P=0.108). Among supra-tentorial tumors, deaths and persistent disease were seenalmost exclusively in the STR group (17 occurrences afterSTR vs. 0 after GTR). There was no difference betweenboys and girls in terms of recurrence or outcome.

Survival characteristics of the study group revealeda number of interesting features. First, there was astatistically significant positive correlation between PFSand OS in the whole group (P<0.001). The mean OS was271.4 months, and the mean PFS was 137.6 months,based on Kaplan-Meier analysis. There was a significantdifference in PFS between posterior fossa and supraten-torial tumors (Fig. 1A). PFS was also significantlydifferent among institutions even though this calculationwas partly limited by the high number of censored casesin 2 of the institutions (Fig. 1B).

PFS was significantly different between tumorsundergoing STR or GTR (STR mean 95mo vs. GTRmean 190mo; log rank P<0.001; Fig. 1C). This differencewas noted even when posterior fossa or supratentorialtumors were analyzed separately. When all tumors withSTR were selected, supratentorial tumors had a slightlyshorter PFS as opposed to tumors in the posterior fossa inunivariate analysis (log rank P=0.0425). However,tumor location was found to be insignificant on multi-variate analysis.

PFS was not influenced by sex or NF-1 status(P=0.654; Fig. 1D). As we had relatively few NF-1cases, we cannot exclude the possibility that NF-1 statusmay influence PFS in a sufficiently large cohort.

One of the interesting findings of the study was thestatistically insignificant negative impact of initial che-motherapy and radiotherapy on PFS after STR. PFS wasshorter in patients who received adjuvant therapycompared with patients who did not undergo adjuvanttherapy when only tumors after STR were considered.This difference could be found for both supratentorialand posterior fossa tumors. However, this statisticallyinsignificant difference probably reflected limited numbers

FIGURE 1. Kaplan-Meier survival analysis of PFS and various prognostic factors in PAs: (A) tumor location; (B) original institution;(C) extent of surgery; (D) NF-1 status.



as opposed to a negative effect of these treatmentmodalities on tumors with STR.

PFS was also calculated for the whole group at80 months (the mean follow-up period) to account fordifferences in follow-up periods among patients andinstitutions, but the results in terms of statistical signifi-cance were essentially identical. When this cutoff point wastaken, the statistical significance of analyses did not changefrom the number obtained using the entire follow-upperiod. This was predominantly because of the fact thatthe majority of recurrences or adverse effects occurredbefore the mean value of 80 months in the early part of thefollow-up period, with few cases of recurrences later in thecourse. The results of univariate and multivariate analysesfor prognostic factors are presented in terms of PFSin Table 4.

Histopathologic FeaturesAll tumors were reviewed by 2 of the authors to

confirm diagnosis and record histologic features. Themajority (57%) of PAs showed a biphasic pattern(Fig. 2A). All tumors except 1 demonstrated eitherRosenthal fibers or eosinophilic granular bodies. In 50cases (43%) there was focal reactive vascular proliferation,often in a linear arrangement, and also granulation tissue-type hypervascularity (Fig. 2B). Extensive vascularhyalinization was also seen in some foci (Fig. 2C).Thirty-four tumors (29%) had conspicuous pleomorphiccells noted in more than 1 focus (Fig. 2D). Forty-twotumors (36%) demonstrated focal or prominent oligoden-droglioma-like areas, and the majority of these tumorswere located in the posterior fossa (Fig. 2E). Mitoticfigures were extremely rare, and the indices were typically<2 per 10 high-power fields in all tumors. Extensivecalcification was seen in a smaller number of tumors(12%) and was occasionally in the form of psammomabodies (Fig. 2F). Anaplastic features were not seen in anyof the tumors. There were no significant differencesbetween posterior fossa and supratentorial tumors interms of harboring nuclear pleomorphism, vascularproliferation, oligodendroglioma-like features, or Ro-senthal fiber formation. The prevalence of the histologicfeatures was also similar in all 4 institutions. Furthermore,tumors from NF-1 patients were not histologicallydifferent from those of non-NF-1 patients.

None of the histologic features analyzed in thisstudy correlated with the outcome measures or clinicalcharacteristics of tumors in this study.

Immunohistochemical FeaturesThe spectrum of immunohistochemical reactivity is

described with respect to individual antigens below. Theoverall staining percentages are presented in Table 1 forcytoplasmic/membranous stains and in Table 2 fornuclear stains.

Glial Markers (GFAP, Olig-2, and Vimentin)As nonspecific markers of glial lineage cells, GFAP

and vimentin were positive in all tumors. Vimentinwas diffusely and strongly positive, whereas GFAP wasvariable. Most tumors had strong GFAP positivity, butin occasional tumors the staining was patchy and focal.Overall, 90 tumors demonstrated category 3 and 4staining. The remaining 4 tumors had either category1 or 2 staining patterns. None of the tumors tested wasentirely negative. Thus, a negative GFAP stain shouldlead to suspicion of the staining results or to a diagnosis ofPA. All tumors stained with the Olig-2 antibody (n=45)showed nuclear positivity in the majority of tumor cells(Fig. 3A).

Synaptophysin, Neurofilament Protein, and Neu-NThe majority of tumors did not show any positivity

with the neuronal markers synaptophysin, neurofilamentprotein, or Neu-N. However, in a small subset of tumors,neurofilament protein was considered to be positivewithin the tumor tissue (Fig. 3B). Neurofilament positiv-ity was recorded in 13 tumors in a category 1 stainingpattern. In most cases this was attributed to neuropiltissue incorporated within the tumors, which made suchtumors appear somewhat infiltrative. Neu-N positivitywas detected in 2 cases focally, and this was alsoassociated with the trapped normal neurons. Thus, mostneuronal staining was interpreted as trapped or incorpo-rated neuropil. Synaptophysin was the only neuronalmarker that was detected in 23 tumors, and some tumorcells demonstrated unambiguous staining (Fig. 3C). Thesynaptophysin positivity was due to adjacent or incorpo-rated neuropil in another 19 cases. All tumors withneurofilament positivity also demonstrated some type ofsynaptophysin positivity.

EGFR and p53 ProteinP53 protein or EGFR immunopositivity, which are

the most common detectable genetic alterations seen inthe infiltrating astrocytomas, were not detected in any ofthe cases tested.

CC3 and bcl-2Focal and isolated tumor cell staining was seen in 23

cases with the CC3 antibody (Fig. 3D). In these cases, thepercentage of tumor nuclei was <8%. In 4 other casesCC3 showed significant staining in 20% (2 cases) and

TABLE 4. Influence of Factors on PFS in PAs

Variables Univariate Analysis Multivariate Analysis

Age younger than 36mo <0.001 0.004*Institute of origin 0.017 0.031*NF-1 status 0.650Tumor location 0.007 0.108Extent of surgery <0.001 0.012*Ki-67 (MIB-1) LI 0.021 0.340BRAF status <0.01 0.181

*Statistically significant factors after multivariate analysis.



40% (2 cases) of tumor cells. In 3 patients with increasedCC3 staining there was a history of radiation orchemotherapy. There was no correlation between age,tumor location, or BRAF status and CC3 staining.

Six tumors showed focal positivity (category 1 and 2)with the bcl-2 antibody (6 of 42; Fig. 3E). Another 36 tumors

showed no positive staining with this antibody. None of thebcl-2-positive tumors had BRAF positivity. The tumorswith bcl-2 positivity did not appear to act moreaggressively than others, but the numbers were too smallto make a conclusive statement or to further characterizethe significance of this staining in our cases.

FIGURE 2. Histologic features seen in PAs. There was no significant correlation between these features and clinical outcomeparameters. A, Typical biphasic pattern; (B) vascular changes in the form of reactive-type vascular proliferation; (C) focal, extensivevascular hyalinization; (E) oligodendroglioma-like pattern; (F) extensive calcifications.

FIGURE 3. Immunohistochemical staining results in PAs. A, Nuclear staining with Olig-2 antibody; (B) neurofilament staining;(C) synaptophysin positivity in tumor cells; (D) nuclear staining for CC3; (E) staining for bcl-2, (F) nuclear staining for Sox-2antibody.



Ki-67 (MIB-1 Antibody)There was substantial variation among tumors and

also within the same tumor for Ki-67 labeling using theMIB-1 antibody. Using the method outlined above, wefound the Ki-67-labeling index to range between 1% and8%. The mean labeling index for the entire group was2.3% (standard deviation±1.9). There was no significantdifference among institutions or between boys and girls.In addition, there was no difference between GTR andSTR, between adjuvant therapy and no adjuvant therapy(radiotherapy or chemotherapy), or between BRAF-positive and BRAF-negative tumors. There was astatistically significant difference in Ki-67 labeling be-tween recurrent tumors and nonrecurrent tumors duringthe entire follow-up period (P=0.021). This significancewas not found on multivariate analysis.

CD34Only endothelial cells in the tumors were immuno-

reactive with CD34. None of the tumors had CD34-positive cells.

NucleosteminFour tumors in the posterior fossa showed category 1

positivity with the nucleostemin antibody. This antibodycould be evaluated in 25 other tumors, including 11supratentorial tumors, none of which showed any positivity.

SOX-2SOX-2 was positive in more than 10% of tumor

nuclei in 42 cases (Fig. 3F). Three tumors showed lowerpercentage of staining, and 2 were negative.

P75NTROnly 1 of the 73 tumors tested with the P75NTR

antibody showed category 1 staining.

CD133CD133 antibody positivity (category 1) was seen in

only 1 of the 76 tumors in which this staining wasperformed. This tumor was a supratentorial PA.

BRAFBRAF staining was performed in 82 tumors (48

posterior fossa and 34 supratentorial tumors) and was incategory 2 or 3 for 42 tumors (Figs. 4A-C). Nine tumorsin the posterior fossa and 18 supratentorial tumors werenegative with the antibody. Category 1 staining was alsopresent in normal CNS tissue, including the cerebellum.There was a strong correlation between BRAF positivityand tumor location in the posterior fossa (P<0.005).There was also a significant negative correlation betweenBRAF positivity and PFS within the first 80 months(P<0.01). This negative correlation was found forsubtotally resected tumors and posterior fossa tumorsbut not for others. This correlation was not significant in

FIGURE 4. Immunohistochemical staining of PAs using the BRAF and pERK antibodies. BRAF staining in (A) case 2 in which FISHanalysis demonstrated duplication and in (B) case 14 in which no BRAF duplication was found. C, The control section from thecerebellum demonstrates weak background staining. pERK staining in (D) case 2 and (E) case 14. pERK staining is similar to BRAFstaining in these cases. F, Control for pERK.



multivariate analysis. There was a positive correlationbetween BRAF and pERK staining, but no other positivecorrelation was found with the other antibodies tested.

pERKStaining was performed in a subset of tumors for

which material was available (73 cases). Staining waspredominantly cytoplasmic and occasionally nuclear,especially in tumors with strong positivity (Figs. 4D-F).Twenty-seven tumors showed category 1 to 3 positivity,whereas 46 tumors were negative. Among the positivetumors, 23 had BRAF duplication by FISH analysis.FISH analysis was not performed in 4 of the pERK-positive tumors. BRAF status was available for only 15 ofthe pERK-negative tumors, and 8 of these tumors hadBRAF duplication. There was a strong correlationbetween pERK positivity and BRAF duplication byFISH (P<0.0001). Similarly, there was a strong positivecorrelation between pERK staining and posterior fossalocation (P<0.001).

FISH FindingsResults for BRAF duplication were available for

49 tumors. Of these tumors, 41 demonstrated duplication,and in 8 tumors there was normal signal. The study couldnot be conducted in the remaining 67 tumors. Thirty-oneof the positive tumors were in the posterior fossa, whereasthe other 10 were supratentorial. There was a significantpositive correlation between posterior fossa tumors andBRAF duplication (P<0.0001). There was a positivecorrelation between BRAF immunohistochemistry andFISH results, but 5 tumors that had duplication on FISHwere negative on immunohistochemistry, and 3 tumorswith positive immunostaining were negative for duplica-tion on FISH. The remaining 38 tumors were concordant.As expected, none of the tumors tested demonstratedlosses or gains in chromosomes 1p and 19q on FISH.Analyses did not reveal any significant positive correla-tions between other immunohistochemical stains testedwith BRAF FISH results.

DISCUSSIONOne of the critical conclusions of our study is the

similarity of PAs from 3 countries in terms of demo-graphic, histopathologic, and molecular features. Ourstudy also confirms the critical importance of the extentof surgery as a determinant of PFS. This finding has beenreported by many other studies, and the recurrence rate inPA in any location is highly dependent on the success ofsurgical resection.19,29 In our study, PFS and OS rateswere positively affected by GTR. The other importantfactor affecting the extent of surgery is tumor location,which at first glance seems to influence survival. However,when the study group was controlled for extent ofresection, tumor location had less influence on PFS. Allof our patients with supratentorial tumors had tumors inthe hypothalamic and chiasmatic region, and the recur-rence rate in this group was higher compared withposterior fossa tumors (52% vs. 19%; P<0.001). This

difference was also observed among tumors that under-went GTR: only 1 of 39 posterior fossa tumors and 3 of11 supratentorial tumors showed recurrence after a GTR.

One critical distinction in terms of tumor location isbetween PA of the anterior visual pathways and tumorsthat involve the hypothalamic/chiasmatic pathway. Thereis substantial evidence since the earlier publications ofBorit and Richardson4 to prove that the PAs affecting theoptic nerve and the orbit may be different from those inthe hypothalamic/chiasmatic region, which have a higherrate of local recurrence.45 There have been numerousanecdotal remarks in neuropathology practice thattumors in these 2 locations also “look” differenthistologically, but a clear account of this distinction isdifficult to come across. Our study did not include anyoptic nerve tumors and may therefore not be comparableto other reports of PAs of the visual pathway that includeboth locations.

The negative correlation between patient age andsurvival probability has been reported in a number ofstudies,27 and this finding was also confirmed in ourstudy. There was a significant difference in PFS betweenthe lowest and the highest quartile of age groups in thisstudy. Patients younger than 36 months at the time ofsurgery also had significantly poorer outcome comparedwith older children. This fact is often considered to be animportant determinant in treatment protocols.

Another finding that creates practical challenges inthe management of PA is the different outcomes we haveobserved in different institutions. Although the resultsshould be interpreted with caution because of the limitednumber of patients in some of the institutions, thecalculated differences were statistically significant. Thisfinding supports the assertion that the treatment outcomeis dependent on the location of care, and access to moreadvanced care can pose significant disparities.24 There isno doubt that improved healthcare standards and ease ofaccess to healthcare are strong determinants of treatmentsuccess and survival parameters. Nevertheless, it is alsoquite possible that biological differences may exist evenbetween tumors of the same histologic type in differentparts of the world. This difference can present significantchallenges to multi-institutional trials and to interpreta-tion of data from different geographical regions.

Recent studies have emphasized the importance oftumor location in terms of molecular alterations. It hasbeen suggested that PAs in different locations may berelated to different cells of origin.21,41,42 An interestingfinding in our study is the presence of BRAF duplicationspredominantly in PAs of the posterior fossa. Althoughsome supratentorial examples also harbored this geneticabnormality, the duplication was more characteristic inthe posterior fossa. Similarly, immunohistochemicalstaining with the pERK antibody also correlated withthe posterior fossa tumors. All these findings suggest thatthe classical posterior fossa PA is a tumor that typicallyharbors the duplication of the BRAF gene, and thisduplication is associated with activation of the RAF/MEK/ERK pathway. This is in accordance with earlier



observations both by our group and by others.2,21

Although it was possible to immunohistochemicallyanalyze BRAF in a retrospective study, it is unlikely thatthis stain can be of practical utility in diagnosis. Themajor reason for this is the low-level positivity in manyCNS tumors and normal tissues. Thus, there is only amoderate qualitative difference in immunopositivitybetween classical PAs, normal tissues, and other neo-plasms that do not harbor BRAF alterations. Theconsistent finding of stronger staining of posterior fossatumors compared with other locations is merely circum-stantial evidence and not a definitive statement on thepresence of BRAF duplications in PAs.

BRAF alterations are the only molecular defectsthat have been detected in PA in a reproduciblemanner.2,26,28,31,43 The activation of the MEK/ERKsignaling pathway has been shown to cause oncogene-induced senescence (OIS).9 OIS is defined as an apparentlypermanent growth arrest initiated by oncogene activa-tion.18 It is thought to occur in melanocytic nevi34 and inother low-grade neoplasms with BRAF mutations.

BRAF alterations in PAs present 2 critical issues thatrequire further investigation. First, BRAF duplication,downstream activation of ERK, and absence of significantcell proliferation suggest that some PAs may be senescent.This supposition is in keeping with the historical reports ofPA that remain dormant or undergo involution afterSTR.39 In addition, a recent study by Raabe et al36

provided further in vitro and in vivo evidence thatinduction of senescence as defined by increased expressionof p16INK4 could explain the indolent behavior of PA. IfOIS plays a role in PA biology, it may explain the clinicaldormancy of some tumors, and it is possible to suggest thattumors with BRAF alterations are not likely to createsignificant problems even after STR.

A second and therapeutically relevant issue involvesthe use of radiation treatment in PAs that harbor BRAFduplications. In experimental systems where BRAFmutation induces senescence, mutation of tumor suppres-sors such as PTEN, TP53, or p16INK4a causes malignantand metastatic tumors presumably through abrogation ofOIS.14,15 It is therefore plausible that additional muta-tions caused by radiation-induced DNA damage mayabrogate senescence in PAs, inducing a more aggressivebehavior. Reported cases of malignant transformation ofPAs after partial resection and radiation treatmentprovide circumstantial support for such a mechanism.46

Although it is well known that radiation can result insenescence and increased apoptosis in many cellularsystems,12 it is not clear how this effect is modified inthe presence of BRAF alterations. Recent evidence alsosuggests that PAs that can escape OIS have worse clinicaloutcomes compared with PAs that show evidence ofOIS.36 We submit that until there is a better under-standing of the biological nature of PAs with BRAFduplications it is wise to be cautious about radiationtreatment of subtotally resected tumors.

A few additional points require special attention interms of the pathologic characteristics of PA. First, the

strong positivity of all tumors with GFAP and nuclearstaining with Olig-2 makes these 2 stains important whendetermining the suitability of tissue for further analysis.Negative staining of both of these stains in an alleged PAsuggests either poor tissue preservation or misdiagnosis.Another important finding is the frequent immunoposi-tivity of this tumor with synaptophysin, which has beenwell known but rarely reported. We believe that thispositivity does not imply neuronal differentiation and istestament to the practical unreliability of the antibodiesto identify neuronal cells. Therefore, conclusions ofstudies in the literature based on synaptophysin stainingalone should be viewed with skepticism.

We also found small numbers of CC3-positiveand bcl-2-positive tumors, which prevented us fromfurther correlating markers of apoptosis to suggestwhether such markers acted in opposition to BRAFduplication and activation of OIS. There is evidence thatBRAF duplication and subsequent OIS can interact withbcl-2 and other regulators of apoptosis.9,17,37 It isinteresting to speculate that tumors expressing bcl-2 orthose with increased CC3 activity may have differentbiological behavior or treatment response to the effects ofBRAF duplication.

We were unable to find some of the immunohisto-chemical correlations we hoped to find in PAs based onearlier observations or reports. First, the putative stem cellmarkers CD34, nucleostemin, p75NTR, and CD133 wereoverwhelmingly negative in PAs. This can be considered aproblem of immunohistochemistry and may not accu-rately reflect the expression levels, but our findings argueagainst any practical value of these markers.

Among the stem cell markers tested, we were onlyable to identify significant immunopositivity with the Sox-2 antibody. The results of stem cell marker expression onparaffin-embedded material should always be interpretedwith caution. The absence of some markers previouslyreported as positive in other astrocytomas could implythat immunohistochemical studies may underestimate thepresence of these markers. There is still much to be doneto characterize PAs in terms of their cells of origin and thestem cell markers they express. Currently, the significanceof finding a positive expression of such markers is of littlepractical benefit. Specifically, it will be important to studythe significance of Sox-2 positivity.

Our results provide further proof of the criticalimportance of age, tumor location, and extent of resectionin PAs and also suggest that PAs in different regions of theworld have different prognostic characteristics. The latteris more likely to be due to healthcare disparities but mayalso reflect genetic or environmental variations in differentpopulations. It is important to consider this difference infuture multi-institutional studies.

Correlation of the histologic features of PA withoutcome revealed some significant associations in uni-variate but not multivariate analysis. It appears thatcommon and rare histologic features were helpful only inconfirming the diagnostic category as PA. Our study alsoconfirms the significance of BRAF gene duplications in



PAs of the posterior fossa and suggests that thisduplication has important consequences in subtotallyresected tumors treated with adjuvant therapy. Thesignificance of Sox-2 staining in PAs is currently unclearin terms of the cell of origin.

In summary, we believe that the recognition ofpertinent diagnostic and prognostic factors will allow usto understand the biology of PA and select bettermanagement strategies. Further progress is best achievedthrough multi-institutional, collaborative studies that canaccurately characterize clinical and genetic aspects ofpathologically well-characterized PAs. There is a direneed for such large-scale, multinational consortia toresolve unanswered questions about PAs, and collabora-tive efforts are likely to provide satisfactory answers tosome of these questions.

ACKNOWLEDGMENTSThe authors thank Ms Gretchen Werner for her

assistance in every step of this study and Mr RodneyCollins for the generation of TMAs and superb technicalassistance. They are also deeply grateful to Dr Arie Perryfor allowing them to perform the BRAF analysis in hislaboratory and to Dr Anthony Karnezis for reviewing thearticle and for helpful suggestions.

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