Photoimmunotherapy and Irradiance Modulation Reduce Chemotherapy Cycles and Toxicity in a Murine Model for Ovarian Carcinomatosis: Perspective and Results

DOI: 10.1002/ijch.201200016

Photoimmunotherapy and Irradiance Modulation ReduceChemotherapy Cycles and Toxicity in a Murine Model forOvarian Carcinomatosis: Perspective and ResultsImran Rizvi,[§, a] Tri A. Dinh,[§, a, b] Weiping Yu,[a] Yuchiao Chang,[c] Margaret E. Sherwood,[a] andTayyaba Hasan*[a, d]

1. Introduction

Overview

This article presents our findings on epidermal growthfactor receptor (EGFR)�targeted treatment of dissemi-nated ovarian cancer using photoimmunotherapy (PIT) incombination with a conventional chemotherapeutic cock-tail (Figure 1). In keeping with the spirit of the specialissue, this article is a combination of a review and originaldata. To orient the reader, we present a relatively com-prehensive discussion of the key barriers to advancedovarian cancer treatment and the role that photodynamictherapy (PDT) might play in the management of this dis-ease. To put PDT and PIT for ovarian cancer in context,we start with a broad overview of current managementstrategies, followed by a discussion of the limitations ofchemotherapy and drug resistance. We then transition toa discussion as to why PDT and PIT might be logical mo-dalities to complement traditional management strategies.

Background and Perspective: Towards Improved Managementof Ovarian Cancer with Targeted Photodynamic Therapy�BasedCombinations

Approximately three quarters of the 140,200 new cases ofepithelial ovarian cancer estimated worldwide[1] will bediagnosed after the disease has metastasized to pelvic

sites or beyond the peritoneal cavity.[2–4] The dissemina-tion pattern for advanced-stage ovarian cancer is charac-terized by metastases to vital peritoneal organs, as well asthe mesothelial lining of the peritoneum, omentum, mes-entery, and diaphragm.[3,5] The grueling toxicities and per-sistently poor outcomes associated with conventionaltreatments for this diffuse metastatic disease[6,7] empha-

Abstract : Significant toxicities from multiple cycles of che-motherapy often cause delays or early termination of treat-ment, leading to poor outcomes in ovarian cancer patients.Complementary modalities that potentiate the efficacy oftraditional agents and lead to fewer cycles and less toxicityare needed. Photodynamic therapy is a mechanistically dis-tinct modality that synergizes with chemical and biologicalagents. Here, a combination regimen with a clinically rele-vant chemotherapy cocktail (cisplatin and paclitaxel) and ep-idermal growth factor receptor�targeted photoimmunother-apy (PIT) is evaluated in a murine model for ovarian carci-

nomatosis. Mice received either one or two cycles of chemo-therapy followed by PIT with a chlorine6-Erbitux photoimmu-noconjugate and 25 J/cm2 light. PIT plus a single cycle ofchemotherapy significantly reduced tumor burden, compara-ble to multiple chemotherapy cycles. Relative to one cycle ofchemotherapy, the addition of PIT did not cause significantmouse weight loss, whereas two cycles of chemotherapy ledto a significant reduction in weight. Irradiance-dependenceon PIT efficacy was a function of the conjugation chemistry,providing an additional variable for optimization of PIT out-come.

Keywords: antitumor agents · cancer · epidermal growth factor receptor · photodynamic therapy · photoimmunoconjugates

[a] I. Rizvi, T. A. Dinh, W. Yu, M. E. Sherwood, T. HasanWellman Center for PhotomedicineDepartment of DermatologyMassachusetts General Hospital, Boston, MA (USA)e-mail: [email protected]

[b] T. A. DinhGillette Center for Gynecologic OncologyMassachusetts General Hospital, Boston, MA (USA)

[c] Y. ChangGeneral Medicine DivisionMassachusetts General Hospital, Boston, MA (USA)

[d] T. HasanCorresponding author:Professor of DermatologyWellman Center for Photomedicine (Bartlett 314)Harvard Medical School, Massachusetts General Hospital40 Blossom Street, Boston, MA 02114 (USA)phone: +001617 7266996

[§] Equal contribution

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size the need for targeted and rationally designed combi-nation regimens that improve the therapeutic index ofconventional therapies.[2,4,8–14]

Following initial surgical staging and operative debulk-ing, the standard of care for advanced-stage ovariancancer has typically involved radiotherapy or intravenousadministration of a chemotherapeutic cocktail of a plati-num agent (cisplatin or carboplatin) and a taxane (pacli-taxel or docetaxel) for up to six treatment cycles.[15] Thisapproach has modestly improved initial response rates,but the disease recurs in 80 % of patients.[4,16] In recentyears, intraperitoneal chemotherapy has been shown toimprove both overall survival and progression-free surviv-al by 20–30% in patients with optimally debulked ad-vanced-stage disease.[15,17,18] One of the key studies dem-onstrating this clinical benefit was GOG 172, conductedby Armstrong et al.[17] Based on this study and others,[15,18]

the National Cancer Institute issued a clinical announce-ment in January 2006 encouraging the use of intraperito-neal cisplatin in patients with optimally cytoreduced ovar-ian cancer.[15] Regardless of the route of administration,treatment-related toxicities and complications limit thenumber of cycles and chemotherapeutic dose intensitythat can be delivered to patients, both of which areemerging as potentially important determinants of treat-ment efficacy in ovarian cancer.[19,20]

Chemotherapy Cycles, Dose Intensity, and Treatment Failure

Recent articles by Fauci et al.[20] and Yen et al.[19] highlightthe prognostic significance of evaluating survival in ovari-an cancer patients within the context of relative dose in-tensity of chemotherapy and the number of deliveredcycles. Relative dose intensity (defined in Eq. 1) is a well-established prognostic indicator for breast cancer andlymphoma, but has not yet been well studied for ovariancancer.[20]

Relative dose intensity ¼ Delivered dose intensity

ðtotal delivered dose=actual time to complete treatmentÞ=Standard dose intensity ðstandard dose=

planned time to complete treatmentÞ ð1Þ

A retrospective analysis of outcomes in chemotherapy-na�ve patients treated with platinum and taxane agentsrevealed that an incomplete or protracted treatmentcourse predicts a poor prognosis and that an optimal ther-apeutic window is critical to providing meaningful im-provements in survival. The essentials of this conclusionwere confirmed in a similar study by Yen et al.[19] The rea-sons for treatment delays or early termination were pri-marily related to the significant toxicities and poor per-formance scores associated with each additional cycle ofchemotherapy.[17,19,20] These findings highlight the criticalneed to identify complementary therapeutic modalitiesthat potentiate the efficacy of chemotherapeutic agentsand improve outcomes with fewer cycles, lower dose in-tensities, and less treatment-related toxicity.

Another reason for the high rate of treatment failureassociated with the standard management of ovariancancer is innate and acquired chemoresistance, which isdriven by a variety of mechanisms.[2,4,11,21–24] Chemothera-pies, with their spectrum of cellular and molecular targets,rely primarily on proliferating cells to be effective.[4]

However, even in rapidly proliferating tumors, a signifi-cant number of cancer cells are quiescent, which confersresistance.[4,24,25] Other key determinants of treatment re-sponse include drug pharmacokinetics and pharmacody-namics, the influence of the tumor microenvironment,and inherent or acquired somatic mutations and epigenet-ic changes that cause chemotherapeutics to fail (e.g., al-terations in DNA repair machinery, increased activity ofdrug inactivation enzymes, upregulation of antiapoptoticproteins such as Bcl-2/Bcl-XL, downregulation of proa-

Figure 1. Orthotopic mouse model for disseminated ovarian cancer and experimental schema. A mouse model for intraperitoneal ovariancarcinomatosis (A), which mimics the complex dissemination pattern of advanced-stage disease, was used to evaluate the efficacy of EGFR-targeted PIT in combination with chemotherapy (B). The impact of irradiance (30 or 180 mW/ cm2) and conjugation chemistry (direct or in-direct PIC) on PIT tumoricidal efficiency was determined. For the combination treatment, mice received either one or two cycles of a clinical-ly relevant chemotherapy cocktail (cisplatin and paclitaxel) followed by a single PIT treatment, and acute tumor burden was evaluated.

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poptotic proteins such as BAX/BAD).[2,4,11,21–23] Overcom-ing these complex resistance mechanisms requires newtherapeutic approaches, which target molecular pathwaysthat are mechanistically distinct from traditional chemo-therapies and cooperatively enhance the efficacy of thesedrugs.[4,6,11,16,21]

Targeted Inhibitors and the Epidermal Growth Factor Receptor

Early indications from preclinical and clinical studiesusing targeted inhibitors in combination with chemother-apy suggest modest, but promising, improvements in out-come compared to standard clinical regimens. The targetsfor these ongoing or recently completed studies includeinhibitors for vascular endothelial growthfactor,[10,11,16,26–32] the folate receptor,[10,11,33–36] the src onco-gene,[11,37] the mTOR/PTEN/PI3K/Akt pathway,[10,11,38–40]

platelet-derived growth factor,[10,32,41–43] poly(ADP-ribose)polymerase,[10,11,44] and the EGFR, an important prognos-tic indicator and therapeutic target in ovariancancer.[10,11,30,45,46]

The EGFR is a member of the ErbB tyrosine kinasefamily and plays a key role in normal ovarian follicle de-velopment.[47] Dysregulation of this pathway increases thegrowth potential of normal ovarian surface epitheliumand contributes to malignant transformation of this peri-toneal lining.[48–50] Studies by Siemens et al.[48] andothers[49,51–55] support the concept that EGFR activationstimulates epithelial-mesenchymal transition�associatedevents in ovarian cancer cells, including disruption of E-cadherin junctions, increased production of matrix metal-loproteinases, and a higher potential for migration and in-vasion. In ovarian tumors, overexpression of EGFR genecopies and protein levels is associated with a high tumorgrade and large residual tumor size, both of which prog-nosticate poor outcomes.[56] Indeed, Psyrri et al. showedthat high EGFR expression in clinical ovarian cancersamples is the most significant prognostic indicator ofpoor overall and disease-free survival.[57] These findings,along with additional studies by our group[12,58–60] andothers,[10,61–65] establish the value of the EGFR as a thera-peutic target. It now remains to be demonstrated howbest to exploit this high value target.

The vast majority of EGFR inhibitors under clinicalevaluation fall into one of two categories: (i) small-mole-cule tyrosine kinase inhibitors (TKIs) that block receptorphosphorylation, or (ii) monoclonal antibodies (mAbs)that inhibit ligand binding and prevent receptor dimeriza-tion. Erbitux (cetuximab) is a clinically approved chimer-ic mouse-human antibody directed against the EGFR. Asdescribed by Li et al.,[66] the epitope for Erbitux residesin domain III of the EGFR, which overlaps with theligand-binding region of the receptor. Interaction of theantibody with the EGFR, therefore, primarily blocksligand binding and prevents the formation of a ligand-sta-bilized extended conformation that is critical to receptor

dimerization.[66] This receptor blockade prevents theEGFR from being activated and leads to induction ofp27, a tumor-suppressor protein that arrests cellulargrowth in the Gap 1 phase.[67,68]

Several anti-EGFR TKIs and mAbs, including Erbitux,have by themselves shown modest clinical benefit and areapproved for the treatment of solid tumors.[10,49,69,70] Thereare also significant toxicities associated with targeted in-hibitors, including anti-EGFR agents.[10,49,69] None of theseagents have performed well enough against ovariancancer to be clinically feasible as monotherapies.[2,4,10,12,49]

Collectively these findings suggest that, as with most anti-cancer therapies, targeted inhibitors are most likely to besuccessful as part of rationally designed combination regi-mens.[71] Our hypothesis has been that the best combina-tions might be those that synergize by acting along nono-verlapping pathways of tumor growth and proliferation,where photodynamic therapy might play a uniquerole.[12,72–74]

Photodynamic Therapy

PDT is a biophysically driven cytotoxic modality that ismechanistically distinct from traditional therapies and hasbeen shown by us and others to reverse chemoresistanceand synergize with chemical and biological agents for thetreatment of ovarian cancer.[12,14,74–79] PDT is based on theactivation of a photosensitizer by light of a specific wave-length to photochemically generate active molecular spe-cies that are locally cytotoxic and have the potential toelicit systemic antitumor effects.

We[12,14,74] and others[80,81] have shown that PDT enhan-ces the efficacy of chemotherapeutics and targeted bio-logics in ovarian cancer. We reported a synergistic en-hancement of Erbitux efficacy by verteporfin-PDT ina clinically relevant mouse model for advanced-stageovarian cancer.[12,82] A greater than 90% reduction inacute tumor burden was observed in mice treated withthe Erbitux plus PDT combination. This dramatic reduc-tion in tumor burden was synergistic relative to the mon-otherapies, and had previously been a difficult result toachieve in this advanced-stage model.[83] Similarly, a syner-gistic enhancement in survival relative to the controls wasobserved in the group that received the Erbitux plus PDTcombination. Four mice remained alive until the end ofthe survival study (day 180 after tumor cell implantation).Three of these four surviving mice received the Erbituxplus PDT combination and did not show evidence ofgross residual disease. All other animals in the study hadvisible tumors present at the time of necropsy.[12] Becausethe Erbitux plus PDT combination regimen potentiatedthe efficacy of the individual modalities, a dramatic re-duction in tumor burden was achieved with fewer PDTtreatments and lower toxicity than had been reportedpreviously by Molpus et al.[83] PDT has also been shownto enhance the efficacy of chemotherapeutic agents. The

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Photoimmunotherapy and Irradiance Modulation Reduce Chemotherapeutic Toxicity


optimal parameters for the interaction between these mo-dalities are dependent on the photosensitizer and chemo-therapeutic agent used.[80] We have shown that PDT de-creases the size and disrupts the structure of ovarian mi-cronodules, and synergizes with carboplatin in a 3Dmodel for micrometastic ovarian cancer.[14,84] These stud-ies have collectively provided promising insights into theuse of PDT to enhance the efficacy of conventional bio-logic and chemotherapies.

Photoimmunotherapy

Motivated by our findings with combinations of PDT andanti-EGFR therapy,[12] we considered delivering the com-bination as a single agent by chemically conjugating thephotosensitizer chlorine6 (Ce6) to Erbitux. The advantageof this approach would be the additional selectivity of thephotosensitizer delivery. This is important because, aswith other treatment modalities, tumor selectivity remainsa key issue in PDT, particularly in complex treatmentsites such as the peritoneal cavity.[85,86] Modest preferen-tial accumulation of photosensitizers in neoplastic tissuehas been demonstrated in several tumor models, includingmurine models for ascites and diffuse ovarian carcinoma-tosis.[83,87–92] To further enhance selectivity for diseasesites, photosensitizers have been conjugated to a varietyof targeted macromolecular carriers includingmAbs.[58,72,74,89,93–109] Photoimmunoconjugates (PICs) werefirst described over 25 years ago,[93–95] and subsequentlya variety of conjugation strategies and targeting moieties,including those directed against the EGFR, have been in-vestigated by our group and others.[58,72,74,89,93–109] Usinga hamster cheek pouch model of squamous cell carcino-ma, Hemming et al. showed that the tumor selectivity ofbenzoporphyrin derivative (BPD) increased from approx-imately 2 :1 in the free state to 26 :1 upon conjugation toan anti-EGFR mAb, and also reported a trend towardsincreased tumoricidal efficacy.[89] Eighty percent of theanimals that received photoimmunotherapy (i.e., PICplus light) in this study were cancer free after one month,as compared to 67 % of those treated with free BPD�PDT, but this difference was not statistically significant.[89]

It is worth noting that the animals treated with PIT wereinjected with one-twentieth of the BPD-equivalent doserelative to the free BPD group. The resulting lower pho-todynamic dose delivered to the PIT group is particularlyimportant in the context of studies by us and others dem-onstrating that photosensitizer photophysical properties,cellular localization patterns, and phototoxic efficacy arealtered upon conjugation to macromolecular carri-ers.[59,60,89,92,103,105,106,110–117] The resulting impact on thera-peutic outcome is dependent on a variety of factors, in-cluding the molecular characteristics of the target diseaseand the trade-off between specificity and cytotoxicity fora particular treatment site.

Studies by Savellano et al. have demonstrated the in-creased selectivity of Erbitux-conjugated BPD, althoughthe factors affecting efficacy appear to be more com-plex.[59,60] Vrouenraets et al. have developed PICs basedon meta-tetra(hydroxyphenyl)chlorin (mTHPC) and alu-minum phthalocyanine tetrasulfonate (AlPcS4) directedagainst a variety of targets, including the EGFR, usingmAbs with differential internalizing properties and bind-ing capacities. Conjugates of the noninternalizing mAbU36 demonstrated very poor phototoxicity, even in a cellline that expressed high levels of the U36-defined anti-gen. However, an AlPcS4 conjugate using the internaliz-ing anti-EGFR mAb 425 was 7500 times more toxic thanthe free AlPcS4 in A431 cells (a human epidermoid carci-noma line that overexpresses the EGFR), and 60 timesmore toxic than the mTHPC�mAb 425 PIC. A morecomprehensive follow-up study that compared mTHPCand AlPcS4, three mAbs, and five cell lines revealed thatthe binding capacity of internalized and surface-boundPICs was a critical determinant of treatment response,and that internalization capacity alone was not correlatedwith efficacy.[103] Since the in vitro data in the above stud-ies was acquired in monolayer cell cultures, the signifi-cance of these findings in vivo needs to considered withinthe context of the experimental systems.

Uptake and phototoxic efficacy of Ce6, the photosensi-tizer used in the present study, has been shown to in-crease upon association with macromolecular carriers.The extent and nature of this increase depends on a varie-ty of factors including the charge, conjugation strategy,and subcellular localization pattern of the macromolecu-lar conjugates.[92,110,118,119] . Soukos et al. have demonstratedthe diagnostic and therapeutic potential of Ce6-ErbituxPICs in oral premalignant lesions.[58] The challenges asso-ciated with leveraging the selectivity and therapeutic ben-efits of PICs for complex treatment sites include limiteduptake and limited mAb specificity due to photosensitizerconjugation near the antigen-binding site. To address thisissue, we developed an alternative conjugation approachusing a poly-l-lysine linker to attach photosensitizers ina site-specific manner to the Fc carbohydrate moiety ofmAbs, distal from the antigen-binding sites.[58,74,98–103,110,120]

In vitro evaluation of differentially charged PICs synthe-sized using this method revealed that uptake and photo-toxicity of Ce6 in OVCAR-5 cells increased upon conjuga-tion to the F(ab�)2 region of an OC125 antibody fragment(directed against CA125, a glycoprotein that is both ex-pressed on the surface of and shed by nonmucinous epi-thelial ovarian cancers).[110] The most significant increasein uptake and cytotoxicity was observed with the cationicPIC, which may have been due to improved internaliza-tion and lysosomal degradation, compared to the anionicPIC and free Ce6. These results were verified with biodis-tribution and treatment response studies in a murinemodel for ovarian carcinomatosis.[101,102] The cationicallycharged PIC had the highest tumor selectivity and deliv-





ered the most Ce6 per gram of tumor than all other con-structs evaluated in the biodistribution study.[101] Consis-tent with these findings, treatment efficacy in the samemodel, as evaluated by median survival, was highest withthe cationic PIC.[102] It is important to note that these re-sults favoring the cationic PIC were based on intraperito-neal administration of the species. Comparable studies ina different tumor model with intravenous administrationled to more favorable results for the anionic conjugate,which is potentially due to a more rapid clearance of thecationic PIC from the blood.[99,100] These promising resultsled to a study by Duska et al. to evaluate the effect ofcombining PIT with cisplatin to treat cisplatin-resistantand cisplatin-sensitive ovarian cancer cells and patienttissue samples.[74] PIT in combination with cisplatin wasshown to reverse chemoresistance and synergisticallyreduce tumor viability.

Present Study

Motivated by the need to reduce chemotherapy cyclesand mitigate toxicity, here we evaluate the efficacy ofEGFR-targeted PIT in combination with a clinically rele-vant chemotherapy cocktail (Figure 1B). An Erbitux-Ce6

conjugate is used for a dual purpose: (i) to selectively de-liver Ce6 to EGFR-overexpressing target tissue, and (ii) tosimultaneously engage the receptor-blocking function ofthe mAb and thereby inhibit EGFR-mediated cell prolif-eration and growth. We initially compare the PIT efficacyof Ce6 directly conjugated to Erbitux (direct PIC) and in-directly conjugated via a poly-l-lysine linker (indirectPIC). The effect of irradiance, which has been shown tobe important for free photosensitizers in PDT[121–124] buthas never been evaluated in PIT (where the macromole-cule-bound photosensitizer may have altered photophysi-cal properties), is also assessed. Finally, the ability ofEGFR-targeted PIT to potentiate the efficacy of cisplatinand paclitaxel in fewer treatment cycles is determined.All studies are conducted in a xenograft murine modelfor ovarian carcinomatosis (Figure 1A) using the NI-H:OVCAR-5 human ovarian cancer cell line.[12,82,83,102]

2. Results and Discussion

Results

To establish the role of conjugation strategy and irradi-ance on PIT efficacy, mice received 1 mg/kg Ce6 equiva-lent of either the direct or indirect PIC. After 24 hours,a total fluence of 25 J/cm2 was delivered equally amongfour quadrants in the peritoneal cavity at either a high(180 mW/cm2) or low (30 mW/cm2) irradiance. A trendtowards irradiance-dependent reduction in residual tumorweight was observed with both the indirect and directPIC (Figure 2). In the indirect PIC group, residual tumorweight was significantly lower with low irradiance PIT

(327.1�74.9 mg, n=8) than with high irradiance PIT(543.2�67.0 mg, n=9) (P<0.05). No statistically signifi-cant difference in residual tumor weight was observed fol-lowing PIT with the direct PIC in the high irradiance(543.3�111.5 mg, n=11) versus low irradiance (471.0�81.1 mg, n=12) groups (P>0.05). Compared to the directPIC, PIT with the indirect PIC did not produce a statisti-cally significant reduction in residual tumor weight ateither the low or high irradiance (P>0.05). Based onthese findings, the high irradiance PIT with the direct PICwas used for subsequent combination studies to minimizethe irradiation times and reduce stress on the animals.

As shown in Figure 3, all treated mice showed a reduc-tion in tumor weight relative to the no treatment group(601.2�202.0 mg, n=19). Tumor weights following treat-ment with PIT alone (472.2�111.0 mg, n=5) or one cycleof chemotherapy alone (Chemo (1 cycle); 580.8�189.0 mg, n=5) were not significantly lower than the un-treated control (i.e. , neither the unadjusted nor adjustedP were less than 0.05). PIT in combination with one cycleof chemotherapy resulted in a residual tumor weight(Chemo (1 Cycle) + PIT; 267.2�252.5 mg, n=5) thatwas significantly lower than one cycle alone (unadjustedP=0.001, adjusted P=0.011). However, there was no sig-nificant evidence of a synergistic effect between thesingle chemotherapy cycle and PIT (ANOVA, P=0.24for treatment interaction). The single chemotherapy cycleplus PIT led to a lower residual tumor weight comparedto two cycles of chemotherapy (Chemo (2 cycles); 314.0�

Figure 2. Role of irradiance and conjugation strategy on PIT effica-cy. An Erbitux-based photoimmunoconjugate with chlorine6 conju-gated either directly to the mAb (direct PIC) or via a poly-l-lysinelinker (indirect PIC) was injected into tumored mice (1 mg/kg Ce6

equivalent). After 24 hours, a total fluence of 25 J/cm2 was equallydistributed in the peritoneal cavity at either a high (180 mW/cm2)or low irradiance (30 mW/cm2). In the indirect PIC group, low irradi-ance PIT resulted in a residual tumor weight (327.1�74.9 mg, n =8) that was significantly lower than high irradiance PIT (543.2�67.0 mg, n = 9) (Student’s t-test, P<0.05). In the direct PIC group,there was a trend towards lower residual tumor weight with low ir-radiance PIT (471.0�81.1 mg, n = 12) compared to high irradiancePIT (543.3�111.5 mg, n = 11), but the difference was not signifi-cant. No statistically significant reduction in residual tumor weightwas observed between the direct and indirect PIC at either the lowor high irradiances.





193.8 mg, n=6), but the difference did not reach statisti-cal significance (unadjusted P=0.68, adjusted P=0.99).The combination of PIT with two cycles of chemotherapy(Chemo (2 cycles) + PIT; 203.6�71.1 mg, n=5) was notstatistically different from the two cycles alone (unadjust-ed P=0.34, adjusted P=0.98). Relative to no treatment,two cycles of chemotherapy plus PIT resulted in thelowest residual disease of all treated groups (unadjustedP<0.001, adjusted P=0.002).

Mouse weights at the time of sacrifice, as a metric fortreatment-related toxicity, are detailed in Table 1. Rela-tive to no treatment (23.0�2.6 g), two cycles of chemo-therapy led to a significant reduction in mouse weightboth alone (�3.6 g, unadjusted P=0.039) and in combi-nation with PIT (19.0�1.4 g, unadjusted P=0.008). How-ever, the statistical significance diminished after adjust-ment for multiple comparisons (adjusted P=0.33 and0.078, respectively). Relative to a single cycle of chemo-therapy (23.5�1.3 g), two cycles showed a significant re-

duction in mouse weight (unadjusted P=0.045, adjustedP=0.37). The addition of PIT did not significantly changethe average mouse weights at sacrifice when used aloneor in combination with one or two cycles of chemothera-py (none of the unadjusted or adjusted P values were lessthan 0.05).

The expression of proliferating cell nuclear antigen(PCNA), a nuclear protein frequently seen in cells under-going division, is used as a marker for cellular prolifera-tion. In our study, tissue from the control mouse showeduniform, strong staining with a mAb against PCNA. Incontrast, tissue from mice treated with the combinationof chemotherapy (one cycle) and PIT showed a weakerand irregular staining pattern. A representative section ofdiaphragmatic tissue is shown in Figure 4.

Discussion

EGFR-targeted treatments using Erbitux or small-mole-cule inhibitors are currently in use for many cancers, withvariable results.[49,63,66,125,126] For ovarian cancer, the effectsare modest and temporary and chemotherapy followingsurgery still remains a mainstay for the management ofthis disease. The main problems associated with chemo-therapy or Erbitux monotherapy are the high toxicity andacquired resistance that result from multiple treatmentcycles.[49,63,66,125,126] The current study, along with previousstudies from our group[12,58,72,74,101,102,108] andothers,[49,71,125,127–139] appears to address some of theseproblems. The present study shows that chemotherapyand PDT doses may be decreased and yet still delivertumor reductions that are comparable to, or greater than,those achieved with higher doses of either monotherapyalone. We suggest that EGFR-targeted PIT be considereda serious contender as a therapeutic strategy to potentiatethe efficacy of standard chemotherapy for disseminatedovarian cancer, with the goal of minimizing treatmentcycles and mitigating treatment-related toxicities.

Paclitaxel and platinum-based agents are widely usedas first-line therapies for ovarian cancer.[2,3,6,15] Althoughthe majority of patients experience clinical remission oftheir disease, the tumors often recur.[4,140] The effective-

Figure 4. Treatment-based expression of proliferating cell nuclearantigen. Weak and irregular PCNA expression was observed follow-ing treatment with the combination of one cycle of chemotherapyplus PIT (right panel), relative to no treatment (left panel).

Table 1. Average mouse weight by treatment group at time of sacri-fice.

Treatment Type Average weight (grams)

No Treatment 23.0�2.6PIT 23.5�2.1Chemotherapy (one cycle) 23.5�1.3Chemotherapy (one cycle) + PIT 22.0�3.7Chemotherapy (two cycles) 20.1�3.6Chemotherapy (two cycles) + PIT 19.0�1.4

Figure 3. PIT in combination with chemotherapy significantly re-duces tumor burden with fewer treatment cycles. PIT in combina-tion with one cycle of chemotherapy resulted in a residual tumorweight (267.2�252.5 mg, n = 5) that was significantly lower thanone cycle of chemotherapy alone (ANOVA, unadjusted P = 0.001,adjusted P = 0.011). A single cycle of chemotherapy plus PITshowed a trend towards a residual tumor weight that was lowerthan two cycles of chemotherapy without PIT (314.0�193.8 mg,n = 6). The addition of PIT to two cycles of chemotherapy resultedin the lowest residual tumor weight (203.6�71.1 mg, n = 5), rela-tive to no treatment (unadjusted P<0.001, adjusted P = 0.002).





ness of cytotoxic therapies is limited by treatment-relatedtoxicities and the development of acquired drug resist-ance.[4,140] Moreover, the development of resistance to anindividual drug is often associated with broad cross-resist-ance to structurally similar drugs, which leads to low re-sponse rates for salvage chemotherapy. Goldie and Cold-man have suggested that most malignant cells have initialintrinsic sensitivity to chemotherapy, but develop sponta-neous chemoresistance at variable rates. Clinically, thisexplains the ability to achieve clinical remission with stan-dard chemotherapy even if the resistant cell lines arepresent.[141]

PDT is mechanistically distinct from chemotherapy andhas been shown to reverse chemoresistance and to syner-gize with chemical and biologic agents.[12,14,72–74,80] Our re-sults show that the combination of PIT and chemotherapysignificantly reduces tumor burden compared with onecycle of chemotherapy alone or PIT alone. The amount oftumor burden reduction achieved with a single cycle ofchemotherapy plus PIT was comparable to (and showedsigns of trending lower than) two cycles of chemotherapy.Additionally, tumor reduction by PIT appears to be great-er after one cycle of chemotherapy compared to the en-hancement after two cycles of chemotherapy. This obser-vation may be due to the experimental design. Since PITwas performed seven days after one cycle of chemothera-py, the effect may have been more pronounced as com-pared with PIT carried out after the second cycle (twodays post-chemotherapy).

Necropsies of mice 24 hours after PIT using the Erbi-tux-Ce6 conjugate did not reveal any significant ascites orhydrothoraces. Previous clinical trials using intraperito-neal PDT have been performed in ovarian cancer patientsunder suboptimal conditions using a nonselective photo-sensitizer (Photofrin; Aptalis Pharma, Birmingham, AL).Delaney et al. reported that 59% of patients developedpleural effusions, and 15% of patients required thora-centesis or prolonged intubation.[142] This postoperativeedema may be related to the mechanism of tumor de-struction via vascular damage from Photofrin-PDT,which, depending on the treatment parameters, maycause vessel constriction and macromolecular vessel leak-age as well as prostaglandin release.[143] Conversely, theCe6 used in the present study causes blood flow stasis dueto platelet aggregation without vessel leakage.[144] Ourstudy takes advantage of this important difference inmechanism of action between the two photosensitizers toovercome the limitations seen in the human studies withPhotofrin.

PIC-mediated PIT provides multiple therapeutic bene-fits including enhanced selectivity for target tissue, in-creased payload delivery, and inherent cytostatic or cyto-toxic potential.[58,83,92,97,101,103,110] We used an Erbitux-basedPIC to deliver Ce6 to selectively target the EGFR-ex-pressing ovarian cancer tissue. EGFR expression is asso-ciated with aggressive ovarian cancer and a poor progno-

sis and therefore serves as a viable PIT target.[12,69,125]

Treating cancer cell lines that express functional EGFRwith Erbitux potentiates the cytotoxic action of conven-tional treatment modalities. Previous experience hasshown that treatment with Erbitux alone in the samemurine xenograft model decreased tumor burden, and thecombination of Erbitux and PDT not only further de-creases tumor burden synergistically, but also increasessurvival times and results in a 30% cure rate.[12]

Chemotherapy leads to permeability of the gut duringtreatment as well as loss of protein and decreased absorp-tion of nutrients. Our study uses the weight of mice as anindirect measure of the toxicity of treatment. Our murinemodel used the NIH:OVCAR-5 cells, and ascites werenot a main finding at 24 days after tumor inoculation;thus, ascites did not give significant input to the weight ofthe mouse at the time of necropsy. Our operative findingat necropsy in mice treated with chemotherapy was signif-icant for the decreased tumor burden. However, this re-duction in tumor burden was offset by increased weightloss. Monotherapy with two cycles of chemotherapy pro-duced mice that appeared more cachectic than thosetreated with only one cycle of chemotherapy, PIT alone,or PIT in combination with one cycle of chemotherapy.PIT, as monotherapy or in combination with chemothera-py, did not cause weight loss. It is probable that the micewould have regained the weight had the study continuedpast 24 days.

Samuels et al. evaluated cancer cachexia in a murinemodel of colon carcinoma and noted that treatment withchemotherapy in nontumored control mice caused a 17 %reduction in intestinal protein mass.[145] This decrease inintestinal protein mass was also seen in untreated tumor-bearing mice. However, treatment of tumors with chemo-therapy did not increase the intestinal protein loss. Com-plete and rapid recovery of intestinal protein mass wasseen after successful treatment with chemotherapy.

3. Conclusions

Our study supports the current trend towards the devel-opment of rationally designed combination therapies byshowing that EGFR-targeted PIT combined with cispla-tin-paclitaxel chemotherapy significantly reduces tumorburden with fewer treatment cycles and lower toxicity ina complex model of ovarian intraperitoneal carcinomato-sis. Based on previous findings that synergistic interactionbetween PDT and conventional therapies is dependent onthe treatment sequence, the nature of the photosensitizer,and the chemical and biologic agents,[12,14,74,80] the currentresults merit further investigation to optimize the order,schedule and dosing of the combination regimen. Addi-tionally, leveraging minimally invasive imaging systemsfor treatment planning, and online monitoring of tumorreduction, will expedite protocol optimization.





4. Experimental Section

Cell Culture

NIH:OVCAR-5 human ovarian cancer cells were kindlyprovided by Dr. Thomas Hamilton at the Fox ChaseCancer Institute (Philadelphia, PA). Cells were grown inRoswell Park Memorial Institute 1640 medium supple-mented with 10% heat-inactivated fetal bovine serum,penicillin (100 U/mL), and streptomycin (100 mg/mL) andmaintained at 37 8C in an atmosphere of 5% carbon diox-ide. For harvesting, cells were grown to 80–90% conflu-ence, disaggregated with trypsin-EDTA (Life Technolo-gies Inc., Gibco-Brl, Gaithersburg, MD). Cells were cen-trifuged at 200 g for ten minutes, resuspended in phos-phate-buffered saline without Ca2+ or Mg2 +, and countedon a hemacytometer.

Ovarian Cancer Mouse Model

All animal experiments were approved by the Massachu-setts General Hospital Subcommittee on ResearchAnimal Care. An orthotopic xenograft mouse model pre-viously developed in our laboratory was used.[82] Six- toeight-week-old female Swiss athymic Nu/Nu mice weigh-ing 20–25 g (Cox Breeding Laboratories, Cambridge,MA) were injected intraperitoneally with 31.5� 106 NI-H:OVCAR-5 cells in 2 mL of sterile phosphate-bufferedsaline. This model reproducibly produced intra-abdominalcarcinomatosis adherent to all peritoneal surfaces within10–14 days post-inoculation (Figure 1A).[82] Mice receivedproper care and maintenance in accordance with institu-tional guidelines. The mice had continuous access to foodand water and were housed in laminar flow racks inpathogen-free conditions. We monitored the mice dailyfor general health status; mice were sacrificed at the endof the study period or if they appeared moribund or hadan excessive tumor burden.

Photoimmunoconjugates

The procedure for preparing PICs has been describedpreviously.[58,74,98–103,110,120] Briefly, poly-l-lysine (averagemolecular weight 25,000 Da) was treated in dimethyl sulf-oxide (DMSO) with the N-succinimidyl ester of Ce6 toform poly-l-lysine-Ce6. The resulting mixture was then re-acted with N-succinimidyl-3-(2-pyridyldithio)propionateto form the functionalized derivative poly-l-lysine-Ce6-N-succinimidyl-3-(2-pyridyldithio)propionate. The mAb Er-bitux was partially reduced for one hour with 5 mM mer-captoethylamine hydrochloride, dialyzed, and then react-ed with the poly-l-lysine-Ce6 pyridyldithiol for 24 hoursto form the photoimmunoconjugate, poly-l-lysine-Ce6-Er-bitux; this photoimmunoconjugate was dialyzed and puri-fied on Sephadex G-200 columns and characterized by

absorption and fluorescence spectrophotometry and poly-acrylamide gel electrophoresis.

Chemotherapy

Paclitaxel (Bristol-Myers Squibb Oncology, Princeton,NJ) and cisplatin (Baxter Healthcare Corporation, NewProvidence, NJ) were obtained from the MassachusettsGeneral Hospital Pharmacy. Paclitaxel was dosed at15 mg/kg, and cisplatin was dosed at 5 mg/kg. Each dosewas diluted in phosphate-buffered saline to give a totalvolume of 0.4–0.6 mL/dose/mouse. Drugs were adminis-tered intraperitoneally. After the drugs were adminis-tered, each mouse was observed for acute toxicity andthen allowed to return to its cage. Chemotherapy was ad-ministered 14 and 19 days after inoculation for mice re-ceiving either one or two cycles of chemotherapy, respec-tively.

Photoimmunotherapy

In vivo PIT was conducted 20 days after tumor inocula-tion; mice were given an intraperitoneal injection con-taining 1 mg/kg Ce6 equivalent. Twenty-four hours later,mice were anesthetized by intraperitoneal administrationof 0.04 mL of an anesthetic cocktail containing 30 mg/mLketamine and 5 mg/mL xylazine. The method of light de-livery has been described previously.[102] Briefly, 2 mL of0.1 % intralipid solution (soybean oil emulsion for intra-venous use; Kabi Pharmacia Inc., Clayton, NC) was in-jected intraperitoneally immediately prior to treatment toenhance light scatter. The mouse was placed in the supineposition. Light was delivered intraperitoneally via an8.0 �0.4 mm cylindrically diffusing tip using a solid statediode laser (BWF 665–1, B&W Tek, Newark, DE) at anirradiance of either 30 or 180 mW/cm2. A total of 25 J/cm2 of light was delivered at a wavelength of 665 nm. Thepower output was measured by an integrating sphere andan oscilloscope. The diffusing fiber was introduced intothe peritoneal cavity via a 22-gauge catheter that trav-ersed the abdominal wall. One fourth of the total lightenergy was delivered to each quadrant. After treatment,the mice were allowed to recover in an animal warmeruntil they awoke and resumed normal activity.

Fourteen days after tumor inoculation, mice were givena small identification tattoo on their abdomen usinga minute (<0.1 mL) amount of a diluted solution of Indiaink. The mice were randomly divided into six groups: (i)control (n=19), (ii) one cycle of chemotherapy (n=5),(iii) two cycles of chemotherapy (n=6), (iv) PIT only(n=5), (v) one cycle of chemotherapy followed by PIT(n=5), and (vi) two cycles of chemotherapy followed byPIT (n=5). Batches of seven to ten tumored mice wererandomly assigned to each treatment group. For everybatch, three mice were assigned to the no treatmentgroup to ensure consistency of tumor growth.





Treatment Evaluation

All mice were weighed at the start of the study and ateach treatment interval, and were sacrificed for necropsyby CO2 inhalation on day 24 of the experiment. Prior tosacrifice, all mice were grouped together and randomlypicked for order of sacrifice. Two investigators (TAD andIR) performed all necropsies together. The first investiga-tor did the necropsy and the second investigator con-firmed completeness of the dissection. Wet tissue weightsof excised tumor were obtained (Mettler AE 163, MettlerInstrument Corp, Hightown, NJ). Treatment response wasassessed by comparing the extent of gross residual diseasein treated animals with the extent of disease in untreatedcontrols. For toxicity studies, mouse weight at the time ofsacrifice was used as an indicator of the mouse tolerancetowards treatment.

After weighing, all resected tissue was fixed in 10 %phosphate-buffered formalin (Mallinckrodt Inc., Paris,KY) and embedded in paraffin. Sections, cut 5 mm thick,were stained with hematoxylin and eosin for microscopicevaluation to confirm the presence of carcinoma. Immu-nohistochemistry was performed on formalin-fixed, paraf-fin-embedded specimen slides using a monoclonal mouseanti�proliferating cell nuclear antigen, clone PC 10(Dako Corporation, Carpinteria, CA). Initial blockingserum and peroxidase-conjugated secondary antibodyfrom the Vectastain ABC Kit-Peroxidase Mouse IgG andVectastain Peroxidase Substrate Kit (Vector Laboratories,Burlingame, CA) were used. Control sections were runconcurrently, following the same method, except usingphosphate-buffered saline in place of the primary anti-body. All sections were lightly stained with Gill�s 3 hema-toxylin (Richard Allen Scientific, Kalamazoo, MI) andcover slipped for evaluation.

Statistical Analysis

All values are expressed as mean� standard deviation. Atwo-tailed Student�s t-test was used to analyze the effectof irradiance and conjugation strategy on tumor reduction(Figure 2). For both tumor burden and weight loss(Figure 3 and Table 1), we first examined the synergisticeffect of the PIT and one cycle of chemotherapy by test-ing the interaction of the two treatments using analysis ofvariance. In addition to comparing between each individ-ual group and the control group, we were also interestedin the following comparisons: one cycle of chemotherapyplus PIT vs. one cycle of chemotherapy alone, two cyclesof chemotherapy plus PIT vs. two cycles of chemotherapyalone, one cycle of chemotherapy alone vs. two cycles ofchemotherapy alone, one cycle of chemotherapy plus PITvs. two cycles of chemotherapy plus PIT, and one cycle ofchemotherapy plus PIT vs. two cycles of chemotherapyalone (a total of ten pair-wise comparisons). The unad-justed P values and Sidak-adjusted P values (to account

for the inflation of type I error from multiple compari-sons) were reported for these prespecified comparisons. Pvalues of less than 0.05 were considered statistically sig-nificant.

Acknowledgements

This work was supported by grant numbers5R01CA160998 and 1R01CA158415 from the NationalCancer Institute, National Institutes of Health, US De-partment of Health and Human Services. Tri A. Dinh,MD, was supported by a Department of Energy Massa-chusetts General Hospital Laser Center Fellowship inGynecologic Oncology from Vincent Memorial Obstetricsand Gynecology Service. We thank Imclone Systems Inc.(New York, NY) for the mAb Erbitux.

References

[1] A. Jemal, F. Bray, M. M. Center, J. Ferlay, E. Ward, D.Forman, Ca-Cancer J. Clin. 2011, 61, 69.

[2] R. C. Bast Jr. , B. Hennessy, G. B. Mills, Nat. Rev. Cancer2009, 9, 415.

[3] K. R. Cho, M. Shih Ie, Annu. Rev. Pathol. 2009, 4, 287.[4] R. Agarwal, S. B. Kaye, Nat. Rev. Cancer 2003, 3, 502.[5] H. Naora, D. J. Montell, Nat. Rev. Cancer 2005, 5, 355.[6] M. Markman, Drugs 2008, 68, 771.[7] M. Markman, Ther. Clin. Risk Manage. 2009, 5, 161.[8] B. A. Chabner, Oncologist 2002, 7 Suppl. 3, 34.[9] P. D. Ryan, B. A. Chabner, Clin. Cancer Res. 2000, 6, 4607.

[10] S. M. Campos, S. Ghosh, J. Oncol. 2010, 2010, 149362.[11] T. A. Yap, C. P. Carden, S. B. Kaye, Nat. Rev. Cancer 2009,

9, 167.[12] M. G. del Carmen, I. Rizvi, Y. Chang, A. C. Moor, E.

Oliva, M. Sherwood, B. Pogue, T. Hasan, J. Natl. CancerInst. 2005, 97, 1516.

[13] L. R. Duska, M. R. Hamblin, J. L. Miller, T. Hasan, J. Natl.Cancer Inst. 1999, 91, 1557.

[14] I. Rizvi, J. P. Celli, C. L. Evans, A. O. Abu-Yousif, A. Muzi-kansky, B. W. Pogue, D. Finkelstein, T. Hasan, Cancer Res.2010, 70, 9319.

[15] B. T. Hennessy, R. L. Coleman, M. Markman, Lancet 2009,374, 1371.

[16] L. P. Martin, R. J. Schilder, Semin. Oncol. 2009, 36, 112.[17] D. K. Armstrong, B. Bundy, L. Wenzel, H. Q. Huang, R.

Baergen, S. Lele, L. J. Copeland, J. L. Walker, R. A.Burger, N. Engl. J. Med. 2006, 354, 34.

[18] W. P. Ceelen, M. F. Flessner, Nat. Rev. Clin. Oncol. 2010, 7,108.

[19] M. S. Yen, N. F. Twu, C. R. Lai, H. C. Horng, K. C. Chao,C. M. Juang, Gynecol. Oncol. 2009, 114, 415.

[20] J. M. Fauci, J. M. Whitworth, K. E. Schneider, A. Subrama-niam, B. Zhang, P. J. Frederick, L. C. Kilgore, J. M.Straughn Jr., Gynecol. Oncol. 2011, 122, 532.

[21] M. M. Shahzad, G. Lopez-Berestein, A. K. Sood, DrugResist. Updat. 2009, 12, 148.

[22] B. Stordal, M. Davey, IUBMB Life 2007, 59, 696.[23] B. Stordal, N. Pavlakis, R. Davey, Cancer Treat. Rev. 2007,

33, 688.





[24] M. A. Shah, G. K. Schwartz, Clin. Cancer Res. 2001, 7,2168.

[25] M. A. Dickson, R. D. Carvajal, A. H. Merrill Jr., M.Gonen, L. M. Cane, G. K. Schwartz, Clin. Cancer Res.2011, 17, 2484.

[26] D. E. Cohn, S. Valmadre, K. E. Resnick, L. A. Eaton, L. J.Copeland, J. M. Fowler, Gynecol. Oncol. 2006, 102, 134.

[27] A. A. Garcia, H. Hirte, G. Fleming, D. Yang, D. D. Tsao-Wei, L. Roman, S. Groshen, S. Swenson, F. Markland, D.Gandara, S. Scudder, R. Morgan, H. Chen, H. J. Lenz,A. M. Oza, J. Clin. Oncol. 2008, 26, 76.

[28] J. D. Wright, A. Hagemann, J. S. Rader, D. Viviano, R. K.Gibb, L. Norris, D. G. Mutch, M. A. Powell, Cancer 2006,107, 83.

[29] J. C. Chura, K. Van Iseghem, L. S. Downs Jr., L. F. Carson,P. L. Judson, Gynecol. Oncol. 2007, 107, 326.

[30] H. S. Nimeiri, A. M. Oza, R. J. Morgan, G. Friberg, K.Kasza, L. Faoro, R. Salgia, W. M. Stadler, E. E. Vokes,G. F. Fleming, Gynecol. Oncol. 2008, 110, 49.

[31] J. P. Micha, B. H. Goldstein, M. A. Rettenmaier, M. Gene-sen, C. Graham, K. Bader, K. L. Lopez, M. Nickle, J. V.Brown 3rd, Int. J. Gynecol. Cancer 2007, 17, 771.

[32] M. D. Anderson Cancer Center, National Cancer Institute,“VEGF Trap and Docetaxel in Treating Patients With Per-sistent or Recurrent Ovarian Epithelial Cancer, PrimaryPeritoneal Cancer, or Fallopian Tube Cancer” [Clinical-Trials.gov identifier NCT00436501], US National Institutesof Health, as accessed on the website: http://clinicaltrials.gov/ct2/show/NCT00436501, last accessed on 07/31/2012.

[33] J. Sehouli, O. Camara, S. Mahner, T. Bauknecht, W. Lichte-negger, I. Runnebaum, K. Look, F. Jaenicke, G. Oskay-Oezcelik, Cancer Chemother. Pharmacol. 2010, 66, 861.

[34] W. A. Spannuth, A. K. Sood, R. L. Coleman, Expert Opin.Biol. Ther. 2010, 10, 431.

[35] University of Arizona, National Cancer Institute, “Peme-trexed Disodium and Docetaxel in Treating Patients WithAdvanced Solid Tumors” [ClinicalTrials.gov identifierNCT01172028], US National Institutes of Health, as ac-cessed on the website: http://clinicaltrials.gov/ct2/show/NCT01172028, last accessed on 07/31/2012.

[36] M. L. Hensley, J. Larkin, M. Fury, S. Gerst, D. F. Tai, P.Sabbatini, J. Konner, M. Orlando, T. L. Goss, C. A. Agha-janian, Clin. Cancer Res. 2008, 14, 6310.

[37] University College London, AstraZeneca, Cancer Re-search UK, “Saracatinib and Paclitaxel in Platinum-resist-ant Ovarian Cancer (SaPPrOC)” [ClinicalTrials.gov identi-fier NCT01196741], US National Institutes of Health, asaccessed on the website: http://clinicaltrials.gov/ct2/show/NCT01196741, last accessed on 07/31/2012.

[38] Washington University School of Medicine, “Phase I Studyof DOXIL and Temsirolimus in Resistant Solid Malignan-cies” [ClinicalTrials.gov identifier NCT00703170], US Na-tional Institutes of Health, as accessed on the website:http://clinicaltrials.gov/ct2/show/NCT00703170, last ac-cessed on 07/31/2012.

[39] Roswell Park Cancer Institute, National Cancer Institute,“PI3K Inhibitor BKM120 and Docetaxel in Treating Pa-tients With Advanced Solid Tumor That is Locally Ad-vanced, Cannot Be Removed By Surgery, or Metastatic”[ClinicalTrials.gov identifier NCT01540253], US NationalInstitutes of Health, as accessed on the website: http://clinicaltrials.gov/ct2/show/NCT01540253, last accessed on07/31/2012.

[40] University of California San Francisco, National CancerInstitute, “Lapatinib and Paclitaxel in Treating PatientsWith Advanced Solid Tumors” [ClinicalTrials.gov identifierNCT00313599], US National Institutes of Health, as ac-cessed on the website: http://clinicaltrials.gov/ct2/show/NCT00313599, last accessed on 07/31/2012.

[41] Hoosier Oncology Group, Novartis Pharmaceuticals,Sanofi-Aventis, Walther Cancer Institute, “Imatinib Mesy-late in Combination With Docetaxel for Advanced, Plati-num-Refractory Ovarian Cancer” [ClinicalTrials.gov iden-tifier NCT00216112], US National Institutes of Health, asaccessed on the website: http://clinicaltrials.gov/ct2/show/NCT00216112, last accessed on 07/31/2012.

[42] Sarah Cannon Research Institute, Bayer, “Paclitaxel andCarboplatin With Or Without Sorafenib In The First-LineTreatment Of Patients With Ovarian Cancer” [Clinical-Trials.gov identifier NCT00390611], US National Institutesof Health, as accessed on the website: http://clinicaltrials.gov/ct2/show/NCT00390611, last accessed on 07/31/2012.

[43] Cancer and Leukemia Group B, National Cancer Institute,“Imatinib Mesylate in Treating Patients With Progressive,Refractory, or Recurrent Stage II or Stage III Testicular orOvarian Cancer” [ClinicalTrials.gov identifierNCT00042952], US National Institutes of Health, as ac-cessed on the website: http://clinicaltrials.gov/ct2/show/NCT00042952, last accessed on 07/31/2012.

[44] Gynecologic Oncology Group, National Cancer Institute,“Carboplatin, Paclitaxel, Bevacizumab, and Veliparib inTreating Patients With Newly Diagnosed Stage II, StageIII, or Stage IV Ovarian Epithelial Cancer, Fallopian TubeCancer, or Primary Peritoneal Cancer” [ClinicalTrials.govidentifier NCT00989651], US National Institutes ofHealth, as accessed on the website: http://clinicaltrials.gov/ct2/show/NCT0098965, last accessed on 07/31/2012.

[45] P. Pautier, F. Joly, P. Kerbrat, P. Bougnoux, P. Fumoleau, T.Petit, O. Rixe, F. Ringeisen, A. T. Carrasco, C. Lhomme,Gynecol. Oncol. 2010, 116, 157.

[46] J. Konner, R. J. Schilder, F. A. DeRosa, S. R. Gerst, W. P.Tew, P. J. Sabbatini, M. L. Hensley, D. R. Spriggs, C. A.Aghajanian, Gynecol. Oncol. 2008, 110, 140.

[47] M. Jamnongjit, A. Gill, S. R. Hammes, Proc. Natl. Acad.Sci. U.S.A. 2005, 102, 16257.

[48] C. H. Siemens, N. Auersperg, J. Cell. Physiol. 1988, 134,347.

[49] R. Zeineldin, C. Y. Muller, M. S. Stack, L. G. Hudson, J.Oncol. 2010, 2010, 414676.

[50] A. Abdollahi, B. N. Gruver, C. Patriotis, T. C. Hamilton,Biochem. Biophys. Res. Commun. 2003, 307, 188.

[51] L. G. Hudson, R. Zeineldin, M. Silberberg, M. S. Stack,Cancer Treat. Res. 2009, 149, 203.

[52] M. V. Barbolina, N. M. Moss, S. D. Westfall, Y. Liu, R. J.Burkhalter, F. Marga, G. Forgacs, L. G. Hudson, M. S.Stack, Cancer Treat. Res. 2009, 149, 319.

[53] K. D. Cowden Dahl, J. Symowicz, Y. Ning, E. Gutierrez,D. A. Fishman, B. P. Adley, M. S. Stack, L. G. Hudson,Cancer Res. 2008, 68, 4606.

[54] L. G. Hudson, R. Zeineldin, M. S. Stack, Clin. Exp. Meta-stasis 2008, 25, 643.

[55] L. G. Hudson, N. M. Moss, M. S. Stack, Future Oncol.2009, 5, 323.

[56] H. Lassus, H. Sihto, A. Leminen, H. Joensuu, J. Isola, N. N.Nupponen, R. Butzow, J. Mol. Med. (Berl.) 2006, 84, 671.





[57] A. Psyrri, M. Kassar, Z. Yu, A. Bamias, P. M. Weinberger,S. Markakis, D. Kowalski, R. L. Camp, D. L. Rimm, M. A.Dimopoulos, Clin. Cancer Res. 2005, 11, 8637.

[58] N. S. Soukos, M. R. Hamblin, S. Keel, R. L. Fabian, T. F.Deutsch, T. Hasan, Cancer Res. 2001, 61, 4490.

[59] M. D. Savellano, T. Hasan, Photochem. Photobiol. 2003,77, 431.

[60] M. D. Savellano, T. Hasan, Clin. Cancer Res. 2005, 11,1658.

[61] F. Ciardiello, R. Bianco, V. Damiano, S. De Lorenzo, S.Pepe, S. De Placido, Z. Fan, J. Mendelsohn, A. R. Bianco,G. Tortora, Clin. Cancer Res. 1999, 5, 909.

[62] F. Ciardiello, R. Caputo, R. Bianco, V. Damiano, G. Fonta-nini, S. Cuccato, S. De Placido, A. R. Bianco, G. Tortora,Clin. Cancer Res. 2001, 7, 1459.

[63] F. Ciardiello, G. Tortora, Clin. Cancer Res. 2001, 7, 2958.[64] S. V. Blank, R. Chang, F. Muggia, Oncology (Williston

Park) 2005, 19, 553.[65] S. L. Gibbs-Strauss, K. S. Samkoe, J. A. O’Hara, S. C.

Davis, P. J. Hoopes, T. Hasan, B. W. Pogue, Acad. Radiol.2010, 17, 7.

[66] S. Li, K. R. Schmitz, P. D. Jeffrey, J. J. Wiltzius, P. Kussie,K. M. Ferguson, Cancer Cell 2005, 7, 301.

[67] Z. Fan, B. Y. Shang, Y. Lu, J. L. Chou, J. Mendelsohn, Clin.Cancer Res. 1997, 3, 1943.

[68] D. Peng, Z. Fan, Y. Lu, T. DeBlasio, H. Scher, J. Mendel-sohn, Cancer Res. 1996, 56, 3666.

[69] G. Lurje, H. J. Lenz, Oncology 2009, 77, 400.[70] S. V. Sharma, D. W. Bell, J. Settleman, D. A. Haber, Nat.

Rev. Cancer 2007, 7, 169.[71] J. Baselga, Oncologist 2002, 7 Suppl. 4, 2.[72] N. Solban, I. Rizvi, T. Hasan, Lasers Surg. Med. 2006, 38,

522.[73] S. Verma, G. M. Watt, Z. Mai, T. Hasan, Photochem. Pho-

tobiol. 2007, 83, 996.[74] L. R. Duska, M. R. Hamblin, J. L. Miller, T. Hasan, J. Natl.

Cancer Inst. 1999, 91, 1557.[75] C. M. Peterson, J. M. Lu, Z. W. Gu, J. G. Shiah, K. Lyth-

goe, C. A. Peterson, R. C. Straight, J. Kopecek, J. Soc. Gy-necol. Investig. 1995, 2, 772.

[76] C. M. Peterson, J. M. Lu, Y. Sun, C. A. Peterson, J. G.Shiah, R. C. Straight, J. Kopecek, Cancer Res. 1996, 56,3980.

[77] J. M. Lu, C. M. Peterson, J. Guo-Shiah, Z. W. Gu, C. A. Pe-terson, R. C. Straight, J. Kopecek, Int. J. Oncol. 1999, 15, 5.

[78] J. G. Shiah, Y. Sun, C. M. Peterson, R. C. Straight, J. Kope-cek, Clin. Cancer Res. 2000, 6, 1008.

[79] J. Hongrapipat, P. Kopeckova, J. Liu, S. Prakongpan, J. Ko-pecek, Mol. Pharm. 2008, 5, 696.

[80] M. F. Zuluaga, N. Lange, Curr. Med. Chem. 2008, 15, 1655.[81] C. Lottner, R. Knuechel, G. Bernhardt, H. Brunner,

Cancer Lett. 2004, 203, 171.[82] K. L. Molpus, D. Koelliker, L. Atkins, D. T. Kato, J.

Buczek-Thomas, A. F. Fuller Jr., T. Hasan, Int. J. Cancer1996, 68, 588.

[83] K. L. Molpus, D. Kato, M. R. Hamblin, L. Lilge, M. Bam-berg, T. Hasan, Cancer Res. 1996, 56, 1075.

[84] J. P. Celli, I. Rizvi, C. L. Evans, A. O. Abu-Yousif, T.Hasan, J. Biomed. Opt. 2010, 15, 051603.

[85] P. Agostinis, K. Berg, K. A. Cengel, T. H. Foster, A. W.Girotti, S. O. Gollnick, S. M. Hahn, M. R. Hamblin, A. Ju-zeniene, D. Kessel, M. Korbelik, J. Moan, P. Mroz, D.Nowis, J. Piette, B. C. Wilson, J. Golab, Ca-Cancer J. Clin.2011, 61, 250.

[86] K. A. Cengel, E. Glatstein, S. M. Hahn, Cancer Treat. Res.2007, 134, 493.

[87] Z. Tochner, Cancer Res. 1985, 45, 2983.[88] Z. Tochner, Br. J. Cancer 1986, 53, 733.[89] A. W. Hemming, N. L. Davis, B. Dubois, N. F. Quenville,

R. J. Finley, Surg. Oncol. 1993, 2, 187.[90] J. Zhang, L. Deng, J. Yao, P. Gu, F. Yang, X. Wang, W.

Liu, Y. Zhang, X. Ke, X. Jing, J. Chen, Bioorg. Med.Chem. 2011, 19, 5520.

[91] B. A. Goff, U. Hermanto, J. Rumbaugh, J. Blake, M. Bam-berg, T. Hasan, Br. J. Cancer 1994, 70, 474.

[92] M. R. Hamblin, J. L. Miller, I. Rizvi, B. Ortel, E. V.Maytin, T. Hasan, Cancer Res. 2001, 61, 7155.

[93] D. Mew, C. K. Wat, G. H. Towers, J. G. Levy, J. Immunol.1983, 130, 1473.

[94] C. K. Wat, D. Mew, J. G. Levy, G. H. Towers, Prog. Clin.Biol. Res. 1984, 170, 351.

[95] D. Mew, V. Lum, C. K. Wat, G. H. Towers, C. H. Sun, R. J.Walter, W. Wright, M. W. Berns, J. G. Levy, Cancer Res.1985, 45, 4380.

[96] M. R. Hamblin, M. Rajadhyaksha, T. Momma, N. S.Soukos, T. Hasan, Br. J. Cancer 1999, 81, 261.

[97] N. S. Soukos, M. R. Hamblin, T. Hasan, Photochem. Photo-biol. 1997, 65, 723.

[98] M. Del Governatore, M. R. Hamblin, E. E. Piccinini, G.Ugolini, T. Hasan, Br. J. Cancer 2000, 82, 56.

[99] M. Del Governatore, M. R. Hamblin, C. R. Shea, I. Rizvi,K. G. Molpus, K. K. Tanabe, T. Hasan, Cancer Res. 2000,60, 4200.

[100] M. R. Hamblin, M. Del Governatore, I. Rizvi, T. Hasan,Br. J. Cancer 2000, 83, 1544.

[101] L. R. Duska, M. R. Hamblin, M. P. Bamberg, T. Hasan, Br.J. Cancer 1997, 75, 837.

[102] K. L. Molpus, M. R. Hamblin, I. Rizvi, T. Hasan, Gynecol.Oncol. 2000, 76, 397.

[103] G. A. van Dongen, G. W. Visser, M. B. Vrouenraets, Adv.Drug Deliv. Rev. 2004, 56, 31.

[104] M. B. Vrouenraets, G. W. Visser, C. Loup, B. Meunier, M.Stigter, H. Oppelaar, F. Stewart, G. B. Snow, G. A. vanDongen, Int. J. Cancer 2000, 88, 108.

[105] M. B. Vrouenraets, G. W. Visser, F. A. Stewart, M. Stigter,H. Oppelaar, P. E. Postmus, G. B. Snow, G. A. van Dongen,Cancer Res. 1999, 59, 1505.

[106] M. B. Vrouenraets, G. W. Visser, M. Stigter, H. Opelaar,G. B. Snow, G. A. van Dongen, Cancer Res. 2001, 61, 1970.

[107] S. A. Sibani, P. A. McCarron, A. D. Woolfson, R. F. Don-nelly, Expert Opin. Drug Deliv. 2008, 5, 1241.

[108] T. Hasan, B. Ortel, N. Solban, B. W. Pogue, in Cancer Med-icine (Eds.: D. Kufe, R. Bast, W. Hait, W. Hong, R. Pol-lock, R. Weichselbaum, J. Holland, E. Frei), 7th edition,B. C. Decker, Inc., Hamilton, Ontario, 2006, pp. 537 –548.

[109] A. M. Bugaj, Photochem. Photobiol. Sci. 2011, 10, 1097.[110] M. R. Hamblin, J. L. Miller, T. Hasan, Cancer Res. 1996,

56, 5205.[111] D. Kessel, Y. Luo, Y. Deng, C. K. Chang, Photochem. Pho-

tobiol. 1997, 65, 422.[112] D. Kessel, Y. Luo, J. Photochem. Photobiol. , B 1998, 42,

89.[113] D. Kessel, Y. Luo, Cell Death Differ. 1999, 6, 28.[114] D. Kessel, J. J. Reiners Jr., Photochem. Photobiol. 2007, 83,

1024.[115] D. Kessel, K. Woodburn, C. J. Gomer, N. Jagerovic, K. M.

Smith, J. Photochem. Photobiol. , B 1995, 28, 13.





[116] Y. Luo, C. K. Chang, D. Kessel, Photochem. Photobiol.1996, 63, 528.

[117] M. R. Hamblin, E. L. Newman, J. Photochem. Photobiol.,B 1994, 23, 3.

[118] R. Bachor, C. R. Shea, R. Gillies, T. Hasan, Proc. Natl.Acad. Sci. U. S. A. 1991, 88, 1580.

[119] R. Bachor, M. Scholz, C. R. Shea, T. Hasan, Cancer Res.1991, 51, 4410.

[120] M. R. Hamblin, M. D. Governatore, I. Rizvi, T. Hasan, Br.J. Cancer 2000, 83, 1544.

[121] S. Iinuma, K. T. Schomacker, G. Wagnieres, M. Rajadhyak-sha, M. Bamberg, T. Momma, T. Hasan, Cancer Res. 1999,59, 6164.

[122] T. H. Foster, D. F. Hartley, M. G. Nichols, R. Hilf, CancerRes. 1993, 53, 1249.

[123] T. H. Foster, R. S. Murant, R. G. Bryant, R. S. Knox, S. L.Gibson, R. Hilf, Radiat. Res. 1991, 126, 296.

[124] M. G. Nichols, T. H. Foster, Phys. Med. Biol. 1994, 39,2161.

[125] J. R. Woodburn, Pharmacol. Ther. 1999, 82, 241.[126] P. A. Bunn Jr., W. Franklin, Semin. Oncol. 2002, 29, 38.[127] T. Goldkorn, Biochim. Biophys. Acta 1997, 1358, 289.[128] J. Baselga, D. Pfister, M. R. Cooper, R. Cohen, B. Burt-

ness, M. Bos, G. D�Andrea, A. Seidman, L. Norton, K.Gunnett, J. Falcey, V. Anderson, H. Waksal, J. Mendel-sohn, J. Clin. Oncol. 2000, 18, 904.

[129] C. Bianco, R. Bianco, G. Tortora, V. Damiano, P. Guerrieri,P. Montemaggi, J. Mendelsohn, S. De Placido, A. R.Bianco, F. Ciardiello, Clin. Cancer Res. 2000, 6, 4343.

[130] C. J. Bruns, M. T. Harbison, D. W. Davis, C. A. Portera, R.Tsan, D. J. McConkey, D. B. Evans, J. L. Abbruzzese, D. J.Hicklin, R. Radinsky, Clin. Cancer Res. 2000, 6, 1936.

[131] F. Ciardiello, R. Bianco, V. Damiano, G. Fontanini, R.Caputo, G. Pomatico, S. De Placido, A. R. Bianco, J. Men-delsohn, G. Tortora, Clin. Cancer Res. 2000, 6, 3739.

[132] M. Jo, D. B. Stolz, J. E. Esplen, K. Dorko, G. K. Michalo-poulos, S. C. Strom, J. Biol. Chem. 2000, 275, 8806.

[133] N. Ahmad, K. Kalka, H. Mukhtar, Oncogene 2001, 20,2314.

[134] G. Lammering, Clin. Cancer Res. 2001, 7, 682.[135] C. Mamot, R. Ritschard, W. K�ng, J. Park, R. Herrmann,

C. Rochlitz, J. Drug Target. 2006, 14, 215.[136] J. N. Sarkaria, B. L. Carlson, M. A. Schroeder, P. Grogan,

P. D. Brown, C. Giannini, K. V. Ballman, G. J. Kitange, A.Guha, A. Pandita, C. D. James, Clin. Cancer Res. 2006, 12,2264.

[137] N. Puri, R. Salgia, J. Carcinog. 2008, 7, 9.[138] Z. Tang, R. Du, S. Jiang, C. Wu, D. S. Barkauskas, J.

Richey, J. Molter, M. Lam, C. Flask, S. Gerson, A. Dowla-ti, L. Liu, Z. Lee, B. Halmos, Y. Wang, J. A. Kern, P. C.Ma, Br. J. Cancer 2008, 99, 911.

[139] P. Chinnaiyan, S. Huang, G. Vallabhaneni, E. Armstrong,S. Varambally, S. A. Tomlins, A. M. Chinnaiyan, P. M.Harari, Cancer Res. 2005, 65, 3328.

[140] M. Markman, Int. J. Gynecol. Cancer 2009, 19 Suppl. 2,S40.

[141] J. H. Goldie, A. J. Coldman, Cancer Treat. Rep. 1979, 63,1727.

[142] T. F. DeLaney, W. F. Sindelar, Z. Tochner, P. D. Smith,W. S. Friauf, G. Thomas, L. Dachowski, J. W. Cole, S. M.Steinberg, E. Glatstein, Int. J. Radiat. Oncol., Biol. , Phys.1993, 25, 445.

[143] V. H. Fingar, T. J. Wieman, P. S. Haydon, Photochem. Pho-tobiol. 1997, 66, 513.

[144] K. S. McMahon, T. J. Wieman, P. H. Moore, V. H. Fingar,Cancer Res. 1994, 54, 5374.

[145] S. E. Samuels, A. L. Knowles, T. Tilignac, E. Debiton, J. C.Madelmont, D. Attaix, Cancer Res. 2000, 60, 4968.

Received: March 16, 2012Accepted: April 10, 2012

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