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Published OnlineFirst July 27, 2010; DOI: 10.1158/1535-7163.MCT-10-0169

Molecular
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cular Medicine in Practice

cular Endothelial Growth Factor Is a Promisingrapeutic Target for the Treatment of Clear Cell

Ther

cinoma of the Ovary

abuchi1, Chiaki Kawase1, Deborah A. Altomare4, Kenichirou Morishige1, Masami Hayashi1,
o Sawada1, Kimihiko Ito7, Yoshito Terai3, Yukihiro Nishio2, Andres J. Klein-Szanto5, t A. Burger4,6, Masahide Ohmichi3, Joseph R. Testa5, and Tadashi Kimura1
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study examines the role of vascular endothelial growth factor (VEGF) as a therapeutic target in clearrcinoma (CCC) of the ovary, which has been regarded as a chemoresistant histologic subtype. Immu-ochemical analysis using tissue microarrays of 98 primary ovarian cancers revealed that VEGF wasly expressed both in early-stage and advanced-stage CCC of the ovary. In early-stage CCCs, patientsad tumors with high levels of VEGF had significantly shorter survival than those with low levels of. In vitro experiments revealed that VEGF expression was significantly higher in cisplatin-refractoryn CCC cells (RMG1-CR and KOC7C-CR), compared with the respective parental cells (RMG1 andC) in the presence of cisplatin. In vivo treatment with bevacizumab markedly inhibited the growthh parental CCC cell–derived (RMG1 and KOC7C) and cisplatin-refractory CCC cell–derived (RMG1-d KOC7C-CR) tumors as a result of inhibition of tumor angiogenesis. The results of the current studyte that VEGF is frequently expressed and can be a promising therapeutic target in the management
indica
of CCC. Bevacizumab may be efficacious not only as a first-line treatment but also as a second-line treatmentof recurrent disease in patients previously treated with cisplatin. Mol Cancer Ther; 9(8); 2411–22. ©2010 AACR.

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duction

rian carcinoma is the fourth most common causecer death among women in the United States, withthan 21,550 new cases diagnosed and 14,600 deathsated for 2009 (1). Cytoreductive surgery followedtinum-based chemotherapy usually combined withaxel is the standard initial treatment and has

ival in patients with epithelial ovarianever, although this treatment regimen is

serouviewthat ppaclitsignifotherAnothof CCCCCmothefor vatherap(6). TCCC,the sementOn

patieagentgioge

s: 1Department of Obstetrics and Gynecology, Osakae School of Medicine; 2Department of Obstetrics anda Police Hospital; 3Department of Obstetrics anda Medical College, Osaka, Japan; 4Women's CancerGenetics and Signaling Program, and 6Departmentase Cancer Center, Philadelphia, Pennsylvania; andbstetrics and Gynecology, Kansai Rosai Hospital,

and C. Kawase contributed equally to this work.

hase Cancer Center/Ovarian Specialized Programs ofce shared facilities were used in the course of thisand Histopathology.

for D.A. Altomare: Burnett School of Biomedicalty of Central Florida, Orlando, FL 32827.

uthor: Seiji Mabuchi, Department of ObstetricsOsaka University Graduate School of Medicine, 2-2, Osaka 565-0871, Japan. Phone: 81-6-6879-3354;59. E-mail: [email protected]

7163.MCT-10-0169

ssociation for Cancer Research.

ls.org

on August 27, 2020. © 20mct.aacrjournals.org d from

lly effective in a high percentage of cases, mostts will ultimately experience a relapse due to theopment of chemoresistance (3).ar cell carcinoma (CCC) of the ovary, which was firstnized by theWorld Health Organization as a distinctogic subtype in 1973 (4), is known to show poorerivity to platinum-based chemotherapy and to be as-ed with a worse prognosis than the more commons adenocarcinoma (SAC). A recent retrospective re-of six randomized phase III clinical trials has shownatients with stage III CCC treated with carboplatin-axel in the setting of first-line chemotherapy had aicantly shorter survival compared with those withhistologic subtypes of epithelial ovarian cancer (5).er important problem in the clinical managementC is the lack of effective chemotherapy for recurrentafter first-line treatment with platinum-based che-rapy. A recent report showed that the response raterious regimens in the setting of second-line chemo-y for recurrent platinum-resistant CCCwas only 1%herefore, to improve the survival of patients withthe development of novel treatment strategies intting of both first-line treatment and salvage treat-for recurrent disease are needed.e possible treatment strategy that may improvent outcome is the use of angiogenesis-targeted
s. Among the variety of potential targets of antian-nesis, vascular endothelial growth factor (VEGF)
2411

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s signaling pathway is reported to be a promisingin anticancer therapy (7–9). In a murine modelrian cancer, Zhang et al. (10) showed that VEGFxpression was associated with tumor growth,genesis, ascites formation, and tumor cell survival.ally, it has been previously reported that VEGFrexpressed in most ovarian tumors (11–13). Highof VEGF have also been found in the serum, plas-nd ascites of ovarian cancer patients and are asso-with poor patient prognosis (14–16). However,se most tumors investigated in previous studieseen from ovarian SACs (11–13), the expression rateGF and its prognostic significance in CCC of theremain unknown. Moreover, although bevacizu-a humanized monoclonal antibody against human, has shown significant single-agent activity inII trials involving patients with recurrent ovarian

r, little information is available regarding its antitu-fficacy in patients with CCC. Thus, the therapeutictial of bevacizumab or other VEGF inhibitors ints with CCC is unknown (7, 8, 17).major limitation in conducting researches on ovar-

CC is the rarity of this histologic subtype. The pre-cidence of CCC is unknown, but it is reported toproximately 5% of all histologic subtypes amongelial ovarian cancers in Western countries (18).ver, in Japan, it is the second most frequent histo-ubtype. More than 20% of ovarian cancers are clas-as CCC (19).ng clinical samples obtained in Japan, we havely reported that mTOR is more frequently activatedrian CCCs than in SACs (87% versus 50%; ref. 20).also been reported that ovarian endometriosis,

which CCC is thought to arise, is characterized byactivation of the AKT-mTOR pathway (21). More-it has been recently reported that hypoxia-inducible1α (HIF-1α) expression levels are significantly

r in CCC than in other histologic subtypes of ovar-ncer (22). Because AKT-mTOR signaling has beento stimulate the expression of HIF-1α and VEGF,

g to tumor angiogenesis essential for tumor growth,on, and metastasis (23), the VEGF pathway holdsise as a target in the therapy of CCC.he current investigation, we examined the expres-f VEGF in both early-stage and advanced-stageand determined its correlation with patient progno-oreover, we investigated the therapeutic potentialacizumab in both cisplatin-sensitive and cisplatin-nt CCC cells in vitro and in vivo.

rials and Methods

ents/antibodiesacizumab was obtained from Genentech, Inc. ECLrn blotting detection reagents were purchased from-Elmer. Anti-VEGF antibody (A-20) was obtained
Santa Cruz Biotechnology. Antibodies recognizingDP-ribose) polymerase (PARP) and β-actin were
negatsemiq

ancer Ther; 9(8) August 2010

on August 27, 2020. © 20mct.aacrjournals.org wnloaded from

ed from Cell Signaling Technology. Anti-CD31/et/endothelial cell adhesion molecule 1 (PECAM-1)dy was obtained from Abcam. Cell Titer 96-welleration assay kit was obtained from Promega.tin was purchased from Sigma.

preparationacizumab was diluted to the appropriate concen-n in PBS before addition to cell culture. For animals, 5 mg/kg bevacizumab were diluted in 200 μL ofefore administration.

cal samplesor samples were obtained from patients undergo-imary cytoreductive surgery at the Osaka MedicaleHospital,WakayamaRosaiHospital, Osaka Policeital, Kansai Rosai Hospital, and Sakai Municipalital before any other therapeutic intervention be-1991 and 2006. All surgical specimens and clinicalere collected and archived according to protocolsved by the institutional review boards of these hos-. Appropriate informed consent was obtained frompatient. Histologic diagnosis was based on Worldh Organization criteria. The tumors included 52and 46 SACs for reference as described previouslyased on the International Federation of Gynecologybstetrics criteria, 27CCCswere stage I-II tumors andre stage III-IV tumors. Among SACs, 22 were stagemors and 24 were stage III-IV tumors. The durationrall survival was measured from the date of diagno-death or censored at the date of last follow-up.

nohistochemistryary ovarian tumors obtained from patients were

in 10% neutral buffered formalin (10% formalde-phosphate-buffered) overnight and then embed-

n paraffin. From each case, a representative tissueconsisting of predominantly viable tumor tissueelected from hematoxylin and eosin (H&E) slides.an cancer tissue microarrays consisting of two coreseach tumor sample were prepared by the TumorFacility at Fox Chase Cancer Center, as describedously (20, 24, 25). For each tumor, 5-μm-sectionstained with H&E were used to locate representa-alignant areas. Two cores (0.6 mm) were punchedthe morphologically representative area of theblock and then placed in the recipient paraffin tis-icroarray block. Fresh 4-μm sections were obtainedeach tissue microarray block, mounted on slides,rocessed for either H&E or immunohistochemicalng. For immunohistochemical studies, sectionsincubated with the primary antibody, followed bypropriate peroxidase-conjugated secondary anti-as reported previously (20, 25). The primary anti-used was anti-VEGF antibody at 1:50 dilution.unding nonneoplastic stroma served as an internal
ive control for each slide. The slides were scoreduantitatively by a pathologist who was blinded to
Molecular Cancer Therapeutics



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inical outcome. A score of 0 indicated no staining,ndicated weak focal staining (less than 10% of theere stained), +1 indicated focal staining (10–50% oflls were stained), +2 indicated clearly positive stain-ore than 50% of the cells were stained), and a scoreindicated intensely positive staining, as describedail elsewhere (20). The slides were examined usinght field microscope by two observers (A.J.K. andwho were blinded to the clinical data of the pa-

. Tumors with a staining of +2 or +3 were groupedtrong-staining group, whereas tumors with a stain-+0.5 or +1 were grouped as a weak-staining group.the two cores from the same tumor sampled different positivity results, the lower score wasered valid.

ultureman ovarian CCC cell lines RMG1 and KOC7Ckindly provided by Dr. H. Itamochi (Tottori Univer-ottori, Japan). These cells were cultured in phenolee Dulbecco's modified Eagle's medium (DMEMs F-12, Gibco Ltd.) with 10% fetal bovine serumas reported previously (26–28). Human umbilicalndothelial cells (HUVEC) were isolated by trypsinion of umbilical veins from fresh umbilical cordsaintained in HuMedia-EG2 medium (Kurabotries) as described previously (29). Subculturesobtained by trypsinization and were used foriments at passages 3 to 5. To expose cells to hypox-ltures were placed into a multigas incubatorYO) that was infused with a mixture of 1% O2,O2, and 94% N2, and incubated at 37°C.

lishment of cisplatin-refractory cell linesa preclinical model of recurrent CCCs after the first-latinum-based chemotherapy, cisplatin-refractoryes from RMG1 and KOC7C were developed inboratory by continuous exposure to cisplatin, asbed previously (20, 30). Briefly, cells of both linesexposed to stepwise increases in cisplatin concen-s. Initial cisplatin exposure was at a concentrationnmol/L. After cells had regained their exponentialth rate, the cisplatin concentration was doubled,hen the procedure was repeated until selection atol/L was attained. The resulting cisplatin-refractorynes, dubbed RMG1-CR and KOC7C-CR, wereltured weekly and treated monthly with 10 μmol/Ltin to maintain a high level of chemoresistance.

roliferation assayMTS assay was used to analyze the effect of VEGFacizumab on cell viability as described (31). Cellscultured overnight in 96-well plates (1 × 104 cellsell). Cell viability was assessed after addition ofizumab or cisplatin at the indicated concentrationshours. The number of surviving cells was assessed
termination of the A490 nm of the dissolved forma-roduct after addition of MTS for 1 hour as described
micethe ri

acrjournals.org


e manufacturer (Promega). Cell viability is ex-d as follows: Aexp group/Acontrol × 100.

ern blot analysisls were treated with either PBS or the indicatedntrations of cisplatin for 24 hours. Cells wered twice with ice-cold PBS and lysed in lysis buffermol/LTris-HCl, 150mmol/LNaCl, 1mmol/LEDTA,ol/L EGTA, 1 mmol/L Na3VO4, 1 mmol/L β-rophosphate, 2.5 mmol/L sodium PPi, 1 mmol/Lminoethyl) benzenesulfonyl fluoride hydrochloride,/mL aprotinin, 1 μg/mL leupeptin, and 1% Triton] for 10 minutes at 4°C. Lysates were centrifuged at0 × g at 4°C for 15 minutes, and protein concen-ns of the supernatants were determined using thead protein assay reagent. Equal amounts of proteinseparated by SDS-PAGE and transferred to nitro-ose membranes. Blocking was done in 5% non-ilk in 1× Tris-buffered saline. Western blot analysesonewith various specific primary antibodies. Immu-ts were visualized with horseradish peroxidase–ed goat anti-rabbit or anti-mouse immunoglobulinng an enhanced chemiluminescenceWestern blotting(Perkin-Elmer).

formation assaye formation assay was done as described previous-). Briefly, the surfaces of 96-well plates were coated0 μL of growth factor–reduced Matrigel matrix (BDiences). Then, 1 × 105 serum-starved HUVECs inL of M199 medium containing 0.5% bovine serumin were plated. Various agents were added at thef plating. After 8-hour incubation, tube formationisualized under an inverted microscope (×40) andages were analyzed.

ro VEGF protein quantitation bye-linked immunosorbent assayG1 or KOC7C cells (5 × 104) were incubated inM Ham's F-12 medium containing 1% FBS forurs. Then, the culture supernatants were collectedevels of VEGF (corrected for cell number) wereined using the Quantikine Human Vascular Endo-l Growth Factor Immunoassay (R&D Systems)ing to the manufacturer's protocol. The remaininglayers were trypsinized, and the cells were countedrmalize VEGF protein values. VEGF values wered from a standard curve of known concentrationsombinant human VEGF. Each sample was analyzedplicate and averaged.

taneous xenograft modelprocedures involving animals and their care wereved by the Institutional Animal Care and Usageittee of Osaka University, in accordance with insti-al and NIH guidelines. Five- to 7-week-old nude
(n = 48) were inoculated subcutaneously (s.c.) intoght flank with 5 × 106 RMG1, RMG1-CR, KOC7C,
Mol Cancer Ther; 9(8) August 2010 2413



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C7C-CR cells in 200 μL of PBS. When tumorsed a size of about 50 mm3, mice were randomlyed into two treatment groups, with 12 mice in each. The first group was treated with PBS twicey. The second group was treated with bevacizumab/kg) twice weekly. Bevacizumab was administerederitoneally as described previously (32). Caliperurements of the longest perpendicular tumorters were done every week to estimate tumor vol-sing the following formula: V = L × W × D × π/6,V is the volume, L is the length,W is the width, and

he depth as described previously (20, 25, 32).

tification of microvessel areacutaneous tumors harvested at autopsy were pro-for immunostaining using anti-CD31/PECAM-1dy at a 1:50 dilution and appropriate peroxidase-gated secondary antibodies. The tissue sectionsviewed at ×100 magnification, and images werered. Four fields per section were analyzed, exclud-ecrotic regions. The percentage of CD31-positivevessel area (MVA) in each field was calculated asibed previously (33). The mean value of MVA inroup was calculated from four tumor samples.

tical analysisl proliferation was analyzed by the Wilcoxon exactifferences in VEGF concentrations and the effectsacizumab on tumor volume and MVA were ana-by Student's t test. Data are expressed as the meanImmunoreactivity was analyzed using Fisher's exacturvival rates were examined using Kaplan-Meierand statistical differences between the survival ratesups were assessed by the log-rank test. A P value ofwas considered significant.

lts

expression in CCCs and SACsunohistochemical analysis of ovarian cancer tissuearrays for VEGF expression was done using 52n CCCs and 46 ovarian SACs as described above.sentative photomicrographs of the CCCs and SACsown in Fig. 1A. VEGF immunoreactivity was

d semiquantitatively (Fig. 1B). When analyzeding to surgical-pathologic stage (Table 1), immuno-

vity for VEGF was greater in advanced-stage CCCsin early-stage CCCs. Among the 27 early-stage, 7 (26%) were scored as +0.5 or +1, 14 (52%) wereas +2, and 6 (22%) were scored as +3. In contrast,g the 25 advanced-stage CCCs, 15 (60%) wereas +2 and 10 (40%) were scored as +3 (Fig. 1C).

r VEGF immunoreactivity was observed in SACs.g the 22 early-stage SACs, 4 (18.2%) were scored asr +1, 15 (68.2%) were scored as +2, and 3 (13.6%)scored as +3. In contrast, among the 24 advanced-
SACs, one (4.2%) was scored as +1, 19 (79.1%) wereas +2, and 4 (16.7%) were scored as +3. When
ing Vactivi



were compared with SACs, the frequency of strongimmunoreactivity was slightly higher in CCCs

in SACs in both early-stage and advanced stage;ver, the differences were not statistically significant.ctively, these results indicate that VEGF can be apeutic target not only in patients with SAC asn previously in clinical trials (7, 8, 17) but also inpatients with CCC.next examined the impact of VEGF expression onal in patients with CCC. Although the correlationen VEGF expression and survival has been intensive-estigated and reported in patients with SAC, to thef our knowledge, it has never been examined ints with CCC. Interestingly, as shown in Fig. 1C,noreactivity for VEGF significantly correlated witht prognosis in patients with early-stage CCC. Thell survival in the groupwithweak expression ofVEGF60 months) was significantly higher than that in thewith high expression of VEGF (mean 40 months). Ints with advanced-stage CCC, all tumor samplesd strong staining for VEGF; thus, we did not observeelation between VEGF immunoreactivity and patientosis in this subgroup.However, the overall survival intswith advanced-stageCCCwas significantly shorterthat observed in the early-stage, weak-expressionor in the early-stage, strong-expression group.

production in CCC cells and antiangiogenicty of bevacizumab in vitroen the frequent VEGF expression found in humantumor specimens (Fig. 1), we evaluated the in vitrossion of VEGF in two human CCC cell lines. Forurpose, we assessed the release of VEGF into thee medium by enzyme-linked immunosorbent assayA). As shown in Fig. 2A, VEGF was secreted in thee medium of both CCC cell lines tested, which istent with immunohistochemical results observed insamples. The concentration of VEGF significantly

sed in response to the exposure to hypoxia in allnes.next examined the proangiogenic activity ofreleased by CCC cells. As shown in Fig. 2B (I)

II), treatment with either recombinant VEGF ored medium containing VEGF secreted by CCCsignificantly stimulated the proliferation ofCs. Moreover, treatment with either recombinantor culture medium containing VEGF enhancedbe formation activity of HUVECs (Fig. 2C). Col-ely, these results suggest that VEGF produced bycells have significant angiogenic activity and thatinhibition may be a promising therapeutic strat-

n the management of CCC. Therefore, we exam-he antiangiogenic activity of bevacizumab in vitro.own in Fig. 2B and C, treatment with bevacizumabt completely inhibited the growth-stimulating activ-d tube formation activity of culturemedium contain-
EGF, consistent with the significant antiangiogenicties of bevacizumab.



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t of bevacizumab on the growth of ovarianin vivoxamine the in vivo growth-inhibitory effect of bev-ab, we used a xenograft model in which athymic

d in the early-stage, weak-expression group (log-rank; P < 0.0001) or in t

were inoculated s.c. with RMG1 or KOC7C cells.tumors reached ∼50 mm3, the mice were random-

(Fig. 3CCCs

acrjournals.org


nto two treatment groups receiving PBS or beva-ab. Drug treatment was well tolerated, withparent toxicity throughout the study. Tumor vol-was measured weekly after the start of treatment

ly-stage, strong-expression group (log-rank; P = 0.0036).

1. VEGF is frequently expressed in both ovarian CCCs and SACs. A, representative photographs of CCC and SAC (H&E staining, ×400). Left,aracterized by clear cells containing abundant cytoplasmic glycogen. Right, SAC characterized by an exophytic proliferation of cellular papillae.sentative photomicrographs of weak, moderate, and strong staining for VEGF are shown. Magnifications: ×100 and ×400 (insets). C, Kaplan-Meiercomparing the overall survival of the patients in all stages. The overall survival in the weak-expression group was significantly longer than that

). Histologically, these subcutaneous tumors were(data not shown). At 4 weeks after the start of




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ent, the mean RMG1-derived tumor burden intreated with bevacizumab was 232.3 mm3 com-with 456.3 mm3 in PBS-treated mice, and meanC-derived tumor burden in animals treated withizumab was 198.8 mm3 compared with 532.9 mm3

-treated mice. These results indicate that bevacizu-as significant antitumor effects as a single agent inCC model.investigate the mechanism by which bevacizumabted the tumor growth in vivo, the endothelial mark-31 in RMG1-derived subcutaneous tumors was as-by immunohistochemistry (Fig. 3C). As shown,

CD31-immunopositive vessels were observed ins from PBS-treated mice, whereas fewer and smal-ssels were observed in tumors from bevacizumab-d mice. There was a significant decrease of MVA inizumab-treated tumors compared with controls (Fig. 3D). To exclude the possibility that bevaci-b directly inhibits the growth of CCC cells, wer examined the effect of bevacizumab on the prolif-n of CCC cells in vitro. Treatment with either VEGF,izumab, or the combination for 72 hours had noon the proliferation of CCC cells (data not shown).results are consistent with the tumor-suppressiveof bevacizumab being mediated primarily throughtion of neovascularization.

expression in cisplatin-refractory cell linesevaluate the preclinical antitumor efficacy of beva-ab on recurrent CCCs after the first-line treatmentplatinum-based chemotherapy, we established

tage III-IV 24 0 1 (4.2

n-refractory CCC cell lines as described above. Toe whether these sublines had acquired resistance to

PBS-treated mice. The antitumor effect of bevacizumabwas similar both in cisplatin-refractory cell–derived

. VEGF production in CCC cells and antiangiogenic activity of bevacizumab in vitro. A, 5 × 104 RMG1 and KOC7C cells were incubated in DMEM-12 containing 1% FBS for 24 h. Levels of secreted VEGF protein in the conditioned medium under normoxic (20% O2) or hypoxic (1% O2)s were measured by ELISA assay. *, P < 0.05, significantly different from control. B, effect of either recombinant VEGF (I) or cultured medium (II)oliferation of HUVECs. HUVECs were treated with VEGF or cultured medium containing VEGF secreted by CCC cells (RMG1 or KOC7Cpoxic conditions) with or without the indicated concentration of bevacizumab in the presence of 5% FBS for 72 h under a normoxic (20% O2). Cell viability was assessed by MTS assay. *, P < 0.05, significantly different from control. C, inhibitory effect of bevacizumab on VEGF-inducedation in vitro. HUVECs were cultured with VEGF or cultured with medium containing VEGF secreted by CCC cells (RMG1 or KOC7C under

tin

2Fnprynrm
conditions), with or without bevacizumab for 8 h under normoxic (20% O2) condit
; b, bevacizumab; c, VEGF; d, VEGF plus bevacizumab. (II, III), three random fieldasured. Columns, mean (n = 4); bars, SD. The concentrations of agents used inL. *, P < 0.05, significantly different from control.



tin, we first evaluated the sensitivity of these cello cisplatin by the MTS assay. As shown in Fig. 4A,differential sensitivity to cisplatin was observed be-parental cells and respective cisplatin-refractoryes. Moreover, treatment with cisplatin inducedge of PARP in parental cells, but not in cisplatin-tory sublines (Fig. 4B). We next examined the pro-n of VEGF in both cisplatin-refractory sublines andtal cells by ELISA. As shown in Fig. 4C, in parentalcells, VEGF production was significantly inhibitedatment with cisplatin. In cisplatin-refractory CCCsignificantly higher concentrations of VEGF wereved than in their respective parental cells. More-VEGF production was not inhibited by treatmentcisplatin.

t of bevacizumab on the cisplatin-refractoryin vivoen the strong VEGF expression found in cisplatin-tory CCC cells, we next examined the in vivo effectvacizumab on cisplatin-refractory CCC. Athymicwere inoculated s.c. with RMG1-CR or KOC7C-ells, and were randomized into two treatments receiving PBS or bevacizumab. Graphs depictingished tumor volumes in bevacizumab-treatedrelative to PBS-treated mice are shown in Fig. 5.RMG1-CR–derived tumor burden in mice treatedbevacizumab was 173.1 mm3 compared withmm3 in PBS-treated mice, and mean KOC7C-erived tumor burden in animals treated with beva-ab was 171.1 mm3 compared with 566.3 mm3 in

19 (79.1) 4 (16.7)

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Molecular Cancer

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No. of patients Immunoreactivity

Zero, n (%) Weak (+0.5 or 1), n (%) Moderate (+2), n (%) Strong (+3), n (%)

e I-II 27 0 7 (26.9) 14 (51.9) 6 (22.2)
e III-IV 25 0 15 (60) (40)
stage I-II 22 0 4 (18.2) 15 (68.2) 3 (13.6)

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Mol Cancer Ther; 9(8) August 2010acrjournals.org 2417

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and in parental cell–derived CCC (Figs. 3 and 5).tively, these in vivo data indicate that inhibitionGF may be a reasonable treatment strategy fortin-refractory CCCs.finally compared the antitumor effect of cisplatinthat of bevacizumab in vivo. As shown in Fig. 6,gh treatment with cisplatin significantly decreased1-derived tumor burden, the antitumor effect oftin was minimal on RMG1-CR–derived tumors,is consistent with the cisplatin-resistant natureG1-CR cells shown in Fig. 4A. Importantly, theof bevacizumab on both RMG1-derived– and

1-CR–derived tumors was more profound thanf cisplatin, which may indicate that bevacizumabically more efficacious for CCCs than cisplatin.

ssion

C of the ovary was originally referred to as “mesone-a” by Schiller in 1939 because it resembled renaloma and was thought to be of mesonephric origin

ti-CD31/PECAM-1 antibody. D, MVA in the RMG1-derived subcutaneousColumns, mean; bars, SD. *, P < 0.05, significantly different from PBS-tre

ubsequent findings of its association with endome-have suggested that CCC is of Mullerian origin (35),

beenovaria



his tumor was recognized as a distinct histologicpe of epithelial ovarian tumors by the World Healthization in 1973 (3). A growing body of evidenceggested that CCC is associated with a diminishedivity to platinum-based chemotherapy and a worseosis than other epithelial ovarian cancers. To im-survival, the development of new treatment strate-at target CCC more effectively is necessary.recent gene expression profiling study, it has been

ted that renal CCC and ovarian CCC have remark-imilar expression patterns, and thus could not beguished statistically (36). Thus, theoretically, a rele-arget molecule in the treatment of renal CCC mayold promise as a therapeutic target in patients withof the ovary (37).ently, bevacizumab, a monoclonal antibody to hu-VEGF, has shown significant antitumor activity inmized clinical trials and has become the standarde for patients with metastatic renal cancer (38),of which is clear cell histology (renal CCC). Al-h significant clinical activity of bevacizumab has

was measured, and percent area was calculated in eachmors.

3. Effect of bevacizumab on growth of ovarian CCC in vivo. Athymic nude mice were inoculated s.c. with RMG1 or KOC7C cells, with 12 micegroup. When tumors reached ∼50 mm3, mice were treated with placebo or 5 mg/kg bevacizumab twice a week for 4 wk. A and B, graphsg weekly tumor volumes (mm3) for each treatment group. Points, mean; bars, SD. *, P < 0.05, significantly different from PBS-treated mice.termine the antiangiogenic activity of bevacizumab, the MVA in the RMG1-derived subcutaneous tumors was assessed by immunohistochemistry

shown in three phase II studies in patients withn cancer (7, 8, 17), most of the patients enrolled




in thewereas a tunkno

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se trials had SAC. Only seven patients with CCCenrolled in these trials; thus, the role of VEGF
herapeutic target in CCC of the ovary is largely comp
n-refractory sublines and parental chemosensitive cells. Levels of secreted VEGFn were measured by ELISA assay. Columns, mean; bars, SD. *, P < 0.05, signif

acrjournals.org


poxia commonly develops within tumors. HIF-1αa key role in helping hypoxic tumor cells to

ensate for hypoxia at the molecular level by in-
wn. creasing the activity or the expression of variety of
4. VEGF expression and its role as a therapeutic target in cisplatin-refractory CCC cells. A and B, establishment of cisplatin-refractory variants. Cisplatin-refractory sublines were established as described in Materials and Methods. A, parental (KOC7C and RMG1) and cisplatin-refractory(KOC7C-CR and RMG1-CR) cells were treated with the indicated concentrations of cisplatin in the presence of 5% FBS for 72 h. Cell viabilitysessed by MTS assay. Points, mean; bars, SD (*, P < 0.05, **, P < 0.01, significantly different from control.). B, effect of cisplatin on cleavage of PARPntal and cisplatin-refractory variant cell lines. KOC7C, KOC7C-CR, RMG1, and RMG1-CR treated with 10 μmol/L cisplatin or bevacizumab forells were harvested, and then lysates were subjected to Western blotting using anti-PARP or anti–β-actin antibody. C, production of VEGF in

protein in the conditioned medium under normoxic or hypoxicicantly different from control.




proteishowhigheovariendomlatessis totumorrationtientsIn

expreStrongquentCCCsdepen

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ns connected with angiogenesis. Recent work hasn that HIF-1α expression levels are significantlyr in CCC than in other histologic subtypes ofan cancer, including serous, mucinous, andetrioid carcinoma (22). Because HIF-1α stimu-

the expression of VEGF and promotes angiogene-meet the metabolic requirements for sustainedgrowth (39), there is a particularly strong clinicalale for blocking VEGF in the treatment for pa-with CCC.the present study, we show that VEGF wasssed in all stage III-IV CCCs and stage I-II CCCs.VEGF immunoreactivity was observed more fre-

ly in advanced-stage CCCs than in early-stage

= 12) or RMG1-CR cells (n = 12) were treated with placebo or 5 mg/kg bevas (mm3) for each treatment group. Points, mean; bars, SD. *, P < 0.05, sig

, suggesting that advanced-stage CCCs are more mor e

/kg cisplatin twice a week for 4 wk. PBS, bevacizumab, and cisplatin were admins (mm3) for each treatment group. Points, mean; bars, SD. *, P < 0.05, significan



CCCs. Importantly, as previously shown in patientsSAC (12, 13), immunoreactivity for VEGF was in-y correlated with patient prognosis in early-stagePatients whose tumor showed strong immunoreac-had significantly shorter survival than those withimmunoreactivity for VEGF (mean, 60 monthss 40 months, respectively).evaluated the efficacy of bevacizumab in vivo,s.c. xenograft models (Fig. 3). As predicted, intra-neal treatment with bevacizumab was well tole-with no apparent toxicity. In mice inoculated s.c.RMG1 or KOC7C cells, treatment with bevacizu-ignificantly inhibited tumor growth. These findingste that bevacizumab could have significant antitu-

ab twice a week for 4 wk. A and B, graphs depicting weekly tumortly different from PBS-treated mice.

5. Effect of bevacizumab on the growth of cisplatin-refractory CCC-derived tumors in vivo. Athymic nude mice inoculated s.c. with KOC7C-CRcizum

ffects as a single agent for CCC in the setting of
dent on VEGF for tumor progression than early- first-line therapy.
6. Comparison between the effect of bevacizumab versus cisplatin on the growth of CCC-derived tumors in vivo. Athymic nude mice were inoculatedh RMG1 or RMG1-CR cells, with 12 mice in each group. When tumors reached ∼50 mm3, mice were treated with PBS, 5 mg/kg bevacizumab,

istered intraperitoneally. A and B, graphs depicting weekly tumortly different from PBS-treated mice.




AnantituCCC.CCCsmajorTherefor rethe cCCCpared4C).For eLewis1.5-focells (nocarexpreity conomarefracmentOu

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additional important finding in our study is themor activity of bevacizumab in cisplatin-refractoryThe lack of effective chemotherapy for recurrentafter first-line platinum-based chemotherapy is aclinical problem in the management of CCC.

fore, identification of new treatment strategiescurrent CCC of the ovary is urgently needed. Inurrent study, we found that cisplatin-refractorycell lines exhibit higher VEGF expression com-with the corresponding parental cell lines (Fig.

Similar findings have been reported by others.xample, VEGF production in cisplatin-resistantlung carcinoma cells has been reported to be

ld higher than that in cisplatin-sensitive parental40). Moreover, 5-fluorouracil–resistant colon ade-cinoma subclones were found to have increasedssion of VEGF and enhanced proangiogenic activ-mpared with corresponding primary adenocarci-cells (41). These results suggest that cisplatin-

tory tumors might be good candidates for treat-with bevacizumab.r cisplatin-refractory CCC cell–derived tumorsed significant sensitivity to bevacizumab in vivo). However, although cisplatin-refractory CCC cellsss higher concentrations of VEGF than parentalthe antitumor effect of bevacizumab on cisplatin-tory cell–derived tumors was similar to that ob-in parental cell–derived tumors. This finding is

tent with recent reports suggesting that VEGF ex-on may not be a reliable biomarker for predictingivity to bevacizumab (42). Importantly, when wely compared bevacizumab with cisplatin, the in vivooplastic effect of bevacizumab on CCC-derived tu-rowth was more profound than that for cisplatin6). The dose of cisplatin used in our experiment/kg) is roughly equivalent to the standard clinical(50–75 mg/mm2) used in patients (43). Moreover,MG1-CR and KOC7C-CR cells used in this studythe clinical situation involving the development
istance to cisplatin. Thus, our results suggest
ically efficacious not onlyRece

publish

epithelial ovarian cancer: a Gynecologic Oncology Group Study.lin Oncol 2007;25:3621–7.tzer DR, Sun CC, Coleman RL, Wolf JK, Levenback CF,

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irst-line treatment but also as a second-line treat-of recurrent disease in patients previously treatedcisplatin.onclusion, our collective findings indicate that bev-ab is a promising agent for the treatment of CCCovary both as a first-line treatment and as a salvageent for recurrence after platinum-based chemo-y. Although bevacizumab is currently being evalu-by the Gynecologic Oncology Group (GOG) incol GOG 218 (9) to evaluate the benefit of bevacizu-n combination with first-line chemotherapy as wellen administered as maintenance therapy, to date,nical studies have been initiated to examine themor effect of bevacizumab specifically in patientsCCC. We believe that our data provide significantale for future clinical trials with bevacizumab in pa-with CCC of the ovary.

osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

owledgments

hank Drs. Min Huang and Yulan Gong in the Tumor Bank Facilityparing tissue microarrays, and Drs. M. Tsujimoto (Department ofgy, Osaka Police Hospital), Y. Tsubota (Department of Pathology,ama Rosai Hospital), Y. Hoshida (Department of Pathology, Kansaiospital), and H. Miwa (Department of Pathology, Sakai Municipall) for providing tumor specimens and clinical information.

Support

publication was supported in part by NIH grants P30 CA006927-77429, and P50 CA83638, and by an appropriation from the Com-ealth of Pennsylvania. Grant-in-aid for Young Scientists no.54 from the Ministry of Education, Culture, Sports, Science andlogy of Japan. D.A. Altomare is supported in part by the Liz TilberisProgram, sponsored by the Ovarian Cancer Research Fund, Inc.costs of publication of this article were defrayed in part by thet of page charges. This article must therefore be hereby markedement in accordance with 18 U.S.C. Section 1734 solely to indicatet.

ived 02/22/2010; revised 05/27/2010; accepted 06/04/2010;ed OnlineFirst 07/27/2010.
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2010;9:2411-2422. Published OnlineFirst July 27, 2010.Mol Cancer Ther Seiji Mabuchi, Chiaki Kawase, Deborah A. Altomare, et al. Target for the Treatment of Clear Cell Carcinoma of the OvaryVascular Endothelial Growth Factor Is a Promising Therapeutic

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Therapeutics Vascular Endothelial Growth Factor Is a ... · cell carcinoma (CCC) of the ovary, which has been regarded as a chemoresistant histologic subtype. Immu-nohistochemical