Ovarian Cancer Patient-derived Xenografts Research Articles

Abstract 324: Targeting RNA-polymerase I using CX-5461 as a mechanism for treating chemotherapy resistant epithelial ovarian cancer

Abstract BACKGROUND: One of the first identified hallmarks of neoplastic transformation was an increased rate of ribosome biogenesis, and increased activity of RNA polymerase I (RNA pol I) is observed in most human cancers. Ribosome biogenesis is an energy intensive and tightly regulated process, and recent studies have shown that CX-5461, a potent and specific RNA pol I inhibitor, has selective toxicity to cancer cells. Here we demonstrate that CX-5461 is an effective anti-proliferative agent in epithelial ovarian carcinoma cells with an increased efficacy in chemoresistant ovarian cancer cells compared to their sensitive parental lines. METHODS AND RESULTS: Ovarian cancer cells were demonstrated to be highly sensitive to RNA pol I inhibition by MTT assay, with an IC50 range from 25nM to 2uM in 13 different ovarian cancer cell lines, and an IC50 of 3uM in the nontransformed immortalized normal surface epithelial line HIO-180. Notably, chemoresistant cell populations were generally more sensitive to CX-5461, compared to matched chemosensitive lines. IC50 in HeyA8MDR was 80nM compared to 2000nM in parental HeyA8, and was 100nM in SKOV3TRip2 compared to 400nM in SKOV3ip1. CX-5461 induces G2/M arrest (averaging 52.7% in G2 compared to 32.5% in controls), primarily through mitotic catastrophe, inducing an accumulation of multinucleated senescent cells. DNA damage pathways are activated, operating through ATM/ATR, as demonstrated by rescue with caffeine and ATR specific inhibitors. In an effort to identify why chemoresistant cells might be more susceptible, ribosomal synthesis processes were examined. Chemoresistant cells were found to have 2-4-fold increased Pol I occupancy on rDNA by ChIP, and increased rRNA synthesis by isotopic incorporation, while ribosome abundance and overall translation efficiency, measured by sucrose gradient fractionation, were unchanged. Treatment of five separate patient-derived xenograft models (PDX) with CX-5461 showed a complete response in one model, partial 55% reduction in tumor size in a second, stable disease in a 3rd, and progression in two models. CONCLUSIONS: CX-5461 shows high activity in ovarian cancer cell lines and patient-derived xenograft models, with a preferential effect on chemoresistant cells. Enhanced sensitivity to the compound is due at least in part to cancer cells’ increased demand for rRNA synthesis. RNA pol I inhibitors represent an exciting new treatment option that may have preferential effect on the chemoresistant population. Citation Format: Robert Cornelison, Zachary C. Dobbin, Ashwini A. Katre, Dae Hoon Jeong, Yulia Petrova, Danielle C. Llaneza, Adam D. Steg, Yinfeng Zhang, David A. Schneider, Charles N. Landen. Targeting RNA-polymerase I using CX-5461 as a mechanism for treating chemotherapy resistant epithelial ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 324.

251 - Ovarian cancer patient-derived xenografts resistant to cisplatin exhibit metabolic changes

251 - Ovarian cancer patient-derived xenografts resistant to cisplatin exhibit metabolic changes

522 - Cediranib affects tumor dissemination and prolongs the survival of mice bearing patient-derived ovarian cancer xenografts (EOC-PDX)

522 - Cediranib affects tumor dissemination and prolongs the survival of mice bearing patient-derived ovarian cancer xenografts (EOC-PDX)

Phase 1b of WNT inhibitor ipafricept (IPA, decoy receptor for WNT ligands) with carboplatin (C) and paclitaxel (P) in recurrent platinum-sensitive ovarian cancer (OC).

2515Background: The WNT pathway is an important oncologic driver of OC. The first-in-class recombinant fusion protein IPA blocks WNT signaling through binding of WNT ligands. In patient-derived OC xenografts, IPA inhibits growth, reduces cancer stem cell frequency, promotes differentiation, synergizes with taxanes and shows greatest efficacy when given 2 to 3 days before the taxane. In Phase 1a, IPA was well tolerated with dysgeusia, fatigue, muscle spasms and anorexia as most common adverse events (AEs). Target modulation was observed in hair follicles at ≥ 2.5 mg/kg every 3 weeks (q3w). Consequent to the clinical experience with, mainly, vantictumab (VAN), a 2nd WNT inhibitor, serum biochemical markers of bone turnover were used to trigger bone-protective zoledronic acid (ZA)therapy, if appropriate. Methods: Dose escalation started with a standard 3+3 design for IPA/C/P with q3w intravenous IPA on Day 1, in cycles 1 to 6 with C (AUC = 5 mg/mL · min) and P (175 mg/m2). For enhanced bone safety the trial ...

Patient-derived ovarian cancer xenografts re-growing after a cisplatinum treatment are less responsive to a second drug re-challenge: a new experimental setting to study response to therapy.

Even if ovarian cancer patients are very responsive to a cisplatinum-based therapy, most will relapse with a resistant disease. New experimental animal models are needed to explore the mechanisms of resistance, to better tailor treatment and improve patient prognosis. To address these aims, seven patient-derived high-grade serous/endometrioid ovarian cancer xenografts were characterized for the antitumor response after one and two cycles of cisplatinum and classified as Very Responsive, Responsive, and Low Responsive to drug treatment. Xenografts re-growing after the first drug cycle were much less responsive to the second one. The expression of epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) genes was investigated in cisplatinum-treated and not-treated tumors. We found that different EMT (TCF3, CAMK2N1, EGFR, and IGFBP4) and CSCs (SMO, DLL1, STAT3, and ITGA6) genes were expressed at higher levels in Low Responsive than in Responsive and Very Responsive xenografts. The expression of STAT3 was found to be associated with lower survival (HR = 13.7; p = 0.013) in the TCGA patient data set. MMP9, CD44, DLL4, FOXP1, MERTK, and PTPRC genes were found more expressed in tumors re-growing after cisplatinum treatment than in untreated tumors. We here describe a new in vivo ovarian carcinoma experimental setting that will be instrumental for specific trials of combination therapy to counteract cisplatinum resistance in order to improve the prognosis of ovarian patients.

Abstract B48: Design of effective combination therapies for high-grade serous ovarian cancer using patient-derived xenograft models.

Abstract Despite advances in understanding the genetics and molecular biology of high grade serous ovarian cancer, there have not been any significant improvements in the outcome of patients treated with approved therapies to date. The overall aim of our studies is to identify synergistic drug combinations for the treatment of high-grade serous ovarian (HGS-Ov) cancer and the biomarkers that predict sensitivity for future translation in clinical trials. In particular, this study focuses on the vulnerabilities of patient-derived ovarian cancer cells to inhibition of anti-apoptotic proteins and the design of novel combination therapies to overcome drug resistance and improve the response of selected patients to a particular set of targeted therapies. Previous studies from our lab provided evidence that inhibition of BCL-2/XL can significantly enhance the sensitivity of tumor cells to targeted therapies in ovarian and breast cancer cell line models (Muranen T, et al. Cancer Cell 2012). In this study, we evaluated the efficacy of this combination treatment in ascites cells derived from 15 high-grade serous patient-derived ovarian cancer xenografts (PDXs). We found that inhibitors of PI3K/mTOR (GNE-493) and BCL-2/XL (ABT-737) act synergistically ex vivo, with an ~15 fold variability among the 15 patient-derived samples. A large scale in vivo experiment is ongoing to evaluate the efficacy of this combined treatment in six PDX models. To identify biomarkers that predict drug sensitivity we performed proteomic Reverse Phase Protein Array (RPPA) and immunoblot analyses. We found that baseline levels of the pro-apoptotic protein BIM correlate with sensitivity to BCL-2/XL inhibition. This motivated us to examine the basis for low BIM expression. In three of the least sensitive PDX models, we found that low levels of BIM correlated with ERK activation based on increased ERK phosphorylation and that inhibition of MEK by PD-0325901 in these models caused upregulation of BIM, as predicted from previous published reports which demonstrated that phosphorylation of BIM by ERK causes its degradation (Ley R, et al. Biol Chem 2003). We then examined the sensitivity of these PDX models to ABT-737 and PD-0325901, MEK162, or CI-1040 ex vivo and observed strong synergy resulting in reduction of cell viability and increased cell death. These results suggest that low BIM/high phosphoERK is a potential biomarker for sensitivity of ovarian tumors to combined MEK and BCL-2/XL inhibition. We now plan to examine the efficacy of drug combinations that target MEK and BCL-2/XL in these PDX models in vivo. Our studies promise to lead to the identification of new drug combination therapies for HGS-Ov cancer treatment and predictive biomarkers to stratify patients that can benefit from these targeted therapies. Citation Format: Claudia Iavarone, Ioannis Zervantonakis, Hsing-Yu Chen, Sangeetha S. Palakurthi, Joyce Fu Liu, Ursula A. Matulonis, Ronny I. Drapkin, Gordon B. Mills, Joel D. Leverson, Deepak Sampath, Joan S. Brugge. Design of effective combination therapies for high-grade serous ovarian cancer using patient-derived xenograft models. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B48.

Abstract B46: Use of a novel orthotopic ovarian cancer transplant patient derived xenograft model as a preclinical platform for bench to bedside research.

Abstract Introduction: Treatment for cancer is moving from a “one size fits all” to a personalized and targeted approach addressing the unique biology of each patients' tumor. Preclinical research in ovarian cancer (OVCA) has relied on established cell lines that have recently been shown to have marked differences in molecular profiles when compared to ovarian tumors from the TCGA. Patient derived xenografts (PDXs) are emerging as a reliable preclinical model that can recapitulate the principal characteristics of the patients' original tumor while remaining biologically stable while passaged in mice. We developed an orthotopic model that involves transplanting tumor directly to the fallopian tube/ovary in order to accurately study ovarian cancer tumorigenicity and metastasis in the native environment. Experimental Procedures: Fresh OVCA tumor was transplanted orthotopically to the fallopian tube/ovary of NSG 5-8 week female mice. Tumor growth was followed over time with ultrasound. Tumors were evaluated by IHC, genomic and proteomic analysis. Alu II probe staining was performed to evaluate human stroma content. DNA sequencing analysis was performed using a 153 ovarian cancer gene panel, which includes all genes relevant to OVCA, including homologous recombination and all actionable genes. Reverse Phase Protein Array Analysis (RPPA) was used to evaluate signaling pathway activation. Several primary ovarian tumor cultures were also developed from the patients' tumor for mechanistic studies. Results: To date, we have transplanted tumor from 18 primary, 4 interval, and 6 recurrent ovarian debulking surgeries using an orthotopic ovarian tumor transplant approach with an 90% success rate in generating tumors in mouse passage 1 (MP1) and 100% in generating MP2 and MP3. The mean time for tumors to reach ~1 cm (first passage in mice) was 8 weeks and ~6 weeks when tumors were then passaged again (MP2/MP3). We have generated 12 BRCA1/2 deleterious mutation positive carrier PDXs. Alu staining of PDXs demonstrated human cells in the stroma. We have generated several primary tumor cell lines from the original tumors/PDXs. These primary cells express ovarian epithelial markers CK7, and PAX8. Targeted DNA sequencing analysis showed that P53 and BRCA1/2 pathogenic mutations were highly conserved from the patient tumor, to the PDXs (MP1-3) and primary tumor cell lines. We evaluated 280 phospho/total proteins in our tumor samples by RPPA. Unsupervised cluster analysis of 24 patient tumors and correlating PDX (MP1-3) and 6 cell lines showed that several parent tumors clustered together with their MP1-3 PDXs. WO-2-1 BRCA 2 mutant PDXs were sensitive to platinum treatment demonstrating a response similar to that noted in the original patient after platinum chemotherapy. Conclusions: Although technically more challenging the orthotopic transplantation technique is feasible in generating ovarian cancer PDX models that more closely resemble the natural environment for ovarian cancer tumor growth and metastasis. PDXs maintain consistent gene alterations and signaling pathway activation to the original patient tumor. Given PDXs maintain the characteristics of the patients' original tumor, they are excellent models to study therapeutic response. Citation Format: Erin George, Hyoung Kim, Clemens Krepler, Janos Tanyi, Zhi Wei, Katrin Sproesser, Patricia Brafford, Marilda Beqiri, Adina-Monica Vultur, Rachel Lee, Mark Morgan, Ronny Drapkin, Tan Ince, Meenhard Herlyn, Fiona Simpkins. Use of a novel orthotopic ovarian cancer transplant patient derived xenograft model as a preclinical platform for bench to bedside research. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B46.

Abstract B50: Antineoplastic activity of Top I inhibitor etirinotecan pegol (NKTR-102) and PARP inhibitor rucaparib (CO-388) in platinum-resistant high-grade serous BRCA WT ovarian cancer PDX models.

Abstract Background: At the time of ovarian cancer platinum resistance, standard therapies have limited activity. New anti-cancer drug combinations may lead to improved clinical outcomes. Activity of topoisomerase I (Top I) inhibitors or poly(ADP-ribose) polymerase (PARP) inhibitors have been reported in ovarian cancer and have demonstrated potentiation in other models. Objective: To investigate the efficacy of the Top I inhibitor etirinotecan pegol (NKTR-102) and the PARP inhibitor rucaparib (CO-338) as single agents and in combination in patient-derived xenograft (PDX) models of ovarian cancer. Methods: Five ovarian cancer PDX models in SCID mice were treated for 28 days with NKTR-102 (10 mg/kg/q7d IV), rucaparib (150 mg/kg/qd PO), or combination NKTR-102 plus rucaparib at the same doses and schedules. Of the five PDX models, four were platinum resistant and one was a platinum-sensitive positive control. Each treatment arm had a total of 10 mice and animals were observed daily for body weight and general condition. Tumor size was assessed weekly by ultrasound. The primary endpoint was change in tumor cross-sectional area (cm2) from pretreatment baseline to final tumor burden. Western blot was performed to investigate potential biomarkers of response. Results: Compared to vehicle-treated controls, statistically significant improvement (p<0.05) was observed in 3 of 5 models in single agent rucaparib treated tumors and in 5 of 5 models in single agent NKTR-102 treated tumors. The combination of NKTR-102 plus rucaparib also demonstrated statistically significant improvement compared to vehicle-treated controls in 5 of 5 models. The combination of NKTR-102 plus rucaparib induced maximal tumor regression in 2 of 5 tumors with a trend towards smaller tumors compared to single agent treatment arms. This was not found to be statistically significant in the 28 day study period under which the experiment was performed. Exploratory analysis to assess candidate drug response determinants (e.g.Top I, PAPR I, and PARP polymer formation levels) was investigated. Conclusion: NKTR-102 and rucaparib are well tolerated in mice and demonstrate activity in platinum-resistant and platinum-sensitive ovarian cancer PDX models. Combination therapy demonstrated a trend towards decreased tumor size when compared to single agent activity during treatment. Current efforts are focused on extending the time on study for the PDX models evaluated to determine if statistical superiority of the NKTR-102/rucaparib combination can be established. Citation Format: Caroline C. Nitschmann, Rachel M. Hurley, Marc A. Becker, Xiaonan Hou, Ute Hoch, Thomas C. Harding, Deborah H. Charych, Scott H. Kaufmann, Paul Haluska, Saravut J. Weroha. Antineoplastic activity of Top I inhibitor etirinotecan pegol (NKTR-102) and PARP inhibitor rucaparib (CO-388) in platinum-resistant high-grade serous BRCA WT ovarian cancer PDX models. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B50.

Studying platinum sensitivity and resistance in high-grade serous ovarian cancer: Different models for different questions.

High-grade serous ovarian cancer (HGSOC) has the highest mortality rate among all gynecological cancers. Patients are generally diagnosed in an advanced stage with the majority of cases displaying platinum resistant relapses. Recent genomic interrogation of large numbers of HGSOC patient samples indicated high complexity in terms of genetic aberrations, intra- and intertumor heterogeneity and underscored their lack of targetable oncogenic mutations. Sub-classifications of HGSOC based on expression profiles, termed 'differentiated', 'immunoreactive', 'mesenchymal' and 'proliferative', were shown to have prognostic value. In addition, in almost half of all HGSOC patients, a deficiency in homologous recombination (HR) was found that potentially can be targeted using PARP inhibitors. Developing precision medicine requires advanced experimental models. In the current review, we discuss experimental HGSOC models in which resistance to platinum therapy and the use of novel therapeutics can be carefully studied. Panels of better-defined primary cell lines need to be established to capture the full spectrum of HGSOC subtypes. Further refinement of cell lines is obtained with a 3-dimensional culture model mimicking the tumor microenvironment. Alternatively, ex vivo ovarian tumor tissue slices are used. For in vivo studies, larger panels of ovarian cancer patient-derived xenografts (PDXs) are being established, encompassing all expression subtypes. Ovarian cancer PDXs grossly retain tumor heterogeneity and clinical response to platinum therapy is preserved. PDXs are currently used in drug screens and as avatars for patient response. The role of the immune system in tumor responses can be assessed using humanized PDXs and immunocompetent genetically engineered mouse models. Dynamic tracking of genetic alterations in PDXs as well as patients during treatment and after relapse is feasible by sequencing circulating cell-free tumor DNA and analyzing circulating tumor cells. We discuss how various models and methods can be combined to delineate the molecular mechanisms underlying platinum resistance and to select HGSOC patients other than BRCA1/2-mutation carriers that could potentially benefit from the synthetic lethality of PARP inhibitors. This integrated approach is a first step to improve therapy outcomes in specific subgroups of HGSOC patients.

MAP3K8/TPL-2/COT is a potential predictive marker for MEK inhibitor treatment in high-grade serous ovarian carcinomas.

Ovarian cancer is a silent disease with a poor prognosis that urgently requires new therapeutic strategies. In low-grade ovarian tumours, mutations in the MAP3K BRAF gene constitutively activate the downstream kinase MEK. Here we demonstrate that an additional MAP3K, MAP3K8 (TPL-2/COT), accumulates in high-grade serous ovarian carcinomas (HGSCs) and is a potential prognostic marker for these tumours. By combining analyses on HGSC patient cohorts, ovarian cancer cells and patient-derived xenografts, we demonstrate that MAP3K8 controls cancer cell proliferation and migration by regulating key players in G1/S transition and adhesion dynamics. In addition, we show that the MEK pathway is the main pathway involved in mediating MAP3K8 function, and that MAP3K8 exhibits a reliable predictive value for the effectiveness of MEK inhibitor treatment. Our data highlight key roles for MAP3K8 in HGSC and indicate that MEK inhibitors could be a useful treatment strategy, in combination with conventional chemotherapy, for this disease.

Biobanking of patient and patient-derived xenograft ovarian tumour tissue: efficient preservation with low and high fetal calf serum based methods.

Using patient-derived xenografts (PDXs) for preclinical cancer research demands proper storage of tumour material to facilitate logistics and to reduce the number of animals needed. We successfully established 45 subcutaneous ovarian cancer PDXs, reflecting all histological subtypes, with an overall take rate of 68%. Corresponding cells from mouse replaced human tumour stromal and endothelial cells in second generation PDXs as demonstrated with mouse-specific vimentin and CD31 immunohistochemical staining. For biobanking purposes two cryopreservation methods, a fetal calf serum (FCS)-based (95%v/v) “FCS/DMSO” protocol and a low serum-based (10%v/v) “vitrification” protocol were tested. After primary cryopreservation, tumour take rates were 38% and 67% using either the vitrification or FCS/DMSO-based cryopreservation protocol, respectively. Cryopreserved tumour tissue of established PDXs achieved take rates of 67% and 94%, respectively compared to 91% using fresh PDX tumour tissue. Genotyping analysis showed that no changes in copy number alterations were introduced by any of the biobanking methods. Our results indicate that both protocols can be used for biobanking of ovarian tumour and PDX tissues. However, FCS/DMSO-based cryopreservation is more successful. Moreover, primary engraftment of fresh patient-derived tumours in mice followed by freezing tissue of successfully established PDXs is the preferred way of efficient ovarian cancer PDX biobanking.

Anti-EFNA4 Calicheamicin Conjugates Effectively Target Triple-Negative Breast and Ovarian Tumor-Initiating Cells to Result in Sustained Tumor Regressions.

Triple-negative breast cancer (TNBC) and ovarian cancer each comprise heterogeneous tumors, for which current therapies have little clinical benefit. Novel therapies that target and eradicate tumor-initiating cells (TIC) are needed to significantly improve survival. A panel of well-annotated patient-derived xenografts (PDX) was established, and surface markers that enriched for TIC in specific tumor subtypes were empirically determined. The TICs were queried for overexpressed antigens, one of which was selected to be the target of an antibody-drug conjugate (ADC). The efficacy of the ADC was evaluated in 15 PDX models to generate hypotheses for patient stratification. We herein identified E-cadherin (CD324) as a surface antigen able to reproducibly enrich for TIC in well-annotated, low-passage TNBC and ovarian cancer PDXs. Gene expression analysis of TIC led to the identification of Ephrin-A4 (EFNA4) as a prospective therapeutic target. An ADC comprising a humanized anti-EFNA4 monoclonal antibody conjugated to the DNA-damaging agent calicheamicin achieved sustained tumor regressions in both TNBC and ovarian cancer PDX in vivo. Non-claudin low TNBC tumors exhibited higher expression and more robust responses than other breast cancer subtypes, suggesting a specific translational application for tumor subclassification. These findings demonstrate the potential of PF-06647263 (anti-EFNA4-ADC) as a first-in-class compound designed to eradicate TIC. The use of well-annotated PDX for drug discovery enabled the identification of a novel TIC target, pharmacologic evaluation of the compound, and translational studies to inform clinical development.

Clostridium perfringens enterotoxin C-terminal domain labeled to fluorescent dyes for in vivo visualization of micrometastatic chemotherapy-resistant ovarian cancer.

Identification of micrometastatic disease at the time of surgery remains extremely challenging in ovarian cancer patients. We used fluorescence microscopy, an in vivo imaging system and a fluorescence stereo microscope to evaluate fluorescence distribution in Claudin-3- and -4-overexpressing ovarian tumors, floating tumor clumps isolated from ascites and healthy organs. To do so, mice harboring chemotherapy-naïve and chemotherapy-resistant human ovarian cancer xenografts or patient-derived xenografts (PDXs) were treated with the carboxyl-terminal binding domain of the Clostridium perfringens enterotoxin (c-CPE) conjugated to FITC (FITC-c-CPE) or the near-infrared (NIR) fluorescent tag IRDye CW800 (CW800-c-CPE) either intraperitoneally (IP) or intravenously (IV). We found tumor fluorescence to plateau at 30 min after IP injection of both the FITC-c-CPE and the CW800-c-CPE peptides and to be significantly higher than in healthy organs (p < 0.01). After IV injection of CW800-c-CPE, tumor fluorescence plateaued at 6 hr while the most favorable tumor-to-background fluorescence ratio (TBR) was found at 48 hr in both mouse models. Importantly, fluorescent c-CPE was highly sensitive for the in vivo visualization of peritoneal micrometastatic tumor implants and the identification of ovarian tumor spheroids floating in malignant ascites that were otherwise not detectable by conventional visual observation. The use of the fluorescent c-CPE peptide may represent a novel and effective optical approach at the time of primary debulking surgery for the real-time detection of micrometastatic ovarian disease overexpressing the Claudin-3 and -4 receptors or the identification of residual disease at the time of interval debulking surgery after neoadjuvant chemotherapy treatment.

Abstract AS25: An adeno-associated virus (AAV) 9 vector delivering modified human mullerian inhibiting substance (MIS) as a gene therapy for ovarian cancer

Abstract Purpose: Ovarian cancer is the most lethal malignancy of the female reproductive tract; as such, effective treatments are needed to improve patient survival. MIS has previously been shown to inhibit growth of a stem-like subset of the total cell population in ovarian cancer cell lines (Wei et al, 2010; Meirelles et al, 2012). We have recently engineered novel peptide modifications to human MIS (LR-MIS), which increase production and potency in vitro and in vivo (Pepin et al, 2013), and inserted it into an AAV vector. AAV delivered gene therapy has undergone a clinical resurgence with a good safety profile and sustained gene expression (Zhang et al, 2011). Therefore, we assessed the efficacy of a single intraperitoneal (ip) dose of AAV9-LR-MIS to inhibit tumor growth of an established cell line for subsequent evaluation in patient-derived ovarian cancer xenografts (PDX). Methods: 6 week old female nude mice were treated with a single ip injection of 3e11 or 1e12 virions of AAV9 LR-MIS or GFP. Serum levels of human MIS were monitored weekly by ELISA. After serum steady state was achieved, the established ovarian cancer cell line, OVCAR5 was xenografted subcutaneously at 1e6 cells per animal. Tumors treated with AAV were measured twice weekly. Additionally, Patient ascites were processed to isolate the cancer cell population, which was specifically amplified in vitro to create low passage primary cell lines. Results: AAV9-LR-MIS treatment resulted in elevated and sustained blood concentrations of MIS (Fig1A) in nude mice without any signs of toxicity, and significantly inhibited OVCAR5 tumor growth (p=0.0003) (Fig1B) in two separate experiments. AAV9-GFP revealed that the pancreas, omentum, gut mesentery, and body wall muscles were the primary sites of infection. Treatment with MIS induced downregulation of CCND1 in tumors. A panel of 15 primary ovarian cancer cell lines, all expressing the MIS type II receptor were generated from patient ascites and cells are being tested for their ability to grow as PDX in Nude, NOD/SCID, and NSG mice. Figure 1.AAV-LR-MIS treatment results in stable expression of MIS and inhibition of tumor growth of OVCAR5 xenografts. A) Weekly MIS ELISA of serum from mice after single doses of 1e10 to 1e12 virions. B) Tumor growth of 1e6 OVCAR5 cells xenografted into nude mice (N=5) pretreated with 3e11/mouse (1.5e13/kg) LR-MIS or GFP virus (p=0.0003 by two-way ANOVA).Figure 1. AAV-LR-MIS treatment results in stable expression of MIS and inhibition of tumor growth of OVCAR5 xenografts. A) Weekly MIS ELISA of serum from mice after single doses of 1e10 to 1e12 virions. B) Tumor growth of 1e6 OVCAR5 cells xenografted into nude mice (N=5) pretreated with 3e11/mouse (1.5e13/kg) LR-MIS or GFP virus (p=0.0003 by two-way ANOVA). Conclusions: AAV9 virally delivered human LR-MIS can produce adequate sustained levels of MIS that inhibited the growth of the human ovarian cancer cell line, OVCAR 5. AAV9 LR MIS provides a well-tolerated, low toxicity treatment of ovarian cancer, which can now be evaluated in a phase 0 trial using PDX models being prepared from human ovarian cancer ascites. Citation Format: David Pepin, Amanda Sosulski, Katherine Hendren, Li Hua Zhang, Fotini Nicolaou, Dan Wang, Guangping Gao, Patricia K Donahoe. An adeno-associated virus (AAV) 9 vector delivering modified human mullerian inhibiting substance (MIS) as a gene therapy for ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS25.

Abstract POSTER-THER-1404: in vivo synergism between chemotherapy and the 3C23K monoclonal antibody directed against the anti-müllerian hormone type II receptor in ovarian cancer patient-derived xenografts

Abstract Purpose of the study: Müllerian inhibiting substance, also called anti-Müllerian hormone (AMH), belongs to the TGF-β family and plays a key role during sexual development. It has been shown that AMH inhibits proliferation and induces apoptosis of AMH type II receptor-positive tumor cells, and particularly progenitor cancer cells. Moreover, various reports have underlined expression of AMHRII in human gynecologic cancers including ovarian tumors. Based on these considerations, a humanized glyco-engineered monoclonal antibody (3C23K) capable of increasing effector cells engagement has been developed and tested in AMHR2 transfected xenografted cell lines. In order to document its activity in naturalistic models, we have initiated a preclinical study to assess AMHRII expression in a panel of ovarian cancer patient-derived xenografts (PDXs) and evaluate in vivo efficacy of 3C23K. Experimental procedures: AMHRII expression was studied in 14 ovarian cancers PDXs, using both flow cytometry (FC) and immunofluorescence (IF) analyses performed with alexaFluor488-conjugated 3C23K antibody on 300µm slices of xenograft fragments. In vivo efficacy of 3C23K alone (10 or 20 mg/kg twice a week, IP) or in combination with standard chemotherapy (CT) (carboplatin 66 mg/kg IP days 1-22 + paclitaxel 30 mg/kg IP days 1-22) has been planned to be evaluated in 4 AMHRII-positive ovarian cancers PDXs. One undifferentiated highly proliferative serous ovarian adenocarcinoma PDX, the OV54 model, has already been treated. On these 4 selected models, expression and quantification of AMHRII was or will be performed before and after treatments, as well as tumor-associated macrophages (TAMs) infiltrate. Summary of the data: Expression studies showed that over the 14 PDX tested, 6 (43%) were positive with at least 25% of tumor cells being positive, among these 3 expressed more than 50% of positive cells. This expression was confirmed by ex vivo IF with a profile comparable to that observed with fresh primary human ovarian tumors resected from stage IV patients. In vivo experiments (OV54 model) showed an intermediate efficacy of the 3C23K antibody administered alone, as well as a high and significant increase of CT antitumor effect. Synergistic effects were characterized by 50% of complete response (CR) when 3C23K (20mg/kg/inj.) was added to CT vs 10% CR with CT alone. The cumulative number of days under CR was 110 with 3C23K + CT vs 7 on CT alone. Impact on survival was consistent with a 90% survival at D37 with 3C23K + CT vs 60% survival on CT alone. We did not observe modification of AMHRII expression in OV54 tumors after 3C23K administration. Stromal FC analyses showed a high TAMs infiltrate of M2 type compatible with induction of Antibody Dependent Cell Phagocytosis by 3C23K. Conclusions: Using a panel of 14 ovarian cancer PDXs, we confirmed the expression of AMHRII in human ovarian cancers. Targeting this receptor by a specific monoclonal antibody, i.e. 3C23K, dramatically increases the efficacy of a standard chemotherapy. Our results therefore suggest that such combination could be further developed in ovarian cancer patients whose prognosis remains dismal. Citation Format: Fariba Némati, Jean-Marc Barret, Gérald Massonnet, Houcine Bougherara, Marie-Aude Le Frère-Belda, Jean-François Prost, Didier Decaudin. in vivo synergism between chemotherapy and the 3C23K monoclonal antibody directed against the anti-müllerian hormone type II receptor in ovarian cancer patient-derived xenografts [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1404.

Abstract 1470: Ovarian cancer PDXs preserve preexisting genetic oligoclonality

Abstract Advanced Epithelial Ovarian Cancer (EOC) is the leading cause of female cancer casualties and its prognosis remains grim, with about 30% of 5-year overall survival. Classically EOCs have been stratified according to 5 histotypes, tumor grading and disease stages. However, worst prognosis is associated to high grade EOCs, which mostly comprise serous ovarian carcinomas (SOC). A large majority of the patients relapse by 18 to 24 months after end of chemotherapy (CT) and eventually develop resistant disease. Research aiming at proposing more effective therapies relies on a limited set of cancer cell lines established several decades ago and whose representativeness of the actual disease has been put in doubt (Domcke et al., 2013; Nature Comm). This strongly calls for the production of novel EOC models to support experimental and preclinical work. In the last decade patient derived xenografts (PDX) have gained considerable interest, because they faithfully reproduce the characteristics of the tumor of origin. We, here, report the establishment of a collection of 35 ovarian cancer PDXs resulting from the original graft of 77 ovarian tumor samples onto immuno-compromised mice. Interestingly, established PDXs covered the diversity of EOC histotypes. We characterized a subset of 14 EOC PDXs at the genetic (array-CGH and transcriptome) and histological levels. This revealed that PDXs perfectly reproduce the original genetic and morphological features of the ovarian tumors they stemmed from. It was of note that PDXs conserved the principal characteristics of the original CNC profiles over several passages. However, a detailed comparison revealed fluctuation of CNC patterns in a fraction of chromosomal regions between the original tumor and the PDXs, that could be attributed to the oligoclonal nature of the original tumor. The analysis by CGH, FISH and exome sequencing of one case, for which several tumor nodules were sampled and grafted, revealed that PDX globally maintained an oligoclonal structure. Despite fluctuations in the relative prevalence of some mutations according to the sample, we did not observe any overgrowth of a particular subclone present in the original tumor. This suggests that xenotransplantation of ovarian tumors and growth as PDX is not associated to a drastic change in selective pressure. We believe these findings are of interest to determine the impact of different therapy regimen on the clonality of EOC. Citation Format: Pierre-Emmanuel Colombo, Stanislas du Manoir, Beatrice Orsetti, Rui Bras-Goncalves, Alan MacKay, Maryou Lambros, Tuan T. Nguyen, Florence Boissiere, Didier Pourquier, Jorge Reis-Filho, Charles G. Theillet. Ovarian cancer PDXs preserve preexisting genetic oligoclonality. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1470. doi:10.1158/1538-7445.AM2015-1470

Abstract 5425: An anti-Ephrin-A4 calicheamicin conjugate effectively targets triple-negative breast and ovarian tumor-initiating cells to result in sustained tumor regression

Abstract Triple-negative breast cancer (TNBC) and ovarian cancer comprise heterogeneous tumors, and neither targeted therapies nor traditional chemotherapies have provided consistent clinical benefit. Novel therapies that target and actively eradicate the subpopulation of tumor cells that mediate drug resistance and tumor relapse could significantly improve patient survival. Tumor-initiating cells (TIC) are functionally defined as the subpopulation of cells that drive long-term tumor growth, resistance to therapy and disease relapse. We herein identified CD324 as a surface antigen able to reproducibly enrich for TIC in well annotated, low passage TNBC and ovarian cancer patient-derived xenografts (PDXs). Gene expression analysis of TIC led to the identification of Ephrin-A4 as a prospective therapeutic TIC target. Humanized Ephrin-A4-specific monoclonal antibodies (mAbs) were generated and demonstrated to internalize to mediate the delivery of potent cytotoxins. An antibody-drug conjugate (ADC) comprising a humanized anti-Ephrin-A4 mAb conjugated to the DNA damaging agent calicheamicin achieved sustained tumor regressions in vivo in both TNBC and ovarian cancer PDX. Anti-Ephrin-A4-ADC (PF-06647263) actively reduced TIC frequency as evidenced by limiting dilution analysis in serial transplantation assays. Unexpectedly, TNBC tumors of the non-Claudin low molecular subtype exhibited higher Ephrin-A4 expression and more robust responses to the ADC than other breast cancer subtypes, which suggests a specific translational application for breast tumor subtype classification. Together these findings demonstrate the potential of the Ephrin-A4-targeted calicheamicin conjugate as a first-in-class compound designed to eradicate TIC and improve long-term survival of cancer patients. PF-06647263 is currently being evaluated in a Phase I clinical trial. Citation Format: Marc Damelin, Alex Bankovich, Albert Park, Jorge Aguilar, Wade Anderson, Marianne Santaguida, Sarah Fong, Monette Aujay, Kiran Khandke, Virginia Pulito, Elana Ernstoff, Paul Escarpe, Jeff Bernstein, Marybeth A. Pysz, Wenyan Zhong, Erik Upeslacis, Judy Lucas, Justin Lucas, Timothy Nichols, Kathryn Loving, Orit Foord, Johannes Hampl, Robert Stull, Frank Barletta, Hadi Falahatpisheh, Puja Sapra, Hans Peter Gerber, Scott J. Dylla. An anti-Ephrin-A4 calicheamicin conjugate effectively targets triple-negative breast and ovarian tumor-initiating cells to result in sustained tumor regression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5425. doi:10.1158/1538-7445.AM2015-5425

Abstract 697: c-MET as a potential target in ovarian clear-cell carcinoma

Abstract Purpose: Recent reports revealed that c-MET activation is associated with epithelial ovarian carcinoma (EOC) and is association with poor prognosis. In this study, we investigated the effects of therapeutic targeting for c-MET in ovarian clear cell carcinoma (OCCC). Experimental Design: Expression levels of c-MET in the epithelial ovarian carcinomas and normal ovarian tissues were evaluated using real-time PCR. To test c-MET inhibitors in CCC cell lines, we performed in vitro experiments including MTT and apoptosis assay. We performed Western blots to evaluate the c-MET expression and down-stream pathway. In addition, we performed in vivo therapy experiments in orthotopic ovarian cancer mice model (RMG1) and patient-derived tumor xenograft (PDX) models of CCC to confirm these effects. Results: The c-MET expression was significantly increased in CCCs compared with serous carcinomas and normal ovarian tissues (P &amp;lt; 0.05). CCC cells treated with c-MET inhibitors (SU11274 or crizotinib) demonstrated significantly decreased cell viability and increased apoptosis. Western blot assay showed that the protein expressions for c-MET signaling pathway were decreased by c-MET inhibitor. Moreover, tumor weight was significant decreased in both in vivo RMG1 and PDX models receiving SU11274 treatment compared with control (P &amp;lt; 0.05). Conclusion: These results show that c-MET inhibitor has the significant anti-tumor effects in ovarian CCC, and suggested that c-MET could have the potential agent in the therapy for this cancer. Citation Format: Jeong-Won Lee, Byoung-Gie Kim, Duk-Soo Bae, Yoo Young Lee. c-MET as a potential target in ovarian clear-cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 697. doi:10.1158/1538-7445.AM2015-697

Abstract 3223: A true orthotopic ovarian cancer patient-derived xenograft (PDX) model

Abstract Introduction: Patient derived xenografts (PDXs) show promise as models to study cancer as they recapitulate the principle characteristics of the patients’ original tumor such as histology, mutational status, DNA copy number, gene expression patterns, and clinical behavior while remaining biologically stable through passaged in mice. We used an orthotopic model transplanting tumor directly to the fallopian tube/ovary in order to accurately study the native environment and the progression of the ovarian cancer (OVCA). Experimental Design: Fresh OVCA tumor was transplanted orthotopically to the fallopian tube/ovary of NSG 5-8 week old mice. Tumor growth was followed over time. Tumors were evaluated by IHC. Alu II probe staining was performed to evaluate human stroma content. RPPA and targeted DNA sequencing are in-process. Drug studies are in progress. Primary tumor lines are being generated. Results: To date, we have transplanted tumor from 18 primary, 2 interval, and 5 recurrent ovarian debulking surgeries using an orthotopic ovarian tumor transplant approach with an 85% success rate in generating tumors in mouse passage 1 (MP1). The median time for tumors to reach ∼2 cm is 3 months when tumors are then passaged again (MP2/MP3). We have generated 8 BRCA 1/2 deleterious mutation positive carrier PDXs. Alu staining of PDXs demonstrates human cells in the stroma. We have generated 8 primary tumor cell lines from original tumors/PDXs. Conclusions: We conclude that although technically more challenging, the orthotopic transplantation technique is feasible in generating ovarian cancer PDXs that most closely resemble the natural environment for ovarian cancer progression. Citation Format: Hongmei Cui, Yingyan He, Clemens Krepler, Janos Tanyi, Mark A. Morgan, Robert A. Burger, Sarah Kim, Emily Ko, Tan Ince, Meenhard Herlyn, Fiona Simpkins. A true orthotopic ovarian cancer patient-derived xenograft (PDX) model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3223. doi:10.1158/1538-7445.AM2015-3223

Abstract 1474: Characterization of 148 ovarian cancer tumografts (Avatars) using BROCA-HR deep sequencing

Abstract Ovarian cancer is the most lethal gynecologic malignancy and the fifth leading cause of cancer deaths among women. The current standard of care post surgical cytoreduction is combination platinum/taxane chemotherapy. The initial response varies widely; subsets of carcinomas demonstrate resistance or sensitivity from the onset. The underlying cause of this response heterogeneity remains incompletely understood. Patient-derived xenografts (PDX) serve as useful in vivo models to study molecular response markers and test the efficacy of targeted therapies. Our group has demonstrated a high engraftment rate (&amp;gt;70%) of ovarian cancer PDXs (Avatars) by injecting treatment naïve patient tumor directly into the peritoneal cavity of an in immunocompromised mouse host in an effort to better mimic the anatomic context by which ovarian cancer naturally develops. We now have 328 engrafted Avatar models representing the wide range of ovarian cancer subtypes, including serous (65%), mixed epithelial (10%), endometrioid (7%), clear cell (4%), mucinous (3%), carcinosarcoma (1%) and other (10%). Using these models, we are performing a massively parallel sequencing strategy referred to as BROCA-HR to detect all mutation classes (e.g. gene rearrangements, copy number variations, etc.) and gene aberrations within the Fanconi Anemia-BRCA homologous recombination (HR), non-homologous end joining (NHEJ), PTEN, and DNA Mismatch repair pathways. To date, 148 Avatar models have undergone BROCA sequencing. Observed deleterious mutations included 12 BRCA1 (8%), 6 BRCA2 (4%), and 4 PIK3CA (3%); additional loss of function mutations were also evident in the following genes: ATM, RAD51C, FANCM, FANCD2, FANCA, CHEK2, PALB2, MHS6, CDK12 and GEN1. These models are representative of mutations seen in the TCGA mutation set. Models harboring said mutations are currently being tested with platinum agents and/or PARP inhibitors. Taken together, the Avatars represent a high throughput model system for pre-clinical testing in vivo that effectively recapitulates ovarian cancer (e.g. mutations in BRCA1, BRCA2 and other DNA damage response proteins). We have been able to establish avatars at a high rate, including those with mutations in BRCA1, BRCA2 and other DNA damage response genes. These models will be a unique resource for future studies of biomarkers and novel therapeutic approaches. This work is funded by the Ovarian Cancer Research Fund (750563), Mayo Ovarian SPORE (CA136393), National Cancer Institute (RO1 CA184502). Note: This abstract was not presented at the meeting. Citation Format: Kunal A. Lodhia, Marc A. Becker, Xiaonan Hou, Kimberly R. Kalli, Maria I. Harrell, Elizabeth M. Swisher, John S. Weroha, Paul Haluska. Characterization of 148 ovarian cancer tumografts (Avatars) using BROCA-HR deep sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1474. doi:10.1158/1538-7445.AM2015-1474