Novel genetic alterations in serous borderline tumors of the ovary revealed by whole exome sequencing

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Abstracts / Gynecologic Oncology 130 (2013) e1–e169

determine the effect of celecoxib on COX-2 expression in the ovarian cancer cell lines. Adhesion was assessed by ELISA assay and invasion was assessed by ChemoTx assay. The K18-gT121+/-;p53fl/fl;Brca1fl/fl (KpB) mice were subjected to a 60% calories derived from fat in a highfat diet (HFD) versus 10% calories from fat in a low-fat diet (LFD) to mimic diet-induced obesity and subsequently exposed to celecoxib or placebo for 4 weeks. Results: Celecoxib inhibited proliferation in all ovarian cancer cell lines (IC50 25 mcM for SKOV3, 25 mcM for HEY, and 50 mcM for IGROV1) (P = 0.0001- 0.0002) after 48 hours of exposure. Treatment with celecoxib resulted in G1 cell cycle arrest and induction of apoptosis. Celecoxib inhibited hTERT mRNA expression in all cell lines. Western immunoblot analysis demonstrated that celecoxib treatment suppressed COX-2 expression within 24 hours of exposure. Cellular adhesion was decreased by 20%-40% in all cell lines at a dose of 25 mM (P = 0.002-0.009) and at 50 mM (P = 0.00001-0.02). Cellular invasion was decreased in all cell lines in a dose-dependent manner (P b 0.001). In the KpB mice fed the HFD and treated with celecoxib, tumor weight decreased by 70% (P = 0.04) when compared with control animals. Among KpB mice fed the LFD, tumor weight decreased by 25% after treatment with celecoxib, but this was not statistically significant. Conclusions: Celecoxib inhibited cell growth via G1 arrest, decreased telomerase activity, increased apoptotic cell death, and decreased cellular adhesion and invasion in human ovarian cancer cells. In vivo studies using the KpB mouse model found that treatment with celecoxib was more efficacious in inhibiting tumor growth among mice fed a HFD. This work suggests that celecoxib may be a novel chemotherapeutic agent for ovarian cancer prevention and treatment and is potentially more beneficial in the obese population. doi:10.1016/j.ygyno.2013.04.360

302 Role of ARID1A for the clinicopathologic characteristics of clear cell carcinoma (CCC) of the ovary H. Abou-Taleb, K. Yamaguchi, M. Mandai, K. Yamanoi, Y. Amano, N. Matsumura, T. Baba, Y. Yoshioka, J. Hamanishi, I. Konishi. Graduate School of Medicine, Kyoto University, Kyoto, Japan. Objective: To study the relation ARID1A expression with CCC molecular and pathologic features as well as the effect of ARID1A mutation on the response to treatment and survival of patients. Methods: Specimens from 123 ovarian cancer cases (56 CCC, 25 endometrioid [EAC], 20 serous [SAC], 22 mucinous [MAC]) were examined for ARID1A, HNF1-beta, ER-alpha, P53, pAKT, and Ki67 by immunohistochemistry, after obtaining informed consent from all patients. mRNA expression microarray analyses were performed on 14 CCC samples merged with GSE2109 and GSE6008 data sets. This study was approved by the institutional review board of Kyoto University. Results: ARID1A showed positive staining in 22 (48%), 21 (84%), 20 (100%), and 22 (100%) cases of CCC, EAC, SAC, and MAC. ARID1A was correlated to HNF1-beta, ER-alpha, P53, and Ki67 but not to pAKT. CCC has 3 architectural patterns: tubulocystic, papillary, and solid. Solid one had higher ARID1A (P = 0.0169) and lower HNF1-beta (P = 0.025) expression than other patterns. Unsupervised hierarchical clustering of microarray data of 14 samples merged with GSE2109 and GSE6008 showed 2 main clusters, with ARID1A accumulated in 1 cluster. Univariate analyses suggested that cases lacking ARID1A had a shorter progression-free survival compared with cases expressing ARID1A, although the difference was not statistically significant. Overall and progression-free survival rates were not affected by ARID1A status. Conclusions: CCC has a unique molecular profile which distinguishes it from the other histologic types of ovarian cancer. There are 2 CCC

subtypes based on ARID1A that are related to the morphologic pattern. ARID1A expression is preserved in the solid pattern of CCC, which may be related to a more aggressive tumor. This suggests that independent therapeutic strategies are needed based on CCC molecular subtypes. doi:10.1016/j.ygyno.2013.04.361

303 Celecoxib and paclitaxel synergistically induce apoptosis in the human ovarian cancer cell line OVCAR-3 H. Kim, G. Yim, Y. Kim. Women’s Life Medical Science, Department of Obstetrics and Gynecology, Yonsei University, College of Medicine, Seoul, Republic of Korea. Objective: Celecoxib, a highly selective cyclooxygenase (COX-2) inhibitor, regulates apoptosis of several human cancer cell types. The aim of this study was to investigate whether celecoxib alone or in combination with paclitaxel modulates apoptosis of ovarian cancer cells and to identify the signal pathway by which celecoxib mediates apoptosis. Methods: OVCAR-3 cells were exposed to paclitaxel (20 mcM) in the absence or presence of celecoxib (10 mcM). Cell viability was evaluated using a cell counting kit-8 (CCK-8) assay. Apoptosis was examined by Annexin-V/7-AAD staining and cellular DNA fragmentation enzymelinked immunosorbent assay (ELISA). Caspase-3 was evaluated using the Caspase-3/CPP32 Colorimetric Assay kit. Caspase-9 and cleavage of poly ADP-ribose polymerase (PARP) were determined by western blotting. Expression of nuclear factor-kappa-B (NFkappa- B) was assessed using Trans AM kits and immunofluorescence. Vascular endothelial growth factor (VEGF) and Akt activation were studied by reverse transcriptasepolymerase chain reaction (RT-PCR) and western blotting. Results: Celecoxib enhanced paclitaxel-induced growth inhibition of OVCAR-3 cells. Celecoxib significantly increased paclitaxel-induced apoptosis of OVCAR-3 cells. Pretreatment with celecoxib also increased activation of caspase-9, -3, and cleaved PARP following paclitaxel-treatment. Exposure of OVCAR-3 cells to celecoxib in combination with paclitaxel resulted in downregulation of NFkappa-B activation and VEGF expression. Furthermore, combining celecoxib and paclitaxel inhibited phosphorylation of Akt. Conclusions: Our data indicated that OVCAR-3 cells were sensitized to paclitaxel-induced apoptosis by celecoxib through downregulation of NFkappa- B and Akt activation, suggesting that celecoxib may be associated with the anticancer effects of paclitaxel via a synergistic role in inhibiting different targets. Combining celecoxib with paclitaxel may provide clinical advantages for the treatment of ovarian cancer. doi:10.1016/j.ygyno.2013.04.362

304 Novel genetic alterations in serous borderline tumors of the ovary revealed by whole exome sequencing J. Boyd, B. Luo, S. Peri, B. Wirchansky, L. Hughes, C. Forsythe, H. Wu, M. Morgan. Fox Chase Cancer Center, Philadelphia, PA. Objective: Serous borderline tumor (SBT) is a unique histopathologic entity of the ovary, believed to be intermediate between benign cystadenoma and invasive low-grade serous carcinoma. While somatic mutations in the KRAS or BRAF, and rarely ERBB2, genes have been well characterized in SBTs, other genetic alterations have not been described. Toward a more comprehensive understanding of the molecular genetic architecture of SBTs, we undertook whole exome sequencing of this tumor type.

Abstracts / Gynecologic Oncology 130 (2013) e1–e169

Methods: Following pathologic review and laser capture microdissection to enrich for tumor cells, whole exomes were prepared from DNA of 2 independent SBTs (SBT-s2 and SBT-s5) and subjected to massively parallel DNA sequencing, using the Illumina Genome Analyzer IIx. Image analysis and base calling were performed using the standard Genome Analyzer pipeline software, Sequencing Control Studio (SCS), and Real-Time Analysis (RTA), respectively. Publicly available software tools, such as BWA aligner, the SamTools package, Picard, and GATK were used for bioinformatical analysis of data. Validation of potential somatic mutations was performed using standard Sanger sequencing techniques. Results: A small number of somatic mutations were identified in these SBTs. Both tumors contained an activating mutation of the BRAF gene. A total of 15 additional somatic mutations were identified, 9 in one tumor and 6 in the other. Eleven were missense mutations and four were nonsense or deletion mutations. Fourteen of the 16 genes found to be mutated in this study have been reported to be mutated in other cancers. Furthermore, 12 of these genes are mutated in invasive ovarian cancers. The FBXW7 and KIAA1462 genes are noteworthy candidates for a genetic role in serous borderline tumorigenesis, insofar as inactivating mutations of these 2 genes have been reported in low-grade serous ovarian carcinomas. Conclusions: A small number of somatic genetic mutations characterize SBTs of the ovary. The mutant genes described herein represent candidates for the pathogenesis of SBT of the ovary; further studies are required to determine their biological relevance. doi:10.1016/j.ygyno.2013.04.363

305 Tumor-specific targeting with novel LXY-30 peptide-linked nanoparticles for paclitaxel delivery in ovarian cancer mouse xenograft model N. Suby1, K. Xiao2, Y. Yi2, W. Xiao2, L. Tiglao2, K. Lam2. 1UC Davis Medical Center, Sacramento, CA, 2University of California - Davis, Sacramento, CA. Objective: Boronate cross-linked micelles (BCM) allow for drug delivery to the tumor and retain the encapsulated drug under physiologic conditions and release due to lower pH values or by a trigger release such as mannitol. In previous work, we demonstrated that nanocarriers linked with the LXY-30 ligand increases in vitro cellular uptake compared to nanocarriers alone in SKOV-2 ovarian cancer cells. In this study, we investigated the therapeutic efficacy and toxicity profiles of the BCMs carrying paclitaxel (PTX) in ovarian cancer xenograft-bearing mice. Methods: The intraperitoneal (IP) xenograft model was established by injecting SKOV-3/LUC cells intra-abdominally into female athymic nude mice at 6-8 weeks of age. SKOV-3/Luciferase is a line that stably expresses the firefly luciferase gene, which was used for intraabdominal imaging of tumor intensity. After 2 months of tumor growth, on day 0, the mice were randomly divided into 5 groups (PBS, PTX, BCM-PTX, BCM-LXY30-PTX, and BCMLXY30- PTX followed by mannitol trigger) and were treated with IP injections of the formulations twice weekly for a total of 3 doses. PTX was given at a dose of 10 mg/kg, which is nearly the maximum tolerated dose (MTD). At 24 hours following the administration of the BCM-PTXLXY-30 nanoparticle, the drug release was triggered by injection of a 20% mannitol solution per mouse. The mice were followed weekly with imaging. Tumor burden was assessed by the relative intensity of the tumor. Toxicities were assessed by analyzing the effects on animal behavior and body weight change. Results: Compared with the PBS and PTX groups, mice in the nanoparticle treatment groups showed significant inhibition of tumor growth (P b 0.05). At the dose of 20 mg PTX/kg, the BCM-PTX and

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BCM-LXY30-PTX exhibited superior tumor growth inhibition and longer survival time compared to PTX. The BCM-LXY30-PTX and BCM-LXY30-PTX with mannitol trigger showed improved tumor growth inhibition and longer survival times than the BCMPTX group. The median survival time was 35 days for PBS, 45.5 days for 10 mg/ kg PTX, 49 days for 20 mg/kg BCM-PTX, 63 days for 20 mg/kg BCMLXY30- PTX, and 66 days for 20 mg/kg BCM-LXY30-PTX with mannitol trigger. Conclusions: We demonstrated that the novel use of the BCM linked with LXY30 was more efficacious than both the free drug at the MTD and BCM without the use of a ligand at equivalent doses of PTX in the ovarian cancer xenograft mouse model. doi:10.1016/j.ygyno.2013.04.364

306 Decreased 53BP1 expression predicts improved survival in sporadic ovarian carcinoma K. Pennington1, A. Wickramanayake1, B. Norquist1, S. Kaufmann2, C. Pennil1, R. Garcia1, K. Agnew1, P. Welcsh1, E. Swisher1. 1University of Washington Medical Center, Seattle, WA, 2Mayo Clinic, Rochester, MN. Objective: 53BP1 is a critical regulator of the balance between homologous recombination (HR) and the more error-prone nonhomologous endjoining (NHEJ) DNA repair. Deletion of 53BP1 in brca1(but not brca2-) null cells rescues embryonic lethality, partially restores HR, and reverses sensitivity to poly-ADP ribose polymerase inhibitors (PARPi). We characterized 53BP1 and BRCA1 expression in a large number of primary and recurrent ovarian carcinomas to determine if 53BP1 expression is associated with clinical outcomes in sporadic and inherited cases. Methods: We evaluated 53BP1 protein expression using immunohistochemistry in 248 ovarian carcinomas comprehensively characterized for germline mutations in BRCA1 and BRCA2, including 54 paired primary and recurrent samples. We evaluated relative 53BP1 and BRCA1 mRNA expression in a subset of 85 cases with quantitative reverse transcriptase-polymerase chain reaction. Results: Both primary and recurrent BRCA1-mutated (but not BRCA2mutated) ovarian carcinomas had significantly higher 53BP1 protein expression than wild type carcinomas. 53BP1 message levels were significantly associated with BRCA1 message levels in wild type and in BRCA1-mutated but not in BRCA2-mutated ovarian carcinomas. In wild type carcinomas, lower 53BP1 message predicted improved survival (median survival 74 vs. 41 months, HR 0.49, 95% CI 0.27-0.88, P b 0.02). Survival was not significantly impacted by BRCA1 message level. Neither 53BP1 protein nor mRNA expression was associated with primary platinum resistance. In 54 paired primary and recurrent cases, 53BP1 protein expression was equally likely to decrease or increase, and there was no association between decreased 53BP1 at recurrence and the development of platinum resistance. Conclusions: BRCA1-mutated ovarian carcinomas have higher 53BP1 protein expression than wild type or BRCA2-mutated carcinomas, in direct contrast to previous findings in breast carcinomas. Higher 53BP1, which promotes NHEJ DNA repair, could explain the high aneuploidy that is characteristic of BRCA1- mutated ovarian carcinomas. In wild type ovarian carcinomas, decreased 53BP1 mRNA expression predicts improved overall survival, an opposite effect to our prediction. We speculate that if 53BP1 expression correlates with a predominant NHEJ phenotype, then low 53BP1 could be a good prognostic factor in sporadic cases secondary to overall decreased aneuploidy and more ordered DNA repair. doi:10.1016/j.ygyno.2013.04.365