Clear Cell Ovarian Cancer Research Articles

Immune Checkpoint Inhibitors in Gynecological Cancers: Update of Literature and Perspectives of Clinical Research.

The presence of tumor infiltrating lymphocytes (TILs) influences the clinical outcome of cancer patients and immune checkpoint inhibitors (ICPI) have been approved for treating different types of malignancies. In this review, we assess the scanty data from literature and the perspectives of clinical research about the use of ICPI in gynecological cancers. These agents have obtained objective response rates ranging from 5.9% to 15% in early phase Ib-II trials, including patients with platinum-resistant ovarian cancer, whereas only anecdotal data are available for patients with recurrent, heavily pretreated endometrial cancer. Several ongoing trials are investigating ICPI alone or in combination with chemotherapy or with other biological agents in untreated and recurrent ovarian cancer, advanced and recurrent endometrial cancer, as well as advanced and recurrent cervical cancer. Breast cancer (BRCA)-mutated high-grade serous ovarian cancers, clear cell ovarian cancers with microsatellite instability (MSI), POLE ultramutated and MSI hypermutated endometrial cancers are likely to be sensitive to programmed cell death (PD-1)/PD-ligand 1 (PD-L1) pathway blockade, since these tumors show increased neoantigen load, increased CD8+ TIL number and PD-1 and PD-L1 overexpression. ICPI could have a role as maintenance treatment in patients with persistent, recurrent or metastatic cervical cancer in response after chemotherapy.

325 - Clinical Outcomes of Patients with Clear Cell and Endometrioid Ovarian Cancer Arising in Endometriosis

Study Objective: The aim of this investigation was to compare outcomes of patients with clear cell carcinoma (CCC) and endometrioid carcinoma (EC) of the ovary arising in endometriosis (ES) to CCC and EC not arising in ES.

Overcoming Clear Cell Ovarian Cancer Resistance to Taxol by an Oligo Arginine Transporter Conjugate

Objectives: Taxol remains first line treatment for all epithelial ovarian cancer (EOC). However, it has been shown that ovarian clear cell carcinoma (CCC), a rare histological subtype of EOC, often becomes resistant to platinum based therapy when at advanced stage. This chemo-resistant phenotype has led to a poorer prognosis in patients with CCC than in patients with serous subtypes. While many factors contribute to resistance; a major contributor is the active export of drugs from cells mediated by transmembrane efflux pumps that prevent drugs from reaching their intracellular targets. Our lab has developed a Taxol-oligoarginine conjugate that prevents export of the drug from serous papillary EOC leading to increased cell death. In this study we aim to assess whether this Taxol conjugate is effective in improving Taxol mediated cell death in platinum resistant Clear cell ovarian cancer.

Candidate synthetic lethality partners to PARP inhibitors in the treatment of ovarian clear cell cancer.

Inhibitors of poly(ADP-ribose) polymerase (PARP) are new types of personalized treatment of relapsed platinum-sensitive ovarian cancer harboring BRCA1/2 mutations. Ovarian clear cell cancer (CCC), a subset of ovarian cancer, often appears as low-stage disease with a higher incidence among Japanese. Advanced CCC is highly aggressive with poor patient outcome. The aim of the present study was to determine the potential synthetic lethality gene pairs for PARP inhibitions in patients with CCC through virtual and biological screenings as well as clinical studies. We conducted a literature review for putative PARP sensitivity genes that are associated with the CCC pathophysiology. Previous studies identified a variety of putative target genes from several pathways associated with DNA damage repair, chromatin remodeling complex, PI3K-AKT-mTOR signaling, Notch signaling, cell cycle checkpoint signaling, BRCA-associated complex and Fanconi's anemia susceptibility genes that could be used as biomarkers or therapeutic targets for PARP inhibition. BRCA1/2, ATM, ATR, BARD1, CCNE1, CHEK1, CKS1B, DNMT1, ERBB2, FGFR2, MRE11A, MYC, NOTCH1 and PTEN were considered as candidate genes for synthetic lethality gene partners for PARP interactions. When considering the biological background underlying PARP inhibition, we hypothesized that PARP inhibitors would be a novel synthetic lethal therapeutic approach for CCC tumors harboring homologous recombination deficiency and activating oncogene mutations. The results showed that the majority of CCC tumors appear to have indicators of DNA repair dysfunction similar to those in BRCA-mutation carriers, suggesting the possible utility of PARP inhibitors in a subset of CCC.

Type and risk of cancer related to endometriosis: ovarian cancer and beyond.

Endometriosis is a common disease in reproductive age women with an incidence of about 10% depending on the definition and cohort characteristics. The pathological diagnosis is a benign, but the disease often recurs and can in rare cases metastasise like a cancer. Endometriosis poses unique surgical challenges and can result in surgical procedures that are more technically difficult than those for malignant disease. Saraswat et al. analysed outcomes of 17 834 women with surgically diagnosed endometriosis and found a hazard ratio of 1.7 for ovarian cancer. The current Scottish nationwide study did not specify the histologic type of ovarian cancer. Endometriosis has been associated with increased risk of several subtypes of epithelial ovarian cancer: low-grade serous, clear cell and endometrioid (Pearce, Lancet Oncol 2012;13:385–94). Aside from ovarian cancer, the risk of several types of cancer, including breast cancer, haematologic malignancies, non-Hodgkin's lymphoma and brain tumours, is increased in patients with endometriosis (Melin et al. Human Reprod. 2006;21:1237–42; Brinton et al. Obstet Gynecol 1997;176:572–9). In the current study, the risk of these cancers was not increased, but that may have been due to the relatively small sample size. In both clinical and research settings, there are several issues to be addressed. First, information regarding the true incidence of endometriosis is lacking and may be underestimated. A laparoscopic biopsy is necessary to confirm the diagnosis and many women may remain asymptomatic or never present for surgical management. Secondly, we do not have enough information to identify which subgroups of women with endometriosis are at risk for endometriosis-related malignant disease. Endometriosis-associated endometrioid ovarian cancer shares symptoms with endometriosis, including dysmenorrhoea (28.0%), dyspareunia (20.7%) and infertility (14.6%) (Lim et al. Cancer Epidemiol Biomarker Prev 2010;19:398–404). These symptoms have also been reported in endometriosis-related ovarian clear cell carcinoma [dysmenorrhoea (31.3%), dyspareunia (37.5%) and infertility (31.3%)] and are newly developed or exacerbated by the presentation of ovarian cancer (Lim et al. Gynecol Endocrinol 2009;25:435–40). The retrospective study design prevents a clear delineation of when symptoms began. Acute or subacute exacerbation of symptoms might suggest endometriosis-related ovarian cancer but should be considered in conjunction with imaging and serum CA125 and HE4 (Zapardiel et al. Int J Gynecol Cancer 2016;26:52–5). Thirdly, the aetiology of cancer in women with endometriosis is not clear: it may result from a shared aetiology or as a secondary effect of endometriosis. A better understanding of the underlying aetiology may allow tailored interventions to prevent endometriosis-related malignant disease. Fourthly, the effect of surgical treatment and long-term use of conservative medical treatment on the endometriosis-related malignancy has not been clearly established. Women with endometriosis have a higher risk of low-grade serous, clear cell and endometrioid ovarian cancer. New development or exacerbation of existing endometriosis-related symptoms such as dysmenorrhoea, dyspareunia and infertility should raise a red flag for the possibility of endometriosis-related ovarian cancer, especially in the presence of an accompanying solid mass and elevated serum HE4. None declared. Completed disclosure of interests form available to view online as supporting information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Use of antidepressants and risk of epithelial ovarian cancer.

Antidepressants are widely prescribed among women to treat depression and anxiety disorders, but studies of their effects on gynecological cancer risk are sparse. We assessed associations between various antidepressants and risk of epithelial ovarian cancer. By using Danish nationwide registers, we identified all women (cases) aged 30-84 years with incident epithelial (serous, endometrioid, clear cell or mucinous) ovarian cancer during 2000-2011 (n = 4,103) and matched each case to 20 population controls (n = 58,706) by risk-set matching. Data on drug use (including tricyclic and related antidepressants, selective serotonin reuptake inhibitors, other antidepressants, and potential confounder drugs), medical and reproductive history and socioeconomic parameters, were obtained from nationwide registries. We used conditional logistic regression models to estimate adjusted odds ratios (ORs) and two-sided 95% confidence intervals (CIs) for epithelial ovarian cancer associated with antidepressive drug use. Compared with non-use, use of selective serotonin reuptake inhibitors was associated with a decreased risk of ovarian cancer (OR, 0.85; 95% CI, 0.74-0.96), whereas the associations for other antidepressants were close to unity [tricyclic and related antidepressants: OR, 0.99 (95% CI, 0.78-1.26); other antidepressants: OR, 1.05 (95% CI, 0.76-1.46)]. For individual types of SSRI, reduced ORs were observed for citalopram OR, 0.78 (95% CI, 0.66-0.93), paroxetine 0.79 (95% CI, 0.56-1.12) and sertraline 0.80 (95% CI, 0.60-1.08). Among postmenopausal women, the inverse association was restricted to users of menopausal hormone therapy. In conclusion, use of selective serotonin reuptake inhibitors was associated with a decreased risk of epithelial ovarian cancer; thereby implying potential chemopreventive properties of these drugs.

Associations between residual disease and survival in epithelial ovarian cancer by histologic type.

Surgical cytoreduction has been postulated to affect survival by increasing the efficacy of chemotherapy in ovarian cancer. We hypothesized that women with high-grade serous ovarian cancer, which usually responds to chemotherapy, would derive greater benefit from complete cytoreduction than those with histologic subtypes that are less responsive to chemotherapy, such as mucinous and clear cell carcinoma. We conducted a retrospective cohort study of patients who underwent primary cytoreductive surgery and adjuvant chemotherapy for stage IIIC or IV epithelial ovarian cancer from 2011 to 2013 using data from the National Cancer Database. We constructed multivariable models to quantify the magnitude of associations between residual disease status (no residual disease, ≤1cm, or >1cm) and all-cause mortality by histologic type among women with clear cell, mucinous, and high-grade serous ovarian cancer. Because 26% of the sample had unknown residual disease status, we used multiple imputations in the primary analysis. We identified 6,013 women with stage IIIC and IV high-grade serous, 307 with clear cell, and 140 with mucinous histology. The association between residual disease status and mortality hazard did not differ significantly among histologic subtypes of ovarian cancer (p for interaction=0.32). In covariate adjusted models, compared to suboptimal cytoreduction, cytoreduction to no gross disease was associated with a hazard reduction of 42% in high-grade serous carcinoma (hazard ratio [HR]=0.58, 95% confidence interval [CI]=0.49-0.68), 61% in clear cell carcinoma (HR=0.39, 95% CI=0.22-0.69), and 54% in mucinous carcinoma (HR=0.46, 95% CI=0.22-0.99). We found no evidence that surgical cytoreduction was of greater prognostic importance in high-grade serous carcinomas than in histologies that are less responsive to chemotherapy.

Integrated genomic analysis of clear cell ovarian cancers identified PRKCI as a potential therapeutic target.

Clear cell ovarian cancer (CCOC) is an epithelial ovarian cancer histotype with unique pathologic, biologic and clinical features. Despite its worse prognosis than serous ovarian cancer (SOC), the genomic landscape of CCOC is less well defined. Integrated genomic analysis of CCOC allows the identification of potential therapeutic targets to improve the treatment of this tumor. Using comparative genomic hybridization and gene expression profiling, we have screened 12 CCOC cell lines and 40 tumors to identify 45 amplified and overexpressed genes. Pathways analysis of these genes identified 19 genes with cancer-related functions. Of these, PRKCI is one of the most frequently amplified and overexpressed genes and its expression induced cancer cell proliferation and migration/invasion in vitro as well as tumor growth in vivo. Targeting PRKCI by small molecule inhibitor, sodium aurothiomalate (ATM), significantly reduced the in vivo tumor growth and may be a new therapeutic strategy to improve the treatment of CCOC.

A key HDAC6 dependency of ARID1A-mutated ovarian cancer.

Mutations in AT rich interactive domain 1A (ARID1A) are common in ovarian cancers. A study now shows that HDAC6 activity is required for survival of ovarian cancer cells bearing ARID1A mutations. HDAC6 inhibition selectively promotes apoptosis of ARID1A-inactivated cells, supporting the use of HDAC6 inhibition in the treatment of clear cell ovarian cancer.

Abstract 3380: Synthetic lethal interaction between ARID1A mutation and BET bromodomain inhibition in ovarian clear cell carcinoma

Abstract Introduction: Current treatment for advanced stage ovarian clear cell cancer is severely hampered by a lack of effective systemic therapy options, leading to a poor outlook for these patients. Given that ARID1A is inactivated by mutation in over 50% of ovarian clear cell carcinomas, we pursued an ARID1A synthetic lethal screening strategy to identify druggable targets in OCCC. Experimental procedures: We performed synthetic lethal kinome short hairpin (shRNA) screens in a large panel (n=14) of OCCC cell lines having different ARID1A mutation status. Hit validation was performed with isogenic ARID1A ko cell line pairs and in (patient-derived) xenograft mouse models. Summary of the data: We show here that BRD2 inhibition is synthetic lethal with ARID1A mutation in ovarian clear cell cancer cells. Importantly, inhibiting the BET family of proteins, to which BRD2 belongs, with small molecules specifically inhibits proliferation of ARID1A mutated cell lines both in vitro and in ovarian clear cell cancer xenografts and patient-derived xenograft models. We demonstrate that ARID1A loss leads to upregulation of the WNT ligand WNT10B, possibly causing a WNT dependency in the ARID1A mutant lines. BET inhibitors cause a reduction in WNT10B expression and WNT target genes such as MYC, JUN and WISP1, providing a potential explanation for the observed synthetic lethal interaction with ARID1A loss. Conclusions: Our study uncovered a new synthetic lethal interaction between ARID1A mutation and BET bromodomain inhibition, suggesting a new treatment strategy for ARID1A mutant ovarian clear cell carcinomas. Citation Format: Katrien Berns, Joseph J. Caumanns, E Marielle Hijmans, Annemiek Gennissen, Bastiaan Evers, Bea A. Wisman, Gert Jan Meersema, Cor Lieftink, Roderick L. Beijersbergen, Hiroaki Itamochi, Ate G. van der Zee, Steven de Jong, René Bernards. Synthetic lethal interaction between ARID1A mutation and BET bromodomain inhibition in ovarian clear cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3380. doi:10.1158/1538-7445.AM2017-3380

Abstract 1397: Antisense oligo nucleotide of Annexin A4 improved platinum resistance in ovarian clear cell cancer

Abstract Introduction: Ovarian cancer in Japan are classified as clear cell carcinoma (CCC) more than 20 %, this percentage is higher than in Europe and United States. Besides, it is well known that CCC of ovary is highly resistant to cancer chemotherapy including carboplatin and paclitaxel treatment. We reported that Annexin A4 protein was overexpressed in ovarian CCC tissues by immunohistochemical analysis. Elevated Annexin A4 level has been detected in various epithelial cancer cell lines and have reported associating with chemoresistance to platinum-based cancer drugs. To overcome the platinum chemoresistance, we thought antisense oligonucleotides (ASOs) to be a good therapeutic option in a way of highly specific therapy for improving chemoresistance by suppressing the expression of Annexin A4 in cancer cells. Methods: We generated ASO targeting Annexin A4 with 2’, 4’-bridged nucleic acid. And we analyzed suppression of Annexin A4 in ASO-transfected RMG-I cell line (CCC) in vitro using real time PCR and western blotting. In 16 types of ASOs targeting Annexin A4, 2 ASOs were eligible. Cells were seeded in 96-well plates (2,000 cells per well). Next day, cells were transfected with ASOs using lipofectamine 2000 and were exposed to various concentrations of cisplatin (0 - 100 μM) for 72 hr. Then, drug concentrations resulting in a 50% inhibition of cell growth (IC50 values) were calculated. Intracellular platinum accumulation in Annexin A4 overexpressing cells was analyzed. To assess the improvement of platinum resistance in vivo, we used ICR nu/nu mice xenografted subcutaneously with RMG-I cells. Intraperitoneal injection of cisplatin 3mg/kg after intratumoral administration of ASO 1mg/kg each twice a week were given to xenograft mice. Results: By realtime PCR analysis, among strong 16 types of ASOs targeting Annexin A4, 2 ASOs showed strong knockdown efficiency (about 80% knockdown) compared to negative control ASOs. Western blotting analysis showing knockdown of Annexin A4 expression was observed in Annexin A4 ASO transfected cells compared to no treatment or control ASOs in vitro. ASO-transfected RMG-I cells was less resistant to cisplatin (IC50 = 3.3μM) compared with control cells (IC50 = 5.2μM) Same result were obtained with carboplatin. Platinum resistance was significantly improved in treated with Annexin A4 ASO and cisplatin compared to control ASO and cisplatin treated group in vivo. Conclusion: By transfection of ASOs targeting Annexin A4, platinum resistance have improved in vivo and in vitro, Annexin A4 have associated with efflux of platinum anti-tumor drug. In conclusion, antisense oligonucleotides for Annexin A4 will be a therapeutic option for ovarian clear cell carcinoma with chemoresistance to platinum antitumor drug. Citation Format: Reisa Kakubari, Satoshi Nakagawa, Tadashi Iwamiya, Eiji Kobayashi, Shinnya Matsuzaki, Yutaka Ueda, Kiyoshi Yoshino, Yuya Kasahara, Satoshi Obika, Tadashi Kimura, Satoshi Serada, Tetsuji Naka, Minoru Fujimoto. Antisense oligo nucleotide of Annexin A4 improved platinum resistance in ovarian clear cell cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1397. doi:10.1158/1538-7445.AM2017-1397

Abstract 4274: The contribution of deleterious germ-line mutations of susceptibility genes to ovarian, fallopian tube, and peritoneal cancers in Japanese

Abstract Different ethnic groups present specific morphological features in ovarian cancer (OC). High-grade serous OC is more frequently found in Caucasian women, in contrast clear cell OC is more frequently found in East Asian women. Although inherited mutations of BRCA1 or BRCA2 (BRCA1/2) and other genes are known to predispose to OC, the contributions of these inherited mutations on disease burden is not well characterized in Japanese OC patients. Using a comprehensive genetic testing panel, the aim of our study was to identify the prevalence of pathogenic germ-line mutations of candidate genes associated with genetic predisposition to OC in Japanese patients with ovarian, fallopian tube, or peritoneal cancer.Samples from 236 individuals with unselected ovarian, fallopian tube, or peritoneal cancer, which were obtained from the Keio Women’s Health Biobank (KWB) from the School of Medicine at Keio University (Tokyo, Japan), were used for this study. Germ-line DNA was enriched using the SureSelect XT Target Enrichment System (Agilent Technologies) designed for 75 or 79 genes as a custom OC panel, followed by sequencing using MiSeq (Illumina). Detected variants, including point mutations, small indels, and a gross deletion, were classified according to the American College of Medical Genetics and Genomics (ACMG) recommendations. Furthermore, BRCA1/2 variants were interpreted using resources from Myriad Genetic Laboratories.Forty-two (17.8%) OC patients had germ-line mutations of cancer predisposition genes. BRCA1/2 pathological germ-line mutations were found in 27 cases (11.4%) while six cases (2.5%) had a mutation in a mismatch repair gene (MLH1, MSH2, MSH6, or PMS2). Furthermore, ten cases (4.2%) had germ-line mutations in the RAD51D, ATM, MRE11A, FANCC, or GABRA2 genes. Of patients with a BRCA1/2 mutation (n=27), we found that the most common histological subtype was high-grade serous OC, but we also found two cases with clear cell carcinoma. Of 42 cases with an inherited mutation, we found that 25% (6/24) had no family history of cancer, and 78% (31/41) were under 60 years old at diagnosis.We found that approximately 18% of unselected Japanese patients with ovarian, fallopian tube, or peritoneal cancer were associated with inherited mutations of cancer-predisposing genes. Our study demonstrates that comprehensive genetic testing is informative for all women with ovarian, fallopian tube, and peritoneal cancers to establish personalized clinical management and genetic counseling, irrespective of the patient's histological subtypes, ages or family histories. Citation Format: Akira Hirasawa, Issei Imoto, Takuya Naruto, Tomoko Akahane, Wataru Yamagami, Nobuyuki Susumu, Hitoshi Tsuda, Daisuke Aoki. The contribution of deleterious germ-line mutations of susceptibility genes to ovarian, fallopian tube, and peritoneal cancers in Japanese [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4274. doi:10.1158/1538-7445.AM2017-4274

Impact of adjuvant chemotherapy in patients with FIGO stage I ovarian clear cell cancer in the platinum era: A Surveillance, Epidemiology, and End Results cohort study, 2000-2013

Impact of adjuvant chemotherapy in patients with FIGO stage I ovarian clear cell cancer in the platinum era: A Surveillance, Epidemiology, and End Results cohort study, 2000-2013

Abstract AP12: CYSTEINE METABOLISM IN CLEAR CELL OVARIAN CANCER

Abstract Our past research has used genomic screens to identify unique features of ovarian cancer subtypes. This has resulted in the description of frequent mutations in ARID1A in both clear cell (CCOC) and endometrioid (ENOC) ovarian cancers and patterns of genomic rearrangements indicative of each subtype. While genomic and RNA expression profiling have been extremely informative, they do not provide a view of the protein expression landscape of cancers. We therefore set out to perform proteomic profiling to better define the key features of the different subtypes and to understand the biology that underpin these diseases. We have developed a novel mass spectrometry proteomic profiling technique based on para–magnetic bead technology called SP3–Clinical Tumor Proteomics (SP3–CTP). This technique is able to perform sensitive proteomic profiling off of a single formalin–fixed paraffin–embedded (FFPE) section. Using this platform, we performed proteomic profiling of three subtypes of ovarian cancer: high grade serous (HGS), ENOC and CCOC. Many proteins known to be enriched in a particular subtype were validated by our screen. WT1 levels were higher in HGS and HNF1B levels were higher in CCOC, as expected. Perhaps more importantly, this screen also revealed that several proteins were enriched in one subtype, which had not previously been described. One such protein was cystathionine gamma lyase (CTH), which was enriched in CCOC compared to the other two subtypes. We validated these results on an ovarian tumor tissue microarray, which confirmed that the majority of CCOC (75%) express high levels of CTH. Conversely, only 10% ENOCa and less than 2% of HGS express high levels of CTH. CTH is a transulfuration enzyme which, along with cystathione beta lyase, form the biosynthetic pathway in which methionine is converted to cysteine. High levels of CTH activity can lead to the generation of hydrogen sulfide (H2S), which has been increasingly implicated as a gaseous intracellular signaling molecule. Overexpression of CTH in CCOC could explain the unique metabolism and other clinical features of this disease. We have found that CTH exhibits a unique staining pattern in the normal endometrium, staining some cells very dark while the rest exhibit little or no CTH staining. It is well documented that endometriosis is the presumed precursor lesion of both CCOC and ENOC. It is not known, however, how such phenotypically different cancers arise from the same precursor. It may be that the metabolic state of cells in the endometrium determines whether it becomes a CCOC or and ENOCa upon receiving a genetic hit. Our initial findings from our proteomic screen have highlighted CTH overexpression as being a feature common in CCOC and comparatively rare in ENOC and HGS. We believe that high CTH expression could provide a unique perspective into the initiation and pathology of CCOC. Citation Format: Dawn R. Cochrane, Christopher S. Hughes, Tayyebeh Nazeran, Anthony N. Karnezis, Melissa K. McConechy, Gregg B. Morin and David G. Huntsman. CYSTEINE METABOLISM IN CLEAR CELL OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP12.

Abstract DPOC-014: BEYOND CODING MUTATIONS: USING RETROTRANSPOSONS TO PREDICT OVARIAN CANCER DEVELOPMENT

Abstract PURPOSE: Women with endometriosis, a painful condition caused by displaced endometrial tissue, have a 3-fold increased risk of developing endometrioid ovarian cancer (ENOC) and clear cell ovarian cancer (CCOC). How two distinct cancers arise from the same precursor lesion is unknown. Sensitive biomarkers are needed to identify women with endometriosis at risk of developing cancer. We performed whole genome sequencing on 29 ENOC and 36 CCOC cases and observed a highly frequent insertion event originating from an active LINE-1 (L1) retrotransposon in the TTC28 gene. L1 retrotransposons are mobile genetic elements that can take downstream DNA pieces and insert them into random genomic locations in a process called 3' transduction. L1s are epigenetically silenced in normal tissues, but are known to become activated in a variety of cancers. A recent study showed a stepwise loss of methylation across various L1 loci between normal endometrium, contiguous endometriosis (endometriosis adjacent to tumor), and ENOC/CCOC tissues. We hypothesize that TTC28 L1 retrotransposon is an early event in the transformation of endometriosis into ENOC and CCOC and such events could be used as biomarkers for endometriosis with high cancer risk. METHODS: We compared the presence of TTC28 L1 3' transductions to six SNVs and frame shifts mutations in normal, endometriosis, and tumor tissues from different anatomical sites in four ENOC and four CCOC cases. PCR followed by Sanger sequencing was used to detect TTC28 L1 insertions, and micro-fluidic PCR assay followed by MiSeq sequencing used to detect SNV/frameshift mutations. To broaden the analysis we will use a target capture sequencing method to track novel TTC28 and other L1 transductions. In these experiments probes tiling 1kbp downstream of L1s will be used to capture DNA fragments containing L1 transductions; the captured fragments will be sequenced on the MiSeq. We will assess the difference in TTC28 L1 methylation status between normal, endometriosis, and tumor tissues via the sequencing of bi-sulfite treated DNA. RESULTS: TTC28 L1 retrotransposition insertion is present at all 5 tumor sites in 75% (6/8) of cases, and is present in 3/5 or 4/5 tumor sites in the remaining cases. Analysis shows that TTC28 L1 insertion preceded some SNV and/or frameshift mutations. Preliminary results show that TTC28 L1 promoters are unmethylated in tumors with L1 insertions. Future experiments involving additional cases with endometriosis tissues will be performed. We expect to see L1 promoter hypomethylation and L1 transductions in endometriosis tissues. CONCLUSION: TTC28 L1 promoter hypomethylation and TTC28 L1 transductions may be early events in the transformation of endometriosis to cancer that can be explored as a method to predict tumor development. The development of a target capture assay to detect novel L1 transductions will be crucial for investigating cases without whole genome sequencing data. Ultimately, we hope to detect L1 insertions in plasma samples, and use L1 insertions as a biomarker to identify high-risk endometriosis cases. Citation Format: Zhouchunyang Xia, Dawn Cochrane, Michael S Anglesio, Tayyebeh M Nazeran, Janine Senz, Amy Lum, Ali Bashashati, Yi Kan Wang, Sohrab P Shah, David Huntsman. BEYOND CODING MUTATIONS: USING RETROTRANSPOSONS TO PREDICT OVARIAN CANCER DEVELOPMENT [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr DPOC-014.

Abstract AP09: GENOMIC ALTERATIONS IN ENDOMETRIOSIS

Abstract BACKGROUND: Endometriosis affects ~10% of reproductive–aged women and is characterized by extra–uterine biphasic growth of uterine endometrial epithelium and stroma. Endometriosis is widely considered to be a hormonally–dependent inflammatory condition and is classified into three anatomically defined types: deep infiltrating endometriosis that locally invades pelvic organs; ovarian endometriotic cysts, and superficial peritoneal endometriosis. A clonal “precursor–cancer” link to co–occurring clear cell and endometrioid ovarian cancers has been established and somatic mutations occur in these cancer–associated endometriotic cysts, including distant benign–appearing lesions. The spectrum of genetic alteration in non–cancer associated endometriosis is not known. METHODS: We screened a pilot series of 10 deep–infiltrating [pelvic] endometriosis lesions, without co–occurrence or history of cancer, for somatic alterations using laser captured endometrial stroma and epithelium. Two overlapping amplicon panels and digital PCR were used to validate mutations. RESULTS: In four of ten cases we validated the presence of activating codon 12 KRAS mutations. Mutations were restricted to the epithelium and no evidence of any alterations was found in the stromal fraction. CONCLUSIONS: We provide incontrovertible evidence of activating KRAS mutations in 40% of deep infiltrating endometriosis. This frequency of KRAS mutations is substantially higher than what has been observed in clear cell (CCOC) or endometrioid (ENOC) ovarian cancers and the occurrence of non–ovarian primary CCOC/ENOC is largely anecdotal. Our evidence suggests that these mutations represent core features of endometriosis itself, rather than a risk of malignant transformation. Overall we propose this as an opportunity to fundamentally shift the study of endometriosis to a spectrum of neoplasms. This may lead to the generation of a molecularly informed classification system and ultimately to improved prognostication and treatment. Citation Format: Michael S. Anglesio, Tayyebeh M. Nazeran, Hugo M. Horlings, Vivian Lac, Amy Lum, Janine Senz, Julie Ho, Amy Wang, Fontayne Wong, Catherine Allaire, Christina Williams, C. Blake Gilks, Paul J. Yong, and David G. Huntsman. GENOMIC ALTERATIONS IN ENDOMETRIOSIS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP09.

Genetic Variants in Epigenetic Pathways and Risks of Multiple Cancers in the GAME-ON Consortium.

Background: Epigenetic disturbances are crucial in cancer initiation, potentially with pleiotropic effects, and may be influenced by the genetic background.Methods: In a subsets (ASSET) meta-analytic approach, we investigated associations of genetic variants related to epigenetic mechanisms with risks of breast, lung, colorectal, ovarian and prostate carcinomas using 51,724 cases and 52,001 controls. False discovery rate-corrected P values (q values < 0.05) were considered statistically significant.Results: Among 162,887 imputed or genotyped variants in 555 candidate genes, SNPs in eight genes were associated with risk of more than one cancer type. For example, variants in BABAM1 were confirmed as a susceptibility locus for squamous cell lung, overall breast, estrogen receptor (ER)-negative breast, and overall prostate, and overall serous ovarian cancer; the most significant variant was rs4808076 [OR = 1.14; 95% confidence interval (CI) = 1.10-1.19; q = 6.87 × 10-5]. DPF1 rs12611084 was inversely associated with ER-negative breast, endometrioid ovarian, and overall and aggressive prostate cancer risk (OR = 0.93; 95% CI = 0.91-0.96; q = 0.005). Variants in L3MBTL3 were associated with colorectal, overall breast, ER-negative breast, clear cell ovarian, and overall and aggressive prostate cancer risk (e.g., rs9388766: OR = 1.06; 95% CI = 1.03-1.08; q = 0.02). Variants in TET2 were significantly associated with overall breast, overall prostate, overall ovarian, and endometrioid ovarian cancer risk, with rs62331150 showing bidirectional effects. Analyses of subpathways did not reveal gene subsets that contributed disproportionately to susceptibility.Conclusions: Functional and correlative studies are now needed to elucidate the potential links between germline genotype, epigenetic function, and cancer etiology.Impact: This approach provides novel insight into possible pleiotropic effects of genes involved in epigenetic processes. Cancer Epidemiol Biomarkers Prev; 26(6); 816-25. ©2017 AACR.

Abstract AP25: INHIBITION OF HDAC ACTIVITY SELECTIVELY INHIBITS ARID1A–MUTATED OVARIAN CLEAR CELL CARCINOMA THROUGH A NOVEL P53 REGULATORY MECHANISM

Abstract PURPOSE OF THE STUDY: ARID1A is mutated ~50% and 30% of clear cell (OCCC) and endometrioid (OEC) ovarian cancers, respectively. Over 90% of the ARID1A mutations observed in ovarian cancer are frame-shift or nonsense mutations that result in loss of ARID1A protein expression. In several cancers including OCCC, ARID1A and p53 mutations are often mutually exclusive. OCCC carries a worse prognosis compared to the other histosubtypes of ovarian cancer. Thus, there is an urgent clinical need for improved therapeutic strategies. EXPERIMENTAL PROCEDURE: To investigate the role of specific HDACs in the context of ARID1A expression, we examined the effect of knocking down HDACs in an isogenic OCCC cell line with and without ARID1A expression. Utilizing a panel of ARID1A-mutated and wildtype OCCC cell lines, we evaluated the effect of HDAC knockdown and inhibition through a variety of 2D and 3D assays. We also examined the consequence of HDAC inhibition on tumor growth, survival, and dissemination in an in vivo mouse model of ovarian cancer. SUMMARY OF THE DATA: We have discovered that ARID1A is involved in the repression of HDACs, and ARID1A-inactivation promotes aberrant transcriptional regulation of HDACs. Importantly, we uncovered a novel HDAC-dependent regulatory mechanism of p53. Several HDAC inhibitors are currently in clinical trials. We found HDAC inhibitors were more selective in ARID1A-mutated OCCC cell lines compared to ARID1A-wildtype cells. We observed that HDAC inhibition led to a significant increase in apoptosis in ARID1A-mutated cells. In an orthotopic intra-bursal xenograft model using ARID1A wildtype and deficient cells, the HDAC inhibitor suppressed primary tumor growth and inhibited tumor cell dissemination only in the ARID1A-mutated tumors. Significantly, HDAC inhibition significantly improved the survival of mice bearing ARID1A-mutated tumors. CONCLUSIONS: These findings imply that HDAC inhibition represents a novel therapeutic strategy for ARID1A-deficient cancers. This study further elucidates the observed mutual exclusivity of ARID1A and p53. Importantly, we have uncovered a novel regulatory mechanism of p53 mediated through ARID1A and HDACs. Clinically, this study describes a precision medicine approach to the treatment of ARID1A-mutated OCCC. Citation Format: Benjamin G. Bitler, Pyoung Hwa Park, Yang Hai, Katherine M. Aird, Yemin Wang, David G. Huntsman, Kathleen R. Cho, David W. Christianson, Rugang Zhang. INHIBITION OF HDAC ACTIVITY SELECTIVELY INHIBITS ARID1A–MUTATED OVARIAN CLEAR CELL CARCINOMA THROUGH A NOVEL P53 REGULATORY MECHANISM [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP25.

Abstract TMEM-033: DEVELOPING A MONKEY MODEL FOR SPONTANEOUS TYPE I OVARIAN CANCER

Abstract The goal of this research project is to investigate if Non-Human Primates (NHP) can serve as model for endometriosis-associated Type I ovarian cancer. Endometriosis is a spontaneously occurring condition in both women and rhesus macaques. Endometriosis is likely caused by aberrant retrograde menstruation of endometrial epithelium that implants and grows on the serosal surfaces of the ovaries, uterus, urinary bladder, intestines and abdominal peritoneum. Several studies have shown direct association between endometriosis and two histological subtypes of Type I ovarian cancer- clear cell ovarian carcinoma (CCOC) and low grade serous endometrioid ovarian cancer (EnOC). Genetically altered mouse models for Type I and II ovarian cancer and are contributing significantly to our understanding of the biology of this malignancy. Since rodents do not have menstrual cycles, there are no mouse models for spontaneous endometriosis and Type I ovarian tumors. On the other hand, between 10-30% of female rhesus macaques of reproductive age develop endometriosis. A survey of pathology reports at the Wisconsin National Primate Research Center (WNPRC) identified cases of ovarian and peritoneal neoplasia in monkeys that also had endometriosis. This is a typical feature of CCOC and EnOC in humans where tumors are found adjacent to atypical endometriotic lesions. We are conducting immunohistological analysis to study the molecular phenotype of endometriosis and the ovarian and peritoneal neoplasia in the rhesus macaque. We will discuss our findings on the expression of important molecular markers of ovarian cancer (CA125, EpCAM, mesothelin, ARID1A, and others) in the non-human primate reproductive organs as well as in endometriotic explants and in ovarian and peritoneal neoplasms from the female rhesus. The data we will present and our on-going efforts to sequence the genome and transcriptome of normal and aberrant reproductive tissue samples of female rhesus monkeys will be essential in determining if the rhesus can be used as a model for CCOC and EnOC. Citation Format: Mildred Felder, Arvinder Kapur, Heather Simmons, David Abbott, and Manish S. Patankar. DEVELOPING A MONKEY MODEL FOR SPONTANEOUS TYPE I OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-033.

Abstract NTOC-119: VCP INHIBITORS INDUCE ENDOPLASMIC RETICULUM (ER) STRESS AND SYNERGISTICALLY KILL OVARIAN CANCER CELLS IN COMBINATION WITH SALUBRINAL OR MIFEPRISTONE

Abstract Valosin-containing protein (VCP) or p97, a member of AAA-ATPase protein family, has been linked to several cellular functions including endoplasmic reticulum associated degradation (ERAD), cell division, golgi-membrane reassembly and autophagy. Recent studies have indicated VCP as one of the vulnerabilities in ovarian cancer. Here, we show that treatment with CB-5083, an orally bioavailable compound derived from previously identified quinazoline scaffold-based VCP inhibitor ML240, contributes to cytotoxicity in high-grade serous or clear cell ovarian cancer cell lines. CB-5083 treatment results in increased unfolded protein response (UPR), accumulation of cell cycle proteins as a result of inhibition of proteosomal degradation, cell cycle arrest at G1, and subsequent cell death mediated by both intrinsic and extrinsic apoptotic pathways. These results support an emerging concept that ER stress pathway can be therapeutically targeted in ovarian cancer. Since CB-5083 induces CHOP-dependent apoptosis, we tested the extent to which VCP inhibitors act synergistically with salubrinal, a compound that inhibits GADD34 and enhances eIF2α phosphorylation. Results from Sulforadamine B and clonogenic assays indicate that VCP inhibitors and salubrinal act synergistically across various concentrations of drug combination. Based on recent studies indicating that mifepristone, a steroidal antagonist to progesterone and glucocorticoid, induce ER stress and subsequent cell death, we also tested the potential drug synergies between VCP inhibitors and mifepristone. Our results indicate these drugs act synergistically across various concentrations in ovarian cancer cells. Analysis of TCGA RNA-sequencing datasets indicate VCP expression is down-regulated in cisplatin-resistant tumor samples compared to cisplatin-sensitive cancer. This data is consistent with the fact that tumors with lower expression of VCP shows significant association with poor progression-free survival and overall survivor. Finally, our results indicate that cells with lower expression of VCP are more sensitive to VCP inhibitors than those with higher expression. Considering that cisplatin-resistant tumor samples have lower expression of VCP, VCP inhibitors may be a good therapeutic candidate to treat cisplatin-resistant ovarian cancer. Collectively, our results provide evidence that VCP inhibitors can be used as a single agent and can be synergized with compounds that prolong or induce ER stress in ovarian cancer. Citation Format: Prabhakar Bastola, Frank J. Schoenen, Jeremy Chien. VCP INHIBITORS INDUCE ENDOPLASMIC RETICULUM (ER) STRESS AND SYNERGISTICALLY KILL OVARIAN CANCER CELLS IN COMBINATION WITH SALUBRINAL OR MIFEPRISTONE [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-119.