High-grade Serous Tumors Research Articles

A distinct innate lymphoid cell population regulates tumor-associated T cells.

Antitumor T cells are subject to multiple mechanisms of negative regulation. Recent findings that innate lymphoid cells (ILCs) regulate adaptive T cell responses led us to examine the regulatory potential of ILCs in the context of cancer. We identified a unique ILC population that inhibits tumor-infiltrating lymphocytes (TILs) from high-grade serous tumors, defined their suppressive capacity in vitro, and performed a comprehensive analysis of their phenotype. Notably, the presence of this CD56+CD3- population in TIL cultures was associated with reduced T cell numbers, and further functional studies demonstrated that this population suppressed TIL expansion and altered TIL cytokine production. Transcriptome analysis and phenotypic characterization determined that regulatory CD56+CD3- cells exhibit low cytotoxic activity, produce IL-22, and have an expression profile that overlaps with those of natural killer (NK) cells and other ILCs. NKp46 was highly expressed by these cells, and addition of anti-NKp46 antibodies to TIL cultures abrogated the ability of these regulatory ILCs to suppress T cell expansion. Notably, the presence of these regulatory ILCs in TIL cultures corresponded with a striking reduction in the time to disease recurrence. These studies demonstrate that a previously uncharacterized ILC population regulates the activity and expansion of tumor-associated T cells.

Abstract C44: Endometrial carcinoma recurrence in black and white women in the NRG Oncology/Gynecologic Oncology Group 210 trial

Abstract BACKGROUND: Black women diagnosed with endometrial carcinoma have a higher risk of recurrence compared with white women. Higher recurrence risk among black women is due, in part, to a greater frequency of aggressive tumor characteristics including non-endometrioid histologies and advanced stage tumors. The risk of recurrence for black and white women within distinct tumor groupings has not been well-examined due to low numbers of black women and under-representation of aggressive histologic subtypes in single institution studies. Therefore, we examined the association between self-reported race and recurrence risk stratified by histologic subtype in the NRG Oncology/Gynecology Oncology Group (GOG) 210, a prospective observational study that enrolled 6,124 newly diagnosed endometrial carcinoma patients between 2003 and 2011. METHODS: We restricted this analysis to 618 black and 4,316 white women with endometrial carcinoma. At study enrollment, women completed a questionnaire that assessed risk factors for gynecologic cancers. Recurrence, defined as evidence of disease following complete response to primary therapy, was abstracted from medical records. Tumor characteristics were available from surgical pathology reports and a centralized review by pathologists with expertise in gynecologic cancers. Cox proportional hazards regression models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for the association between race (black vs. white) and recurrence risk in models stratified by histologic subtype (low-grade endometrioid, high-grade endometrioid, serous, mixed epithelial, carcinosarcoma, clear cell) and adjusted for stage (I, II, III, IV), age at diagnosis (continuous), and adjuvant therapy (none, chemotherapy, radiation, chemotherapy and radiation, unknown). We also examined income ($20,000-$39,999, $40,000-$69,999, ≥$70,000) and education (high school/GED, some college/technical school, college graduate/beyond) as potential mediators of the association between race and endometrial carcinoma recurrence risk using the Baron-Kenny method. Median follow-up time was 5 years. RESULTS: Black women were more frequently diagnosed with non-endometrioid (52.9% vs. 31.8%) and advanced stage (Stage III or IV: 29.0% vs. 21.1%) tumors compared with white women. Recurrence was documented in 26% of black and 17% of white endometrial carcinoma patients. In histologic subtype-stratified models adjusted for stage, age, and adjuvant therapy, a higher risk of recurrence was noted in black women with low-grade endometrioid (HR=1.65, 95% CI=1.07-2.54), mixed epithelial (HR=1.75, 95% CI=1.13-2.71), or carcinosarcomas (HR=1.55, 95% CI=1.03-2.31) compared with white women. No significant race-recurrence relationships were observed among women diagnosed with high-grade endometrioid, serous, or clear cell tumors. After adjustment for income and education, the association of black race and recurrence risk remained elevated, but was not statistically significant: low-grade endometrioid (HR=1.52, 95% CI=0.97-2.38), mixed epithelial (HR=1.56, 95% CI=0.98-2.50), and carcinosarcoma (HR=1.48, 95% CI=0.98-2.25). Further, neither income nor education were significantly associated with recurrence risk in the multivariable models. CONCLUSIONS: Our study provides evidence that racial disparities in endometrial carcinoma recurrence risk vary by histologic subtype. Socioeconomic factors do not explain black-white differences in endometrial carcinoma recurrence risk. Future studies exploring the role of biological factors and post-diagnosis surveillance contributing to racial differences in endometrial carcinoma recurrence are warranted. Citation Format: Ashley S. Felix, Theodore Brasky, David Cohn, Scott McMeekin, David Mutch, Creasman William, Premal Thaker, Joan Walker, Richard Moore, Shashikant Lele, Saketh Guntupalli, Levi Downs, Christa Nagel, John Boggess, Michael Pearl, Olga Ioffe, Kay Park, Shamshad Ali, Louise Brinton. Endometrial carcinoma recurrence in black and white women in the NRG Oncology/Gynecologic Oncology Group 210 trial. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr C44.

Evaluation of Prognostic and Predictive Significance of Circulating MicroRNAs in Ovarian Cancer Patients.

Ovarian cancer patients are recognized with poor prognosis. This study aimed to identify microRNAs in plasma for predicting response to treatment and outcome. We have investigated microRNAs in plasma from ovarian cancer patients enrolled in a large multicenter study (ICON7), investigating the effect of adding bevacizumab to standard chemotherapy in patients diagnosed with epithelial ovarian cancer. Patients with different histology, grade, and FIGO stages were included (n = 207) in this study. Screening of 754 unique microRNAs was performed in the discovery phase (n = 91) using TaqMan Low Density Arrays. The results were validated using single assays and RT-qPCR. Low levels of miR-200b, miR-1274A (tRNALys5), and miR-141 were significantly associated with better survival, confirmed with log-rank test in the validation set. The level of miR-1274A (tRNALys5) correlated with outcome was especially pronounced in the high-grade serous tumors. Interestingly, low level of miR-200c was associated with 5-month prolongation of PFS when treated with bevacizumab compared to standard chemotherapy. We found prognostic significance of miR-200b, miR-141, and miR-1274A (tRNALys5) in all histological types, where miR-1274A (tRNALys5) may be a specific marker in high-grade serous tumors. The level of miR-200c may be predictive of effect of treatment with bevacizumab. However, this needs further validation.

Diagnostic and Prognostic Significance of Ki-67 Immunohistochemical Expression in Surface Epithelial Ovarian Carcinoma.

The Surface Epithelial Ovarian Carcinoma (SEOC) at the moment of diagnosis, the disease is extended beyond the structures of the pelvis. Ki-67 is one of the prognostic marker which determines the growth fraction of a tumour and its over expression is associated with malignancy, tumour aggression, reserved prognosis and metastasis. To evaluate the proliferative activity using Ki-67 immuno-staining in SEOC and to correlate with histological subtype, grade, Federation of Gynecology and Obstetrics (FIGO) stage, CA125 levels for diagnostic and prognostic purpose. The study was conducted in JSS Medical College and Hospital, JSS University, Mysuru. It was a descriptive cross-sectional study involving 40 cases of SEOC over a period of two years. The proliferation expression related to Ki-67 antigen was evaluated by immunohistochemical monoclonal MIB-1 antibody. In each case, the Ki-67 labeling index (Ki-67 LI) was articulated as percentage of positively stained cells using high power objective of the microscope (x400). Among the 40 carcinomas, 26 were serous, five mucinous, four each of clear cell and undifferentiated and one transitional cell carcinoma. A total of 75% were high grade tumours. High Ki-67 LI was associated with high grade tumours (69.9%), high grade serous tumours (65.34%) and advanced FIGO staging (70.6%) with the p-value of <0.001. CA 125 levels did not have a significant correlation with Ki-67 LI. Ki-67 is an exceptionally a cost effective marker to determine the growth fraction of a tumour cell population. In SEOC histological grade and FIGO stage when combined with Ki-67 LI in histopathology report would help in diagnostic differentiation of subtypes, prognostication, deciding the need for adjuvant chemotherapy and in predicting survival analysis.

CLINICAL AND MORPHOLOGICAL FEATURES OF HEREDITARY OVARIAN CANCER

Germ-line mutations in BRCA1 and BRCA2 genes are the most established risk factors for hereditary breast and ovarian cancers. The purpose of the study was to analyze BRCA1/2 testing in ovarian cancer patients. Materials and methods. We analyzed 222 patients with ovarian cancer (OC) who underwent genetic testing. Results. Recurrent Slavic mutations in these genes were detected in 60/222 (27%) patients.104 patients lacked any clinical signs of hereditary form of the disease, however BRCA1/2 genetic defects were identified among 11 (11%) of these women. BRCA1/2-associated carcinomas were characterized by more advanced stage at diagnosis and predominance of high-grade serous histological tumor subtype. Conclusion. These results emphasize the need for BRCA1/2 testing for all patients with OC. BRCA1/2-associated carcinomas have clinical and pathological cgaracteristics, which should be considered while planning therapy.

Long and irregular menstrual cycles, polycystic ovary syndrome, and ovarian cancer risk in a population-based case-control study.

Long and irregular menstrual cycles, a hallmark of polycystic ovary syndrome (PCOS), have been associated with higher androgen and lower sex hormone binding globulin levels and this altered hormonal environment may increase the risk of specific histologic subtypes of ovarian cancer. We investigated whether menstrual cycle characteristics and self-reported PCOS were associated with ovarian cancer risk among 2,041 women with epithelial ovarian cancer and 2,100 controls in the New England Case-Control Study (1992-2008). Menstrual cycle irregularity, menstrual cycle length, and PCOS were collected through in-person interview. Unconditional logistic regression models were used to calculate odds ratios (OR) and 95% confidence intervals (95% CIs) for ovarian cancer risk overall, and polytomous logistic regression to evaluate whether risk differed between histologic subtypes. Overall, we observed no elevation in ovarian cancer risk for women who reported periods that were never regular or for those reporting a menstrual cycle length of >35 days with ORs of 0.87 (95% CI = 0.69-1.10) and 0.83 (95% CI = 0.44-1.54), respectively. We observed no overall association between self-reported PCOS and ovarian cancer (OR = 0.97; 95% CI = 0.61-1.56). However, we observed significant differences in the association with menstrual cycle irregularity and risk of ovarian cancer subtypes (pheterogeneity = 0.03) as well as by BMI and OC use (pinteraction < 0.01). Most notable, menstrual cycle irregularity was associated with a decreased risk of high grade serous tumors but an increased risk of serous borderline tumors among women who had never used OCs and those who were overweight. Future research in a large collaborative consortium may help clarify these associations.

Cadherin-6 type 2, K-cadherin (CDH6) is regulated by mutant p53 in the fallopian tube but is not expressed in the ovarian surface.

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy and may arise in either the fallopian tube epithelium (FTE) or ovarian surface epithelium (OSE). A mutation in p53 is reported in 96% of HGSOC, most frequently at R273 and R248. The goal of this study was to identify specific gene targets in the FTE that are altered by mutant p53, but not in the OSE. Gene analysis revealed that both R273 and R248 mutant p53 reduces CDH6 expression in the oviduct, but CDH6 was not detected in murine OSE cells. p53R273H induced SLUG and FOXM1 while p53R248W did not induce SLUG and only modestly increased FOXM1, which correlated with less migration as compared to p53R273H. An oviduct specific PAX8Cre/+/p53R270H/+ mouse model was created and confirmed that in vivo mutant p53 repressed CDH6 but was not sufficient to stabilize p53 expression alone. Overexpression of mutant p53 in the p53 null OVCAR5 cells decreased CDH6 levels indicating this was a gain-of-function. SLUG knockdown in murine oviductal cells with p53R273H restored CDH6 repression and a ChIP analysis revealed direct binding of mutant p53 on the CDH6 promoter. NSC59984, a small molecule that degrades mutant p53R273H, rescued CDH6 expression. In summary, CDH6 is expressed in the oviduct, but not the ovary, and is repressed by mutant p53. CDH6 expression with further validations may aide in establishing markers that inform upon the cell of origin of high grade serous tumors.

Case 2 – Advanced low-grade ovarian cancer with peritoneal implants

Low-grade serous carcinomas are a less common subtype of serous epithelial ovarian cancer. They differ from high-grade serous tumors in their immuno-histochemical profile, epidemiologic features, clinical behavior and molecular characteristics. This case report describes the clinical history of a young woman who underwent multiple surgical and medical treatments.

Abstract 1761: Serous ovarian cancer risk factors by grade: Evidence for etiologic heterogeneity

Abstract Background: Growing evidence suggests that high grade serous ovarian carcinomas may arise from the fallopian tube, while low-grade serous ovarian cancer may develop on the ovaries. Previous studies have suggested differences in ovarian cancer risk factors by histologic subtypes, but few have evaluated differences in risk factors for high- vs. low-grade serous cancers. In the Ovarian Cancer Cohort Consortium (OC3), a pooling project of individual-level data from 23 prospective cohort studies, we evaluated associations of hormonal, reproductive, demographic and lifestyle factors, and family history of cancer with for serous ovarian cancer subtypes. Methods: Among 16 studies that abstracted grade information from pathology reports, 3,095 serous ovarian cancers were identified during follow-up (125 well differentiated, 506 moderately differentiated, 1671 poorly differentiated, 793 undetermined). We used competing risks Cox proportional hazards regression to compute relative risks (RRs) and 95% confidence intervals (CIs) for differences in association by grade. Models were stratified on study and year of birth and adjusted for age, parity and OC use; subtype heterogeneity was evaluated by likelihood ratio tests. Results: Although sample sizes were small for low-grade tumors, there was evidence for heterogeneity in the associations for oral contraceptive (OC) use, age at menopause, endometriosis, and family history of ovarian cancer. For example, each 5-year increase in OC use was associated with a 21% decrease in risk of low-grade serous ovarian cancer (RR: 0.79; 95% CI: 0.62-1.00), but only a 10% decrease in risk of high-grade serous cancers (RR: 0.90; 95% CI: 0.84-0.96; p-heterogeneity = 0.09). Consistent with a prior report in the Ovarian Cancer Association Consortium, endometriosis was associated with increased risk of low-grade serous tumors (RR: 3.77; 95% CI: 1.24-11.48), but not with high-grade serous tumors (RR: 1.11; 95% CI: 0.70-1.74; p-heterogeneity = 0.12). Conclusion: Our results demonstrate heterogeneous associations of risk factors with subtypes of serous ovarian cancer, supporting the idea that the high- and low-grade serous tumors develop through different pathways. Despite the small sample size for low-grade serous tumors, most risk factors were more strongly associated with low-grade tumors compared to high-grade serous carcinomas, suggesting that risk prediction may be more challenging for the most fatal subtype. Identifying subtype-specific risk factor and biomarkers is important both for better understanding ovarian cancer etiology and for targeted development of novel prevention approaches. These results underscore the importance of consortial projects to evaluate rare subtypes (low-grade serous cancers) for the better understanding of etiologic heterogeneity of this deadly disease. Citation Format: Elizabeth M. Poole, Nicolas A. Wentzensen, Britton Trabert, Shelley S. Tworoger, The Ovarian Cancer Cohort Consortium. Serous ovarian cancer risk factors by grade: Evidence for etiologic heterogeneity. [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 1761.

Abstract 2638: Cardiovascular comorbidity and ovarian cancer patient survival: history of hypertension, heart disease, and diabetes, evidence from the Ovarian Cancer Association Consortium

Abstract Background: The role of previously diagnosed comorbidities in relation to prognosis for ovarian cancer patients has not been studied extensively, particularly examining histological subtype. We explored the association between pre-existing hypertension, heart disease, and diabetes and overall survival (OS) and progression-free survival (PFS) among women diagnosed with ovarian cancer in a large international study. Methods: Using pooled data from 14 case-control studies participating in the Ovarian Cancer Association Consortium (n = 7076), we examined associations between self-reported pre-existing hypertension, heart disease, and diabetes, and OS and PFS among patients diagnosed with invasive epithelial ovarian carcinoma. We used Cox proportional hazards regression models adjusted for age and stage to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). We also examined associations separately for the main histological subtypes, high-grade serous, low-grade serous, mucinous, endometrioid, and clear cell tumors. Analyses were also conducted in strata of menopausal status, body mass index (BMI) categories, stage of disease, and ever use of medications specific for each condition. Results: No significant associations were observed for overall cancer for risk of death with history of hypertension (n = 5847; HR = 0.93; 95% CI 0.85-1.02), history of heart disease (n = 4178; HR = 1.11; 95%CI 0.91-1.36) or history of diabetes (n = 7043; HR = 1.07; 95%CI 0.95-1.20). Similarly, there was no association of these comorbidities with PFS. However, among cases with endometrioid tumors, history of hypertension was associated with lower risk of both OS and PFS (n = 869; HR = 0.70; 95%CI 0.51-0.96, and n = 246, HR = 0.54; 95%CI 0.33-0.90 correspondingly). There were no significant associations observed between history of the comorbidities and PFS among the other histological subtypes. None of the examined associations differed in strata defined by menopausal status, BMI categories, stage of disease, or categories of medications intake. Conclusion: Pre-existing hypertension may be inversely associated with survival among women diagnosed with invasive ovarian cancer, particularly for those with endometrioid tumors. Understanding of the mechanism for this observation could provide for treatment strategies. Future studies are needed to explore the role of chronic diseases and their management in relation to prognosis of ovarian cancer patients and investigate the difference of tumor microenvironment among various histologic subtypes. Citation Format: Albina Minlikeeva, Jo L. Freudenheim, Kirsten B. Moysich, Ovarian Cancer Association Consortium. Cardiovascular comorbidity and ovarian cancer patient survival: history of hypertension, heart disease, and diabetes, evidence from the Ovarian Cancer Association Consortium. [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 2638.

Irregular menses predicts ovarian cancer: Prospective evidence from the Child Health and Development Studies.

We tested the hypothesis that irregular menstruation predicts lower risk for ovarian cancer, possibly due to less frequent ovulation. We conducted a 50-year prospective study of 15,528 mothers in the Child Health and Development Studies cohort recruited from the Kaiser Foundation Health Plan from 1959 to 1966. Irregular menstruation was classified via medical record and self-report at age 26. We identified 116 cases and 84 deaths due to ovarian cancer through 2011 via linkage to the California Cancer Registry and Vital Statistics. Contrary to expectation, women with irregular menstrual cycles had a higher risk of ovarian cancer incidence and mortality over the 50-year follow-up. Associations increased with age (p <0.05). We observed a 2-fold increased incidence and mortality by age 70 (95% confidence interval [CI] = 1.1, 3.4) rising to a 3-fold increase by age 77 (95% CI = 1.5, 6.7 for incidence; 95% CI = 1.4, 5.9 for mortality). We also found a 3-fold higher risk of mortality for high-grade serous tumors (95% CI = 1.3, 7.6) that did not vary by age. This is the first prospective study to show an association between irregular menstruation and ovarian cancer-we unexpectedly found higher risk for women with irregular cycles. These women are easy to identify and many may have polycystic ovarian syndrome. Classifying high-risk phenotypes such as irregular menstruation creates opportunities to find novel early biomarkers, refine clinical screening protocols and potentially develop new risk reduction strategies. These efforts can lead to earlier detection and better survival for ovarian cancer.

A metabolic labeling approach for glycoproteomic analysis reveals altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells

Epithelial ovarian cancer (EOC) is a disease responsible for more deaths among women in the Western world than all other gynecologic malignancies. There is urgent need for new therapeutic targets and a better understanding of EOC initiation and progression. We have previously identified the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene, a member of the GalNAc-transferases (GalNAc-Ts) gene family, as hypomethylated and overexpressed in high-grade serous EOC tumors, compared to low malignant potential EOC tumors and normal ovarian tissues. This data also suggested for a role of GALNT3 in aberrant EOC glycosylation, possibly implicated in disease progression. To evaluate differential glycosylation in EOC caused by modulations in GALNT3 expression, we used a metabolic labeling strategy for enrichment and mass spectrometry-based characterization of glycoproteins following GALNT3 gene knockdown (KD) in A2780s EOC cells. A total of 589 differentially expressed glycoproteins were identified upon GALNT3 KD. Most identified proteins were involved in mechanisms of cellular metabolic functions, post-translational modifications, and some have been reported to be implicated in EOC etiology.The GALNT3-dependent glycoproteins identified by this metabolic labeling approach support the oncogenic role of GALNT3 in EOC dissemination and may be pursued as novel EOC biomarkers and/or therapeutic targets. Biological significanceKnowledge of the O-glycoproteome has been relatively elusive, and the functions of the individual polypeptide GalNAc-Ts have been poorly characterized. Alterations in GalNAc-Ts expression were shown to provide huge variability in the O-glycoproteome in various pathologies, including cancer. The application of a chemical biology approach for the metabolic labeling and subsequent characterization of O-glycoproteins in EOC using the Ac4GalNAz metabolite has provided a strategy allowing for proteomic discovery of GalNAc-Ts specific functions. Our study supports an essential role of one of the GalNAc-Ts — GALNT3, in EOC dissemination, including its implication in modulating PTMs and EOC metabolism. Our approach validates the use of the applied metabolic strategy to identify important functions of GalNAc-Ts in normal and pathological conditions.

The mannose receptor LY75 (DEC205/CD205) modulates cellular phenotype and metastatic potential of ovarian cancer cells.

The molecular basis of epithelial ovarian cancer (EOC) dissemination is still poorly understood. Previously, we identified the mannose receptor LY75 gene as hypomethylated in high-grade (HG) serous EOC tumors, compared to normal ovarian tissues. LY75 represents endocytic receptor expressed on dendritic cells and so far, has been primarily studied for its role in antigen processing and presentation. Here we demonstrate that LY75 is overexpressed in advanced EOC and that LY75 suppression induces mesenchymal-to-epithelial transition (MET) in EOC cell lines with mesenchymal morphology (SKOV3 and TOV112), accompanied by reduction of their migratory and invasive capacity in vitro and enhanced tumor cell colonization and metastatic growth in vivo. LY75 knockdown in SKOV3 cells also resulted in predominant upregulation of functional pathways implicated in cell proliferation and metabolism, while pathways associated with cell signaling and adhesion, complement activation and immune response were mostly suppressed. Moreover, LY75 suppression had an opposite effect on EOC cell lines with epithelial phenotype (A2780s and OV2008), by directing epithelial-to-mesenchymal transition (EMT) associated with reduced capacity for in vivo EOC cell colonization, as similar/identical signaling pathways were reversely regulated, when compared to mesenchymal LY75 knockdown EOC cells.To our knowledge, this is the first report of a gene displaying such pleiotropic effects in sustaining the cellular phenotype of EOC cells and points to novel functions of this receptor in modulating EOC dissemination. Our data also support previous findings regarding the superior capacity of epithelial cancer cells in metastatic colonization of distant sites, compared to cancer cells with mesenchymal-like morphology.

Abstract B24: Personal history of endometriosis and survival in ovarian cancer patients: A pooled analysis of 10 studies from the Ovarian Cancer Association Consortium.

Abstract Background: There is mounting evidence that a personal history of endometriosis is associated with an increased risk of ovarian cancer. Previous studies have shown that ovarian cancer patients with a prior diagnosis of endometriosis had more favorable outcomes than women without this condition, likely due to increased surveillance resulting in earlier detection. However, most of these studies were relatively small in size and did not consider histological subtypes in their analyses. Given the increased recognition of the importance of histology in ovarian cancer prognosis, we examined the association between history of endometriosis and overall survival among women diagnosed with ovarian cancer in a large multi-national sample. Methods: We utilized data from 10 case-control studies participating in Ovarian Cancer Association Consortium and used age- and stage-adjusted Cox- proportional hazards models to estimate associations between history of endometriosis and overall survival among patients diagnosed with invasive epithelial ovarian carcinoma. Analyses were also conducted separately for histological subtypes of ovarian cancer (low grade serous, high grade serous, mucinous, endometriod and clear cell). We also examined the associations within strata of menopausal status and body mass index (BMI) categories. Results: A personal history of endometriosis was inversely associated with risk of death (hazard ratio (HR)=0.84; 95% confidence interval (CI) 0.74-0.95) among all ovarian cancer patients (n=7033). We observed strong risk reductions among patients with low-grade serous disease (n=214; HR=0.48; 95% CI 0.24-0.95), but not among patients with high-grade serous tumors (n=3273; HR=0.97; 95% CI 0.83-1.14). Furthermore, patients with clear cell tumors (n=555) had a 40% risk reduction (HR=0.60; 95% CI 0.39-0.91). No significant associations were observed for patients with mucinous (n=356) or endometriod (n=1072) ovarian cancer, although the HRs were well below the null value (HR=0.82; 95% CI 0.33-1.05, and HR=0.70; 95% CI 0.47-1.05, respectively). When we considered BMI as a potential effect modifier, we observed that the survival benefit associated with a personal history of endometriosis was restricted to normal weight women (HR=0.70; 95% CI 0.58-0.85) and absent among overweight (HR=1.02; 95% CI 0.82-1.28) and obese (HR=1.08; 95% CI 0.82-1.40) patients. The relationship between a personal history of endometriosis and ovarian cancer outcomes did not differ between premenopausal and postmenopausal patients. Conclusion: Our results suggest that history of endometriosis is associated with overall survival among women diagnosed with invasive ovarian cancer, particularly among clear cell and low grade serous carcinomas, including after control for stage of disease. Despite being underpowered to adequately assess between endometriosis and outcomes for patients with mucinous and endometrioid tumors, there was a trend towards improved survival for those with a personal history of endometriosis. These findings underscore the importance of examining prognostic indicators for ovarian cancer among distinct histological subgroups, as well as to distinguish between low-grade serous and high-grade serous disease. Moreover, the constellation of changes caused by endometriosis to induce ovarian cancer may alter the tumor microenvironment and change the ultimate disease prognosis. Future studies are needed to determine the role of endometriosis in the tumor environment and ovarian cancer prognosis. Citation Format: Albina N. Minlikeeva, Jo L. Freudenheim, C. Leigh Pearce, J.Brian Szender, Ovarian Cancer Association Consortium, Kirsten B. Moysich. Personal history of endometriosis and survival in ovarian cancer patients: A pooled analysis of 10 studies from the Ovarian Cancer Association Consortium. [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 B24.

Abstract PR12: Mutant p53 drives early events in fallopian tube tumorigenesis through mesenchyme-associated autocrine production of matrix that supports survival and mesothelial intercalation.

Abstract High-grade serous ovarian cancer (HGS-OvCa) harbor p53 mutations and predominantly originates from the secretory epithelial cell compartment of the fallopian tube fimbriae. From this site, tumor cells detach, survive in the peritoneal cavity, and form cellular clusters that clear the mesothelium and expand to form peritoneal masses. To examine the contribution of mutant p53 to any of these phenotypic alterations associated with HGS-OvCA tumor progression, we developed live-cell microscopy assays that recapitulate these phenotypes and examined dynamic processes regulated by depletion of p53 or gain-of-function (GOF) mutations in p53 in cultured secretory fallopian tube epithelial cells. Expression of GOF mutant variants of p53, but not depletion of endogenous wild-type p53, promoted survival and cell-cell aggregation under conditions of cell detachment, leading to the formation of spheroids with mesothelium-clearing capacity. These phenotypes were blocked by mutant p53 reactivation and depended on the expression of the mesenchyme transcription factor TWIST1, inhibition of p63 and de novo formation of a unique cell-matrix adhesion niche supported by autocrine fibronectin production and activation of integrins in detached cells. Inhibition of fibronectin or integrin α5 in suspension blocked cell-to-cell adhesion leading to HGS-OvCa spheroid disintegration and ultimately anoikis. These results indicate the detached FTE cells-expressing GOF mutants of p53 undergo a mesenchymal transition under conditions of matrix detachment leading to autocrine production of matrix and anchorage-independent survival and proliferation, as well as mesothelial clearance activity. These findings provide important new insights into activities of mutant p53 that may play a critical role in early events in tumorigenesis of high-grade ovarian serous tumors and provide a rationale for the development of therapeutics that target ovarian cancer cells transiting through the peritoneal cavity. This abstract is also presented as Poster B03. Citation Format: Marcin P. Iwanicki, Hsing-Yu Chen, Ioannis Zervantonakis, Marian Novak, Taru Muranen, Tan A. Ince, Ronny Drapkin, Joan S. Brugge. Mutant p53 drives early events in fallopian tube tumorigenesis through mesenchyme-associated autocrine production of matrix that supports survival and mesothelial intercalation. [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 PR12.

Targeted therapy and immunotherapy in ovarian cancer

Epithelial ovarian cancer is one of the last treatment areas to be influenced by the 'personalized medicine' bandwagon. Some anti-angiogenic agents, poly (ADP-ribose) polymerase (PARP), and immune checkpoint inhibitors have shown efficacy in early stages of development for the treatment of epithelial ovarian cancer. As a result of vigorous clinical trials, bevacizumab, cediranib, pazopanib, olaparib, and rucaparib, either used alone or in adjunct to conventional cytotoxic agents, have all been shown to improve progression-free survival in first-line/maintenance, platinum-resistant or sensitive recurrent settings. A biomarker for bevacizumab is currently elusive. Olaparib is the first drug approved for the treatment of high-grade serous tumors and requires routine testing for BRCA mutation. Trial results of PARP inhibitors in the homologous recombination-deficient (non BRCA mutation) population are awaited and the introduction of these agents into the clinic will require robust methods of detecting homologous recombination-deficiency. Second-generation studies combining anti-angiogenic agents with PARP inhibitors are in progress and early results in the recurrent setting are encouraging. Trials with immune checkpoint inhibitors such as nivolumab produced prolonged responses in a small set of ovarian cancer cases and need further exploration, for example in combination with anti-angiogenic agents. The application of targeted agents and immunologics, alone or in combination, to induce a survival advantage in patients with epithelial ovarian cancer should be continued.

Statement by the Kommission Ovar of the AGO: The New FIGO and WHO Classifications of Ovarian, Fallopian Tube and Primary Peritoneal Cancer.

More than 25 years after the last revision, in 2012 the FIGO Oncology Committee began revising the FIGO classification for staging ovarian, Fallopian tube and primary peritoneal cancers. The new classification has become effective with its publication at the beginning of 2014. Following recent findings on the pathogenesis of ovarian, Fallopian tube and primary peritoneal cancer and reflecting standard clinical practice, the three entities have now been classified uniformly. The histological subtype is included (high-grade serous - HGSC; low-grade serous - LGSC; mucinous - MC; clear cell - CCC; endometrioid - EC). Stages III and IV have been fundamentally changed: stage IIIA now refers to a localized tumor limited to the pelvis with (only) retroperitoneal lymph node metastasis (formerly classified as IIIC). Stage IV has been divided into IVA and IVB, with IVA defined as malignant pleural effusion and IVB as parenchymatous or extra-abdominal metastasis including inguinal and mediastinal lymph node metastasis as well as umbilical metastasis. A new WHO classification was published almost concurrently. The classification of serous tumors addresses the issue of the tubal carcinogenesis of serous ovarian cancer, even if no tubal precursor lesions are found for up to 30 % of serous high-grade cancers. The number of subgroups was reduced and subgroups now include only high-grade serous, low-grade serous, mucinous, seromucinous, endometrioid, clear cell and Brenner tumors. The category "transitional cell carcinomas" has been dropped and the classification "seromucinous tumors" has been newly added. More attention has been focused on the role of borderline tumors as a stage in the progression from benign to invasive lesions.

Novel high-grade serous epithelial ovarian cancer cell lines that reflect the molecular diversity of both the sporadic and hereditary disease

Few cell line models of epithelial ovarian cancer (EOC) have been developed for the high-grade serous (HGS) subtype, which is the most common and lethal form of gynaecological cancer. Here we describe the establishment of six new EOC cell lines spontaneously derived from HGS tumors (TOV2978G, TOV3041G and TOV3291G) or ascites (OV866(2), OV4453 and OV4485). Exome sequencing revealed somatic TP53 mutations in five of the cell lines. One cell line has a novel BRCA1 splice-site mutation, and another, a recurrent BRCA2 nonsense mutation, both of germline origin. The novel BRCA1 mutation induced abnormal splicing, mRNA instability, resulting in the absence of BRCA1 protein. None of the cell lines harbor mutations in KRAS or BRAF, which are characteristic of other EOC subtypes. SNP arrays showed that all of the cell lines exhibited structural chromosomal abnormalities, copy number alterations and regions of loss of heterozygosity, consistent with those described for HGS. Four cell lines were able to produce 3D-spheroids, two exhibited anchorage-independent growth, and three (including the BRCA1 and BRCA2 mutated cell lines) formed tumors in SCID mice. These novel HGS EOC cell lines and their detailed characterization provide new research tools for investigating the most common and lethal form of EOC.

Abstract AS31: MEK inhibition reverses antiestrogen resistance in ovarian cancer (OVCA) via alteration of cell cycle pathways and MAPK/estrogen regulated gene expression

Abstract BACKGROUND: It is estimated that 67% of epithelial OVCAs are estrogen receptor (ER) positive. However, the response to anti-estrogen therapy in OVCA remains marginal. The Ras/Raf/MEK/MAPK pathway is hyperactivated in 40% of OVCAs. We have previously shown that estrogens further activate kinases such as Src, ER and Src kinase binding in the cytoplasm suggesting a non-genomic role or ER in OVCA. We postulated that estrogens further activate MAPK signaling and combination ER blockade with MEK inhibition would block cross-talk and increase the efficacy of ER blockade. METHODS: The effects of treatment with MEK inhibitor (AZD6244) and anti-estrogen (Fulvestrant), each alone or together, on cell cycle and cell survival were evaluated in ER+ OVCA lines in vitro. Drug effects on xenograft tumor growth were assayed in vivo in NOD/SCIDs. Reverse phase protein lysate array (RPPA) analysis and gene expression analysis (GEA) were performed to evaluate biomarkers of drug response. Finally, a previously reported MAPK gene signature identified in breast cancer was analyzed in the OVCA lines treated with AZD6244 and combination treatment. And using alteration of gene expression upon MEKi treatment as suggestive of MAPK regulation, we define a MAPK gene signature originating from ovarian cancer cells. RESULTS: RPPA analysis of high grade serous tumors from the TCGA (n=408) demonstrates that over 70% of tumors have phosphorylated MEK and MAPK, and patients with ER+ cancers and high pMAPK or pMEK (top50%), have a worse overall survival than those with low pMAPK or pMEK. Estrogen (E2) increases phosphorylation of MEK in ER+ OVCA cells. Fulvestrant caused minimal growth arrest after treatment demonstrating intrinsic resistance. AZD6244 caused loss of pMAPK, partial G1 cell cycle arrest and a modest increase in p27 levels in a dose dependent manner after treatment. However, responsiveness of OVCA cells to fulvestrant increased by addition of AZD6244 in vitro, with synergistic cell cycle arrest mediated by p27 binding to Cyclin E/cdk2 and much greater inhibition of MAPK activity. Gene enrichment analysis showed an increase in the ERB4/MAPK gene set with Fulv alone and the addition of AZD6244 showed that the top 20 gene sets downregulated were all related to replication and cell cycle (ie FOXM1, CyclinE). RPPA confirmed that combination was more effective in decreasing cell cycle promoting proteins (ie FOXM1, Cyclin B1) and upregulating p27. AZD6244 treatment of OVCA lines resulted in differential expression of about ¼ of the breast cancer defined MAPK gene expression signature, and of these, fulvestrant addition to MEK inhibition (MEKi) differentially affected 19 genes, reflective of these being E2 regulated genes. Similarly, of the total MEKi affected genes, a subset were differentially regulated by the addition of fulvestrant indicating putative E2 regulation underlying these genes. Xenograft data showed the greatest decrease in tumor volume with the drug combination compared to either drug alone. CONCLUSION: Given the majority of primary OVCAs express high MEK/MAPK activity may underlie failure of anti-estrogen therapy. MEK inhibition reverses anti-estrogen resistance in our OVCA models. These data support further pre-clinical and clinical evaluation of combined fulvestrant and MEK inhibition in OVCA. Citation Format: K. Hew, P. Miller, J. Sun, Z. Wei, G. Zhang, Y. Lu, G. Mills, J. Slingerland, MD, PhD, D. El-Ashry, F. Simpkins. MEK inhibition reverses antiestrogen resistance in ovarian cancer (OVCA) via alteration of cell cycle pathways and MAPK/estrogen regulated gene expression [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 AS31.

Abstract 2964: Histology-specific patterns of DNA methylation in Lynch-associated and sporadic ovarian cancer

Abstract Epithelial ovarian cancer is a heterogeneous group of cancers, and molecular tools are urgently needed for a better understanding and targeted management of this often lethal disease. Since epigenetic methods can offer new tools for the management of ovarian cancer, our aim was to investigate epigenetic mechanisms in ovarian tumorigenesis representing different histological types. Expression profiling of ovarian and endometrial cancer cell lines treated with demethylating agents as well as literature were used to select gene candidates for epigenetic regulation. A methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay was constructed for thirteen genes to study methylation in 104 (85 sporadic and 19 Lynch syndrome-associated) ovarian carcinomas. Increased methylation (hypermethylation) was characteristic of ovarian cancer tissues relative to the corresponding normal tissues and hypermethylation was consistently more prominent in non-serous than serous tumors. The highest frequencies of hypermethylation were detected in Lynch-associated clear cell ovarian carcinomas. An interesting finding among endometrioid ovarian carcinomas was a significant association of lower levels of methylation in HOXA9, RSK4 and SPARC with higher tumor grade; thus, the methylation patterns of high-grade endometrioid ovarian carcinomas resembled epigenetic characteristics of high-grade serous tumors. Overall, non-serous ovarian tumors of Lynch and sporadic origin (as opposed to serous tumors) showed frequent epigenetic inactivation in RSK4, SPARC, PROM1, HOXA10, HOXA9, WT1-AS, SFRP2, SFRP5, OPCML, and MIR34B. Our findings provide important new information regarding some critical genes affected by epigenetic dysregulation in ovarian tumorigenesis. The results will be utilized in future experiments aiming to define the genetic and epigenetic origins of ovarian carcinomas of different types. Citation Format: Anni Niskakoski, Sippy Kaur, Synnove Staff, Laura Renkonen-Sinisalo, Heini Lassus, Heikki J. Järvinen, Jukka-Pekka Mecklin, Ralf Bützow, Päivi Peltomäki. Histology-specific patterns of DNA methylation in Lynch-associated and sporadic ovarian cancer. [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 2964. doi:10.1158/1538-7445.AM2015-2964