Talhouk, Aline

Abstract Background: Ovarian high-grade serous carcinomas (HGSC) comprise four distinct molecular subtypes based on mRNA expression patterns, with differential survival. Understanding risk factor associations is important to elucidate the etiology of HGSC. We investigated associations between different epidemiologic risk factors and HGSC molecular subtypes. Methods: We pooled data from 11 case–control studies with epidemiologic and tumor gene expression data from custom NanoString CodeSets developed through a collaboration within the Ovarian Tumor Tissue Analysis consortium. The PrOTYPE-validated NanoString-based 55-gene classifier was used to assign HGSC gene expression subtypes. We examined associations between epidemiologic factors and HGSC subtypes in 2,070 cases and 16,633 controls using multivariable-adjusted polytomous regression models. Results: Among the 2,070 HGSC cases, 556 (27%) were classified as C1.MES, 340 (16%) as C5.PRO, 538 (26%) as C2.IMM, and 636 (31%) as C4.DIF. The key factors, including oral contraceptive use, parity, breastfeeding, and family history of ovarian cancer, were similarly associated with all subtypes. Heterogeneity was observed for several factors. Former smoking [OR = 1.25; 95% confidence interval (CI) = 1.03, 1.51] and genital powder use (OR = 1.42; 95% CI = 1.08, 1.86) were uniquely associated with C2.IMM. History of endometriosis was associated with C5.PRO (OR = 1.46; 95% CI = 0.98, 2.16) and C4.DIF (OR = 1.27; 95% CI = 0.94, 1.71) only. Family history of breast cancer (OR = 1.44; 95% CI = 1.16, 1.78) and current smoking (OR = 1.40; 95% CI = 1.11, 1.76) were associated with C4.DIF only. Conclusions: This study observed heterogeneous associations of epidemiologic and modifiable factors with HGSC molecular subtypes. Impact: The different patterns of associations may provide key information about the etiology of the four subtypes.

Abstract Purpose: The purpose of this study was to evaluate RB1 expression and survival across ovarian carcinoma histotypes and how co-occurrence of BRCA1 or BRCA2 (BRCA) alterations and RB1 loss influences survival in tubo-ovarian high-grade serous carcinoma (HGSC). Experimental Design: RB1 protein expression was classified by immunohistochemistry in ovarian carcinomas of 7,436 patients from the Ovarian Tumor Tissue Analysis consortium. We examined RB1 expression and germline BRCA status in a subset of 1,134 HGSC, and related genotype to overall survival (OS), tumor-infiltrating CD8+ lymphocytes, and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cells with and without BRCA1 alterations to model co-loss with treatment response. We performed whole-genome and transcriptome data analyses on 126 patients with primary HGSC to characterize tumors with concurrent BRCA deficiency and RB1 loss. Results: RB1 loss was associated with longer OS in HGSC but with poorer prognosis in endometrioid ovarian carcinoma. Patients with HGSC harboring both RB1 loss and pathogenic germline BRCA variants had superior OS compared with patients with either alteration alone, and their median OS was three times longer than those without pathogenic BRCA variants and retained RB1 expression (9.3 vs. 3.1 years). Enhanced sensitivity to cisplatin and paclitaxel was seen in BRCA1-altered cells with RB1 knockout. Combined RB1 loss and BRCA deficiency correlated with transcriptional markers of enhanced IFN response, cell-cycle deregulation, and reduced epithelial–mesenchymal transition. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1. Conclusions: Co-occurrence of RB1 loss and BRCA deficiency was associated with exceptionally long survival in patients with HGSC, potentially due to better treatment response and immune stimulation.

Abstract Purpose: Endometrioid ovarian carcinoma (ENOC) is the second most-common type of ovarian carcinoma, comprising 10%–20% of cases. Recently, the study of ENOC has benefitted from comparisons to endometrial carcinomas including defining ENOC with four prognostic molecular subtypes. Each subtype suggests differential mechanisms of progression, although tumor-initiating events remain elusive. There is evidence that the ovarian microenvironment may be critical to early lesion establishment and progression. However, while immune infiltrates have been well studied in high-grade serous ovarian carcinoma, studies in ENOC are limited. Experimental Design: We report on 210 ENOC, with clinical follow-up and molecular subtype annotation. Using multiplex IHC and immunofluorescence, we examine the prevalence of T-cell lineage, B-cell lineage, macrophages, and populations with programmed cell death protein 1 or programmed death-ligand 1 across subtypes of ENOC. Results: Immune cell infiltrates in tumor epithelium and stroma showed higher densities in ENOC subtypes with known high mutation burden (POLEmut and MMRd). While molecular subtypes were prognostically significant, immune infiltrates were not (overall survival P > 0.2). Analysis by molecular subtype revealed that immune cell density was prognostically significant in only the no specific molecular profile (NSMP) subtype, where immune infiltrates lacking B cells (TILB minus) had inferior outcome (disease-specific survival: HR, 4.0; 95% confidence interval, 1.1–14.7; P < 0.05). Similar to endometrial carcinomas, molecular subtype stratification was generally superior to immune response in predicting outcomes. Conclusions: Subtype stratification is critical for better understanding of ENOC, in particular the distribution and prognostic significance of immune cell infiltrates. The role of B cells in the immune response within NSMP tumors warrants further study.

Abstract Background: Better understanding of prognostic factors in tubo-ovarian high-grade serous carcinoma (HGSC) is critical, as diagnosis confers an aggressive disease course. Variation in tumor DNA methylation shows promise predicting outcome, yet prior studies were largely platform-specific and unable to evaluate multiple molecular features. Methods: We analyzed genome-wide DNA methylation in 1,040 frozen HGSC, including 325 previously reported upon, seeking a multi-platform quantitative methylation signature that we evaluated in relation to clinical features, tumor characteristics, time to recurrence/death, extent of CD8+ tumor-infiltrating lymphocytes (TIL), gene expression molecular subtypes, and gene expression of the ATP-binding cassette transporter TAP1. Results: Methylation signature was associated with shorter time to recurrence, independent of clinical factors (N = 715 new set, hazard ratio (HR), 1.65; 95% confidence interval (CI), 1.10–2.46; P = 0.015; N = 325 published set HR, 2.87; 95% CI, 2.17–3.81; P = 2.2 × 10−13) and remained prognostic after adjustment for gene expression molecular subtype and TAP1 expression (N = 599; HR, 2.22; 95% CI, 1.66–2.95; P = 4.1 × 10−8). Methylation signature was inversely related to CD8+ TIL levels (P = 2.4 × 10−7) and TAP1 expression (P = 0.0011) and was associated with gene expression molecular subtype (P = 5.9 × 10−4) in covariate-adjusted analysis. Conclusions: Multi-center analysis identified a novel quantitative tumor methylation signature of HGSC applicable to numerous commercially available platforms indicative of shorter time to recurrence/death, adjusting for other factors. Along with immune cell composition analysis, these results suggest a role for DNA methylation in the immunosuppressive microenvironment. Impact: This work aids in identification of targetable epigenome processes and stratification of patients for whom tailored treatment may be most beneficial.

AbstractBackgroundCyclin E1 (CCNE1) is a potential predictive marker and therapeutic target in tubo‐ovarian high‐grade serous carcinoma (HGSC). Smaller studies have revealed unfavorable associations for CCNE1 amplification and CCNE1 overexpression with survival, but to date no large‐scale, histotype‐specific validation has been performed. The hypothesis was that high‐level amplification of CCNE1 and CCNE1 overexpression, as well as a combination of the two, are linked to shorter overall survival in HGSC.MethodsWithin the Ovarian Tumor Tissue Analysis consortium, amplification status and protein level in 3029 HGSC cases and mRNA expression in 2419 samples were investigated.ResultsHigh‐level amplification (>8 copies by chromogenic in situ hybridization) was found in 8.6% of HGSC and overexpression (>60% with at least 5% demonstrating strong intensity by immunohistochemistry) was found in 22.4%. CCNE1 high‐level amplification and overexpression both were linked to shorter overall survival in multivariate survival analysis adjusted for age and stage, with hazard stratification by study (hazard ratio [HR], 1.26; 95% CI, 1.08‐1.47, p = .034, and HR, 1.18; 95% CI, 1.05‐1.32, p = .015, respectively). This was also true for cases with combined high‐level amplification/overexpression (HR, 1.26; 95% CI, 1.09‐1.47, p = .033). CCNE1 mRNA expression was not associated with overall survival (HR, 1.00 per 1‐SD increase; 95% CI, 0.94‐1.06; p = .58). CCNE1 high‐level amplification is mutually exclusive with the presence of germline BRCA1/2 pathogenic variants and shows an inverse association to RB1 loss.ConclusionThis study provides large‐scale validation that CCNE1 high‐level amplification is associated with shorter survival, supporting its utility as a prognostic biomarker in HGSC.