Exome Sequencing Identifies Carriers of the Autosomal Dominant Cancer Predisposition Disorders Beyond Current Practice Guideline Recommendations.

PURPOSE: The autosomal dominant cancer predisposition disorders hereditary breast and ovarian cancer (HBOC) and Lynch syndrome (LS) are genetic conditions for which early identification and intervention have a positive effect on the individual and public health. The goals of this study were to determine whether germline genetic screening using exome sequencing could be used to efficiently identify carriers of HBOC and LS. METHODS: Participants were recruited from three geographically and racially diverse sites in the United States (Rochester, MN; Phoenix, AZ; Jacksonville, FL). Participants underwent Exome+ sequencing (Helix Inc, San Mateo, CA) and return of results for specific genetic findings: HBOC (BRCA1 and BRCA1) and LS (MLH1, MSH2, MSH6, PMS2, and EPCAM). Chart review was performed to collect demographics and personal and family cancer history. RESULTS: To date, 44,306 participants have enrolled in Tapestry. Annotation and interpretation of all variants in genes for HBOC and LS resulted in the identification of 550 carriers (prevalence, 1.24%), which included 387 with HBOC (27.2% BRCA1, 42.8% BRCA2) and 163 with LS (12.3% MSH6, 8.8% PMS2, 4.5% MLH1, 3.8% MSH2, and 0.2% EPCAM). More than half of these participants (52.1%) were newly diagnosed carriers with HBOC and LS. In all, 39.2% of HBOC/LS carriers did not satisfy National Comprehensive Cancer Network (NCCN) criteria for genetic evaluation. NCCN criteria were less commonly met in underrepresented minority populations versus self-reported White race (51.5% v 37.5%, P = .028). CONCLUSION: Our results emphasize the need for wider utilization of germline genetic sequencing for enhanced screening and detection of individuals who have LS and HBOC cancer predisposition syndromes.

Methylation Signature Implicated in Immuno-Suppressive Activities in Tubo-Ovarian High-Grade Serous Carcinoma.

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.

Molecular Subclasses of Clear Cell Ovarian Carcinoma and Their Impact on Disease Behavior and Outcomes.

PURPOSE: To identify molecular subclasses of clear cell ovarian carcinoma (CCOC) and assess their impact on clinical presentation and outcomes. EXPERIMENTAL DESIGN: We profiled 421 primary CCOCs that passed quality control using a targeted deep sequencing panel of 163 putative CCOC driver genes and whole transcriptome sequencing of 211 of these tumors. Molecularly defined subgroups were identified and tested for association with clinical characteristics and overall survival. RESULTS: We detected a putative somatic driver mutation in at least one candidate gene in 95% (401/421) of CCOC tumors including ARID1A (in 49% of tumors), PIK3CA (49%), TERT (20%), and TP53 (16%). Clustering of cancer driver mutations and RNA expression converged upon two distinct subclasses of CCOC. The first was dominated by ARID1A-mutated tumors with enriched expression of canonical CCOC genes and markers of platinum resistance; the second was largely comprised of tumors with TP53 mutations and enriched for the expression of genes involved in extracellular matrix organization and mesenchymal differentiation. Compared with the ARID1A-mutated group, women with TP53-mutated tumors were more likely to have advanced-stage disease, no antecedent history of endometriosis, and poorer survival, driven by their advanced stage at presentation. In women with ARID1A-mutated tumors, there was a trend toward a lower rate of response to first-line platinum-based therapy. CONCLUSIONS: Our study suggests that CCOC consists of two distinct molecular subclasses with distinct clinical presentation and outcomes, with potential relevance to both traditional and experimental therapy responsiveness. See related commentary by Lheureux, p. 4838.

MCM3 is a novel proliferation marker associated with longer survival for patients with tubo-ovarian high-grade serous carcinoma.

Tubo-ovarian high-grade serous carcinomas (HGSC) are highly proliferative neoplasms that generally respond well to platinum/taxane chemotherapy. We recently identified minichromosome maintenance complex component 3 (MCM3), which is involved in the initiation of DNA replication and proliferation, as a favorable prognostic marker in HGSC. Our objective was to further validate whether MCM3 mRNA expression and possibly MCM3 protein levels are associated with survival in patients with HGSC. MCM3 mRNA expression was measured using NanoString expression profiling on formalin-fixed and paraffin-embedded tissue (N = 2355 HGSC) and MCM3 protein expression was assessed by immunohistochemistry (N = 522 HGSC) and compared with Ki-67. Kaplan-Meier curves and the Cox proportional hazards model were used to estimate associations with survival. Among chemotherapy-naïve HGSC, higher MCM3 mRNA expression (one standard deviation increase in the score) was associated with longer overall survival (HR = 0.87, 95% CI 0.81-0.92, p < 0.0001, N = 1840) in multivariable analysis. MCM3 mRNA expression was highest in the HGSC C5.PRO molecular subtype, although no interaction was observed between MCM3, survival and molecular subtypes. MCM3 and Ki-67 protein levels were significantly lower after exposure to neoadjuvant chemotherapy compared to chemotherapy-naïve tumors: 37.0% versus 46.4% and 22.9% versus 34.2%, respectively. Among chemotherapy-naïve HGSC, high MCM3 protein levels were also associated with significantly longer disease-specific survival (HR = 0.52, 95% CI 0.36-0.74, p = 0.0003, N = 392) compared to cases with low MCM3 protein levels in multivariable analysis. MCM3 immunohistochemistry is a promising surrogate marker of proliferation in HGSC.

DNA Methylation Profiles of Ovarian Clear Cell Carcinoma.

BACKGROUND: Ovarian clear cell carcinoma (OCCC) is a rare ovarian cancer histotype that tends to be resistant to standard platinum-based chemotherapeutics. We sought to better understand the role of DNA methylation in clinical and biological subclassification of OCCC. METHODS: We interrogated genome-wide methylation using DNA from fresh frozen tumors from 271 cases, applied nonsmooth nonnegative matrix factorization (nsNMF) clustering, and evaluated clinical associations and biological pathways. RESULTS: Two approximately equally sized clusters that associated with several clinical features were identified. Compared with Cluster 2 (N = 137), Cluster 1 cases (N = 134) presented at a more advanced stage, were less likely to be of Asian ancestry, and tended to have poorer outcomes including macroscopic residual disease following primary debulking surgery (P < 0.10). Subset analyses of targeted tumor sequencing and IHC data revealed that Cluster 1 tumors showed TP53 mutation and abnormal p53 expression, and Cluster 2 tumors showed aneuploidy and ARID1A/PIK3CA mutation (P < 0.05). Cluster-defining CpGs included 1,388 CpGs residing within 200 bp of the transcription start sites of 977 genes; 38% of these genes (N = 369 genes) were differentially expressed across cluster in transcriptomic subset analysis (P < 10-4). Differentially expressed genes were enriched for six immune-related pathways, including IFNα and IFNγ responses (P < 10-6). CONCLUSIONS: DNA methylation clusters in OCCC correlate with disease features and gene expression patterns among immune pathways. IMPACT: This work serves as a foundation for integrative analyses that better understand the complex biology of OCCC in an effort to improve potential for development of targeted therapeutics.

Identification of a Locus Near ULK1 Associated With Progression-Free Survival in Ovarian Cancer.

BACKGROUND: Many loci have been found to be associated with risk of epithelial ovarian cancer (EOC). However, although there is considerable variation in progression-free survival (PFS), no loci have been found to be associated with outcome at genome-wide levels of significance. METHODS: We carried out a genome-wide association study (GWAS) of PFS in 2,352 women with EOC who had undergone cytoreductive surgery and standard carboplatin/paclitaxel chemotherapy. RESULTS: We found seven SNPs at 12q24.33 associated with PFS (P < 5 × 10-8), the top SNP being rs10794418 (HR = 1.24; 95% CI, 1.15-1.34; P = 1.47 × 10-8). High expression of a nearby gene, ULK1, is associated with shorter PFS in EOC, and with poor prognosis in other cancers. SNP rs10794418 is also associated with expression of ULK1 in ovarian tumors, with the allele associated with shorter PFS being associated with higher expression, and chromatin interactions were detected between the ULK1 promoter and associated SNPs in serous and endometrioid EOC cell lines. ULK1 knockout ovarian cancer cell lines showed significantly increased sensitivity to carboplatin in vitro. CONCLUSIONS: The locus at 12q24.33 represents one of the first genome-wide significant loci for survival for any cancer. ULK1 is a plausible candidate for the target of this association. IMPACT: This finding provides insight into genetic markers associated with EOC outcome and potential treatment options.See related commentary by Peres and Monteiro, p. 1604.

Molecular signatures of X chromosome inactivation and associations with clinical outcomes in epithelial ovarian cancer.

X chromosome inactivation (XCI) is a key epigenetic gene expression regulatory process, which may play a role in women's cancer. In particular tissues, some genes are known to escape XCI, yet patterns of XCI in ovarian cancer (OC) and their clinical associations are largely unknown. To examine XCI in OC, we integrated germline genotype with tumor copy number, gene expression and DNA methylation information from 99 OC patients. Approximately 10% of genes showed different XCI status (either escaping or being subject to XCI) compared with the studies of other tissues. Many of these genes are known oncogenes or tumor suppressors (e.g. DDX3X, TRAPPC2 and TCEANC). We also observed strong association between cis promoter DNA methylation and allele-specific expression imbalance (P = 2.0 × 10-10). Cluster analyses of the integrated data identified two molecular subgroups of OC patients representing those with regulated (N = 47) and dysregulated (N = 52) XCI. This XCI cluster membership was associated with expression of X inactive specific transcript (P = 0.002), a known driver of XCI, as well as age, grade, stage, tumor histology and extent of residual disease following surgical debulking. Patients with dysregulated XCI (N = 52) had shorter time to recurrence (HR = 2.34, P = 0.001) and overall survival time (HR = 1.87, P = 0.02) than those with regulated XCI, although results were attenuated after covariate adjustment. Similar findings were observed when restricted to high-grade serous tumors. We found evidence of a unique OC XCI profile, suggesting that XCI may play an important role in OC biology. Additional studies to examine somatic changes with paired tumor-normal tissue are needed.

Association of p16 expression with prognosis varies across ovarian carcinoma histotypes: an Ovarian Tumor Tissue Analysis consortium study.

We aimed to validate the prognostic association of p16 expression in ovarian high-grade serous carcinomas (HGSC) and to explore it in other ovarian carcinoma histotypes. p16 protein expression was assessed by clinical-grade immunohistochemistry in 6525 ovarian carcinomas including 4334 HGSC using tissue microarrays from 24 studies participating in the Ovarian Tumor Tissue Analysis consortium. p16 expression patterns were interpreted as abnormal (either overexpression referred to as block expression or absence) or normal (heterogeneous). CDKN2A (which encodes p16) mRNA expression was also analyzed in a subset (n = 2280) mostly representing HGSC (n = 2010). Association of p16 expression with overall survival (OS) was determined within histotypes as was CDKN2A expression for HGSC only. p16 block expression was most frequent in HGSC (56%) but neither protein nor mRNA expression was associated with OS. However, relative to heterogeneous expression, block expression was associated with shorter OS in endometriosis-associated carcinomas, clear cell [hazard ratio (HR): 2.02, 95% confidence (CI) 1.47-2.77, p < 0.001] and endometrioid (HR: 1.88, 95% CI 1.30-2.75, p = 0.004), while absence was associated with shorter OS in low-grade serous carcinomas (HR: 2.95, 95% CI 1.61-5.38, p = 0.001). Absence was most frequent in mucinous carcinoma (50%), and was not associated with OS in this histotype. The prognostic value of p16 expression is histotype-specific and pattern dependent. We provide definitive evidence against an association of p16 expression with survival in ovarian HGSC as previously suggested. Block expression of p16 in clear cell and endometrioid carcinoma should be further validated as a prognostic marker, and absence in low-grade serous carcinoma justifies CDK4 inhibition.

Genomic Analysis Using Regularized Regression in High-Grade Serous Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is a complex disease in which initiation and progression have been associated with copy number alterations, epigenetic processes, and, to a lesser extent, germline variation. We hypothesized that, when summarized at the gene level, tumor methylation and germline genetic variation, alone or in combination, influence tumor gene expression in HGSOC. We used Elastic Net (ENET) penalized regression method to evaluate these associations and adjust for somatic copy number in 3 independent data sets comprising tumors from more than 470 patients. Penalized regression models of germline variation, with or without methylation, did not reveal a role in HGSOC gene expression. However, we observed significant association between regional methylation and expression of 5 genes (WDPCP, KRT6C, BRCA2, EFCAB13, and ZNF283). CpGs retained in ENET model for BRCA2 and ZNF283 appeared enriched in several regulatory elements, suggesting that regularized regression may provide a novel utility for integrative genomic analysis.

Pooled Clustering of High-Grade Serous Ovarian Cancer Gene Expression Leads to Novel Consensus Subtypes Associated with Survival and Surgical Outcomes.

Purpose: Here we assess whether molecular subtyping identifies biological features of tumors that correlate with survival and surgical outcomes of high-grade serous ovarian cancer (HGSOC).Experimental Design: Consensus clustering of pooled mRNA expression data from over 2,000 HGSOC cases was used to define molecular subtypes of HGSOCs. This de novo classification scheme was then applied to 381 Mayo Clinic HGSOC patients with detailed survival and surgical outcome information.Results: Five molecular subtypes of HGSOC were identified. In the pooled dataset, three subtypes were largely concordant with prior studies describing proliferative, mesenchymal, and immunoreactive tumors (concordance > 70%), and the group of tumors previously described as differentiated type was segregated into two new types, one of which (anti-mesenchymal) had downregulation of genes that were typically upregulated in the mesenchymal subtype. Molecular subtypes were significantly associated with overall survival (P < 0.001) and with rate of optimal surgical debulking (≤1 cm, P = 1.9E-4) in the pooled dataset. Among stage III-C or IV Mayo Clinic patients, molecular subtypes were also significantly associated with overall survival (P = 0.001), as well as rate of complete surgical debulking (no residual disease; 16% in mesenchymal tumors compared with >28% in other subtypes; P = 0.02).Conclusions: HGSOC tumors may be categorized into five molecular subtypes that associate with overall survival and the extent of residual disease following debulking surgery. Because mesenchymal tumors may have features that were associated with less favorable surgical outcome, molecular subtyping may have future utility in guiding neoadjuvant treatment decisions for women with HGSOC. Clin Cancer Res; 23(15); 4077-85. ©2017 AACR.

Assessing health disparities in children using a modified housing-related socioeconomic status measure: a cross-sectional study.

OBJECTIVES: Socioeconomic status (SES) is a well-established risk factor for many health outcomes. Recently, we developed an SES measure based on 4 housing-related characteristics (termed HOUSES) and demonstrated its ability to assess health disparities. In this study, we aimed to evaluate whether fewer housing-related characteristics could be used to provide a similar representation of SES. STUDY SETTING AND PARTICIPANTS: We performed a cross-sectional study using parents/guardians of children aged 1-17 years from 2 US Midwestern counties (n=728 in Olmsted County, Minnesota, and n=701 in Jackson County, Missouri). PRIMARY AND SECONDARY OUTCOME MEASURES: For each participant, housing-related characteristics used in the formulation of HOUSES (assessed housing value, square footage, number of bedrooms and number of bathrooms) were obtained from the local government assessor's offices, and additional SES measures and health outcomes with known associations to SES (obesity, low birth weight and smoking exposure) were collected from a telephone survey. Housing characteristics with the greatest contribution for predicting the health outcomes were added to formulate a modified HOUSES index. RESULTS: Among the 4 housing characteristics used in the original HOUSES, the strongest contributions for predicting health outcomes were observed from assessed housing value and square footage (combined contribution ranged between 89% and 96%). Based on this observation, these 2 were used to calculate a modified HOUSES index. Correlation between modified HOUSES and other SES measures was comparable to the original HOUSES for both locations. Consistent with the original HOUSES formula, the strongest association with modified HOUSES was observed with smoking exposure (OR=0.24 with 95% CI 0.11 to 0.49 for comparing participants in highest HOUSES vs lowest group; overall p<0.001). CONCLUSIONS: The modified HOUSES requires only 2 readily available housing characteristics thereby improving the feasibility of using this index as a proxy for SES in multiple communities, especially in the US Midwestern region.

Expression signature distinguishing two tumour transcriptome classes associated with progression-free survival among rare histological types of epithelial ovarian cancer.

BACKGROUND: The mechanisms of recurrence have been under-studied in rare histologies of invasive epithelial ovarian cancer (EOC) (endometrioid, clear cell, mucinous, and low-grade serous). We hypothesised the existence of an expression signature predictive of outcome in the rarer histologies. METHODS: In split discovery and validation analysis of 131 Mayo Clinic EOC cases, we used clustering to determine clinically relevant transcriptome classes using microarray gene expression measurements. The signature was validated in 967 EOC tumours (91 rare histological subtypes) with recurrence information. RESULTS: We found two validated transcriptome classes associated with progression-free survival (PFS) in the Mayo Clinic EOC cases (P=8.24 × 10(-3)). This signature was further validated in the public expression data sets involving the rare EOC histologies, where these two classes were also predictive of PFS (P=1.43 × 10(-3)). In contrast, the signatures were not predictive of PFS in the high-grade serous EOC cases. Moreover, genes upregulated in Class-1 (with better outcome) were showed enrichment in steroid hormone biosynthesis (false discovery rate, FDR=0.005%) and WNT signalling pathway (FDR=1.46%); genes upregulated in Class-2 were enriched in cell cycle (FDR=0.86%) and toll-like receptor pathways (FDR=2.37%). CONCLUSIONS: These findings provide important biological insights into the rarer EOC histologies that may aid in the development of targeted treatment options for the rarer histologies.

Leveraging an Electronic Health Record-Linked Biorepository to Generate a Metformin Pharmacogenomics Hypothesis.

Metformin is a first-line antihyperglycemic agent commonly prescribed in type 2 diabetes mellitus (T2DM), but whose pharmacogenomics are not clearly understood. Further, due to accumulating evidence highlighting the potential for metformin in cancer prevention and treatment efforts it is imperative to understand molecular mechanisms of metformin. In this electronic health record(EHR)-based study we explore the potential association of the flavin-containing monooxygenase(FMO)-5 gene, a biologically plausible biotransformer of metformin, and modifying glycemic response to metformin treatment. Using a cohort of 258 T2DM patients who had new metformin exposure, existing genetic data, and longitudinal electronic health records, we compared genetic variation within FMO5 to change in glycemic response. Gene-level and SNP-level analysis identified marginally significant associations for FMO5 variation, representing an EHR-driven pharmacogenetics hypothesis for a potential novel mechanism for metformin biotransformation. However, functional validation of this EHR-based hypothesis is necessary to ascertain its clinical and biological significance.

Using EHR-Linked Biobank Data to Study Metformin Pharmacogenomics.

Metformin is a commonly prescribed diabetes medication whose mechanism of action is poorly understood. In this study we utilized EHR-linked biobank data to elucidate the impact of genomic variation on glycemic response to metformin. Our study found significant gene- and SNP-level associations within the beta-2 subunit of the heterotrimeric adenosine monophosphate-activated protein kinase complex. Using EHR phenotypes where were able to add additional clarity to ongoing metformin pharmacogenomic dialogue.

Genome-wide investigation of regional blood-based DNA methylation adjusted for complete blood counts implicates BNC2 in ovarian cancer.

Due to its potential as a biomarker for early cancer detection, blood-based DNA methylation (DNAm) is of interest in cancer research. Specifically, highly predictive mechanisms for early detection of epithelial ovarian cancer (EOC) are desired, so previous studies have compared DNAm between EOC cases and controls. However, case-control studies are confounded by the distribution of white blood cell types through an immune response induced by the cancer. Rather than determining the distribution of the cell types manually or investigating isolated cell types, an alternative approach involves the use of complete blood count (CBC), which is routinely collected. In the analysis of an EOC case-control study of DNAm, we incorporate CBC measures to adjust for this confounding and compare DNAm between 242 EOC cases and 181 age-matched controls (assayed on the Illumina Infinium HumanMethylation27 or HumanMethylation450 Beadchips), at both the individual CpG and CpG island levels. We found that adjustment for leukocyte distribution using CBC measurements dramatically reduced confounding, with 62 single CpG sites found to be associated with EOC status after adjustment (P < 5E-8). Additionally, regional DNAm was assessed by applying principal components analysis to CpG islands. The top associated CpG island (P = 7E-6) was located in the promoter/transcription start site of the human basonuclin 2 gene (BNC2), a known susceptibility gene for EOC risk identified through GWAS. Follow-up studies are necessary to establish the role of BNC2 in blood-based DNA and EOC, including prospective studies to validate this region as a potential biomarker and predictor of EOC susceptibility.

Methylation of leukocyte DNA and ovarian cancer: relationships with disease status and outcome.

BACKGROUND: Genome-wide interrogation of DNA methylation (DNAm) in blood-derived leukocytes has become feasible with the advent of CpG genotyping arrays. In epithelial ovarian cancer (EOC), one report found substantial DNAm differences between cases and controls; however, many of these disease-associated CpGs were attributed to differences in white blood cell type distributions. METHODS: We examined blood-based DNAm in 336 EOC cases and 398 controls; we included only high-quality CpG loci that did not show evidence of association with white blood cell type distributions to evaluate association with case status and overall survival. RESULTS: Of 13,816 CpGs, no significant associations were observed with survival, although eight CpGs associated with survival at p < 10-3, including methylation within a CpG island located in the promoter region of GABRE (p = 5.38 x 10-5, HR = 0.95). In contrast, 53 CpG methylation sites were significantly associated with EOC risk (p <5 x10-6). The top association was observed for the methylation probe cg04834572 located approximately 315 kb upstream of DUSP13 (p = 1.6 x10-14). Other disease-associated CpGs included those near or within HHIP (cg14580567; p =5.6x10-11), HDAC3 (cg10414058; p = 6.3x10-12), and SCR (cg05498681; p = 4.8x10-7). CONCLUSIONS: We have identified several CpGs in leukocytes that are differentially methylated by case-control status. Since a retrospective study design was used, we cannot differentiate whether DNAm was etiologic or resulting from EOC; thus, prospective studies of EOC-associated loci are the critical next step.

Tumor hypomethylation at 6p21.3 associates with longer time to recurrence of high-grade serous epithelial ovarian cancer.

To reveal biologic mechanisms underlying clinical outcome of high-grade serous (HGS) epithelial ovarian carcinomas (EOC), we evaluated the association between tumor epigenetic changes and time to recurrence (TTR). We assessed methylation at approximately 450,000 genome-wide CpGs in tumors of 337 Mayo Clinic (Rochester, MN) patients. Semi-supervised clustering of discovery (n=168) and validation (n=169) sets was used to determine clinically relevant methylation classes. Clustering identified two methylation classes based on 60 informative CpGs, which differed in TTR in the validation set [R vs. L class, P=2.9×10(-3), HR=0.52; 95% confidence interval (CI), 0.34-0.80]. Follow-up analyses considered genome-wide tumor mRNA expression (n=104) and CD8 T-cell infiltration (n=89) in patient subsets. Hypomethylation of CpGs located in 6p21.3 in the R class associated with cis upregulation of genes enriched in immune response processes (TAP1, PSMB8, PSMB9, HLA-DQB1, HLA-DQB2, HLA-DMA, and HLA-DOA), increased CD8 T-cell tumor infiltration (P=7.6×10(-5)), and trans-regulation of genes in immune-related pathways (P=1.6×10(-32)). This is the most comprehensive assessment of clinical outcomes with regard to epithelial ovarian carcinoma tumor methylation to date. Collectively, these results suggest that an epigenetically mediated immune response is a predictor of recurrence and, possibly, treatment response for HGS EOC.

Epigenome-wide ovarian cancer analysis identifies a methylation profile differentiating clear-cell histology with epigenetic silencing of the HERG K+ channel.

Ovarian cancer remains the leading cause of death in women with gynecologic malignancies, despite surgical advances and the development of more effective chemotherapeutics. As increasing evidence indicates that clear-cell ovarian cancer may have unique pathogenesis, further understanding of molecular features may enable us to begin to understand the underlying biology and histology-specific information for improved outcomes. To study epigenetics in clear-cell ovarian cancer, fresh frozen tumor DNA (n = 485) was assayed on Illumina Infinium HumanMethylation450 BeadChips. We identified a clear-cell ovarian cancer tumor methylation profile (n = 163) which we validated in two independent replication sets (set 1, n = 163; set 2, n = 159), highlighting 22 CpG loci associated with nine genes (VWA1, FOXP1, FGFRL1, LINC00340, KCNH2, ANK1, ATXN2, NDRG21 and SLC16A11). Nearly all of the differentially methylated CpGs showed a propensity toward hypermethylation among clear-cell cases. Several loci methylation inversely correlated with tumor gene expression, most notably KCNH2 (HERG, a potassium channel) (P = 9.5 × 10(-7)), indicating epigenetic silencing. In addition, a predicted methylation class mainly represented by the clear-cell cases (20 clear cell out of 23 cases) had improved survival time. Although these analyses included only 30 clear-cell carcinomas, results suggest that loss of expression of KCNH2 (HERG) by methylation could be a good prognostic marker, given that overexpression of the potassium (K(+)) channel Eag family members promotes increased proliferation and results in poor prognosis. Validation in a bigger cohort of clear-cell tumors of the ovary is warranted.

Identification and molecular characterization of a new ovarian cancer susceptibility locus at 17q21.31.

Epithelial ovarian cancer (EOC) has a heritable component that remains to be fully characterized. Most identified common susceptibility variants lie in non-protein-coding sequences. We hypothesized that variants in the 3' untranslated region at putative microRNA (miRNA)-binding sites represent functional targets that influence EOC susceptibility. Here, we evaluate the association between 767 miRNA-related single-nucleotide polymorphisms (miRSNPs) and EOC risk in 18,174 EOC cases and 26,134 controls from 43 studies genotyped through the Collaborative Oncological Gene-environment Study. We identify several miRSNPs associated with invasive serous EOC risk (odds ratio=1.12, P=10(-8)) mapping to an inversion polymorphism at 17q21.31. Additional genotyping of non-miRSNPs at 17q21.31 reveals stronger signals outside the inversion (P=10(-10)). Variation at 17q21.31 is associated with neurological diseases, and our collaboration is the first to report an association with EOC susceptibility. An integrated molecular analysis in this region provides evidence for ARHGAP27 and PLEKHM1 as candidate EOC susceptibility genes.

GWAS meta-analysis and replication identifies three new susceptibility loci for ovarian cancer.

Genome-wide association studies (GWAS) have identified four susceptibility loci for epithelial ovarian cancer (EOC), with another two suggestive loci reaching near genome-wide significance. We pooled data from a GWAS conducted in North America with another GWAS from the UK. We selected the top 24,551 SNPs for inclusion on the iCOGS custom genotyping array. We performed follow-up genotyping in 18,174 individuals with EOC (cases) and 26,134 controls from 43 studies from the Ovarian Cancer Association Consortium. We validated the two loci at 3q25 and 17q21 that were previously found to have associations close to genome-wide significance and identified three loci newly associated with risk: two loci associated with all EOC subtypes at 8q21 (rs11782652, P = 5.5 × 10(-9)) and 10p12 (rs1243180, P = 1.8 × 10(-8)) and another locus specific to the serous subtype at 17q12 (rs757210, P = 8.1 × 10(-10)). An integrated molecular analysis of genes and regulatory regions at these loci provided evidence for functional mechanisms underlying susceptibility and implicated CHMP4C in the pathogenesis of ovarian cancer.