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.

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.

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.

Although ovarian cancer is the most lethal of gynecologic malignancies, wide variation in outcome following conventional therapy continues to exist. The presence of tumor-infiltrating regulatory T cells (Tregs) has a role in outcome of this disease, and a growing body of data supports the existence of inherited prognostic factors. However, the role of inherited variants in genes encoding Treg-related immune molecules has not been fully explored. We analyzed expression quantitative trait loci (eQTL) and sequence-based tagging single nucleotide polymorphisms (tagSNPs) for 54 genes associated with Tregs in 3,662 invasive ovarian cancer cases. With adjustment for known prognostic factors, suggestive results were observed among rarer histological subtypes; poorer survival was associated with minor alleles at SNPs in RGS1 (clear cell, rs10921202, p=2.7×10(-5)), LRRC32 and TNFRSF18/TNFRSF4 (mucinous, rs3781699, p=4.5×10(-4), and rs3753348, p=9.0×10(-4), respectively), and CD80 (endometrioid, rs13071247, p=8.0×10(-4)). Fo0r the latter, correlative data support a CD80 rs13071247 genotype association with CD80 tumor RNA expression (p=0.006). An additional eQTL SNP in CD80 was associated with shorter survival (rs7804190, p=8.1×10(-4)) among all cases combined. As the products of these genes are known to affect induction, trafficking, or immunosuppressive function of Tregs, these results suggest the need for follow-up phenotypic studies.

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.

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.

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.

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.

HNF1B is overexpressed in clear cell epithelial ovarian cancer, and we observed epigenetic silencing in serous epithelial ovarian cancer, leading us to hypothesize that variation in this gene differentially associates with epithelial ovarian cancer risk according to histological subtype. Here we comprehensively map variation in HNF1B with respect to epithelial ovarian cancer risk and analyse DNA methylation and expression profiles across histological subtypes. Different single-nucleotide polymorphisms associate with invasive serous (rs7405776 odds ratio (OR)=1.13, P=3.1 × 10(-10)) and clear cell (rs11651755 OR=0.77, P=1.6 × 10(-8)) epithelial ovarian cancer. Risk alleles for the serous subtype associate with higher HNF1B-promoter methylation in these tumours. Unmethylated, expressed HNF1B, primarily present in clear cell tumours, coincides with a CpG island methylator phenotype affecting numerous other promoters throughout the genome. Different variants in HNF1B associate with risk of serous and clear cell epithelial ovarian cancer; DNA methylation and expression patterns are also notably distinct between these subtypes. These findings underscore distinct mechanisms driving different epithelial ovarian cancer histological subtypes.

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.