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Background: BRCA1 methylation has been associated with homologous recombination deficiency, a biomarker of platinum sensitivity. Studies evaluating BRCA1-methylated tubal and ovarian cancer (OC) do not consistently support improved survival following platinum chemotherapy. We examine the characteristics of BRCA1-methylated OC in a meta-analysis of individual participant data., Methods: Data of 2636 participants across 15 studies were analyzed. BRCA1-methylated tumors were defined according to their original study. Associations between BRCA1 methylation and clinicopathological characteristics were evaluated. The effects of methylation on overall survival (OS) and progression-free survival (PFS) were examined using mixed-effects models. All statistical tests were 2-sided., Results: 430 (16.3%) tumors were BRCA1-methylated. BRCA1 methylation was associated with younger age and advanced-stage, high-grade serous OC. There were no survival differences between BRCA1-methylated and non-BRCA1-methylated OC (median PFS = 20.0 vs 18.5 months, hazard ratio [HR] = 1.01, 95% CI = 0.87 to 1.16; P = .98; median OS = 46.6 vs 48.0 months, HR = 1.02, 95% CI = 0.87 to 1.18; P = .96). Where BRCA1/2 mutations were evaluated (n = 1248), BRCA1 methylation displayed no survival advantage over BRCA1/2-intact (BRCA1/2 wild-type non-BRCA1-methylated) OC. Studies used different methods to define BRCA1 methylation. Where BRCA1 methylation was determined using methylation-specific polymerase chain reaction and gel electrophoresis (n = 834), it was associated with improved survival (PFS: HR = 0.80, 95% CI = 0.66 to 0.97; P = .02; OS: HR = 0.80, 95% CI = 0.63 to 1.00; P = .05) on mixed-effects modeling., Conclusion: BRCA1-methylated OC displays similar clinicopathological features to BRCA1-mutated OC but is not associated with survival. Heterogeneity within BRCA1 methylation assays influences associations. Refining these assays may better identify cases with silenced BRCA1 function and improved patient outcomes., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: DNA repair pathways are potential targets of molecular therapy in cancer patients. The FANCD2, BRIP1, BRCA1/2, and FANCF genes are involved in homologous recombination DNA repair, which implicates their possible role in cell response to DNA-damaging agents. We evaluated a clinical significance of pre-treatment expression of these genes at mRNA level in 99 primary, advanced-stage ovarian carcinomas from patients, who later received taxane-platinum (TP) or platinum-cyclophosphamide (PC) treatment., Methods: Gene expression was determined with the use of Real-Time PCR. The BRCA2 and BRIP1 gene sequence was investigated with the use of SSCP, dHPLC, and PCR-sequencing., Results: Increased FANCD2 expression occurred to be a negative prognostic factor for all patients (PC+TP:HR 3.85, p = 0.0003 for the risk of recurrence; HR 1.96, p = 0.02 for the risk of death), and this association was even stronger in the TP-treated group (HR 6.7, p = 0.0002 and HR 2.33, p = 0.01, respectively). Elevated BRIP1 expression was the only unfavorable molecular factor in the PC-treated patients (HR 8.37, p = 0.02 for the risk of recurrence). Additionally, an increased FANCD2 and BRCA1/2 expression levels were associated with poor ovarian cancer outcome in either TP53-positive or -negative subgroups of the TP-treated patients, however these groups were small. Sequence analysis identified one protein truncating variant (1/99) in BRCA2 and no mutations (0/56) in BRIP1., Conclusions: Our study shows for the first time that FANCD2 overexpression is a strong negative prognostic factor in ovarian cancer, particularly in patients treated with TP regimen. Moreover, increased mRNA level of the BRIP1 is a negative prognostic factor in the PC-treated patients. Next, changes in the BRCA2 and BRIP1 genes are rare and together with other analyzed FA genes considered as homologous recombination deficiency may not affect the expression level of analyzed genes.

Large-scale genome-wide association studies (GWAS) have identified approximately 35 loci associated with epithelial ovarian cancer (EOC) risk. The majority of GWAS-identified disease susceptibility variants are located in noncoding regions, and causal genes underlying these associations remain largely unknown. Here, we performed a transcriptome-wide association study to search for novel genetic loci and plausible causal genes at known GWAS loci. We used RNA sequencing data (68 normal ovarian tissue samples from 68 individuals and 6,124 cross-tissue samples from 369 individuals) and high-density genotyping data from European descendants of the Genotype-Tissue Expression (GTEx V6) project to build ovarian and cross-tissue models of genetically regulated expression using elastic net methods. We evaluated 17,121 genes for their cis -predicted gene expression in relation to EOC risk using summary statistics data from GWAS of 97,898 women, including 29,396 EOC cases. With a Bonferroni-corrected significance level of P < 2.2 × 10 -6 , we identified 35 genes, including FZD4 at 11q14.2 (Z = 5.08, P = 3.83 × 10 -7 , the cross-tissue model; 1 Mb away from any GWAS-identified EOC risk variant), a potential novel locus for EOC risk. All other 34 significantly associated genes were located within 1 Mb of known GWAS-identified loci, including 23 genes at 6 loci not previously linked to EOC risk. Upon conditioning on nearby known EOC GWAS-identified variants, the associations for 31 genes disappeared and three genes remained ( P < 1.47 × 10 -3 ). These data identify one novel locus (FZD4 ) and 34 genes at 13 known EOC risk loci associated with EOC risk, providing new insights into EOC carcinogenesis. Significance: Transcriptomic analysis of a large cohort confirms earlier GWAS loci and reveals FZD4 as a novel locus associated with EOC risk. Cancer Res; 78(18); 5419-30. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer mortality in American women. Normal ovarian physiology is intricately connected to small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran) which govern processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. We hypothesized that common germline variation in genes encoding small GTPases is associated with EOC risk. We investigated 322 variants in 88 small GTPase genes in germline DNA of 18,736 EOC patients and 26,138 controls of European ancestry using a custom genotype array and logistic regression fitting log-additive models. Functional annotation was used to identify biofeatures and expression quantitative trait loci that intersect with risk variants. One variant, ARHGEF10L (Rho guanine nucleotide exchange factor 10 like) rs2256787, was associated with increased endometrioid EOC risk (OR = 1.33, p = 4.46 x 10-6). Other variants of interest included another in ARHGEF10L, rs10788679, which was associated with invasive serous EOC risk (OR = 1.07, p = 0.00026) and two variants in AKAP6 (A-kinase anchoring protein 6) which were associated with risk of invasive EOC (rs1955513, OR = 0.90, p = 0.00033; rs927062, OR = 0.94, p = 0.00059). Functional annotation revealed that the two ARHGEF10L variants were located in super-enhancer regions and that AKAP6 rs927062 was associated with expression of GTPase gene ARHGAP5 (Rho GTPase activating protein 5). Inherited variants in ARHGEF10L and AKAP6, with potential transcriptional regulatory function and association with EOC risk, warrant investigation in independent EOC study populations., Competing Interests: The authors have declared that no competing interests exist.

EMSY, a BRCA2-associated protein, is amplified and overexpressed in various sporadic cancers. This is the first study assessing the clinical impact of its expression and polymorphisms on ovarian cancer (OvCa) outcome in the context of the chemotherapy regimen used. In 134 frozen OvCa samples, we assessed EMSY mRNA expression with Reverse Transcription-quantitative PCR, and also investigated the EMSY gene sequence using SSCP and/or PCR-sequencing. Clinical relevance of changes in EMSY mRNA expression and DNA sequence was evaluated in two subgroups treated with either taxane/platinum (TP, n=102) or platinum/cyclophosphamide (PC, n=32). High EMSY expression negatively affected overall survival (OS), disease-free survival (DFS) and sensitivity to treatment (PS) in the TP-treated subgroup (p-values: 0.001, 0.002 and 0.010, respectively). Accordingly, our OvCa cell line studies showed that the EMSY gene knockdown sensitized A2780 and IGROV1 cells to paclitaxel. Interestingly, EMSY mRNA expression in surviving cells was similar as in the control cells. Additionally, we identified 24 sequence alterations in the EMSY gene, including the previously undescribed: c.720G>C, p.(Lys240Asn); c.1860G>A, p.(Lys620Lys); c.246-76A>G; c.421+68A>C. In the PC-treated subgroup, a heterozygous genotype comprising five SNPs (rs4300410, rs3814711, rs4245443, rs2508740, rs2513523) negatively correlated with OS (p-value=0.009). The same SNPs exhibited adverse borderline associations with PS in the TP-treated subgroup. This is the first study providing evidence that high EMSY mRNA expression is a negative prognostic and predictive factor in OvCa patients treated with TP, and that the clinical outcome may hinge on certain SNPs in the EMSY gene as well., Competing Interests: CONFLICTS OF INTEREST The authors have no conflicts of interest to disclose.

Background: Observational studies suggest greater height is associated with increased ovarian cancer risk, but cannot exclude bias and/or confounding as explanations for this. Mendelian randomisation (MR) can provide evidence which may be less prone to bias., Methods: We pooled data from 39 Ovarian Cancer Association Consortium studies (16,395 cases; 23,003 controls). We applied two-stage predictor-substitution MR, using a weighted genetic risk score combining 609 single-nucleotide polymorphisms. Study-specific odds ratios (OR) and 95% confidence intervals (CI) for the association between genetically predicted height and risk were pooled using random-effects meta-analysis., Results: Greater genetically predicted height was associated with increased ovarian cancer risk overall (pooled-OR (pOR) = 1.06; 95% CI: 1.01-1.11 per 5 cm increase in height), and separately for invasive (pOR = 1.06; 95% CI: 1.01-1.11) and borderline (pOR = 1.15; 95% CI: 1.02-1.29) tumours., Conclusions: Women with a genetic propensity to being taller have increased risk of ovarian cancer. This suggests genes influencing height are involved in pathways promoting ovarian carcinogenesis.

Objective: TP53 mutation is the most frequent molecular event in BRCA1-associated ovarian carcinomas. TP53 status may be a confounding factor in the evaluation of clinical importance of other proteins. We aimed to evaluate the clinical significance of BRCA1 mutations with respect to the TP53 accumulation status in 159 high-grade ovarian carcinomas., Methods: Statistical analyses were done with the Kaplan-Meier method, log-rank test, the Cox's and logistic regression models for all patients, and in subgroups with and without TP53 accumulation (TP53+ and TP53-, respectively)., Results: Forty of 159 ovarian carcinomas (25.2%) were diagnosed in patients with BRCA1 germline mutations; 102 tumors (64.2%) were TP53+ and 57 (37.8%) were TP53-. Among patients with TP53+ carcinomas, BRCA1 carriers had increased odds of recurrence compared with sporadic cases (HR 2.25, P=0.003; median disease-free survival time 7.7 vs. 18.4months, respectively). In the smaller TP53- subgroup, BRCA1 mutation reduced the risk of death by 46% (HR 0.54, P=0.099, median overall survival time 42.7 vs. 28.1months), but beyond the border of significance. When the TP53 status was not taken into account, BRCA1 mutations did not show any significance, however, there was a trend toward increased odds of complete remission for women with BRCA1 mutations compared to non-carriers (OR 2.47, P=0.064). Taxane-platinum therapy showed advantage over the platinum-cyclophosphamide one in the entire group of patients and in the TP53+ subgroup., Conclusions: Our results suggest that the TP53 accumulation status determines the prognosis of BRCA1 mutation carriers with high-grade ovarian carcinomas., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Regulatory T (Treg) cells, a subset of CD4+ T lymphocytes, are mediators of immunosuppression in cancer, and, thus, variants in genes encoding Treg cell immune molecules could be associated with ovarian cancer., Methods: In a population of 15,596 epithelial ovarian cancer (EOC) cases and 23,236 controls, we measured genetic associations of 1,351 SNPs in Treg cell pathway genes with odds of ovarian cancer and tested pathway and gene-level associations, overall and by histotype, for the 25 genes, using the admixture likelihood (AML) method. The most significant single SNP associations were tested for correlation with expression levels in 44 ovarian cancer patients., Results: The most significant global associations for all genes in the pathway were seen in endometrioid ( p = 0.082) and clear cell ( p = 0.083), with the most significant gene level association seen with TGFBR2 ( p = 0.001) and clear cell EOC. Gene associations with histotypes at p < 0.05 included: IL12 ( p = 0.005 and p = 0.008, serous and high-grade serous, respectively), IL8RA ( p = 0.035, endometrioid and mucinous), LGALS1 ( p = 0.03, mucinous), STAT5B ( p = 0.022, clear cell), TGFBR1 ( p = 0.021 endometrioid) and TGFBR2 ( p = 0.017 and p = 0.025, endometrioid and mucinous, respectively)., Conclusions: Common inherited gene variation in Treg cell pathways shows some evidence of germline genetic contribution to odds of EOC that varies by histologic subtype and may be associated with mRNA expression of immune-complex receptor in EOC patients.

Purpose: Ovarian cancer is typically diagnosed at late stages, and thus, patients' prognosis is poor. Improvement in treatment outcomes depends, at least partly, on better understanding of ovarian cancer biology and finding new molecular markers and therapeutic targets., Methods: An unsupervised method of data analysis, singular value decomposition, was applied to analyze microarray data from 101 ovarian cancer samples; then, selected genes were validated by quantitative PCR., Results: We found that the major factor influencing gene expression in ovarian cancer was tumor histological type. The next major source of variability was traced to a set of genes mainly associated with extracellular matrix, cell motility, adhesion, and immunological response. Hierarchical clustering based on the expression of these genes revealed two clusters of ovarian cancers with different molecular profiles and distinct overall survival (OS). Patients with higher expression of these genes had shorter OS than those with lower expression. The two clusters did not derive from high- versus low-grade serous carcinomas and were unrelated to histological (ovarian vs. fallopian) origin. Interestingly, there was considerable overlap between identified prognostic signature and a recently described invasion-associated signature related to stromal desmoplastic reaction. Several genes from this signature were validated by quantitative PCR; two of them-DSPG3 and LOX-were validated both in the initial and independent sets of samples and were significantly associated with OS and disease-free survival., Conclusions: We distinguished two molecular subgroups of serous ovarian cancers characterized by distinct OS. Among differentially expressed genes, some may potentially be used as prognostic markers. In our opinion, unsupervised methods of microarray data analysis are more effective than supervised methods in identifying intrinsic, biologically sound sources of variability. Moreover, as histological type of the tumor is the greatest source of variability in ovarian cancer and may interfere with analyses of other features, it seems reasonable to use histologically homogeneous groups of tumors in microarray experiments.

Epithelial-mesenchymal transition (EMT) is a process whereby epithelial cells assume mesenchymal characteristics to facilitate cancer metastasis. However, EMT also contributes to the initiation and development of primary tumors. Prior studies that explored the hypothesis that EMT gene variants contribute to epithelial ovarian carcinoma (EOC) risk have been based on small sample sizes and none have sought replication in an independent population. We screened 15,816 single-nucleotide polymorphisms (SNPs) in 296 genes in a discovery phase using data from a genome-wide association study of EOC among women of European ancestry (1,947 cases and 2,009 controls) and identified 793 variants in 278 EMT-related genes that were nominally (P < 0.05) associated with invasive EOC. These SNPs were then genotyped in a larger study of 14,525 invasive-cancer patients and 23,447 controls. A P-value <0.05 and a false discovery rate (FDR) <0.2 were considered statistically significant. In the larger dataset, GPC6/GPC5 rs17702471 was associated with the endometrioid subtype among Caucasians (odds ratio (OR) = 1.16, 95% CI = 1.07-1.25, P = 0.0003, FDR = 0.19), whereas F8 rs7053448 (OR = 1.69, 95% CI = 1.27-2.24, P = 0.0003, FDR = 0.12), F8 rs7058826 (OR = 1.69, 95% CI = 1.27-2.24, P = 0.0003, FDR = 0.12), and CAPN13 rs1983383 (OR = 0.79, 95% CI = 0.69-0.90, P = 0.0005, FDR = 0.12) were associated with combined invasive EOC among Asians. In silico functional analyses revealed that GPC6/GPC5 rs17702471 coincided with DNA regulatory elements. These results suggest that EMT gene variants do not appear to play a significant role in the susceptibility to EOC., (© 2015 WILEY PERIODICALS, INC.)

Purpose: Chemotherapy resistance remains a major challenge in the treatment of ovarian cancer. We hypothesize that germline polymorphisms might be associated with clinical outcome., Experimental Design: We analyzed approximately 2.8 million genotyped and imputed SNPs from the iCOGS experiment for progression-free survival (PFS) and overall survival (OS) in 2,901 European epithelial ovarian cancer (EOC) patients who underwent first-line treatment of cytoreductive surgery and chemotherapy regardless of regimen, and in a subset of 1,098 patients treated with ≥ 4 cycles of paclitaxel and carboplatin at standard doses. We evaluated the top SNPs in 4,434 EOC patients, including patients from The Cancer Genome Atlas. In addition, we conducted pathway analysis of all intragenic SNPs and tested their association with PFS and OS using gene set enrichment analysis., Results: Five SNPs were significantly associated (P ≤ 1.0 × 10(-5)) with poorer outcomes in at least one of the four analyses, three of which, rs4910232 (11p15.3), rs2549714 (16q23), and rs6674079 (1q22), were located in long noncoding RNAs (lncRNAs) RP11-179A10.1, RP11-314O13.1, and RP11-284F21.8, respectively (P ≤ 7.1 × 10(-6)). ENCODE ChIP-seq data at 1q22 for normal ovary show evidence of histone modification around RP11-284F21.8, and rs6674079 is perfectly correlated with another SNP within the super-enhancer MEF2D, expression levels of which were reportedly associated with prognosis in another solid tumor. YAP1- and WWTR1 (TAZ)-stimulated gene expression and high-density lipoprotein (HDL)-mediated lipid transport pathways were associated with PFS and OS, respectively, in the cohort who had standard chemotherapy (pGSEA ≤ 6 × 10(-3))., Conclusions: We have identified SNPs in three lncRNAs that might be important targets for novel EOC therapies., (©2015 American Association for Cancer Research.)

Genome-wide association studies have identified 20 genomic regions associated with risk of epithelial ovarian cancer (EOC), but many additional risk variants may exist. Here, we evaluated associations between common genetic variants [single nucleotide polymorphisms (SNPs) and indels] in DNA repair genes and EOC risk. We genotyped 2896 common variants at 143 gene loci in DNA samples from 15 397 patients with invasive EOC and controls. We found evidence of associations with EOC risk for variants at FANCA, EXO1, E2F4, E2F2, CREB5 and CHEK2 genes (P ≤ 0.001). The strongest risk association was for CHEK2 SNP rs17507066 with serous EOC (P = 4.74 x 10(-7)). Additional genotyping and imputation of genotypes from the 1000 genomes project identified a slightly more significant association for CHEK2 SNP rs6005807 (r (2) with rs17507066 = 0.84, odds ratio (OR) 1.17, 95% CI 1.11-1.24, P = 1.1×10(-7)). We identified 293 variants in the region with likelihood ratios of less than 1:100 for representing the causal variant. Functional annotation identified 25 candidate SNPs that alter transcription factor binding sites within regulatory elements active in EOC precursor tissues. In The Cancer Genome Atlas dataset, CHEK2 gene expression was significantly higher in primary EOCs compared to normal fallopian tube tissues (P = 3.72×10(-8)). We also identified an association between genotypes of the candidate causal SNP rs12166475 (r (2) = 0.99 with rs6005807) and CHEK2 expression (P = 2.70×10(-8)). These data suggest that common variants at 22q12.1 are associated with risk of serous EOC and CHEK2 as a plausible target susceptibility gene., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Genome-wide association studies (GWAS) have so far reported 12 loci associated with serous epithelial ovarian cancer (EOC) risk. We hypothesized that some of these loci function through nearby transcription factor (TF) genes and that putative target genes of these TFs as identified by coexpression may also be enriched for additional EOC risk associations., Methods: We selected TF genes within 1 Mb of the top signal at the 12 genome-wide significant risk loci. Mutual information, a form of correlation, was used to build networks of genes strongly coexpressed with each selected TF gene in the unified microarray dataset of 489 serous EOC tumors from The Cancer Genome Atlas. Genes represented in this dataset were subsequently ranked using a gene-level test based on results for germline SNPs from a serous EOC GWAS meta-analysis (2,196 cases/4,396 controls)., Results: Gene set enrichment analysis identified six networks centered on TF genes (HOXB2, HOXB5, HOXB6, HOXB7 at 17q21.32 and HOXD1, HOXD3 at 2q31) that were significantly enriched for genes from the risk-associated end of the ranked list (P < 0.05 and FDR < 0.05). These results were replicated (P < 0.05) using an independent association study (7,035 cases/21,693 controls). Genes underlying enrichment in the six networks were pooled into a combined network., Conclusion: We identified a HOX-centric network associated with serous EOC risk containing several genes with known or emerging roles in serous EOC development., Impact: Network analysis integrating large, context-specific datasets has the potential to offer mechanistic insights into cancer susceptibility and prioritize genes for experimental characterization., (©2015 American Association for Cancer Research.)

Genome-wide association studies have reported 11 regions conferring risk of high-grade serous epithelial ovarian cancer (HGSOC). Expression quantitative trait locus (eQTL) analyses can identify candidate susceptibility genes at risk loci. Here we evaluate cis-eQTL associations at 47 regions associated with HGSOC risk (P≤10(-5)). For three cis-eQTL associations (P<1.4 × 10(-3), FDR<0.05) at 1p36 (CDC42), 1p34 (CDCA8) and 2q31 (HOXD9), we evaluate the functional role of each candidate by perturbing expression of each gene in HGSOC precursor cells. Overexpression of HOXD9 increases anchorage-independent growth, shortens population-doubling time and reduces contact inhibition. Chromosome conformation capture identifies an interaction between rs2857532 and the HOXD9 promoter, suggesting this SNP is a leading causal variant. Transcriptomic profiling after HOXD9 overexpression reveals enrichment of HGSOC risk variants within HOXD9 target genes (P=6 × 10(-10) for risk variants (P<10(-4)) within 10 kb of a HOXD9 target gene in ovarian cells), suggesting a broader role for this network in genetic susceptibility to HGSOC.

Background: Defective cellular transport processes can lead to aberrant accumulation of trace elements, iron, small molecules and hormones in the cell, which in turn may promote the formation of reactive oxygen species, promoting DNA damage and aberrant expression of key regulatory cancer genes. As DNA damage and uncontrolled proliferation are hallmarks of cancer, including epithelial ovarian cancer (EOC), we hypothesized that inherited variation in the cellular transport genes contributes to EOC risk., Methods: In total, DNA samples were obtained from 14,525 case subjects with invasive EOC and from 23,447 controls from 43 sites in the Ovarian Cancer Association Consortium (OCAC). Two hundred seventy nine SNPs, representing 131 genes, were genotyped using an Illumina Infinium iSelect BeadChip as part of the Collaborative Oncological Gene-environment Study (COGS). SNP analyses were conducted using unconditional logistic regression under a log-additive model, and the FDR q<0.2 was applied to adjust for multiple comparisons., Results: The most significant evidence of an association for all invasive cancers combined and for the serous subtype was observed for SNP rs17216603 in the iron transporter gene HEPH (invasive: OR = 0.85, P = 0.00026; serous: OR = 0.81, P = 0.00020); this SNP was also associated with the borderline/low malignant potential (LMP) tumors (P = 0.021). Other genes significantly associated with EOC histological subtypes (p<0.05) included the UGT1A (endometrioid), SLC25A45 (mucinous), SLC39A11 (low malignant potential), and SERPINA7 (clear cell carcinoma). In addition, 1785 SNPs in six genes (HEPH, MGST1, SERPINA, SLC25A45, SLC39A11 and UGT1A) were imputed from the 1000 Genomes Project and examined for association with INV EOC in white-European subjects. The most significant imputed SNP was rs117729793 in SLC39A11 (per allele, OR = 2.55, 95% CI = 1.5-4.35, p = 5.66x10-4)., Conclusion: These results, generated on a large cohort of women, revealed associations between inherited cellular transport gene variants and risk of EOC histologic subtypes.

Background: SMARCA4 mutations have recently been identified as driving lesions of the ovarian small cell carcinoma of hypercalcemic type (SCCHT). Familial occurrence of this neoplasm was described previously., Methods: We looked for germline SMARCA4 alterations in eight patients with the SCCHT. DNA was extracted from probands' and their relatives' blood. The SMARCA4 coding sequence, previously found altered in all the tumors, was PCR amplified and sequenced in the germline DNA., Results: Two patients carried a heterozygous germline SMARCA4 alteration: c.3760G > T and c.2352insG, respectively. The analysis of the probands' next of kins revealed that the c.3760G > T mutation was inherited by the proband and her sister from their father, and the sisters' four children also carried the mutation. The proband's sister was diagnosed with a carcinoma of the parotid gland at age 2. A brother of the other proband was tested negative., Conclusions: Our study suggests that some women develop the ovarian SCCHT due to the inherited or possibly de novo-occurring germline alterations in the SMARCA4 gene, however, its penetrance appears limited. Nevertheless, because of high aggressiveness of the SCCHT, a molecular diagnostics of the SMARCA4 gene and careful follow-up should be offered to patients with this cancer and their families.

Genome-wide association studies (GWAS) have identified 12 epithelial ovarian cancer (EOC) susceptibility alleles. The pattern of association at these loci is consistent in BRCA1 and BRCA2 mutation carriers who are at high risk of EOC. After imputation to 1000 Genomes Project data, we assessed associations of 11 million genetic variants with EOC risk from 15,437 cases unselected for family history and 30,845 controls and from 15,252 BRCA1 mutation carriers and 8,211 BRCA2 mutation carriers (3,096 with ovarian cancer), and we combined the results in a meta-analysis. This new study design yielded increased statistical power, leading to the discovery of six new EOC susceptibility loci. Variants at 1p36 (nearest gene, WNT4), 4q26 (SYNPO2), 9q34.2 (ABO) and 17q11.2 (ATAD5) were associated with EOC risk, and at 1p34.3 (RSPO1) and 6p22.1 (GPX6) variants were specifically associated with the serous EOC subtype, all with P < 5 × 10(-8). Incorporating these variants into risk assessment tools will improve clinical risk predictions for BRCA1 and BRCA2 mutation carriers.

Disruption in circadian gene expression, whether due to genetic variation or environmental factors (e.g., light at night, shiftwork), is associated with increased incidence of breast, prostate, gastrointestinal and hematologic cancers and gliomas. Circadian genes are highly expressed in the ovaries where they regulate ovulation; circadian disruption is associated with several ovarian cancer risk factors (e.g., endometriosis). However, no studies have examined variation in germline circadian genes as predictors of ovarian cancer risk and invasiveness. The goal of the current study was to examine single nucleotide polymorphisms (SNPs) in circadian genes BMAL1, CRY2, CSNK1E, NPAS2, PER3, REV1 and TIMELESS and downstream transcription factors KLF10 and SENP3 as predictors of risk of epithelial ovarian cancer (EOC) and histopathologic subtypes. The study included a test set of 3,761 EOC cases and 2,722 controls and a validation set of 44,308 samples including 18,174 (10,316 serous) cases and 26,134 controls from 43 studies participating in the Ovarian Cancer Association Consortium (OCAC). Analysis of genotype data from 36 genotyped SNPs and 4600 imputed SNPs indicated that the most significant association was rs117104877 in BMAL1 (OR = 0.79, 95% CI = 0.68-0.90, p = 5.59 × 10 -4 ]. Functional analysis revealed a significant down regulation of BMAL1 expression following cMYC overexpression and increasing transformation in ovarian surface epithelial (OSE) cells as well as alternative splicing of BMAL1 exons in ovarian and granulosa cells. These results suggest that variation in circadian genes, and specifically BMAL1 , may be associated with risk of ovarian cancer, likely through disruption of hormonal pathways.

Ovarian carcinoma is the most lethal type of gynecologic malignancy in the Western world. Majority of early stage ovarian cancers are asymptomatic and this is the main reason that more than two-thirds of patients are diagnosed with advanced disease. Ovarian tumors are heterogeneous and the different histologic subtypes are further classified as benign, borderline (low-grade) and malignant (high-grade) to reflect their behavior. The aim of the study was to analyze gene expression profiles in three histologic types of ovarian carcinoma in an attempt to find the molecular differences among serous, endometrioid and clear cell subtypes. The analysis of gene expression was performed on 57 samples of ovarian carcinoma. RNA was isolated from the ovarian cancer tissues. The gene expression changes were determined by microarray analysis and quantitative real time polymerase chain reaction (qRT-PCR). Measurement of relative gene expression levels was used to identify molecular differences among three histologic types of ovarian carcinoma (clear-cell, endometrioid and serous). Unsupervised statistical analysis revealed four biological subtypes among three histotypes under study. The endometrioid ovarian carcinoma was divided into two molecular subtypes. The biggest molecular differences were observed between clear-cell and serous carcinomas (1070 genes, FDR 0.05), the smallest between endometrioid and serous carcinomas (81 genes, FDR 0.05). The biggest group of differentially expressed genes was involved in transport and metabolism. This finding can explain the differences in the response to chemotherapy observed among different histologic types of ovarian carcinomas. In conclusion, we found TCF2 (HNF1B) gene as a suitable marker for ovarian clear cell carcinoma. Gene expression profiling also shed light on the molecular mechanisms of different chemoresistance among the analyzed histotypes.

Survival in epithelial ovarian cancer (EOC) is influenced by the host immune response, yet the key genetic determinants of inflammation and immunity that affect prognosis are not known. The nuclear factor-κB (NF-κB) transcription factor family plays an important role in many immune and inflammatory responses, including the response to cancer. We studied common inherited variation in 210 genes in the NF-κB family in 10,084 patients with invasive EOC (5,248 high-grade serous, 1,452 endometrioid, 795 clear cell, and 661 mucinous) from the Ovarian Cancer Association Consortium. Associations between genotype and overall survival were assessed using Cox regression for all patients and by major histology, adjusting for known prognostic factors and correcting for multiple testing (threshold for statistical significance, P < 2.5 × 10(-5)). Results were statistically significant when assessed for patients of a single histology. Key associations were with caspase recruitment domain family, member 11 (CARD11) rs41324349 in patients with mucinous EOC [HR, 1.82; 95% confidence interval (CI), 1.41-2.35; P = 4.13 × 10(-6)] and tumor necrosis factor receptor superfamily, member 13B (TNFRSF13B) rs7501462 in patients with endometrioid EOC (HR, 0.68; 95% CI, 0.56-0.82; P = 2.33 × 10(-5)). Other associations of note included TNF receptor-associated factor 2 (TRAF2) rs17250239 in patients with high-grade serous EOC (HR, 0.84; 95% CI, 0.77-0.92; P = 6.49 × 10(-5)) and phospholipase C, gamma 1 (PLCG1) rs11696662 in patients with clear cell EOC (HR, 0.43; 95% CI, 0.26-0.73; P = 4.56 × 10(-4)). These associations highlight the potential importance of genes associated with host inflammation and immunity in modulating clinical outcomes in distinct EOC histologies., (©2014 American Association for Cancer Research.)

Epithelial ovarian cancer (EOC) is a heterogeneous cancer with both genetic and environmental risk factors. Variants influencing the risk of developing the less-common EOC subtypes have not been fully investigated. We performed a genome-wide association study (GWAS) of EOC according to subtype by pooling genomic DNA from 545 cases and 398 controls of European descent, and testing for allelic associations. We evaluated for replication 188 variants from the GWAS [56 variants for mucinous, 55 for endometrioid and clear cell, 53 for low-malignant potential (LMP) serous, and 24 for invasive serous EOC], selected using pre-defined criteria. Genotypes from 13,188 cases and 23,164 controls of European descent were used to perform unconditional logistic regression under the log-additive genetic model; odds ratios (OR) and 95 % confidence intervals are reported. Nine variants tagging six loci were associated with subtype-specific EOC risk at P < 0.05, and had an OR that agreed in direction of effect with the GWAS results. Several of these variants are in or near genes with a biological rationale for conferring EOC risk, including ZFP36L1 and RAD51B for mucinous EOC (rs17106154, OR = 1.17, P = 0.029, n = 1,483 cases), GRB10 for endometrioid and clear cell EOC (rs2190503, P = 0.014, n = 2,903 cases), and C22orf26/BPIL2 for LMP serous EOC (rs9609538, OR = 0.86, P = 0.0043, n = 892 cases). In analyses that included the 75 GWAS samples, the association between rs9609538 (OR = 0.84, P = 0.0007) and LMP serous EOC risk remained statistically significant at P < 0.0012 adjusted for multiple testing. Replication in additional samples will be important to verify these results for the less-common EOC subtypes.

The presence of regulatory T cells (Treg) in solid tumors is known to play a role in patient survival in ovarian cancer and other malignancies. We assessed inherited genetic variations via 749 tag single-nucleotide polymorphisms (SNP) in 25 Treg-associated genes (CD28, CTLA4, FOXP3, IDO1, IL10, IL10RA, IL15, 1L17RA, IL23A, IL23R, IL2RA, IL6, IL6R, IL8, LGALS1, LGALS9, MAP3K8, STAT5A, STAT5B, TGFB1, TGFB2, TGFB3, TGFBR1, TGRBR2, and TGFBR3) in relation to ovarian cancer survival. We analyzed genotype and overall survival in 10,084 women with invasive epithelial ovarian cancer, including 5,248 high-grade serous, 1,452 endometrioid, 795 clear cell, and 661 mucinous carcinoma cases of European descent across 28 studies from the Ovarian Cancer Association Consortium (OCAC). The strongest associations were found for endometrioid carcinoma and IL2RA SNPs rs11256497 [HR, 1.42; 95% confidence interval (CI), 1.22-1.64; P = 5.7 × 10(-6)], rs791587 (HR, 1.36; 95% CI, 1.17-1.57; P = 6.2 × 10(-5)), rs2476491 (HR, = 1.40; 95% CI, 1.19-1.64; P = 5.6 × 10(-5)), and rs10795763 (HR, 1.35; 95% CI, 1.17-1.57; P = 7.9 × 10(-5)), and for clear cell carcinoma and CTLA4 SNP rs231775 (HR, 0.67; 95% CI, 0.54-0.82; P = 9.3 × 10(-5)) after adjustment for age, study site, population stratification, stage, grade, and oral contraceptive use. The rs231775 allele associated with improved survival in our study also results in an amino acid change in CTLA4 and previously has been reported to be associated with autoimmune conditions. Thus, we found evidence that SNPs in genes related to Tregs seem to play a role in ovarian cancer survival, particularly in patients with clear cell and endometrioid epithelial ovarian cancer.

The introduction of microarray techniques to cancer research brought great expectations for finding biomarkers that would improve patients' treatment; however, the results of such studies are poorly reproducible and critical analyses of these methods are rare. In this study, we examined global gene expression in 97 ovarian cancer samples. Also, validation of results by quantitative RT-PCR was performed on 30 additional ovarian cancer samples. We carried out a number of systematic analyses in relation to several defined clinicopathological features. The main goal of our study was to delineate the molecular background of ovarian cancer chemoresistance and find biomarkers suitable for prediction of patients' prognosis. We found that histological tumor type was the major source of variability in genes expression, except for serous and undifferentiated tumors that showed nearly identical profiles. Analysis of clinical endpoints [tumor response to chemotherapy, overall survival, disease-free survival (DFS)] brought results that were not confirmed by validation either on the same group or on the independent group of patients. CLASP1 was the only gene that was found to be important for DFS in the independent group, whereas in the preceding experiments it showed associations with other clinical endpoints and with BRCA1 gene mutation; thus, it may be worthy of further testing. Our results confirm that histological tumor type may be a strong confounding factor and we conclude that gene expression studies of ovarian carcinomas should be performed on histologically homogeneous groups. Among the reasons of poor reproducibility of statistical results may be the fact that despite relatively large patients' group, in some analyses one has to compare small and unequal classes of samples. In addition, arbitrarily performed division of samples into classes compared may not always reflect their true biological diversity. And finally, we think that clinical endpoints of the tumor probably depend on subtle changes in many and, possibly, alternative molecular pathways, and such changes may be difficult to demonstrate.

Ovarian tumors from two patients, compatible by histological and immunohistochemical criteria with small cell carcinoma of hypercalcemic type (SCCHT) (WT1+, EMA dispersed+, synaptophysin+ or dispersed+), were extensively sampled in order to find clues to their histogenesis. Subsequently, small foci of immature teratoma were found in both of them (in 1/122 and in 3/80 tumor sections). In one case, microfoci of yolk sac tumor were also present within the teratoma area as well as in the background of the small cell tumor population - in the primary tumor and in omental metastasis. We found a resemblance of the microscopic patterns of SCCHT and atypical teratoid/rhabdoid tumor (AT/RT) of the central nervous system, and this prompted us to evaluate INI-1 and SMARCA4 immunohistochemical expression, because their alternative loss is regarded as a molecular hallmark of AT/RT. INI-1 expression was retained, while that of SMARCA4 was lost. We therefore analyzed tumor DNA by PCR amplification and sequencing for mutations in the SMARCA4 gene (NG&#95;011556.1), which were identified in both tumors (c.2184&#95;2206del; nonsense c.3277C>T - both in one tumor; nonsense c.3760G>T in another tumor). These data suggest that SCCHT is most likely an embryonal tumor originating from immature teratoma and related to malignant rhabdoid tumor. Further analyses are necessary to determine whether the tumors diagnosed as SCCHT constitute a homogeneous group or represent more than one entity.

p19(INK4d) (CDKN2D) is a negative regulator of the cell cycle. Little is known of its role in cancer development and prognosis. We aimed to evaluate the clinical significance of p19(INK4d) expression in ovarian carcinomas with respect to the TP53 accumulation status, as well as the frequency of CDKN2D mutations. p19(INK4d) and TP53 expression was evaluated immunohistochemically in 445 ovarian carcinomas: 246 patients were treated with platinum-cyclophosphamide (PC/PAC), while 199 were treated with taxane-platinum agents (TP). CDKN2D gene expression (mRNA) was examined in 106 carcinomas, while CDKN2D mutations in 68 tumors. Uni- and multivariate statistical analyses (logistic regression and the Cox proportional hazards model) were performed for patient groups divided according to the chemotherapeutic regimen administered, and in subgroups with and without TP53 accumulation. High p19(INK4d) expression increased the risk of death, but only in patients with the TP53-negative carcinomas (HR 1.61, P = 0.049 for PC/PAC-treated patients, HR 2.00, P = 0.015 for TP-treated patients). This result was confirmed by the mRNA analysis (HR 4.24, P = 0.001 for TP-treated group). High p19(INK4d) protein expression associated with adverse clinicopathological factors. We found no alterations in the CDKN2D gene; the c.90C>G (p.R30R; rs1968445) polymorphism was detected in 10% of tumors. Our results suggest that p19(INK4d) expression is a poor prognostic factor in ovarian cancer patients. Analyses of tumor groups according to the TP53 accumulation status facilitate the identification of cancer biomarkers.

Background: Ovarian cancer is a leading cause of cancer-related death among women. In an effort to understand contributors to disease outcome, we evaluated single-nucleotide polymorphisms (SNP) previously associated with ovarian cancer recurrence or survival, specifically in angiogenesis, inflammation, mitosis, and drug disposition genes., Methods: Twenty-seven SNPs in VHL, HGF, IL18, PRKACB, ABCB1, CYP2C8, ERCC2, and ERCC1 previously associated with ovarian cancer outcome were genotyped in 10,084 invasive cases from 28 studies from the Ovarian Cancer Association Consortium with over 37,000-observed person-years and 4,478 deaths. Cox proportional hazards models were used to examine the association between candidate SNPs and ovarian cancer recurrence or survival with and without adjustment for key covariates., Results: We observed no association between genotype and ovarian cancer recurrence or survival for any of the SNPs examined., Conclusions: These results refute prior associations between these SNPs and ovarian cancer outcome and underscore the importance of maximally powered genetic association studies., Impact: These variants should not be used in prognostic models. Alternate approaches to uncovering inherited prognostic factors, if they exist, are needed.

TERT-locus SNPs and leukocyte telomere measures are reportedly associated with risks of multiple cancers. Using the Illumina custom genotyping array iCOGs, we analyzed ∼480 SNPs at the TERT locus in breast (n = 103,991), ovarian (n = 39,774) and BRCA1 mutation carrier (n = 11,705) cancer cases and controls. Leukocyte telomere measurements were also available for 53,724 participants. Most associations cluster into three independent peaks. The minor allele at the peak 1 SNP rs2736108 associates with longer telomeres (P = 5.8 × 10(-7)), lower risks for estrogen receptor (ER)-negative (P = 1.0 × 10(-8)) and BRCA1 mutation carrier (P = 1.1 × 10(-5)) breast cancers and altered promoter assay signal. The minor allele at the peak 2 SNP rs7705526 associates with longer telomeres (P = 2.3 × 10(-14)), higher risk of low-malignant-potential ovarian cancer (P = 1.3 × 10(-15)) and greater promoter activity. The minor alleles at the peak 3 SNPs rs10069690 and rs2242652 increase ER-negative (P = 1.2 × 10(-12)) and BRCA1 mutation carrier (P = 1.6 × 10(-14)) breast and invasive ovarian (P = 1.3 × 10(-11)) cancer risks but not via altered telomere length. The cancer risk alleles of rs2242652 and rs10069690, respectively, increase silencing and generate a truncated TERT splice variant.

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.

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.

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.

BRCA1 dysfunction may occur by different mechanisms that are rarely evaluated concomitantly. We aimed to analyze BRCA1 germline mutations, loss of heterozygosity (LOH) and promoter methylation in unselected ovarian carcinomas in the context of their clinicopathologic characteristics and other molecular changes. BRCA1 mutations were analyzed in 257 carcinomas using single-strand conformation polymorphism (SSCP), heteroduplex, and sequencing methods. LOH at the BRCA1 locus was screened for in 180 cancers. Methylation analysis was performed for 241 tumors using quantitative methylation specific PCR (qMSP). BRCA1 alterations, comprising germline mutations, allelic loss, and/or aberrant promoter methylation, were found in 77.6% (125/161) of ovarian carcinomas. Patients with germline mutations were younger than non-carriers (P < 0.0001). Germline mutations and LOH were associated with advanced stages (P=0.009, P < 0.0001), high tumor grade (P=0.005, P < 0.0001), and TP53 mutations (P=0.003, P < 0.0001, for mutations and LOH, respectively). LOH was also associated with the serous histological type (P=0.004) and PIK3CA amplification (P=0.003). Aberrant promoter methylation was associated with LOH (P=0.017) and absence of germline mutations (P=0.037). The high frequency of LOH at the BRCA1 locus suggests that LOH may be an important mechanism of BRCA1 deficiency in ovarian carcinomas. Tumors with various BRCA1 alterations have a similar phenotype of high-grade, high-stage carcinomas with frequent TP53 mutations., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Survivin is an inhibitor of apoptosis and a regulator of mitotic progression. TP53 protein is a negative transcriptional regulator of survivin. The aim of our study was to evaluate the clinical significance of survivin expression in advanced stages ovarian cancer with respect to the TP53 status., Methods: Survivin and TP53 expression was evaluated immunohistochemically in 435 archival samples of ovarian carcinomas (244 patients were treated with platinum/cyclophosphamide-PC/PAC; 191-with taxane-platinum (TP) agents). Univariate and multivariate statistical analyses were performed in patients groups divided according to the administered chemotherapeutic regimen, and in subgroups with and without TP53 accumulation (TP53+ and TP53-, respectively)., Results: Nuclear and cytoplasmic survivin expression was observed in 92% and 74% of the carcinomas, respectively. In patients treated with TP, high nuclear survivin expression decreased the risk of disease recurrence and death, and increased the probability of high platinum sensitivity (p < 0.01), but only in the TP53(+) group, and not in the TP53(-) group., Conclusions: It appears that TP53 status determines the clinical importance of nuclear survivin expression in taxane-platinum treated ovarian cancer patients.