Narod, Steven A.

AbstractBackground:Germline DNA copy number variation (CNV) is a ubiquitous source of genetic variation and remains largely unexplored in association with epithelial ovarian cancer (EOC) risk.Methods:CNV was quantified in the DNA of approximately 3,500 cases and controls genotyped with the Illumina 610k and HumanOmni2.5M arrays. We performed a genome-wide association study of common (>1%) CNV regions (CNVRs) with EOC and high-grade serous (HGSOC) risk and, using The Cancer Genome Atlas (TCGA), performed in silico analyses of tumor-gene expression.Results:Three CNVRs were associated (P < 0.01) with EOC risk: two large (∼100 kb) regions within the 610k set and one small (<5 kb) region with the higher resolution 2.5M data. Large CNVRs included a duplication at LILRA6 (OR = 2.57; P = 0.001) and a deletion at CYP2A7 (OR = 1.90; P = 0.007) that were strongly associated with HGSOC risk (OR = 3.02; P = 8.98 × 10−5). Somatic CYP2A7 alterations correlated with EGLN2 expression in tumors (P = 2.94 × 10−47). An intronic ERBB4/HER4 deletion was associated with reduced EOC risk (OR = 0.33; P = 9.5 × 10−2), and somatic deletions correlated with ERBB4 downregulation (P = 7.05 × 10−5). Five CNVRs were associated with HGSOC, including two reduced-risk deletions: one at 1p36.33 (OR = 0.28; P = 0.001) that correlated with lower CDKIIA expression in TCGA tumors (P = 2.7 × 10−7), and another at 8p21.2 (OR = 0.52; P = 0.002) that was present somatically where it correlated with lower GNRH1 expression (P = 5.9 × 10−5).Conclusions:Though CNV appears to not contribute largely to EOC susceptibility, a number of low-to-common frequency variants may influence the risk of EOC and tumor-gene expression.Impact:Further research on CNV and EOC susceptibility is warranted, particularly with CNVs estimated from high-density arrays.

Thymidylate synthase (TYMS) is a crucial enzyme for DNA synthesis. TYMS expression is regulated by its antisense mRNA, ENOSF1. Disrupted regulation may promote uncontrolled DNA synthesis and tumor growth. We sought to replicate our previously reported association between rs495139 in the TYMS-ENOSF1 3′ gene region and increased risk of mucinous ovarian carcinoma (MOC) in an independent sample. Genotypes from 24,351 controls to 15,000 women with invasive OC, including 665 MOC, were available. We estimated per-allele odds ratios (OR) and 95% confidence intervals (CI) using unconditional logistic regression, and meta-analysis when combining these data with our previous report. The association between rs495139 and MOC was not significant in the independent sample (OR = 1.09; 95% CI = 0.97–1.22; p = 0.15; N = 665 cases). Meta-analysis suggested a weak association (OR = 1.13; 95% CI = 1.03–1.24; p = 0.01; N = 1019 cases). No significant association with risk of other OC histologic types was observed (p = 0.05 for tumor heterogeneity). In expression quantitative trait locus (eQTL) analysis, the rs495139 allele was positively associated with ENOSF1 mRNA expression in normal tissues of the gastrointestinal system, particularly esophageal mucosa (r = 0.51, p = 1.7 × 10−28), and nonsignificantly in five MOC tumors. The association results, along with inconclusive tumor eQTL findings, suggest that a true effect of rs495139 might be small.

Background The rarity of mutations in PALB2, CHEK2 and ATM make it difficult to estimate precisely associated cancer risks. Population-based family studies have provided evidence that at least some of these mutations are associated with breast cancer risk as high as those associated with rare BRCA2 mutations. We aimed to estimate the relative risks associated with specific rare variants in PALB2, CHEK2 and ATM via a multicentre case-control study. Methods We genotyped 10 rare mutations using the custom iCOGS array: PALB2 c.1592delT, c.2816T>G and c.3113G>A, CHEK2 c.349A>G, c.538C>T, c.715G>A, c.1036C>T, c.1312G>T, and c.1343T>G and ATM c.7271T>G. We assessed associations with breast cancer risk (42 671 cases and 42 164 controls), as well as prostate (22 301 cases and 22 320 controls) and ovarian (14 542 cases and 23 491 controls) cancer risk, for each variant. Results For European women, strong evidence of association with breast cancer risk was observed for PALB2 c.1592delT OR 3.44 (95% CI 1.39 to 8.52, p=7.1×10−5), PALB2 c.3113G>A OR 4.21 (95% CI 1.84 to 9.60, p=6.9×10−8) and ATM c.7271T>G OR 11.0 (95% CI 1.42 to 85.7, p=0.0012). We also found evidence of association with breast cancer risk for three variants in CHEK2, c.349A>G OR 2.26 (95% CI 1.29 to 3.95), c.1036C>T OR 5.06 (95% CI 1.09 to 23.5) and c.538C>T OR 1.33 (95% CI 1.05 to 1.67) (p≤0.017). Evidence for prostate cancer risk was observed for CHEK2 c.1343T>G OR 3.03 (95% CI 1.53 to 6.03, p=0.0006) for African men and CHEK2 c.1312G>T OR 2.21 (95% CI 1.06 to 4.63, p=0.030) for European men. No evidence of association with ovarian cancer was found for any of these variants. Conclusions This report adds to accumulating evidence that at least some variants in these genes are associated with an increased risk of breast cancer that is clinically important.

ABSTRACTEpithelial‐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.

Abstract 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 &lt; 0.05 and FDR &lt; 0.05). These results were replicated (P &lt; 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. Cancer Epidemiol Biomarkers Prev; 24(10); 1574–84. ©2015 AACR.

Abstract Since most GWAS hits fall in non-coding regions of the genome and long non-coding RNAs (lncRNAs) are emerging as drivers of carcinogenesis, we hypothesized that SNPs in lncRNAs influence epithelial ovarian cancer (EOC) risk. A comprehensive lncRNA database (lncRNA db) that contained 104 human lncRNAs when downloaded in November 2011 was used to identify 1,737 variants in 63 unique lncRNAs that were represented in three genome-wide association studies from North America, the United Kingdom, and Poland (3,995 EOC cases and 3,277 controls of European background). SNPs with MAF &lt;5% were excluded, and missing genotypes were inferred with Mach using the HapMap CEU population. Unconditional logistic regression treating alternate alleles as an ordinal variable was used to evaluate individual SNP-EOC risk associations. Subgroup analysis was conducted for serous adenocarcinomas (N= 2,066), the main histologic subtype of EOC. Models were adjusted for study and European ancestry using the first two principal components (PCs). We reduced correlated signals (r2 ≥ 0.8) within each lncRNA and kept the most statistically significant variants based on Wald tests, resulting in 651 SNPs for further analyses. Fisher's product method was used to aggregate evidence of multiple variants within each lncRNA using 10,000 permutations. To adjust for multiple comparisons, a false discovery rate (FDR) of 10% was used. A replication phase was carried out for top-ranked SNPs from an additional 9,854 EOC cases (5,802 serous) and 17,633 controls genotyped through an international effort known as the Collaborative Oncological Gene-Environment study (COGS). Four lncRNAs, WT1-AS (p = 0.005, n = 2 SNPs), Hoxa11as (p = 0.013, n = 2), AK082072 (p = 0.016, n = 4) and H19 (p = 0.020, n = 2), were significantly associated with EOC risk among all histologies (FDR ≤ 10%). The SNP in WT1-AS most significantly associated with EOC risk was rs3809061 (T&gt;C) (minor allele frequency= 36%, odds ratio (OR) = 0.91, CI: 0.84-0.97, p = 0.008). Among serous cases, rs3809061 was also protective (OR = 0.92, p = 0.04). These findings were replicated by a correlated SNP rs2301250 (r2 = 0.84) evaluated in COGS among serous cases (OR = 0.94, CI: 0.90-0.99, p = 0.009) and less significant among all histologies (OR = 0.96, CI: 0.93-1.00, p = 0.054). We further interrogated its functional role by performing an expression quantitative trait locus analysis on WTI-AS mRNA expression using the Cancer Genome Atlas (TCGA) data (N = 462 serous cases). WT1-AS expression was significantly higher among carriers of the variant allele (compared to TT homozygotes) using the Mann-Whitney U test (p = 0.001). WT1-AS is the antisense and possible regulator of WT1, a tumor suppressor gene reported to be prognostic in advanced EOC. These findings implicate germline lncRNA variants associated with EOC risk that merit further investigation. Citation Format: Yian Ann Chen, Zhihua Chen, Jennifer Permuth-Wey, Ya-Yu Tsai, Hui-Yi Lin, Xiaotao Qu, Kate Lawrenson, David Fenstermacher, Catherine M. Phelan, Alvaro Monteiro, Simon A. Gayther, Steven A. Narod, Rebecca Sutphen, Michael J. Birrer, Nicolas Wentzensen, Joellen M. Schildkraut, Ellen L. Goode, Paul Pharoah, Thomas Sellers. Variants in long non-coding RNAs are associated with epithelial ovarian cancer risk in a pooled analysis of three genome-wide association studies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4579. doi:10.1158/1538-7445.AM2013-4579

ScopeWe reevaluated previously reported associations between variants in pathways of one‐carbon (1‐C) (folate) transfer genes and ovarian carcinoma (OC) risk, and in related pathways of purine and pyrimidine metabolism, and assessed interactions with folate intake.Methods and resultsOdds ratios (OR) for 446 genetic variants were estimated among 13 410 OC cases and 22 635 controls, and among 2281 cases and 3444 controls with folate information. Following multiple testing correction, the most significant main effect associations were for dihydropyrimidine dehydrogenase (DPYD) variants rs11587873 (OR = 0.92; p = 6 × 10−5) and rs828054 (OR = 1.06; p = 1 × 10−4). Thirteen variants in the pyrimidine metabolism genes, DPYD, DPYS, PPAT, and TYMS, also interacted significantly with folate in a multivariant analysis (corrected p = 9.9 × 10−6) but collectively explained only 0.2% of OC risk. Although no other associations were significant after multiple testing correction, variants in SHMT1 in 1‐C transfer, previously reported with OC, suggested lower risk at higher folate (pinteraction = 0.03–0.006).ConclusionVariation in pyrimidine metabolism genes, particularly DPYD, which was previously reported to be associated with OC, may influence risk; however, stratification by folate intake is unlikely to modify disease risk appreciably in these women. SHMT1 SNP‐by‐folate interactions are plausible but require further validation. Polymorphisms in selected genes in purine metabolism were not associated with OC.