Tyrer, Jonathan P.

Abstract Introduction: Identifying dysregulated gene expression in ovarian cancers has been limited by a deficit of available normal tissues. Here, we generated the largest set of high-grade serous ovarian cancer (HGSOC) tumors with normal precursor tissues for transcriptome analyses to identify lncRNA expression patterns, provide insight into their cellular contexts and functions, and assess their relevance to HGSOC pathogenesis and genetic susceptibility. Methods: We performed RNA sequencing on 220 primary HGSOCs and 116 benign epithelia (micro-dissected fallopian tube, ovarian surface, and inclusion cyst epithelia), and combined samples with 428 HGSOCs from TCGA, 60 HGSOCs from a prior study, and 180 bulk ovary tissues from GTEx. Reads were processed with a uniform bioinformatic and quality control pipeline, combined and batch corrected. Normalized TPM expression values were compared using the Wilcoxon rank sum test with >2-fold change and FDR <1% considered statistically significant. We computed weighted gene co-expression networks for each tissue type and tested for enrichment of differentially expressed genes (DEGs) and gene ontologies within co-expression modules. Results: Transcriptomes included 27,700 expressed genes (8,202 lncRNAs) across 706 HGSOCs, 180 bulk ovary, and 88 ovarian epithelia tissues. Most genes were expressed in all tissues, though 5% each showed HGSOC- or normal tissue-specific expression which comprised ≥50% lncRNA. Comparing HGSOCs to normal tissues identified 11,804 DEGs with 4,522 lncRNAs (DElncRNA) of which ~50% were tissue-specific. DEGs included MUC16 and multiple GWAS/TWAS implicated susceptibility genes including RAD51, BRIP1, BNC2, TIPARP-AS1, PRC1, KANSL1, ANKLE1, CHMP4C, ESRP2, and CCNE1. The most highly expressed DElncRNA in HGSOC were upregulated RMRP (P=1.4 × 10−39), SNHG1 (P=3.0 × 10−27), and HAGLR (P=2.0 × 10−24) at the HOXD risk locus. Lower proportions of lncRNA were seen in the HGSOC co-expression modules compared to those in normal tissues and less (3% vs. 15%) HGSOC modules were considered strongly lncRNA regulated (>50% lncRNA in module). Tissue-specific DElncRNA tended toward upregulation in HGSOC compared to ovarian epithelia with enriched modules associated with cell cycle regulation (P=1.5 × 10−38); and toward downregulation in HGSOC compared to bulk ovary with enriched modules associated with chemokine signaling/response (P=1.7 × 10−113). Conclusions: The lower proportion of lncRNA involved in HGSOC co-expression networks indicates a marked loss of regulation by lncRNA. Preliminary interrogation of HGSOC-dysregulated lncRNA expression revealed tissue-specific differences that highlight unique biological pathways in precursor epithelia and the ovarian microenvironment that contribute to HGSOC pathogenesis. Our results provide additional support for previously nominated risk genes. eQTL analysis has recently been completed and integration with GWAS are underway to elucidate lncRNA and lncRNA-regulated susceptibility genes and will be presented at the conference. Citation Format: Brett M. Reid, Ann Chen, Zhihua Chen, Florian A. Karreth, Peter Kanetsky, Jennifer B. Permuth, Ozlen Saglam, Jamie K. Teer, Xiaoqing Yu, Simon Gayther, Ellen Goode, Paul Pharoah, Thomas A. Sellers, Kate Lawrenson, Jonathan Tyrer. Patterns of lncRNA-regulated gene expression in high-grade serous ovarian carcinomas [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A004.

AbstractBackgroundKnown risk alleles for epithelial ovarian cancer (EOC) account for approximately 40% of the heritability for EOC. Copy number variants (CNVs) have not been investigated as EOC risk alleles in a large population cohort.MethodsSingle nucleotide polymorphism array data from 13 071 EOC cases and 17 306 controls of White European ancestry were used to identify CNVs associated with EOC risk using a rare admixture maximum likelihood test for gene burden and a by-probe ratio test. We performed enrichment analysis of CNVs at known EOC risk loci and functional biofeatures in ovarian cancer–related cell types.ResultsWe identified statistically significant risk associations with CNVs at known EOC risk genes; BRCA1 (PEOC = 1.60E-21; OREOC = 8.24), RAD51C (Phigh-grade serous ovarian cancer [HGSOC] = 5.5E-4; odds ratio [OR]HGSOC = 5.74 del), and BRCA2 (PHGSOC = 7.0E-4; ORHGSOC = 3.31 deletion). Four suggestive associations (P < .001) were identified for rare CNVs. Risk-associated CNVs were enriched (P < .05) at known EOC risk loci identified by genome-wide association study. Noncoding CNVs were enriched in active promoters and insulators in EOC-related cell types.ConclusionsCNVs in BRCA1 have been previously reported in smaller studies, but their observed frequency in this large population-based cohort, along with the CNVs observed at BRCA2 and RAD51C gene loci in EOC cases, suggests that these CNVs are potentially pathogenic and may contribute to the spectrum of disease-causing mutations in these genes. CNVs are likely to occur in a wider set of susceptibility regions, with potential implications for clinical genetic testing and disease prevention.

Abstract 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

Abstract BackgroundCopy number variants (CNVs) are pervasive in the human genome but potential disease associations with rare CNVs have not been comprehensively assessed in large datasets. We analysed rare CNVs in genes and non-coding regions for 86,788 breast cancer cases and 76,122 controls of European ancestry with genome-wide array data.ResultsGene burden tests detected the strongest association for deletions in BRCA1 (P= 3.7E-18). Nine other genes were associated with a p-value < 0.01 including known susceptibility genes CHEK2 (P= 0.0008), ATM (P= 0.002) and BRCA2 (P= 0.008). Outside the known genes we detected associations with p-values < 0.001 for either overall or subtype-specific breast cancer at nine deletion regions and four duplication regions. Three of the deletion regions were in established common susceptibility loci.ConclusionsThis is the first genome-wide analysis of rare CNVs in a large breast cancer case-control dataset. We detected associations with exonic deletions in established breast cancer susceptibility genes. We also detected suggestive associations with non-coding CNVs in known and novel loci with large effects sizes. Larger sample sizes will be required to reach robust levels of statistical significance.

Abstract Blood lipids have been associated with the development of a range of cancers, including breast, lung and colorectal cancer. For endometrial cancer, observational studies have reported inconsistent associations between blood lipids and cancer risk. To reduce biases from unmeasured confounding, we performed a bidirectional, two‐sample Mendelian randomization analysis to investigate the relationship between levels of three blood lipids (low‐density lipoprotein [LDL] and high‐density lipoprotein [HDL] cholesterol, and triglycerides) and endometrial cancer risk. Genetic variants associated with each of these blood lipid levels ( P < 5 × 10 −8 ) were identified as instrumental variables, and assessed using genome‐wide association study data from the Endometrial Cancer Association Consortium (12 906 cases and 108 979 controls) and the Global Lipids Genetic Consortium (n = 188 578). Mendelian randomization analyses found genetically raised LDL cholesterol levels to be associated with lower risks of endometrial cancer of all histologies combined, and of endometrioid and non‐endometrioid subtypes. Conversely, higher genetically predicted HDL cholesterol levels were associated with increased risk of non‐endometrioid endometrial cancer. After accounting for the potential confounding role of obesity (as measured by genetic variants associated with body mass index), the association between genetically predicted increased LDL cholesterol levels and lower endometrial cancer risk remained significant, especially for non‐endometrioid endometrial cancer. There was no evidence to support a role for triglycerides in endometrial cancer development. Our study supports a role for LDL and HDL cholesterol in the development of non‐endometrioid endometrial cancer. Further studies are required to understand the mechanisms underlying these findings.

Purpose The known epithelial ovarian cancer (EOC) susceptibility genes account for less than 50% of the heritable risk of ovarian cancer suggesting that other susceptibility genes exist. The aim of this study was to evaluate the contribution to ovarian cancer susceptibility of rare deleterious germline variants in a set of candidate genes. Methods We sequenced the coding region of 54 candidate genes in 6385 invasive EOC cases and 6115 controls of broad European ancestry. Genes with an increased frequency of putative deleterious variants in cases versus controls were further examined in an independent set of 14 135 EOC cases and 28 655 controls from the Ovarian Cancer Association Consortium and the UK Biobank. For each gene, we estimated the EOC risks and evaluated associations between germline variant status and clinical characteristics. Results The ORs associated for high-grade serous ovarian cancer were 3.01 for PALB2 (95% CI 1.59 to 5.68; p=0.00068), 1.99 for POLK (95% CI 1.15 to 3.43; p=0.014) and 4.07 for SLX4 (95% CI 1.34 to 12.4; p=0.013). Deleterious mutations in FBXO10 were associated with a reduced risk of disease (OR 0.27, 95% CI 0.07 to 1.00, p=0.049). However, based on the Bayes false discovery probability, only the association for PALB2 in high-grade serous ovarian cancer is likely to represent a true positive. Conclusions We have found strong evidence that carriers of PALB2 deleterious mutations are at increased risk of high-grade serous ovarian cancer. Whether the magnitude of risk is sufficiently high to warrant the inclusion of PALB2 in cancer gene panels for ovarian cancer risk testing is unclear; much larger sample sizes will be needed to provide sufficiently precise estimates for clinical counselling.

Abstract DNA methylation is instrumental for gene regulation. Global changes in the epigenetic landscape have been recognized as a hallmark of cancer. However, the role of DNA methylation in epithelial ovarian cancer (EOC) remains unclear. In this study, high-density genetic and DNA methylation data in white blood cells from the Framingham Heart Study (N = 1,595) were used to build genetic models to predict DNA methylation levels. These prediction models were then applied to the summary statistics of a genome-wide association study (GWAS) of ovarian cancer including 22,406 EOC cases and 40,941 controls to investigate genetically predicted DNA methylation levels in association with EOC risk. Among 62,938 CpG sites investigated, genetically predicted methylation levels at 89 CpG were significantly associated with EOC risk at a Bonferroni-corrected threshold of P &lt; 7.94 × 10−7. Of them, 87 were located at GWAS-identified EOC susceptibility regions and two resided in a genomic region not previously reported to be associated with EOC risk. Integrative analyses of genetic, methylation, and gene expression data identified consistent directions of associations across 12 CpG, five genes, and EOC risk, suggesting that methylation at these 12 CpG may influence EOC risk by regulating expression of these five genes, namely MAPT, HOXB3, ABHD8, ARHGAP27, and SKAP1. We identified novel DNA methylation markers associated with EOC risk and propose that methylation at multiple CpG may affect EOC risk via regulation of gene expression. Significance: Identification of novel DNA methylation markers associated with EOC risk suggests that methylation at multiple CpG may affect EOC risk through regulation of gene expression.

As a follow‐up to genome‐wide association analysis of common variants associated with ovarian carcinoma (cancer), our study considers seven well‐known ovarian cancer risk factors and their interactions with 28 genome‐wide significant common genetic variants. The interaction analyses were based on data from 9971 ovarian cancer cases and 15,566 controls from 17 case–control studies. Likelihood ratio and Wald tests for multiplicative interaction and for relative excess risk due to additive interaction were used. The top multiplicative interaction was noted between oral contraceptive pill (OCP) use (ever vs. never) and rs13255292 (p value = 3.48 × 10−4). Among women with the TT genotype for this variant, the odds ratio for OCP use was 0.53 (95% CI = 0.46–0.60) compared to 0.71 (95%CI = 0.66–0.77) for women with the CC genotype. When stratified by duration of OCP use, women with 1–5 years of OCP use exhibited differential protective benefit across genotypes. However, no interaction on either the multiplicative or additive scale was found to be statistically significant after multiple testing correction. The results suggest that OCP use may offer increased benefit for women who are carriers of the T allele in rs13255292. On the other hand, for women carrying the C allele in this variant, longer (5+ years) use of OCP may reduce the impact of carrying the risk allele of this SNP. Replication of this finding is needed. The study presents a comprehensive analytic framework for conducting gene–environment analysis in ovarian cancer.

Abstract Genome-wide association studies have identified 40 ovarian cancer risk loci. However, the mechanisms underlying these associations remain elusive. In this study, we conducted a two-pronged approach to identify candidate causal SNPs and assess underlying biological mechanisms at chromosome 9p22.2, the first and most statistically significant associated locus for ovarian cancer susceptibility. Three transcriptional regulatory elements with allele-specific effects and a scaffold/matrix attachment region were characterized and, through physical DNA interactions, BNC2 was established as the most likely target gene. We determined the consensus binding sequence for BNC2 in vitro, verified its enrichment in BNC2 ChIP-seq regions, and validated a set of its downstream target genes. Fine-mapping by dense regional genotyping in over 15,000 ovarian cancer cases and 30,000 controls identified SNPs in the scaffold/matrix attachment region as among the most likely causal variants. This study reveals a comprehensive regulatory landscape at 9p22.2 and proposes a likely mechanism of susceptibility to ovarian cancer. Significance: Mapping the 9p22.2 ovarian cancer risk locus identifies BNC2 as an ovarian cancer risk gene. See related commentary by Choi and Brown, p. 439

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.