Tyrer, Jonathan P.

Abstract Identifying genetic variants with pleiotropic associations can uncover common pathways influencing multiple cancers. We took a two-stage approach to conduct genome-wide association studies for lung, ovary, breast, prostate, and colorectal cancer from the GAME-ON/GECCO Network (61,851 cases, 61,820 controls) to identify pleiotropic loci. Findings were replicated in independent association studies (55,789 cases, 330,490 controls). We identified a novel pleiotropic association at 1q22 involving breast and lung squamous cell carcinoma, with eQTL analysis showing an association with ADAM15/THBS3 gene expression in lung. We also identified a known breast cancer locus CASP8/ALS2CR12 associated with prostate cancer, a known cancer locus at CDKN2B-AS1 with different variants associated with lung adenocarcinoma and prostate cancer, and confirmed the associations of a breast BRCA2 locus with lung and serous ovarian cancer. This is the largest study to date examining pleiotropy across multiple cancer-associated loci, identifying common mechanisms of cancer development and progression. Cancer Res; 76(17); 5103–14. ©2016 AACR.

Abstract Background: Many epithelial ovarian cancer (EOC) risk factors relate to hormone exposure and elevated estrogen levels are associated with obesity in postmenopausal women. Therefore, we hypothesized that gene–environment interactions related to hormone-related risk factors could differ between obese and non-obese women. Methods: We considered interactions between 11,441 SNPs within 80 candidate genes related to hormone biosynthesis and metabolism and insulin-like growth factors with six hormone-related factors (oral contraceptive use, parity, endometriosis, tubal ligation, hormone replacement therapy, and estrogen use) and assessed whether these interactions differed between obese and non-obese women. Interactions were assessed using logistic regression models and data from 14 case–control studies (6,247 cases; 10,379 controls). Histotype-specific analyses were also completed. Results: SNPs in the following candidate genes showed notable interaction: IGF1R (rs41497346, estrogen plus progesterone hormone therapy, histology = all, P = 4.9 × 10−6) and ESR1 (rs12661437, endometriosis, histology = all, P = 1.5 × 10−5). The most notable obesity–gene–hormone risk factor interaction was within INSR (rs113759408, parity, histology = endometrioid, P = 8.8 × 10−6). Conclusions: We have demonstrated the feasibility of assessing multifactor interactions in large genetic epidemiology studies. Follow-up studies are necessary to assess the robustness of our findings for ESR1, CYP11A1, IGF1R, CYP11B1, INSR, and IGFBP2. Future work is needed to develop powerful statistical methods able to detect these complex interactions. Impact: Assessment of multifactor interaction is feasible, and, here, suggests that the relationship between genetic variants within candidate genes and hormone-related risk factors may vary EOC susceptibility. Cancer Epidemiol Biomarkers Prev; 25(5); 780–90. ©2016 AACR.

Abstract Background: While numerous susceptibility loci for epithelial ovarian cancer (EOC) have been identified, few associations have been reported with overall survival. In the absence of common prognostic genetic markers, we hypothesize that rare coding variants may be associated with overall EOC survival and assessed their contribution in two exome-based genotyping projects of the Ovarian Cancer Association Consortium (OCAC). Methods: The primary patient set (Set 1) included 14 independent EOC studies (4,293 patients) and 227,892 variants, and a secondary patient set (Set 2) included six additional EOC studies (1,744 patients) and 114,620 variants. Because power to detect rare variants individually is reduced, gene-level tests were conducted. Sets were analyzed separately at individual variants and by gene, and then combined with meta-analyses (73,203 variants and 13,163 genes overlapped). Results: No individual variant reached genome-wide statistical significance. A SNP previously implicated to be associated with EOC risk and, to a lesser extent, survival, rs8170, showed the strongest evidence of association with survival and similar effect size estimates across sets (Pmeta = 1.1E−6, HRSet1 = 1.17, HRSet2 = 1.14). Rare variants in ATG2B, an autophagy gene important for apoptosis, were significantly associated with survival after multiple testing correction (Pmeta = 1.1E−6; Pcorrected = 0.01). Conclusions: Common variant rs8170 and rare variants in ATG2B may be associated with EOC overall survival, although further study is needed. Impact: This study represents the first exome-wide association study of EOC survival to include rare variant analyses, and suggests that complementary single variant and gene-level analyses in large studies are needed to identify rare variants that warrant follow-up study. Cancer Epidemiol Biomarkers Prev; 25(3); 446–54. ©2016 AACR.

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 The known genetic variance for epithelial ovarian cancer (EOC) accounts for less than half the familial risks of the disease. BRCA1 and BRCA2 are the major high-penetrance EOC susceptibility genes. More recently genes that interact with BRCA1 and BRCA2 in the same biological pathways, but conferring more moderate penetrance (e.g. RAD51C, RAD51D) have also been identified. Mutations in these genes result in defects in homologous recombination and double strand break repair (HR/DSB). We hypothesized that other genes operating in these and other DNA repair pathways may also confer EOC susceptibility. Whole exome sequencing data, available from more than 400 high-grade serous ovarian cancers, were used to identify 24 candidate genes harboring germline truncating mutations in at least 2 cases, followed by replication analysis using targeted resequencing in ~3,000 cases and 3,000 controls. Subjects were also sequenced for the known susceptibility genes listed above. The data confirmed the previously reported contributions of BRCA1, BRCA2, RAD51C and RAD51D to EOC. In addition, we identified BRIP1, and FANCM as candidate EOC susceptibility genes. Surprisingly, for several genes we found truncating mutations were equally prevalent in cases as they were in controls. These mutations may still represent rare low-penetrance susceptibility alleles for EOC, which we are too limited in sample size and power to confirm; or more likely, it indicates that cells can tolerate heterozygous loss-of-function mutations in apparently critical DNA repair genes without any impact on neoplastic development. These findings may have implications for risk prediction and prevention strategies for EOC and possibly for the identification of novel therapeutic targets. Citation Format: Susan J. Ramus, Honglin Song, Ed Dicks, Maria Intermaggio, Jane Hayward, Jonathan P. Tyrer, Jacek Gronwald, Susanne Krüger Kjær, Alice Wittemore, Beth Karlan, Kunle Odunsi, Usha Menon, David D. Bowtell, Ellen L. Goode, Paul D.P. Pharoah, Simon A. Gayther. Whole exome and targeted resequencing, of population based ovarian cancer cases and controls, identifies susceptibility genes for ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-CTRL-1213.

Abstract Genome-wide association studies (GWAS) have identified several susceptibility regions that confer risk of two or more cancers. A region at 19p13 is associated with both breast and ovarian cancer risk in the general population as well as in BRCA1/BRCA2 mutation carriers. Using the iCOGs custom genotyping array, we analyzed 438 SNPs in this region in 7,435 ER-negative breast cancer cases and 42,599 controls from BCAC, and 15,347 ovarian cancer cases and 30,845 controls from OCAC and 15,252 BRCA1 mutation carriers. We identified two significant peaks of association. Peak 1, consisting of 13 highly correlated SNPs (r2&gt;0.95), spans a 19.4kb region harboring the BABAM1, ANKLE1 and ABHD8 genes. Peak 1 SNPs are associated with serous ovarian cancer (top hit rs4808075, P = 9.2×10-20), ER-negative breast cancer (rs61494113, P = 1.1×10-13) and breast cancer in BRCA1 mutation carriers (rs61494113, P = 7.7×10-16). Peak 2 spans over 100kb and contains 7 SNPs associated with breast cancer in BRCA1 mutation carriers only (rs4464206, P = 8.9×10-8). Multiple assays were used to establish the putative functional roles of risk SNPs and candidate susceptibility genes in this region. Expression quantitative trait locus (eQTL) analyses identified significant genotype-gene expression correlations between genotype at the Peak 1 SNP rs480816 and ABHD8 expression in ovarian tumors (P = 3.0×10-5) and normal breast tissues (P = 2.8×10-3). The Peak 2 SNP, rs7246243, was significantly associated with BABAM1 expression (P = 5×10-3) in normal breast tissue only. Overexpression of BABAM1 in normal ovarian and breast cells did not modulate neoplastic phenotypes, whereas overexpression of ABHD8 induced a significant reduction in migration in both cell types in vitro. Differentially expressed genes in ABHD8 models, identified by RNAseq, were associated with cellular movement (P&lt;1.82×10-44). Chromosome conformation capture (3C) identified an interaction between the ABHD8 promoter and a region containing 4 risk SNPs (rs4808075, rs10419397, rs56069439 and rs4808076) in breast and ovarian normal and cancer cells but only rs56069439 coincides with epigenetic enhancer marks in breast and ovarian cells. CRISPR/Cas9 genome editing was used to delete a 57bp region containing rs56069439 and expression of BABAM1, ANKLE1 and ABHD8 were measured. Taken together, these data suggest a common functional mechanism underlying risk of breast and ovarian cancer at this locus, mediated by rs56069439 variants differentially regulating expression of ABHD8, a gene involved in cell motility. Citation Format: Kate Lawrenson, Siddhartha Kar, Karoline Kuckenbaeker, Stacey Edwards, Qiyuan Li, Jonathan Tyrer, Jonathan Beesley, Kyriaki Michailidou, Susan Ramus, Alison Dunning, Fergus Couch, Alvaro Monteiro, Jacques Simard, Matthew Freedman, Douglas Easton, Georgia Chenevix-Trench, Paul Pharoah, Antonis Antoniou, Simon Gayther, OCAC, BCAC and CIMBA. Common functional mechanisms underlying pleiotropy at the 19p13.1 breast and ovarian cancer cusceptibility locus. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2783. doi:10.1158/1538-7445.AM2015-2783

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 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. Clin Cancer Res; 21(23); 5264–76. ©2015 AACR.

Abstract Primary and acquired chemotherapy resistance remains a major challenge in the treatment of ovarian cancer. We hypothesize that heritable polymorphisms might be associated with clinical outcome. Through the Ovarian Cancer Association Consortium, we have collected detailed clinical data from a large number of ovarian cancer patients. We have conducted association analyses of progression-free survival (PFS) and overall survival (OS) for 2,901 European women with ovarian carcinoma for ∼8 million imputed SNPs with minor allele frequency ≥ 0.02 and good imputation quality (imputation r2 ≥ 0.3) from the iCOGS experiment (Pharoah et al Nat Genet. 2013). Cox regression models adjusted for known prognostic factors were used to assess the log-additive effects of SNPs on PFS and OS in 2,825 patients who underwent cytoreductive surgery and received any chemotherapy (‘all chemo’) and in the subset of 1,098 patients who underwent cytoreductive surgery followed by ≥ 4 cycles of carboplatin AUC 5 or 6 and paclitaxel 175 or 135 mg/m2 intravenously (‘standard chemo’). We identified five SNPs at four loci that were significantly associated with outcome (p ≤ 10-7) in at least one of four analyses (Table). Analysis of expression data for genes within a 1Mb region flanking each lead SNP indicates significant associations between nearby genes and ovarian cancer survival. Of particular interest is the heat shock protein crystalline αB (CRYAB) at 11q23.1. We have also identified significant associations with KIAA1598 and RAVER2. Further analysis of these loci in a larger genotyped sample is necessary to definitively identify SNP(s) associated with outcome. Our study adds to the growing evidence that genetic variants are associated with ovarian cancer prognosis. Table: SNPs associated with ovarian cancer outcome and gene expression Overall Survival (P)*Progression-Free Survival (P)*Lead SNPChromosome LocusImputation R²Expression AnalysisAll chemoStandard chemoAll chemoStandard chemors1429768671p31.30.742.14E-0512.69E-044.68E-055.11E-073.64E-09rs559375217q32.30.600.198.58E-082.32E-052.80E-031.85E-03rs7885346210q26.110.602.23E-0724.57E-036.57E-081.65E-016.85E-03rs1089121911q23.10.841.53E-0531.51E-059.50E-081.50E-042.32E-04rs3579103211q23.10.811.53E-0531.66E-055.88E-086.46E-051.87E-04*P-values are adjusted for known confounders of ovarian cancer outcomes; FIGO stage, grade, histology, residual disease, and additionally for age at diagnosis for OS.1 RAVER2; 2 KIAA1598; 3 CRYAB Citation Format: Sharon E. Johnatty, Jonathan Tyrer, Jonathan Beesley, Bo Gao, Yi Lu, Stuart MacGregor, Anna deFazio, Paul Pharoah, Ellen Goode, Georgia Chenevix-Trench. Identification of genetic loci associated with ovarian cancer prognosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3286. doi:10.1158/1538-7445.AM2014-3286

Abstract An ovarian cancer Genome Wide Association Study (GWAS) identified 9p22.2 as a novel susceptibility locus with the most statistically significant single nucleotide polymorphisms (SNPs) located in an intergenic region near the Basonuclin 2 (BNC2) gene, which codes for a putative transcription regulator containing three pairs of zinc finger (ZF) domains. The minor alleles are protective in terms of ovarian cancer susceptibility. However, the molecular mechanisms by which these SNPs modify susceptibility remain largely unknown. The significant SNPs in the 9p22.2 locus lie in non-coding regions and therefore are hypothesized to affect the activity of regulatory elements and modify the expression of a target gene(s). In order to test this hypothesis we conducted fine mapping for the locus which delimited a ∼74kb region containing SNPs with a p-value less than 10-8. FAIRE-Seq and ChIP-Seq experiments for histone markers conducted in immortalized ovarian surface epithelial cells (IOSE) and fallopian tube epithelial cells (IFTE) were used to prioritize functional SNP candidates which overlap with regulatory elements. Luciferase assays tested the ability of these regulatory elements to activate transcription. Chromosome conformation capture (3C) experiments demonstrate a physical interaction between the BNC2 promoter and candidate regulatory elements containing risk-associated SNPs. Methylation Quantification at Trait Loci (mQTL) revealed an association between decreased methylation at the BNC2 promoter and the protective minor alleles. Expression analysis shows decreased expression of BNC2 in cancer versus normal tissue implicating tumor suppressor function of BNC2. Therefore the protective minor allele likely increases expression of BNC2 which in turn contains tumor suppressing properties that decrease ovarian cancer risk. BNC2 protein is in complex with transcriptional regulatory proteins, in particular the NURD complex components. Moreover, BNC2 acts as a transcriptional repressor in in vitro transfection assays indicating that it functions in transcriptional repression. We then used protein binding microarrays and CHIP-Seq experiments to identify its putative DNA binding motifs and its downstream target genes. Analysis of this data suggests that BNC2 functions in a regulatory transcription network that impacts on genes implicated in ovarian cancer with enrichment for genes in the TGF-beta response pathway. Citation Format: Melissa A. Buckley, Howard C. Shen, Gustavo A. Mendoza-Fandino, Nicholas T. Woods, Anxhela Gjyshi, Juliet French, Kate Lawrenson, Honglin Song, Jonathan Tyrer, Renato S. Carvalho, Alexandra Valle, Ann Chen, Sean Yoder, Gregory Bloom, Ya-Yu Tsai, Ally Yang, Timothy R. Hughes, Xiaotao Qu, Mine Cicek, Melissa Larson, Ellen Goode, Brooke Fridley, Susan Ramus, Georgia Chenevix-Trench, Paul Pharoah, Thomas A. Sellers, Simon Gayther, Alvaro N.A. Monteiro, Ovarian Cancer Association Consortium. Functional analysis of the 9p22 locus implicates the transcriptional regulation of BNC2 as a mechanism in ovarian cancer predisposition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3285. doi:10.1158/1538-7445.AM2014-3285