Fogarty, Zachary C.

MicroRNAs (miRNAs) regulate up to one‐third of all protein‐coding genes including genes relevant to cancer. Variants within miRNAs have been reported to be associated with prognosis, survival, response to chemotherapy across cancer types, in vitro parameters of cell growth, and altered risks for development of cancer. Five miRNA variants have been reported to be associated with risk for development of colorectal cancer (CRC). In this study, we evaluated germline genetic variation in 1,123 miRNAs in 899 individuals with CRCs categorized by clinical subtypes and in 204 controls. The role of common miRNA variation in CRC was investigated using single variant and miRNA‐level association tests. Twenty‐nine miRNAs and 30 variants exhibited some marginal association with CRC in at least one subtype of CRC. Previously reported associations were not confirmed (n = 4) or could not be evaluated (n = 1). The variants noted for the CRCs with deficient mismatch repair showed little overlap with the variants noted for CRCs with proficient mismatch repair, consistent with our evolving understanding of the distinct biology underlying these two groups. © 2016 The Authors Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc.

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.

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.

Abstract Background: Epithelial ovarian cancer (EOC) is composed of five major histologic types: 1) high-grade serous carcinoma (HGSC), accounting for most cases (∼70%); and the rarer 2) clear cell, 3) endometrioid, 4) mucinous, and 5) low- grade serous carcinoma (LGSC). As EOC risk factors are histology specific, as are the site of precursor lesions, accurate subtyping is critical to understanding EOC prognostic factors and etiology. Our aim was to compare two currently employed histology assignment strategies using gene expression, DNA methylation, and clinical outcome data Methods: Histology assignment based on: a) pathologist, and b) an integrated pathologist and IHC prediction algorithm (pathIHC) (using ARID1A, CDKN2A, DKK1, HNF1B, MDM2, PGR, TP53, TFF3, VIM, and WT1 staining patterns), was compared for tumors of all histologies at the Mayo Clinic, Rochester. The 500 most variable probes from Illumina Methylation450 BeadChips (N = 259) and Agilent 4×44K expression arrays (N = 245) were used to perform unsupervised hierarchical clustering. Fisher's Exact test was used to test the association of clusters with histology. Cox proportional hazards regression analysis was used to test the association of clusters and histology with time to progression (TTP) and time to death (TTD). Results: Eighty-two percent of tumors were concordant between histology assignment strategies. Clustering based on methylation data produced two distinct clusters. PathIHC produced more homogenous clusters (p-value = 2.8×10-16) than pathology alone (p = 1.3×10-9). Cluster M1, characterized by high levels of methylation across almost all probes, was enriched for the rarer EOC subtypes; cluster M2, characterized by moderate levels of methylation across 50%-75% of probes, was predominantly HGSC. These patterns were observed irrespective of histology assignment strategy; however, when using histology by pathologist, cluster M2 had more endometrioid and LGSC tumors interspersed with HGSC tumors. Clustering based on expression data also produced two clusters. PathIHC produced slightly more homogenous clusters (p = 9.0×10-11, pathology alone, p = 2.3×10-10). Cluster E1 was enriched for the rarer EOC subtypes; cluster E2 was primarily serous lineage tumours (HGSC and LGSC), particularly when using histology by pathIHC. Considering only clinical outcomes, histologies by pathology were more significantly different in terms of TTP (p = 1.1×10-6) and TTD (p = 1.7×10-3) than histologies by pathIHC (TTP, p = 5.4×10-6; TTD, p = 5.7×10-3). Conclusions: Histology by pathIHC produced more homogeneous clusters in both the methylation and expression data; thus, molecular data supports this strategy. Differences in clinical outcomes (TTP and TTD) between histology groups were more pronounced when using assignment by pathologist; thus, clinical data supports this strategy. Citation Format: M A. Earp, S J. Winham, S M. Armasu, B L. Fridley, M C. Larson, Z C. Fogarty, K R. Kalli, C Wang, G L. Keeney, J M. Cunningham, S Ramus, M Kobel, E L. Goode. Comparison of pathology versus IHC-based ovarian carcinoma histology assignment using gene expression, DNA methylation, and clinical outcome data. [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 4246. doi:10.1158/1538-7445.AM2015-4246

Abstract Many germline genetic variants have been found to be associated with susceptibility to epithelial ovarian cancer (EOC), but currently few associations have been identified for EOC outcomes such as overall survival. In the absence of evidence for common germline markers, it is possible that rare variants not captured in GWAS arrays may be associated with overall survival. We hypothesize that rare variants, either individually or combined across a gene, may be associated with overall survival in EOC. We utilized data from two genotyping projects of the Ovarian Cancer Association Consortium (OCAC) that used commercial exome-based arrays. The primary sample, genotyped on the Affymetrix Axiom Exome Array at 227,892 standard and custom variants, consisted of 14 independent EOC studies (4293 cases; 2257 deaths). The secondary sample, genotyped on the Illumina Infinium HumanExome BeadChip at 114,620 variants, consisted of six additional EOC studies (1744 cases; 1027 deaths). Both sets were analyzed using Cox proportional hazards regression to determine the association of each variant with overall survival time. Meta-analysis was conducted across samples at 73,203 overlapping variants. Gene-level tests of over 18,000 genes were also conducted using burden tests and Sequencing Kernel Association Tests (SKAT) adapted for Cox proportional hazards models. Gene-level tests were conducted separately for the primary and secondary cases and then were meta-analyzed. Models were adjusted for age, site, and three principle components. Six individual variants had meta-analysis p-values < 1.0E-4, five of which were rare (MAF < 0.003). Common variant rs8170 (MAF = 0.2) in BABAM1 encoding BRISC and BRCA1 A complex member 1 - previously reported to be associated with EOC risk_showed evidence of association with overall survival (Pmeta = 1.5E-5) and consistent estimates of effect size across both samples (HR1 = 1.18, HR2 = 1.19). In imputed genotype data from over 18,000 subjects from OCAC genotyped on the COGS array, this association reaches genome-wide significance (HR = 1.15, P = 4.7E-9). Five genes had gene-level meta-analysis p-values < 1.0E-4, including top ranked ATG2B (p = 1.1E-6), an autophagy gene important for apoptosis; zinc finger protein PEG3 (p = 1.8E-5), playing a role in autophagy and down-regulated in EOC; helicase-like transcription factor HLTF (p = 1.9E-5), involved in post-replication DNA repair; mitochondrial fission regulator 1-like gene FAM54B (p = 3.1E-5); and T-cell surface glycoprotein CD1E (p = 8.8E-5). With over 6000 EOC cases in the primary and secondary samples, this is the largest genome-wide assessment of rare variant genetic associations with EOC overall survival. Variant rs8170 in BABAM1, a necessary component of the BRCA1 complex, is associated with overall survival. Single variant and gene-level analyses provide complementary approaches, suggesting additional candidates that warrant follow-up study. Citation Format: Stacey J. Winham, Brooke L. Fridley, Melissa C. Larson, Zachary Fogarty, Andrew Berchuck, Yian Ann Chen, Hui-Yi Lin, Georgia Chenevix-Trench, Jenny Permuth-Wey, Thomas A. Sellers, Ailith Pirie, Ellen L. Goode, Ovarian Cancer Association Consortium. Investigation of exome variants associated with overall survival in ovarian cancer. [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 4636. doi:10.1158/1538-7445.AM2015-4636

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 Tumor infiltrating regulatory T cells (Tregs) appear to play a role in survival in a number of solid tumors, including ovarian cancer. We assessed genetic variation via tag SNPs (N=749) 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. SNPs were selected based on r2 ≥ 0.8, MAF ≥ 0.05, 5 kb upstream and downstream from the gene. We had SNP and outcome data for ovarian cancer from 10,084 combined ovarian cancer cases, including 5,248 high grade serous, 1,452 endometrioid, 661 mucinous, and 795 clear cell cases of European descent across multiple studies from the Ovarian Cancer Association Consortium (OCAC). Cox proportional hazards regression modeling was used to estimate per-allele hazard ratios (HRs) and 95% confidence intervals (CIs) for associations with overall survival, adjusting for age at diagnosis, population substructure PCs, study site, histology (for the all case analysis), tumor stage, tumor grade and oral contraceptive use. The strongest associations were found for endometrioid histologic subtype and IL2RA SNPs (maximum r2=0.67) rs11256497 [HR=1.42, 95% CI: 1.22-1.64; p=5.7 x 10−6], rs791587 [HR=1.36, 95% CI: 1.17-1.57; p=6.2 x 10−5], rs2476491 [HR=1.40, 95% CI: 1.19-1.64; p=5.6 x 10−5], and rs10795763 [HR=1.35, 95% CI: 1.17-1.57; p=7.9 x 10−5], as well as clear cell ovarian cancer and CTLA4 SNP rs231775 [HR=0.67, 95% CI: 0.54-0.82; p=9.3 x 10−5]. No other associations were observed at p<1 x 10−4. Overall, this study provided evidence that variation in genes related to Tregs, particularly IL2RA and CTLA4, influence ovarian cancer survival. Citation Format: Bridget Charbonneau, Keith Knutson, Robert A. Vierkant, Julie M. Cunningham, Zachary C. Fogarty, Kimberly R. Kalli, Matthew J. Maurer, Ann L. Oberg, Brooke L. Fridley, Claudia C. Preston, Paul D.P. Pharoah, Susan J. Ramus, Catherine M. Phelan, Kunle Odunsi, Kirsten Moysich, Ellen L. Goode. Polymorphisms in regulatory T cell related genes and ovarian cancer survival. [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 4844. doi:10.1158/1538-7445.AM2013-4844

Abstract Colorectal cancer (CRC) is estimated to be the third most common cancer in both men and women in the US in 2012, accounting for over 50,000 deaths. Familial CRC accounts for approximately 30% of all CRC, yet only 5% of familial CRCs can be attributed to mutations in known hereditary colon cancer (HCC) genes. Many families with a strong family history of CRC do not have mutations in known CRC genes. Finding the genes underpinning CRC in these families may improve screening for disease and allow the development of more targeted therapeutics. We completed targeted sequencing of over 2,000 genes and miRNAs in 1,500 cases of CRC from the Colon Cancer Family Registry (CCFR) in an effort to identify rare variants associated with CRC risk and to identify mutations in known CRC genes missed by conventional sequencing. Several categories of genes were included: 18 known HCC genes; 18 candidate HCC genes; 185 genes in previously reported CRC linkage peaks; 1,423 miRNAs; and 363 genes identified through whole exome sequencing of 40 affected cases in 16 families with hereditary CRC (n=2,007 total genes sequenced). Genomic DNA samples were indexed, then libraries were made using Agilent's SureSelect Targeted Enrichment kit. Samples were pooled (96 samples/pool), then each pool of samples was run on five lanes on an Illumina HiSeq2000 (average of 19.2 samples/lane). GenomeGPS, an in-house developed workflow, was used for read alignment, variant calling, and annotation. In early analyses of the first 161 cases, the mean number of mapped reads exceeded 20 million per sample and over 90% of the target region exceeded 40x coverage. A total of 26,484 SNPs were identified and passed quality filtering. Sequencing identified known mutations in MMR genes in positive control cases as well as mutations in HCC genes in previously untested cases. Analysis is currently on-going on the remaining samples and we will report results on the full set of 1500 cases at the meeting. Citation Format: Melissa S. DeRycke, Shanaka R. Gunawardena, Sumit Middha, Shannon K. McDonnell, Shaun M. Riska, Zachary C. Fogarty, Bruce W. Eckloff, Daniel J. Schaid, Ellen L. Goode, Stephen N. Thibodeau. Targeted sequencing to identify rare variants in colorectal cancer. [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 2548. doi:10.1158/1538-7445.AM2013-2548