Song, Honglin

Abstract Mi(cro)RNAs are short non-coding RNA molecules that play a key role in carcinogenesis by regulating tumor suppressors and oncogenes. Published data has implicated miRNAs in ovarian cancer (OC) development and progression, and we hypothesize that single nucleotide polymorphisms (SNPs) in sites of miRNA: messenger RNA (mRNA) binding may influence OC risk by altering expression levels of targeted mRNAs. To evaluate associations between SNPs in miRNA binding sites and OC risk, we used bioinformatics tools and public databases to identify miRNA binding SNPs relevant to ovarian cancer. We then evaluated the frequency of approximately 1,100 SNPs in ∼20,000 epithelial OC cases and 20,000 controls represented in the international Collaborative Oncological Gene-environment Study. Unconditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI) between genotypes and case status, with adjustment for the first five principal components representing European ancestry. Log-additive genetic models were applied to each SNP, modeling the number of copies of the minor allele. Subgroup analysis was conducted for serous adenocarcinomas, the most predominant histologic subtype of epithelial OC. Preliminary analysis of 11,215 invasive epithelial OCs and 15,910 controls included four SNPs with P<10−6 and 1 SNP with P<10−5. Analysis of 6,215 invasive serous adenocarcinomas revealed two SNPs with P<10−7 and three SNPs with P<10−6. Noteworthy is rs6104808, a C>T SNP predicted to reside in a highly conserved miRNA binding site of BTBD3 (BTB POZ domain containing 3), a transcription factor suggested to be involved in cell proliferation, cancer progression, and response to platinum-based chemotherapy. rs6104808 was associated with a decreased risk of OC overall (OR (95% CI): 0.42 (0.28-0.62), P=1.26 x 10−5)), and the association was stronger among cases with serous adenocarcinoma (OR (95% CI): 0.30 (0.17-0.50), P=9.89 x 10−6)). This represents the largest, most comprehensive epidemiologic study to date to evaluate associations between miRNA binding site SNPs and OC susceptibility. Although based on a preliminary analysis, these data suggest a significant role and putative biological mechanism for common germline variants in miRNA binding sites leading to cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2927. doi:1538-7445.AM2012-2927

Abstract Ovarian cancer is the leading cause of death in gynecologic malignancies in the United States with most patients presenting with late stage disease; patients receive the same treatment regardless of histology or tumor characteristics. The mismatch repair (MMR) molecular pathway is well-defined and known to be involved in up to 20% of ovarian cancers; however, association of deficient v proficient MMR status (dMMR v pMMR status) with risk factors and outcome is not well understood. We are conducting extensive molecular testing of the MMR genes MLH1, MSH2, MSH3, MSH6, and PMS2 on Mayo Clinic invasive epithelial ovarian cancer cases. First, the prevalence of dMMR tumors will be estimated in ovarian cancer cases (n=496) based on loss of expression of MLH1, MSH2, MSH3, MSH6, and PMS2 determined by immunohistochemistry staining of primary antibodies using negative and positive controls. Staining of tissue microarrays (TMAs) is complete; triplicate cores will be scored independently for presence or loss of protein expression by a board-certified pathologist. Second, for those dMMR cases, the possible cause of MMR deficiency will be evaluated considering germline and somatic mutations. The loss of expression of each protein will be characterized as germline if a deleterious mutation is present in peripheral blood or somatic if the pathogenic mutation is only present in tumor tissue. In cases with MLH1 loss somatic hypermethylation will also be considered. Analysis of germline mutation data will include all 496 cases and somatic mutations data will include a subset of cases tested with frozen tumor DNA (n=292) using Fluidigm access array targeted resequencing. Third, the clinical features of dMMR disease will be compared to those of pMMR; in particular, age at diagnosis, histopathology, and disease outcome. Among cases in this analysis, the majority have high-grade serous disease with a mean age at diagnosis of 61 years, about half had recurrence or progression (n=243) of which two-thirds (n=167) are deceased. Finally, known and suspected ovarian cancer risk factors (family history, smoking, body mass index, parity, use of hormone therapy, and genetic variation in susceptibility loci at 2q31, 3q25, 8q24, 9p22, 17q21, and 19p13) will be analyzed by MMR phenotype. Risk factor questionnaire data is available on 462 cases (93%), as well as 737 population controls with germline genome-wide genotype data. About half of the cases (n=231) report a 1st or 2nd degree family member with breast or ovarian cancer. Given the limited knowledge about ovarian cancer etiology and the poor prognosis of advanced disease, it is very important to study risk factors with this molecular subtype. The ovarian cancer dMMR phenotype may have distinct clinicopathological features that could guide risk stratification and be useful in diagnostics and therapeutics. This is the largest study characterizing MMR in an ovarian cancer population to date. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2619. doi:1538-7445.AM2012-2619

Abstract As pathways of the immune system play an important role in multiple malignancies, we assessed the association between single-nucleotide polymorphisms (SNPs) in inflammation-related genes and risk of ovarian cancer in a multi-site case-control study. Using a custom array, we genotyped 930 epithelial ovarian cancer cases and 1,037 controls for SNPs in ALOX12, ALOX15, ALOX5, CCL11, CCL2, CCL3, CCR3, CRP, CXCL16, IL10, IL15RA, IL18, IL1A, IL1B, IL1RN, IL4R, IL6, IL6R, IL7R, IL8RA, IL8RB, IL9, NOS3, PTGS1, PTGS2, TLR2, and TNF. After analysis by logistic regression, SNPs with p<0.10 were evaluated in an independent data set of 3,143 cases and 2,102 controls from the Follow-up of Ovarian Cancer Genetic Association and Interaction Studies (FOCI) collaboration. Combined analysis revealed association with interleukin 1, alpha (IL1A) SNPs (rs17561 and rs4848300, r2=0.990, p=0.005) which varied by histological subtype (heterogeneity p=0.03). For example, IL1A rs17561, which results in a missense change in amino acid at position 114 and correlates with multiple inflammatory phenotypes, was inversely associated with risk of clear cell, mucinous, and endometrioid subtypes, but not with the serous subtype (Table 1). Additional data on IL1A rs17561 in an even larger collection of ovarian cancer cases and controls are pending and will be analyzed soon. We also found that genotype at rs1864414 in ALOX5 (arachidonate 5-lipoxygenase) was associated with decreased risk combined across subtypes (OR 0.86, 95% CI 0.78-0.95; p=0.002). Thus, inherited variation in IL1A and ALOX5 appears to have a role in ovarian cancer risk, though it is limited to rarer subtypes for IL1A. These data add to existing literature on the importance of inflammation in tumorigenesis and growing evidence of subtype-specific traits in ovarian cancer and may provide important clues on prevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2603. doi:1538-7445.AM2012-2603

Abstract Background: Genome-wide association studies (GWAS) for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy, have identified novel susceptibility loci. GWAS for survival after EOC have had more limited success. The association of each single-nucleotide polymorphism (SNP) individually may not be well suited to detect small effects of multiple SNPs, such as those operating within the same biologic pathway. Gene set analysis (GSA) overcomes this limitation by assessing overall evidence for association of a phenotype with all measured variation in a set of genes. Methods: To determine gene sets associated with EOC overall survival, we conducted GSA using data from two large GWAS (N cases = 2,813, N deaths = 1,116), with a novel Principal Component-Gamma GSA method. Analysis was completed for all cases and then separately for high-grade serous histologic subtype. Results: Analysis of the high-grade serous subjects resulted in 43 gene sets with P < 0.005 (1.7%); of these, 21 gene sets had P < 0.10 in both GWAS, including intracellular signaling pathway (P = 7.3 × 10−5) and macrolide binding (P = 6.2 × 10−4) gene sets. The top gene sets in analysis of all cases were meiotic mismatch repair (P = 6.3 × 10−4) and macrolide binding (P = 1.0 × 10−3). Of 18 gene sets with P < 0.005 (0.7%), eight had P < 0.10 in both GWAS. Conclusion: This research detected novel gene sets associated with EOC survival. Impact: Novel gene sets associated with EOC survival might lead to new insights and avenues for development of novel therapies for EOC and pharmacogenomic studies. Cancer Epidemiol Biomarkers Prev; 21(3); 529–36. ©2012 AACR.

Abstract The importance of inflammation pathways to the development of many human cancers prompted us to examine the associations between single-nucleotide polymorphisms (SNP) in inflammation-related genes and risk of ovarian cancer. In a multisite case–control study, we genotyped SNPs in a large panel of inflammatory genes in 930 epithelial ovarian cancer cases and 1,037 controls using a custom array and analyzed by logistic regression. SNPs with P < 0.10 were evaluated among 3,143 cases and 2,102 controls from the Follow-up of Ovarian Cancer Genetic Association and Interaction Studies (FOCI) post-GWAS collaboration. Combined analysis revealed association with SNPs rs17561 and rs4848300 in the interleukin gene IL1A which varied by histologic subtype (Pheterogeneity = 0.03). For example, IL1A rs17561, which correlates with numerous inflammatory phenotypes, was associated with decreased risk of clear cell, mucinous, and endometrioid subtype, but not with the most common serous subtype. Genotype at rs1864414 in the arachidonate 5-lipoxygenase ALOX5 was also associated with decreased risk. Thus, inherited variation in IL1A and ALOX5 seems to affect ovarian cancer risk which, for IL1A, is limited to rarer subtypes. Given the importance of inflammation in tumorigenesis and growing evidence of subtype-specific features in ovarian cancer, functional investigations will be important to help clarify the importance of inherited variation related to inflammation in ovarian carcinogenesis. Cancer Res; 72(5); 1064–9. ©2012 AACR.

Abstract Background: Single nucleotide polymorphisms (SNP) in microRNA-related genes have been associated with epithelial ovarian cancer (EOC) risk in two reports, yet associated alleles may be inconsistent across studies. Methods: We conducted a pooled analysis of previously identified SNPs by combining genotype data from 3,973 invasive EOC cases and 3,276 controls from the Ovarian Cancer Association Consortium. We also conducted imputation to obtain dense coverage of genes and comparable genotype data for all studies. In total, 226 SNPs within 15 kb of 4 miRNA biogenesis genes (DDX20, DROSHA, GEMIN4, and XPO5) and 23 SNPs located within putative miRNA binding sites of 6 genes (CAV1, COL18A1, E2F2, IL1R1, KRAS, and UGT2A3) were genotyped or imputed and analyzed in the entire dataset. Results: After adjustment for European ancestry, no overall association was observed between any of the analyzed SNPs and EOC risk. Conclusions: Common variants in these evaluated genes do not seem to be strongly associated with EOC risk. Impact: This analysis suggests earlier associations between EOC risk and SNPs in these genes may have been chance findings, possibly confounded by population admixture. To more adequately evaluate the relationship between genetic variants and cancer risk, large sample sizes are needed, adjustment for population stratification should be carried out, and use of imputed SNP data should be considered. Cancer Epidemiol Biomarkers Prev; 20(8); 1793–7. ©2011 AACR.

Abstract Defective microRNA (miRNA) biogenesis contributes to the development and progression of epithelial ovarian cancer (EOC). In this study, we examined the hypothesis that single nucleotide polymorphisms (SNP) in miRNA biogenesis genes may influence EOC risk. In an initial investigation, 318 SNPs in 18 genes were evaluated among 1,815 EOC cases and 1,900 controls, followed up by a replicative joint meta-analysis of data from an additional 2,172 cases and 3,052 controls. Of 23 SNPs from 9 genes associated with risk (empirical P < 0.05) in the initial investigation, the meta-analysis replicated 6 SNPs from the DROSHA, FMR1, LIN28, and LIN28B genes, including rs12194974 (G>A), an SNP in a putative transcription factor binding site in the LIN28B promoter region (summary OR = 0.90, 95% CI: 0.82–0.98; P = 0.015) which has been recently implicated in age of menarche and other phenotypes. Consistent with reports that LIN28B overexpression in EOC contributes to tumorigenesis by repressing tumor suppressor let-7 expression, we provide data from luciferase reporter assays and quantitative RT-PCR to suggest that the inverse association among rs12194974 A allele carriers may be because of reduced LIN28B expression. Our findings suggest that variants in LIN28B and possibly other miRNA biogenesis genes may influence EOC susceptibility. Cancer Res; 71(11); 3896–903. ©2011 AACR.

Abstract Genome-wide association analyses have identified an ovarian cancer susceptibility locus near BNC2 at 9p22.2 (OR 0.82, p 5.1 × 10−19, Nat Genet 41:996-1000, 2009), based on 1,772 and 2,354 Stage I cases and controls, 4,803 and 5,237 Stage 2 cases and controls, and extensive Stage 3 analysis in independent samples. Here, we report on Stage 3 analysis of nine additional regions with Stage 2 results at p < 10−4. Stage 1 samples were genotyped using the Illumina 610Quad and imputed at up to 2 million SNPs; Stage 2 samples were genotyped on an Illumina iSelect of 22,790 SNPs. Stage 3 samples from 4,335 cases (including 2,197 serous subtype) and 5,951 controls (14 studies) were genotyped using Illumina or Sequenom at up to 32 SNPs on chromosomes 1, 2, 3, 7, 8, 11, and 17. No evidence of heterogeneity by study site was observed. Logistic regression revealed four regions with ordinal p ≤ 10−7 in all cases or serous subtype only. The most significant finding for all cases was for rs2072590 at 2q31.1 (all cases OR 1.15, p=8.1 × 10−13; serous only OR 1.19 p=9.0 × 10−13) which is located 3.8 kb 3-prime of HOXD3, a homeobox gene which also had an intronic SNP (rs2301301) with p=3.1 × 10−5 (all cases). Second, we observed a locus at 8q24.21 highlighted by rs10088218 (all cases OR 0.84, p=1.3 × 10−9; serous only OR 0.75 p=2.2 × 10−14). This locus resides on the opposite side of MYC and 1.3 Mb from the 8q24 regions harboring loci for other cancers, and is independent of the other cancer-associated variants (r2 < 0.02). Third, at 3q25.31, promising results were observed at rs2665390, a SNP intronic to TIPARP (TiPARP, TCDD-inducible poly(ADP-ribose) polymerase; all cases OR 1.19, p=8.7 × 10−7; serous only OR 1.26, p=3.2 × 10−8). Finally, at 17q21.32, rs9303542 was associated with risk particularly among serous cases (all cases OR 1.13, p=1.5 × 10−6; serous only OR 1.16 p=4.3 × 10−7); this SNP is intronic to SKAP1 (src kinase associated phosphoprotein 1). We evaluated the functional role of genes at these loci in ovarian cancer and normal ovarian epithelial cells and in an in vitro model of neoplastic initiation and progression from normal ovarian epithelial cells after CYMC and KRAS activation. Both BNC2 and TiPARP were down regulated in cancer versus normal cells (p=5 × 10−9 and p=3.4 × 10−5, respectively) suggesting that loss of function is required for cancer development. Over-expressing CMYC (at 8q24) induced neoplastic transformation of normal ovarian cells. Both BNC2 and TiPARP were down-regulated following CMYC over-expression and showed significant loss of function with further transformation after mutant KRAS activation (p=7.8 × 10−7 and p=6.3 × 10−4, respectively). These results emphasize the importance of genome-wide association studies and consortia for the identification of moderate-risk alleles in rare cancers. Importantly, our findings unravel the genetic components of this disease and suggest functionally-significant novel targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3860.

Abstract Aberrant glycosylation is a well-described hallmark of cancer. In a previous ovarian cancer case control study that examined polymorphisms in 26 glycosylation-associated genes, we found strong statistical evidence (P = 0.00017) that women who inherited two copies of a single-nucleotide polymorphism in the UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase, GALNT1, had decreased ovarian cancer risk. The current study attempted to replicate this observation. The GALNT1 single-nucleotide polymorphism rs17647532 was genotyped in 6,965 cases and 8,377 controls from 14 studies forming the Ovarian Cancer Association Consortium. The fixed effects estimate per rs17647532 allele was null (odds ratio, 0.99; 95% confidence interval, 0.92-1.07). When a recessive model was fit, the results were unchanged. Test for hetero geneity of the odds ratios revealed consistency across the 14 replication sites but significant differences compared with the original study population (P = 0.03). This study underscores the need for replication of putative findings in genetic association studies. Cancer Epidemiol Biomarkers Prev; 19(2); 600–4

Abstract We genotyped 13 single nucleotide polymorphisms (SNPs) in the estrogen receptor alpha gene (ESR1) region in three population-based case-control studies of epithelial ovarian cancer conducted in the United States, comprising a total of 1,128 and 1,866 non-Hispanic white invasive cases and controls, respectively. A SNP 19 kb downstream of ESR1 (rs2295190, G-to-T change) was associated with invasive ovarian cancer risk, with a per-T-allele odds ratio (OR) of 1.24 [95% confidence interval (CI), 1.06-1.44, P = 0.006]. rs2295190 is a nonsynonymous coding SNP in a neighboring gene called spectrin repeat containing, nuclear envelope 1 (SYNE1), which is involved in nuclear organization and structural integrity, function of the Golgi apparatus, and cytokinesis. An isoform encoded by SYNE1 has been reported to be downregulated in ovarian and other cancers. rs2295190 was genotyped in an additional 12 studies through the Ovarian Cancer Association Consortium, with 5,279 invasive epithelial cases and 7,450 controls. The per-T-allele OR for this 12-study set was 1.09 (95% CI, 1.02-1.17; P = 0.017). Results for the serous subtype in the 15 combined studies were similar to those overall (n = 3,545; OR, 1.09; 95% CI, 1.01-1.18; P = 0.025), and our findings were strongest for the mucinous subtype (n = 447; OR, 1.32; 95% CI, 1.11-1.58; P = 0.002). No association was observed for the endometrioid subtype. In an additional analysis of 1,459 borderline ovarian cancer cases and 7,370 controls, rs2295190 was not associated with risk. These data provide suggestive evidence that the rs2295190 T allele, or another allele in linkage disequilibrium with it, may be associated with increased risk of invasive ovarian cancer. Cancer Epidemiol Biomarkers Prev; 19(1); 244–50