Sellers, Thomas A.

Abstract B39 Background The breast glandular epithelium is composed of lobules that undergo age-related involution that increases as women age and has been associated with lowered breast cancer risk. Mammographic density (MD) is an established risk factor for breast cancer that has been shown to decrease with age. This study examined the association of age-related lobular involution with mammographic breast density. Materials and Methods The study sample was selected from approximately 20,000 healthy women who were enrolled in a screening cohort at the Mayo Clinic (Mayo Mammography Health Study) from 2003 to 2006 and had a benign breast biopsy (BBD) within 2 years of the enrollment mammogram. A quantitative measure of mammographic density, percent density (PD) was estimated on the enrollment mammogram, in addition to its components dense and nondense area (NDA). All biopsies were reviewed by a breast pathologist for the type of benign breast disease (BBD) as well as lobular involution status. Involution was defined as the proportion of terminal ductal lobular units (TDLUs) in the background tissue on biopsy that showed at least 50% decrease in size and number of acini from normal lobules and was subjectively assessed as: No- 0% involution, Partial- 1 to 74%, or Complete- >75% involution. Mean density was estimated for the 3 categories of involution using generalized linear models and square root transformation. Confounders were assessed including age, parity, body mass index (BMI), type of BBD, menopausal status, age at first birth and family history Results A total of 317 women from the MMHS had BBD within 2 years of enrollment. Mean age at BBD diagnosis in this sample was 55 years. Age- and BMI- adjusted analyses suggested an inverse association of involution and percent density with adjusted mean PD values of 23.3 (95%CI [17.2, 30.3]) for no involution, 21.9 (95%CI [20.0, 23.9]) for partial involution, and 17.8 (95% CI [14.7, 21.3]) for complete involution (p for trend 0.05). This trend was driven primarily by the positive association of involution with nondense area with adjusted mean NDA values of 103.1cm2 (95% CI [86.1, 121.7]) for no involution, 104.9 cm2 (95% CI [99.4, 110.7]) for partial involution, and 126.2 cm2 (95% CI [115.0, 137.9]) for complete involution (p for trend 0.004). Discussion Our findings suggest that MD is inversely associated with involution of breast tissues, providing a plausible mechanism through which MD influences breast cancer risk. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B39.

Abstract Background: Overexpression of mitotic kinases has been associated with prognosis, histologic grade, and clinical stage in ovarian cancer, but the relationship between inherited variation in these genes and ovarian cancer risk has not been well defined. Methods: We measured associations between 397 single nucleotide polymorphisms (SNPs) from 67 mitotic kinases and invasive epithelial ovarian cancer risk in two case–control studies (n = 671 cases; n = 939 controls). Thirty-six candidate SNPs (P < 0.05) were assessed in a replication analysis consisting of three additional studies (n = 1,094 cases; n = 829 controls). Results: In initial analysis, thirty-six SNPs were suggestive of association with risk of serous ovarian cancer, all subtypes of ovarian cancer, or both (P < 0.05). Replication analyses suggested an association between rs2125846 in the Nemo-like kinase (NLK) gene and ovarian cancer (serous OR = 1.36, 95% CI: 1.11–1.67, P = 1.77 × 10−3; all subtypes OR = 1.30, 95% CI: 1.08–1.56, P = 2.97 × 10−3). Furthermore, rs2125846 was associated with risk in the combined discovery and replication sets (serous OR = 1.33, 95% CI: 1.15–1.54; all subtypes OR = 1.27, 95% CI: 1.12–1.45). Conclusions: Variation in NLK may be associated with risk of invasive epithelial ovarian cancer. Further studies are needed to confirm and understand the biologic relationship between this mitotic kinase and ovarian cancer risk. Impact: An association between SNPs in NLK and ovarian cancer may provide biologic insight into the development of this disease. Cancer Epidemiol Biomarkers Prev; 21(3); 523–8. ©2012 AACR.

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: Invasive ovarian cancer is a significant cause of gynecologic cancer mortality. Methods: We examined whether this mortality was associated with inherited variation in approximately 170 candidate genes/regions [993 single-nucleotide polymorphisms (SNPs)] in a multistage analysis based initially on 312 Mayo Clinic cases (172 deaths). Additional analyses used The Cancer Genome Atlas (TCGA; 127 cases, 62 deaths). For the most compelling gene, we immunostained Mayo Clinic tissue microarrays (TMA, 326 cases) and conducted consortium-based SNP replication analysis (2,560 cases, 1,046 deaths). Results: The strongest initial mortality association was in HGF (hepatocyte growth factor) at rs1800793 (HR = 1.7, 95% CI = 1.3–2.2, P = 2.0 × 10−5) and with overall variation in HGF (gene-level test, P = 3.7 × 10−4). Analysis of TCGA data revealed consistent associations [e.g., rs5745709 (r2 = 0.96 with rs1800793): TCGA HR = 2.4, CI = 1.4–4.1, P = 2.2 × 10−3; Mayo Clinic + TCGA HR = 1.6, CI = 1.3–1.9, P = 7.0 × 10−5] and suggested genotype correlation with reduced HGF mRNA levels (P = 0.01). In Mayo Clinic TMAs, protein levels of HGF, its receptor MET (C-MET), and phospho-MET were not associated with genotype and did not serve as an intermediate phenotype; however, phospho-MET was associated with reduced mortality (P = 0.01) likely due to higher expression in early-stage disease. In eight additional ovarian cancer case series, HGF rs5745709 was not associated with mortality (HR = 1.0, CI = 0.9–1.1, P = 0.87). Conclusions: We conclude that although HGF signaling is critical to migration, invasion, and apoptosis, it is unlikely that HGF genetic variation plays a major role in ovarian cancer mortality. Furthermore, any minor role is not related to genetically-determined expression. Impact: Our study shows the utility of multiple data types and multiple data sets in observational studies. Cancer Epidemiol Biomarkers Prev; 20(8); 1638–48. ©2011 AACR.

Abstract Background: Mitochondria contribute to oxidative stress, a phenomenon implicated in ovarian carcinogenesis. We hypothesized that inherited variants in mitochondrial-related genes influence epithelial ovarian cancer (EOC) susceptibility. Methods: Through a multicenter study of 1,815 Caucasian EOC cases and 1,900 controls, we investigated associations between EOC risk and 128 single nucleotide polymorphisms (SNPs) from 22 genes/regions within the mitochondrial genome (mtDNA) and 2,839 nuclear-encoded SNPs localized to 138 genes involved in mitochondrial biogenesis (BIO, n = 35), steroid hormone metabolism (HOR, n = 13), and oxidative phosphorylation (OXP, n = 90) pathways. Unconditional logistic regression was used to estimate OR and 95% CI between genotype and case status. Overall significance of each gene and pathway was evaluated by using Fisher's method to combine SNP-level evidence. At the SNP level, we investigated whether lifetime ovulation, hormone replacement therapy (HRT), and cigarette smoking were confounders or modifiers of associations. Results: Interindividual variation involving BIO was most strongly associated with EOC risk (empirical P = 0.050), especially for NRF1, MTERF, PPARGC1A, ESRRA, and CAMK2D. Several SNP-level associations strengthened after adjustment for nongenetic factors, particularly for MTERF. Statistical interactions with cigarette smoking and HRT use were observed with MTERF and CAMK2D SNPs, respectively. Overall variation within mtDNA, HOR, and OXP was not statistically significant (empirical P > 0.10). Conclusion: We provide novel evidence to suggest that variants in mitochondrial biogenesis genes may influence EOC susceptibility. Impact: A deeper understanding of the complex mechanisms implicated in mitochondrial biogenesis and oxidative stress may aid in developing strategies to reduce morbidity and mortality from EOC. Cancer Epidemiol Biomarkers Prev; 20(6); 1131–45. ©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 Mitochondria have been implicated in carcinogenesis because of their central role in apoptosis, free radical production, and cellular energy metabolism. Although studies have identified somatic mitochondrial DNA mutations in ovarian cancers, little attention has been directed to the possible role of germline mitochondrial variants in mitochondrial dysfunction and subsequent ovarian cancer development. Through an ongoing epithelial ovarian cancer genome-wide association study of 1,856 invasive cases (59% with serous carcinomas) and 1,912 controls (frequency-matched on age, race, and study site), we genotyped 138 SNPs tagging variation in the maternally-inherited mitochondrial genome (mtDNA) to evaluate the hypothesis that single nucleotide polymorphisms (SNPs) in mtDNA are associated with ovarian cancer risk. All subjects were non-Hispanic, non-Jewish Caucasians, and were genotyped with the Illumina 610-quad Beadchip. Samples and SNPs with call rates less than 95% were excluded. Subjects with ambiguous gender, unresolved identical genotypes, and less than 80% European ancestry were excluded. For each SNP, logistic regression was used to estimate odds ratios (OR) and corresponding 95% confidence intervals (CI) between carriers of the minor versus major maternally-inherited allele and case status. Subgroup analysis was conducted to estimate allele-specific risks between serous-only cases and controls. Haplotype analysis was performed for regions containing statistically significant SNPs (p<0.05). Two rare SNPs, C16329T in the displacement loop gene (MT-D-loop) (OR: 6.72, 95%CI: 1.5-29.8, p=0.004; minor allele frequency (MAF)=0.001) and C15905T in the tRNA threonine gene (MT-TT) (OR: 1.60, 95%CI: 1.1-2.3, p=0.009; MAF=0.027), were associated with increased ovarian cancer risk; whereas T6777C in the cytochrome c oxidase subunit 1 gene (MT-CO1) (OR: 0.68, 95%CI: 0.51-0.91, p=0.009; MAF=0.063) was associated with decreased risk. All three SNPs remained statistically significant after adjustment for multiple comparisons within the mitochondrial SNPs using permutation testing. C16329T and T6777C were also significantly associated among cases with serous histology, with ORs (95% CIs) of 6.13 (1.2-29.6) and 0.61 (0.43-0.87), respectively. Haplotype analysis for each of the three regions investigated revealed significant haplotype-disease associations; however, findings suggest that the respective associations were driven by the most significant SNP(s) listed above. This is the first large-scale epidemiologic study to provide evidence that SNPs in mitochondrial genes may be novel risk factors for ovarian cancer. Future investigation of additional mitochondrial variants (i.e. nuclear-encoded mitochondrial proteins) is necessary to more comprehensively investigate this association. Furthermore, replication of the most promising SNPs in a larger population is warranted to validate these findings. 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 2835.

Abstract As the leading cause of mortality among gynecologic cancers, it is critical to increase understanding of ovarian cancer pathogenesis, which remains largely unknown. Several xenobiotic metabolizing enzymes are responsible for processing and inactivating carcinogens that could initiate ovarian carcinogenesis. Here, we hypothesize that single-nucleotide polymorphisms (SNPs) in genes encoding xenobiotic metabolizing enzymes may be associated with differential risk of ovarian cancer. Cases with epithelial ovarian cancer (N=930) and controls (N=1,037), frequency-matched on age and geographic residence, were enrolled at Mayo Clinic and Duke University, and genotyped at 163 tagSNPs in EPHX1, SULT1E1, ADH5, ADH4, ADH6, ADH1A, ADH1B, ADH1C, NQO2, NAT1, NAT2, GSTP1, CYP19A1, CYP1A1, CYP1A2, and NQO1 using an Illumina GoldenGate™ BeadArray array. Logistic regression was used to estimate odds ratios (ORs) and adjust for population structure and other potential confounders, including oral contraceptive use, parity, and family history. SNP-specific statistical analyses identified several noteworthy results including an association with increased risk of ovarian cancer with a SNP (rs2917666) in NAD(P)H dehydrogenase, quinone 1 (NQO1) (per-allele OR 1.16, 95% confidence interval (CI) 1.01-1.33, p=0.04). The SNP rs1051740 in epoxide hydrolase 1, microsomal (xenobiotic) (EPHX1) was suggestive of increased risk of invasive serous disease (TT v CT OR 0.85, 95% CI 0.66-1.09, TT v CC 1.29, 95% CI 0.89-1.88, p=0.09). In an effort to replicate these observations, a subset of 11 SNPs with a p-value < 0.10 were then genotyped in 727 invasive cancer cases and 1,065 controls from an Australian ovarian cancer study using a Sequenom iPlex assay. Results of combined analyses confirmed an association between EPHX1 rs1051740 and increased invasive serous ovarian cancer risk (TT v CT OR 0.99, 95% CI 0.77-1.29, TT v CC OR 1.61, 95% CI 1.23-2.11, p=0.001; recessive OR 1.61, 95% CI 1.25-2.09, p=0.001). This gene is involved in the activation and detoxification of tobacco-derived carcinogens, and smoking has been associated with the mucinous histologic subtype of ovarian cancer. Analyses by histological subtype and gene-environment interaction analyses considering genotype in conjunction with tobacco use and alcohol intake are underway. Due to the important role of EPHX1 in metabolizing pro-carcinogens and the observed association between rs1051740 and risk of invasive serous disease, an examination of this SNP in a larger ovarian cancer consortium is warranted. 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 914.

Abstract Using data from patients with invasive epithelial ovarian cancer, we sought to examine whether overall survival was associated with common inherited variation in candidate genes and genomic regions including those involved in angiogenesis, mitosis, and regions of interest from genome-wide association and expression studies. We conducted a two-stage analysis based on Mayo Clinic patients with replication of peak results in the Cancer Genome Atlas (TCGA). Ovarian cancer patients (N=312) seen at the Mayo Clinic diagnosed from 1999 to 2006 with median follow-up of 3.7 years (range, 0.1 − 9.5 years) and 172 observed deaths were analyzed at 993 single nucleotide polymorphisms (SNPs) in 179 genes. For all patients and for serous patients only (N=192 with 129 observed deaths), Cox regression was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for each SNP, adjusted for other prognostic factors. Twenty-nine SNPs in ten genes revealed p < 0.01 among all cases or among serous cases only, including 21 independent SNP at r2=0.9; analyses within four genes (HGF, PLG, PRKACB, and DCTN5) yielded p < 0.001. The strongest result was in HGF at rs1800793 (all cases: HR 1.7, 95% CI 1.3 − 2.2, p-trend 1.9 E-5; serous cases: HR 1.6, 95% CI 1.2 − 2.2, p-trend 1.8 E-3). A correlated SNP rs2214825 also showed association (r2=0.76; all cases: HR 1.4, 95% CI 1.1 − 1.8, p-trend 3.3 E-3; serous cases: HR 1.4, 95% CI 1.0 − 1.9, p-trend 0.03), and PCA gene-level tests of the HGF locus were also significantly associated with survival (all cases, p=3.7 E-4; serous cases, p=0.01). Among all cases, differential survival was also associated with DCTN5 rs12447304 (HR 1.8, 95% CI 1.3-2.6; p=7.1 E-4), and among serous cases, with PLG rs783173 (HR 1.6, 95% CI 1.2 − 2.0, p = 8.1 E-4) and PRKACB rs1402694 (HR 1.7, 95% CI 1.3 − 2.2, p=3.2 E-4). We then examined SNPs in the most suggestive gene, HGF, using data from TCGA which had genotyped four overlapping HGF SNPs in 350 invasive cases (188 deaths). Covariate-adjusted TCGA analysis at rs2214825 revealed consistent results (HR 1.7, 95% CI 1.3-2.2, p=1.4 E-4) as did analyses of three other HGF SNPs (HR range 1.3 to 1.5, p range 3.4 E-3 to 0.049); correlation between SNP genotypes and HGF expression was also observed (p=1.4 E-3, rs2214825, probe 210997_at). While this is the first report of SNPs in HGF, it has long been known that the HGF signaling pathway, activated by HGF ligation of c-MET, plays a key role ovarian cancer cell growth, migration, and invasion. c-MET is overexpressed in 40%-60% of ovarian tumors, and high overexpression is associated with lower overall survival. In addition, HGF has been shown to enhance the response of ovarian cancer cells to low doses of paclitaxel and cisplatin by activating the intrinsic apoptotic pathway, leading to cancer cell death. These results now suggest that inherited differences in HGF among women with invasive disease may play a role in survival, holding potential clues for tailored avenues of treatment. 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 897.