Fridley, Brooke L.

Abstract Purpose: Inherited variability in the prognosis of lung cancer patients treated with platinum-based chemotherapy has been widely investigated. However, the overall contribution of genetic variation to platinum response is not well established. To identify novel candidate single nucleotide polymorphisms (SNP)/genes, we carried out a genome-wide association study (GWAS) for cisplatin cytotoxicity by using lymphoblastoid cell lines (LCL), followed by an association study of selected SNPs from the GWAS with overall survival (OS) in lung cancer patients. Experimental Design: A GWAS for cisplatin was conducted with 283 ethnically diverse LCLs. A total of 168 top SNPs were genotyped in 222 small cell lung cancer (SCLC) and 961 non-SCLC (NSCLC) patients treated with platinum-based therapy. Association of the SNPs with OS was determined by using the Cox regression model. Selected candidate genes were functionally validated by siRNA knockdown in human lung cancer cells. Results: Among 157 successfully genotyped SNPs, 9 and 10 SNPs were top SNPs associated with OS for patients with NSCLC and SCLC, respectively, although they were not significant after adjusting for multiple testing. Fifteen genes, including 7 located within 200 kb up or downstream of the 4 top SNPs and 8 genes for which expression was correlated with 3 SNPs in LCLs were selected for siRNA screening. Knockdown of DAPK3 and METTL6, for which expression levels were correlated with the rs11169748 and rs2440915 SNPs, significantly decreased cisplatin sensitivity in lung cancer cells. Conclusions: This series of clinical and complementary laboratory-based functional studies identified several candidate genes/SNPs that might help predict treatment outcomes for platinum-based therapy of lung cancer. Clin Cancer Res; 17(17); 5801–11. ©2011 AACR.

Abstract Purpose: Cell-based approaches were used to identify genetic markers predictive of patients' risk for poor response prior to chemotherapy. Experimental Design: We conducted genome-wide association studies (GWAS) to identify single-nucleotide polymorphisms (SNP) associated with cellular sensitivity to carboplatin through their effects on mRNA expression using International HapMap lymphoblastoid cell lines (LCL) and replicated them in additional LCLs. SNPs passing both stages of the cell-based study were tested for association with progression-free survival (PFS) in patients. Phase 1 validation was based on 377 ovarian cancer patients receiving at least four cycles of carboplatin and paclitaxel from the Australian Ovarian Cancer Study (AOCS). Positive associations were then assessed in phase 2 validation analysis of 1,326 patients from the Ovarian Cancer Association Consortium and The Cancer Genome Atlas. Results: In the initial GWAS, 342 SNPs were associated with carboplatin-induced cytotoxicity, of which 18 unique SNPs were retained after assessing their association with gene expression. One SNP (rs1649942) was replicated in an independent LCL set (Bonferroni adjusted P < 0.05). It was found to be significantly associated with decreased PFS in phase 1 AOCS patients (Pper-allele = 2 × 10−2), with a stronger effect in the subset of women with optimally debulked tumors (Pper-allele = 4 × 10−3). rs1649942 was also associated with poorer overall survival in women with optimally debulked tumors (Pper-allele = 9 × 10−3). However, this SNP was not significant in phase 2 validation analysis with patients from numerous cohorts. Conclusion: This study shows the potential of cell-based, genome-wide approaches to identify germline predictors of treatment outcome and highlights the need for extensive validation in patients to assess their clinical effect. Clin Cancer Res; 17(16); 5490–500. ©2011 AACR.

Abstract Background: Recent reports support an association between chronic inflammation and progression to pancreatic cancer (PC). Methods: This case–control, candidate gene association study evaluated 1,354 Caucasian patients with pancreatic ductal adenocarcinoma and 1,189 healthy Caucasian controls. We genotyped 1,538 single nucleotide polymorphism (SNP) in 102 genes from inflammatory pathways involving NF-κB. Primary tests of association assumed a multiplicative (log-additive) genotype effect; secondary analyses examined dominant, additive, and recessive SNP effects. Results: After adjusting for known risk factors for PC, single SNP analysis revealed an association between four SNPs in NOS1 and one in the CD101 gene with PC risk. These results, however, were not replicated in a PC case–control and cohort population. Conclusion:NOS1 and CD101 may be associated with a risk of PC; however, these findings did not replicate in other PC populations. Future research is needed into the possible role of NOS1 and CD101 for PC. Impact: This research shows a lack of association between genetic variation in 102 inflammation-related genes and PC. Future research is needed into the possible role of other inflammation-related genes and PC risk. Cancer Epidemiol Biomarkers Prev; 20(6); 1251–4. ©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 Background Taxane, a class of common anticancer agent, is widely used to treat many cancers. Among the taxanes, paclitaxel and docetaxel are the most commonly used agents. The mechanism of action for taxane drugs is through stabilizing microtubules by enhancing tubulin assembly and against depolymerization, thereby disrupting the dynamics of the microtubule. Large inter-individual variation has been observed in response to taxane treatment. Therefore, in the present study, we conducted a genome-wide association (GWA) study to identify genetic polymorphisms that might contribute to variation in response to taxanes drugs using the “Human Variation Panel” lymphoblastoid cell line (LCL) model system that consists of 287 ethnically defined cell lines. Method Genome-wide SNP and basal gene expression data were obtained for 287 LCLs using Illumina HumanHap 550K, 510S BeadChips, Affymetix SNP Array 6.0 and Affymetrix U133 plus 2.0 GeneChip. Cytotoxicity assay was also performed in the same cell lines for both paclitaxel and docetaxel. Candidate genes were selected based on an integrated analysis of genome-wide SNPs, expression array data and IC50 values (a cytotoxicity phenotype indicating the drug concentration required for 50% inhibition of maximal cell growth) for paclitaxel and docetaxel. SiRNA screening was performed to functionally validate those top candidates, followed by MTS assay in a breast cancer cell line, MDA-MB231. Results Of the 54,000 probe sets considered, 390 probe sets, representing 272 unique annotated genes, were found to be associated with docetaxel cytotoxicity (IC50 value) with p value <10−3, while 176 probe sets, representing 124 unique genes, were associated with paclitaxel IC50. Of these probe sets, 50 probe sets were associated with IC50 for both drugs. Analysis of 1.3 million SNPs identified 11 loci, defined as a region containing at least 1 SNP of p value <10−5 and 1 SNP of p value <10−4 within 50kb, associated with docetaxel IC50, while 7 loci were associated with paclitaxel IC50. One locus was found to be associated with both drugs. Previous studies suggested that gene expression is regulated in a cell-type specific manner. Therefore, based on our analysis and biological implication, we selected 16 top candidate genes that were expressed in our LCLs to perform functional validation studies using siRNA, followed by MTS assay in a breast cancer cell line, MDA-MB231. Functional studies showed that knockdown of SLC44A1 and HSPA5 significantly desensitized the cancer cell to both taxanes treatment. Conclusion GWAS using LCL model system could provide potential pharmacogenomic candidates for future clinical translational studies. Functional validation in tumor cell lines may help determine the genomic mechanism contributing to the inter-individual variation of taxane response. 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 2599.

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.

Abstract The advancement in genotyping technology and large SNP arrays has led to an increase in the number of genome-wide association studies (GWAS) for various complex traits. These GWAS have resulted in the implication of over 1500 SNPs for association with disease traits. However, the SNPs identified from these GWAS are not necessarily the functional variants. Therefore, the next phase in GWAS will involve the validation, augmentation and refining of these putative regions or loci. One possible “next phase” approach would be to catalog all variants found in the study subjects within the loci of interest using sequencing technology, followed by the association analysis. However, sequencing a locus in a large number of subjects is still relatively expensive. By sequencing only a portion of the samples for the follow-up of signals from GWAS, followed by imputation in the remaining samples, one can significantly reduced the cost to localize the punitive variant involved in the etiology of complex phenotypes. A potentially attractive alternative option would be to impute based only on the 1000 Genomes Project; however, this has the drawbacks of using a reference population that does not necessarily match with disease status and LD pattern. Therefore, we conducted a study to investigate the ability to carry-out genotype imputation using a reference panel consisting of sequencing data for a fraction of the study participants. The various approaches were implemented using data from the candidate gene sequencing study conducted at the Mayo Clinic and the 1000 Genomes Project. Our results show that imputation of untyped markers based on sequencing a portion of the study participants is a reasonable, cost-saving, approach for disease mapping and the refinement of putative regions detected with GWAS. We recommend a few guidelines in designing sequencing studies to take advantage of the recent advances in genotype imputation: (1) Select the largest and most diverse reference panel for sequencing, with respect to both haplotypes and phenotypes. One can also use the 1000 Genomes Project in addition to the sequencing of a portion of the study participants; (2) Genotype as many “anchor” markers as possible, in that, the number of markers genotyped on all subjects impacts accuracy. Therefore, additional genotyping of a few common SNP markers not already genotyped on all subjects using a cost effective platform may be needed if the GWAS SNP array does not provide adequate coverage in the locus to be sequenced. 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 4738.