Cunningham, Julie M.

Abstract A substantial proportion of familial colorectal cancer (CRC) is not a consequence of known susceptibility loci, such as mismatch repair (MMR) genes, supporting the existence of additional loci. To identify novel CRC loci, we conducted a genome-wide linkage scan in 356 white families with no evidence of defective MMR (i.e., no tumors with loss of expression of MMR proteins; no microsatellite instability (MSI)-high tumors, and no evidence of linkage to MMR genes). This represents the largest linkage analysis of proficient MMR (pMMR) familial CRC to date. Families were ascertained via the NCI-supported Colon Cancer Family Registry multi-site (Colon CFR) consortium, the City of Hope, and Memorial University of Newfoundland. A total of 1,612 individuals (average 5.0 per family, range 2-10 with an average of 2.2 affected and 2.8 unaffected individuals) were genotyped using genome-wide single nucleotide polymorphism linkage arrays; parametric and non-parametric linkage analysis used MERLIN in a priori-defined family groups. Five lod scores greater than 3.0 were observed, accounting for heterogeneity, in four chromosomal regions. The greatest lod scores were at 4q21.1 among families with mean age of diagnosis less than 50 years (dominant HLOD=4.51, α=0.84, 145.40 cM, rs10518142) and at 12q24.32 among all families (dominant HLOD=3.60, α=0.48, 285.15 cM, rs952093). Among families with four or more affected individuals and among clinic-based families, a common peak was observed at 15q22.31 (101.40 cM, rs1477798; dominant HLOD=3.07, α=0.29; dominant HLOD=3.03, α=0.32, respectively). Analysis of families with only two affected individuals yielded a peak at 8q13.2 (recessive HLOD=3.02, α=0.51, 132.52 cM, rs1319036). These linkage regions comprise previously unreported loci, supporting the hypothesis that novel loci contribute to CRC in familial pMMR cases. These loci are also largely distinct from those identified in case-control GWAS. To follow up on these findings, a custom Agilent Sure Select Target Enrichment panel has been designed for genes in three of these regions, in order to sequence probands using an Illumina HiSeq 2000. This study demonstrates the utility of family-based data and provides evidence for additional alleles for familial CRC. 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 2930. doi:1538-7445.AM2012-2930

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 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

AbstractTo further explore reports of association of alcohol dependence and response to acamprosate treatment with the GATA4 rs13273672 single nucleotide polymorphism (SNP), we genotyped this and 10 other GATA4 SNPs in 816 alcohol‐dependent cases and 1248 controls. We tested for association of alcohol dependence with the 11 SNPs individually and performed a global test for association using a principle components analysis. Our analyses demonstrate significant association between GATA4 and alcohol dependence at the gene level (P = 0.009) but no association with rs13273672. Further studies are needed to identify potential causal GATA4 variation(s) and the functional mechanism(s) contributing to this association.

Abstract Introduction: Short constitutional telomere length is associated with aging and cancer, but the correlation of telomere length and age at diagnosis of CRC has not been evaluated. Methods: We measured telomere length using quantitative PCR of peripheral blood leukocyte DNA in 1026 individuals diagnosed with MSS CRC who were ≤60 years of age and were chemo-radiotherapy naïve at the time of blood draw. We compared telomere length with 1972 unrelated healthy controls who were frequency matched for gender and age at time of blood collection. We used constructed percentiles of telomere length using the data from the controls. Results: Overall, regardless of cancer status, there was a weak negative correlation of telomere length with age such that as age increased telomere length decreased (Pearson correlation coefficient =−0.1, p< 0.0001); and men had shorter telomeres than women (p=0.0002). The odds ratio (OR) for MSS CRC increased with longer telomere length (OR 1.62 [CI: 1.31-1.99] for those with telomere lengths in the 90th percentile compared to those with telomere lengths in the 50th percentile. In multivariate analysis with adjustment for gender, BMI, smoking, alcohol, and diabetes, telomere length remained an independent risk factor for CRC (p< 0.001), with interaction between telomere length and age (≤50, longer telomeres increased CRC risk; > 50 years old, increasing telomere length associated with decreased CRC risk). Risk was higher for MSS CRC ≤50 years for patients with telomere lengths in the 90th percentile compared to those in the 50th percentile (OR 2.31; CI:1.65-3.23). For subjects diagnosed 50-60 yrs, longer telomere length was associated with lower risk of CRC, and shorter telomere length was associated with the development of CRC (OR 1.22 for telomere lengths in the 10th percentile compared to those in the 50th percentile (OR 1.22:CI; 1.05-1.43). Conclusion: Telomere dynamics appear to influence MSS CRC risk and may differ on the basis of age of cancer diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2994. doi:10.1158/1538-7445.AM2011-2994

Abstract Background: NHL is a malignancy of lymphocytes, and there is growing evidence for a role of immune dysfunction in the etiology of this malignancy. We conducted a 2-stage analysis of single nucleotide polymorphisms (SNPs) from 1,253 genes using the ParAllele (now Affymetrix) Immune and Inflammation Panel with risk of NHL. Methods: In Stage 1, we successfully genotyped 7,670 SNPs in a clinic-based study of 425 NHL cases and 465 frequency matched controls seen at the Mayo Clinic. Genes were tagged with SNPs from HapMap to provide coverage at r2 = 0.8 and minor allele frequency (MAF) >0.05. In Stage 2, we genotyped the top 768 SNPs on an additional 584 cases and 768 controls from the Mayo Clinic Study, and then conducted a pooled analysis of all 1009 cases and 1233 controls. The association of individual SNPs with NHL risk was assessed using the Odds Ratios (ORs) and 95% confidence intervals, adjusted for age and gender. The most prevalent homozygous genotype was used as the reference group, and each SNP was modeled individually as having a log-additive effect. Results: Overall, the mean age at diagnosis was 62.0 years for cases and the mean age at enrollment for controls was 61.1 years. The most common subtypes were chronic lymphocytic leukemia (CLL, N=343), follicular lymphoma (FL, N=245), and diffuse large B-cell lymphoma (DLBCL, N=178). In the pooled analysis, there were 3 SNPs at p<0.0004. The top SNP was rs241447 (MAF=0.26) from TAP2 at 6p21.3 (OR=1.34, 95% CI 1.17-1.53, p=0.00003). This SNP is a cSNP that leads to a missense change (Ala→Thr). The second SNP was rs2857597 (MAF=0.26) from AIF1, also at 6p21.3 (OR=0.77, 95% CI 0.67-0.88, p=0.0002). The region spanning these two SNPs had multiple risk SNPs with p<0.01, including SNPs in BAT2 (rs3132453), BAT3 (rs2242656), C2 (rs7746553), HLA-DRA (rs8084, rs7192), and TAP2 (rs1894408). Of these SNPs, only rs241447 (TAP2) and rs7192 (HLA-DRA) displayed any substantial LD (r2=0.84, all other r2<0.25). The third SNP was rs754505 (MAF=0.07) from NFATC1 at 18q23 (OR=0.63, 95% CI 0.49-0.81, p=0.0003). There were 4 additional SNPs in this gene with a p<0.05 (rs7240932, rs396127, rs177820, rs183374). In subtype analysis, the SNPs from 6p21.3 showed the strongest associations with FL (including the TAP2 SNP rs241447, p=6.9 × 10−8), although there were also associations with CLL (except for the TAP2 and C2 SNPs) and DLBCL (except for the HLA-DRA SNPs). For NFATC1 the associations were similar across the main NHL subtypes. Conclusions: We identified strong associations with NHL risk in the 6p21.3 region, which includes the MHC locus. Our results were particularly striking for FL and supports recent GWAS and candidate gene studies that implicate this region. We also identified NFATC1 as a susceptibility gene. NFATC1 is known to regulate the expression of growth and survival genes including MYC, TNF, CD40L, and BAFF, all of which have also been linked to lymphomagenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2761. doi:10.1158/1538-7445.AM2011-2761

Abstract Epithelial ovarian cancer (EOC) is the 6th most commonly diagnosed cancer among women in the world, accounting for 4% of all female cancers. On a worldwide basis, an estimated 204,000 new cases are diagnosed and 125,000 women die of EOC annually, with 5-year survival rate at 45%. Metastasis and its complications are the usual cause of EOC-related deaths. Thus, factors related to tumor aggressiveness, response to therapy, and the underlying health state of the patient often predict survival. Candidate gene and genome-wide association studies (GWAS) for survival from EOC have discovered only a few the genetic variants, accounting for only a small proportion of the heritability. One explanation for the missing heritability is that the common analysis approach _ assessing the effect of each single nucleotide polymorphism (SNP) individually _ is not well suited to the detection of small effects of multiple SNPs. Gene set analysis (GSA) overcomes this limitation by assessing the overall evidence of association of a phenotype with all measured variation in a set of genes or pathway. Therefore, we conducted a GSA using data from an EOC GWAS, using the Illumina 610-quad SNP array, to determine novel gene sets (GSs) associated with overall survival (OS) from EOC (N cases = 1057, N deaths = 495). All subjects were non-Hispanic, non-Jewish Caucasians, with sample and SNP call rates >95%. Subjects with ambiguous gender, unresolved identical genotypes, and less than 80% European ancestry were also excluded. SNPs were mapped to genes within 20 kb, with genes then mapped to GSs defined by the following: KEGG, Gene Ontology (biological, cellular, molecular) and PharmGKB. GSA was completed in which the association of each gene with OS was assessed using a principal component gene-level test (adjusting for study site, age and population sub-structure based on the first eigenvector from EigenStrat), followed by the aggregation of the gene-level p-values using the Gamma method with a soft truncation threshold of 0.15. Empirical p-values were determined using permutation methods. Of the 2,573 GSs assessed, 30 GSs had a p-value < 0.01. The top GSs were related to: mismatch repair (p = 3.0E-4), platelet formation (p=0.001), leukocyte adhesive activation (p=0.002), antigen processing and presentation of endogenous antigens (p = 0.002) or peptide antigens (p = 0.002), platinum pathway (p = 0.003), aging and lifespan (p = 0.004, p = 0.007), immune system development (p = 0.004) and somatic diversification (p=0.007). These results illustrate the usefulness of GSA for the development of novel hypotheses that can be followed-up with additional clinical or functional studies. Moreover, application of GSA to survival from EOC allowed the detection of significant GSs (and the corresponding genes/SNPs within these GSs) that impact OS that were not detected by assessing each SNP individually, where the smallest p-value from single SNP analysis of the GWAS for OS was 1.3E-05. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2738. doi:10.1158/1538-7445.AM2011-2738

Abstract The etiology of ovarian cancer is poorly understood but there is clearly a heritable component. Efforts to identify susceptibility alleles rarely consider interactions among alleles or their joint effects because it quickly becomes computationally intractable. In this study, we sought to identify multi-SNP effects jointly with a pathway-based analysis (GSEA-SNP) of 1,952 EOC cases and 2,042 frequency-matched controls genotyped with the Illumina 610K array. All subjects were self-reported non-Hispanic non-Jewish Caucasians, with SNP and sample call rates > 95%. Subjects with ambiguous gender, unresolved identical genotypes and < 80% European ancestry were excluded. SNPs with MAF < 1% were excluded. Missing genotypes were inferred using Mach based on the HapMap CEU population. SNPs not within introns were annotated to genes within 100 bp. We retrieved the following databases of gene sets (GSs) from a compiled database, MsigDB: human chromosome and cytogenetic band, chemical and genetic perturbations, canonical pathways, microRNA binding targets, transcription factor targets (TFT), cancer gene neighborhood (CGN), Cancer modules and Gene Ontology. The GSEA-SNP approach calculates and rank orders the trend statistic for association between each SNP and EOC risk. The Enrichment Score (ES) estimates the overrepresentation of top-ranked SNPs for each GS; the statistical significance was estimated using 10,000 permutations. The ES was normalized according to the size of GS to yield Normalized Enrichment Score (NES). The false discovery (FDR) rate was estimated using NES to adjust for multiple hypothesis testing. A total of 5181 gene-sets were included in the analysis. When controlling the FDR at 15%, 14 of the GSs were highly enriched with association signals, including two chromosomal regions, 8q13 (p = 0.0055, FDR = 0.15) and 6p24 (p = 0.0012, FDR = 0.08). BIOCARTA_LYM_PATHWAY, a pathway related to cell adhesion and diapedesis of lymphocytes, was also significant (p = 0.0002, FDR = 0.12). A TFT GS, composed of genes with promoter regions around transcription start sites containing the motif GGCNRNWCTTYS, was associated with risk (p = 0.0004, FDR = 0.08). Currently, no known transcription factors bind to this computationally predicted motif. Molecular function of double stranded RNA binding was also enriched (p = 0.005, FDR = 0.10). The remaining enriched sets were CGN sets, for which the neighborhoods around the cancer genes were originally defined using correlation of gene expression from 4 large data sets of various cancer types. The enriched CGN included: CD48 (p = 0.011), CD53 (p = 0.009), CD97 (p = 0.011), INPP5D (p = 0.010), PTPN6 (p = 0.008), VAV1 (p = 0.011), ITGAL (p = 0.012), PTPRC (p = 0.012), and STAT6 (p = 0.010). In summary, these analyses detected biologically plausible GSs related to etiology of EOC, highlighting SNPs in core enrichment groups that were not identified using individual SNP tests. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4729. doi:10.1158/1538-7445.AM2011-4729

Abstract Background. The hepatocyte growth factor (HGF) signaling pathway regulates key cellular functions related to tumor invasion and metastasis including cell motility, cell proliferation, and morphogenesis. We previously found that minor alleles at a single-nucleotide polymorphism (SNP; rs5745709) in the gene encoding the cytokine HGF was associated with poorer overall survival in women with invasive epithelial ovarian cancer (EOC) enrolled at Mayo Clinic (MAY) and The Cancer Genome Atlas (TCGA) (HR 1.6, 95% CI 1.3-1.9, p=8.9×10−5). In addition, we found that that genotype correlated with HGF expression in ovarian cancer tissue from TCGA women, suggesting that genetically-regulated expression of HGF may play a role in post-diagnosis survival. To further examine this possibility, we evaluated intensity of antibody immunostaining for HGF, MET (HGF's transmembrane tyrosine kinase receptor), and phospho-MET (pMET; MET's activated form) in tumor cells using tissue microarrays (TMAs) created from genotyped primary peritoneal and EOC cases, and correlated these expression levels with overall survival and HGF SNP genotypes. Methods. Participants were 326 white non-Hispanic women over 20 years of age with histologically-confirmed invasive EOC diagnosed between 1998 and 2009. All cases were enrolled at MAY within one year of diagnosis. Five μm sections of TMAs including three 0.6 mm cores per case were immunostained with HGF, MET, and pMET primary antibodies. Staining levels were classified according to combinations of staining extent and intensity as negative (< 10% cells stained), weak, moderate, or strong. The maximal expression value recorded over the multiple cores for each case was used. Cox proportional hazards regression was used to examine associations of overall survival with HGF, MET and pMET expression levels, adjusted for relevant clinical variables. Associations between expression levels and HGF SNP rs5745709 genotypes were examined using linear regression models. Results. HGF and MET expression were not associated with overall survival in our series of EOC cases (p=0.48 and 0.90, respectively). pMET expression was associated with survival, but in the opposite direction of that hypothesized (HR 0.77, 95% CI 0.39-1.52 for moderate expression; HR 0.55, 95% CI 0.27-1.10 for strong expression; each referent to negative/weak, p=0.01). Expression levels were not significantly associated with HGF rs5745709 genotypes (p=0.80 for HGF expression, p=0.89 for MET, p=0.41 for pMET). Conclusion. HGF expression levels were not associated with overall survival or with HGF SNP genotypes that correlated with survival in a previous study. Functional studies such as this one are crucial as a follow-up to discovery-based studies in order to assess biologic plausibility of observed results. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 893. doi:10.1158/1538-7445.AM2011-893

Abstract Background: The role of dietary fruit, vegetable, fiber, and whole grain intake is implicated as a potential protective factor against pancreatic cancer, but studies are inconsistent. A possible explanation for the variability is that individuals who carry constitutional metabolism gene variants (minor alleles) may differentially benefit compared to those who are homozygous for the major allele, which over a life-time could lead to altered pancreatic cancer risk. Methods: We genotyped 70 SNPs that tag ten candidate metabolism genes (CAT, GAA, GCK, MT1E, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT2B4, and UGT2B7) involved in the metabolism of components of fruits, vegetables, fibers, and whole grains to test if differential associations exist with pancreatic adenocarcinoma. We used a clinic-based case-control design, excluding participants who reported changing their diet within 5 years prior to entering the study. Three hundred eighty-four rapidly ascertained cases and nine hundred eighty-three non-cases from a primary care clinic (frequency matched on age at recruitment (+ 5 years), race, sex, and region of residence) provided blood samples for DNA and completed epidemiologic surveys plus a 144-item food frequency questionnaire developed by the National Cancer Institute. We used a dominant model to code SNPs and split dietary intake categories based on the sex-specific median intake level among non-cases. Logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals, adjusted for age, sex, family-history of pancreatic cancer, smoking (never/ former/ current), body mass index, energy intake, and alcohol consumption. The reference group was defined as carriers of no minor alleles in the lowest intake category. Results: The greatest benefit was observed for carriers of no minor alleles, for rs11032702 (CAT) with higher insoluble dietary fiber intake (OR [95%CI]: 0.515 [0.37, 0.71]); for rs735670 (GCK) with higher soluble dietary intake (0.473 [0.34, 0.67]); for rs2070971 (GCK) and rs2268569 (GCK) with higher fruit intake not from citrus, melon, and berries (0.513 [0.36, 0.73] and 0.554 [0.40, 0.78]); and for rs17832252 (GCK) with higher whole grain intake (0.530 [0.37, 0.76]); all comparisons had a reference group of no minor alleles with low dietary intake and a p-value for interaction <0.001. Conclusions: Inter-individual variation in metabolism of fruits, vegetables, fiber, and whole grain via metabolism gene variants may influence pancreatic cancer risk. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3749. doi:10.1158/1538-7445.AM2011-3749