Casey, Graham

Abstract Telomeres are complex structures that cap chromosome ends, protecting them from degradation, double strand breaks and end-to-end fusions. Telomeres are maintained by the enzyme telomerase, which is made up of a reverse transcriptase encoded by TERT, and an RNA template encoded by TERC. The telomere structure itself is composed of proteins encoded by ACD, ACYP2, BICD1, DKC1, DCLRE1B, MPHOSPH6, NAF1, NOLA1, NOLA2, NOLA3, OBFC1, PIK3C3, POT1, RTEL1, TEP1, TERF1, TERF2, TERF21P, TINF2, TNKS/PINX1, TPP1 and ZNF208. Several single nucleotide polymorphisms (SNPs) in the TERT and adjoining CLPTM1L gene region are associated with multiple cancer types, and some are associated with both increased and decreased risks across different cancer types. We systematically characterized the patterns of association between variants in these 25 telomere structure and maintenance genes and risk across five cancer types in the Genetic Association and Mechanisms in Oncology (GAME-ON) consortium. We performed a subset-based meta-analysis (ASSET) of 209,367 directly measured and imputed SNPs, one megabase up- and downstream of these genes, across genome-wide association studies of colorectal (5,100 cases, 4,831 controls), lung (12,160 cases, 16,838 controls), breast (15,748 cases, 18,084 controls), ovarian (4,369 cases, 9,123 controls) and prostate (14,160 cases, 12,724 controls) cancers. Correlations (r2) between SNPs were examined in Haploview using the 1000 Genomes Project CEU population. A total of 87 TERT, 123 TERC and 26 DCLRE1B SNPs were associated with cancer risk at gene-level Bonferroni-corrected p-values of 4.2-7.8×10−6. Patterns of association were similar for prostate and colorectal cancers in DCLRE1B. Much stronger associations were observed in TERT and TERC, with 63 and 24 SNPs reaching genome-wide significance (p<5.0×10−8), respectively. Of these, 9 TERT and 9 TERC SNPs were correlated at r2<0.75. The most strongly associated SNPs in TERT (rs37004, p = 2.6×10−11) and TERC (rs76925190, p = 1.5×10−15) are in regions containing documented risk loci for lung and prostate cancers, respectively. TERT rs37004 (p = 1.2×10−13) and 6 of the other 9 SNPs in TERT were associated only with lung cancer risk. For the other 2 SNPs, there was a suggestion that patterns of risk were opposite for lung and prostate cancers. While TERC rs76925190 was strongly associated with prostate cancer risk (p = 5.4 x10−17), it was also suggestively associated with colorectal cancer risk (p<0.005). This pattern was similar for another 2 of the 9 SNPs, while for the rest, associations were limited to prostate cancer. The complex patterns of association in telomere structure and maintenance genes observed across cancer types may provide insight about the mechanisms through which telomere dysfunction in different tissues influences cancer risk. Citation Format: Sara Karami, Younghun Han, Fredrick R. Schumacher, Zsofia Kote-Jarai, Sara Lindstrom, John S. Witte, Iona Cheng, Shenying Fang, Jiali Han, Peter Kraft, Fengju Song, Rayjean J. Hung, James McKay, Stephen J. Chanock, Mala Pande, Angela Risch, Hongbing Shen, Christopher A. Haiman, Lisa Boardman, Cornelia M. Ulrich, Graham Casey, Ulrike Peters, Nilanjan Chatterjee, Brandon Pierce, Wei Zheng, Christopher I. Amos, Jennifer A. Doherty. Risk loci in telomere structure and maintenance genes across five cancer types: GAME-ON Consortium. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4609. doi:10.1158/1538-7445.AM2015-4609

Abstract Background: Microsatellite instability (MSI) and BRAF mutation status are associated with colorectal cancer survival, whereas the role of body mass index (BMI) is less clear. We evaluated the association between BMI and colorectal cancer survival, overall and by strata of MSI, BRAF mutation, sex, and other factors. Methods: This study included 5,615 men and women diagnosed with invasive colorectal cancer who were followed for mortality (maximum: 14.7 years; mean: 5.9 years). Prediagnosis BMI was derived from self-reported weight approximately one year before diagnosis and height. Tumor MSI and BRAF mutation status were available for 4,131 and 4,414 persons, respectively. Multivariable hazard ratios (HR) and 95% confidence intervals (CI) were estimated from delayed-entry Cox proportional hazards models. Results: In multivariable models, high prediagnosis BMI was associated with higher risk of all-cause mortality in both sexes (per 5-kg/m2; HR, 1.10; 95% CI, 1.06–1.15), with similar associations stratified by sex (Pinteraction: 0.41), colon versus rectum (Pinteraction: 0.86), MSI status (Pinteraction: 0.84), and BRAF mutation status (Pinteraction: 0.28). In joint models, with MS-stable/MSI-low and normal BMI as the reference group, risk of death was higher for MS-stable/MSI-low and obese BMI (HR, 1.32; P value: 0.0002), not statistically significantly lower for MSI-high and normal BMI (HR, 0.86; P value: 0.29), and approximately the same for MSI-high and obese BMI (HR, 1.00; P value: 0.98). Conclusions: High prediagnosis BMI was associated with increased mortality; this association was consistent across participant subgroups, including strata of tumor molecular phenotype. Impact: High BMI may attenuate the survival benefit otherwise observed with MSI-high tumors. Cancer Epidemiol Biomarkers Prev; 24(8); 1229–38. ©2015 AACR.

Abstract Background: The CpG island methylator phenotype (CIMP) represents a subset of colorectal cancers characterized by widespread aberrant DNA hypermethylation at select CpG islands. The risk factors and environmental exposures contributing to etiologic heterogeneity between CIMP and non-CIMP tumors are not known. Methods: We measured the CIMP status of 3,119 primary population-based colorectal cancer tumors from the multinational Colon Cancer Family Registry. Etiologic heterogeneity was assessed by a case–case study comparing risk factor frequency of colorectal cancer cases with CIMP and non-CIMP tumors using logistic regression to estimate the case–case odds ratio (ccOR). Results: We found associations between tumor CIMP status and MSI-H (ccOR = 7.6), BRAF V600E mutation (ccOR = 59.8), proximal tumor site (ccOR = 9; all P < 0.0001), female sex [ccOR = 1.8; 95% confidence interval (CI), 1.5–2.1], older age (ccOR = 4.0 comparing over 70 years vs. under 50; 95% CI, 3.0–5.5), and family history of CRC (ccOR = 0.6; 95% CI, 0.5–0.7). While use of NSAIDs varied by tumor CIMP status for both males and females (P = 0.0001 and P = 0.02, respectively), use of multivitamin or calcium supplements did not. Only for female colorectal cancer was CIMP status associated with increased pack-years of smoking (Ptrend < 0.001) and body mass index (BMI; Ptrend = 0.03). Conclusions: The frequency of several colorectal cancer risk factors varied by CIMP status, and the associations of smoking and obesity with tumor subtype were evident only for females. Impact: Differences in the associations of a unique DNA methylation–based subgroup of colorectal cancer with important lifestyle and environmental exposures increase understanding of the molecular pathologic epidemiology of this heavily methylated subset of colorectal cancer. Cancer Epidemiol Biomarkers Prev; 24(3); 512–9. ©2015 AACR.

AbstractBackground: It is widely accepted that chronic inflammation plays a role in the etiology of colorectal cancer. Using a two-stage design, we examined the associations between colorectal cancer and common variation in 37 key genes in the inflammation and innate immunity pathways.Methods: In the discovery stage, 2,322 discordant sibships (2,535 cases, 3,915 sibling controls) from the Colorectal Cancer Family Registry were genotyped for more than 600 tagSNPs and 99 single-nucleotide polymorphisms (SNP) were selected for further examination based on strength of association. In the second stage, 351 SNPs tagging gene regions covered by the 99 SNPs were tested in 4,783 Multiethnic Cohort subjects (2,153 cases, 2,630 controls).Results: The association between rs9858822 in the PPARG gene and colorectal cancer was statistically significant at the end of the second stage (OR per allele = 1.36, Bonferroni-adjusted P = 0.045), based on the “effective” number of markers in stage II (n = 306). The risk allele C was common (frequency 0.3) in African Americans but rare (frequency < 0.03) in whites, Japanese Americans, Latinos, and Native Hawaiians. No statistically significant heterogeneity of effects across race/ethnicity, body mass index (BMI) levels, regular aspirin use, or pack-years of smoking was detected for this SNP. Suggestive associations were also observed for several SNPs in close vicinity to rs9858822.Conclusions: Our results provide new evidence of association between PPARG variants and colorectal cancer risk.Impact: Further replication in independent samples is warranted. Cancer Epidemiol Biomarkers Prev; 22(11); 2094–101. ©2013 AACR.

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 Introduction: Genome-wide association studies of colorectal cancer have identified 18 risk variants that explain ∼25% of the hereditable risk of colorectal cancer. These variants are located in intronic, inter-genic or gene-desert regions. To uncover whether some of these variants may have a regulatory function on the expression of near-by genes, we conducted expression quantitative trait loci (eQTL) analyses of these 18 regions. Methods: DNA and RNA were extracted from 40 fresh-frozen paired colon tumor (microsatellite stable and CpG island methylator phenotype-negative) and adjacent normal tissue samples collected by the Colon Cancer Family Registry. Whole genome gene expression assessment was performed in the tumors and adjacent normal tissue with the Affymetrix GeneChip Human Exon 1.0 ST array. Genome-wide SNP data was generated with DNA extracted from the same normal tissue samples using the Affymetrix Genome-Wide Human SNP 6.0 array. Partek software was used to compare the expression levels by genotype for each gene spanning a 2 Mb region up- and down- stream of each of the 18 established risk variants for colorectal cancer. Results: Three of the 18 risk variants (rs10795668, rs4444235, rs9929218) demonstrated significant cis-eQTLs (FDR q-values<0.05). Specifically, differential gene expression was observed in genes neighboring rs10795668 at 10p14 (ATP5C1, q=0.024), rs4444235 at 14q22.2 (DLGAP5, q=0.041), and rs9929218 at 16q22.1 (NOL3, q=0.017; DDX28, q=0.046). In particular, DLGAP5 (discs, large (Drosophila) homolog-associated protein 5) at 14q22.2 and NOL3 (nucleolar protein 3) at 16q22 are interesting candidate genes for colorectal cancer. DLGAP5 encodes for a microtubule-associated protein and functions as a mitotic regulator that has been reported to be overexpressed in colon tumors. NOL3 (nucleolar protein 3) encodes for an anti-apoptotic protein that down-regulates the expression of caspase 2 and 8, and p53. NOL3 also appears to be overexpressed in colorectal tumors. Conclusion: In summary, these findings suggest putative functional activities for three established colorectal risk variants and highlight potentially interesting candidate genes for this malignancy. If confirmed, these results may help explain the total genetic risk of the disease, as well as further our understanding of the underlying biology of colorectal cancer. 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 4732. doi:10.1158/1538-7445.AM2011-4732

Background Lynch syndrome is caused by germline mismatch repair (MMR) gene mutations. The PREMM 1,2,6 model predicts the likelihood of a MMR gene mutation based on personal and family cancer history. Objective To compare strategies using PREMM 1,2,6 and tumour testing (microsatellite instability (MSI) and/or immunohistochemistry (IHC) staining) to identify mutation carriers. Design Data from population-based or clinic-based patients with colorectal cancers enrolled through the Colon Cancer Family Registry were analysed. Evaluation included MSI, IHC and germline mutation analysis for MLH1 , MSH2 , MSH6 and PMS2 . Personal and family cancer histories were used to calculate PREMM 1,2,6 predictions. Discriminative ability to identify carriers from non-carriers using the area under the receiver operating characteristic curve (AUC) was assessed. Predictions were based on logistic regression models for (1) cancer assessment using PREMM 1,2,6 , (2) MSI, (3) IHC for loss of any MMR protein expression, (4) MSI+IHC, (5) PREMM 1,2,6 +MSI, (6) PREMM 1,2,6 +IHC, (7) PREMM 1,2,6 +IHC+MSI. Results Among 1651 subjects, 239 (14%) had mutations (90 MLH1 , 125 MSH2 , 24 MSH6 ). PREMM 1,2,6 discriminated well with AUC 0.90 (95% CI 0.88 to 0.92). MSI alone, IHC alone, or MSI+IHC each had lower AUCs: 0.77, 0.82 and 0.82, respectively. The added value of IHC+PREMM 1,2,6 was slightly greater than PREMM 1,2,6 +MSI (AUC 0.94 vs 0.93). Adding MSI to PREMM 1,2,6 +IHC did not improve discrimination. Conclusion PREMM 1,2,6 and IHC showed excellent performance in distinguishing mutation carriers from non-carriers and performed best when combined. MSI may have a greater role in distinguishing Lynch syndrome from other familial colorectal cancer subtypes among cases with high PREMM 1,2,6 scores where genetic evaluation does not disclose a MMR mutation.

Abstract Colon cancer is the result of a multi-step process involving the accumulation of genetic and epigenetic alterations leading to the transformation of normal colon epithelium to adenocarcinoma. Identification of characteristic genetic alterations is of critical importance to increase our understanding of the transformation process. We focused our analysis on the most common molecular sub-type of colon cancers, microsatellite stable (MSS) and CpG island methylator phenotype (CIMP)-negative, to identify characteristic copy number alteration (CNA) events and associated gene expression profiles using high-resolution genome-wide microarrays. DNA and RNA were extracted from 41 fresh-frozen paired colon tumors and adjacent normal tissue collected by the Colon Cancer Family Registry. Genomic profiles, such as CNAs and loss of heterozygosity (LOH), were identified with the Affymetrix Genome-Wide Human SNP 6.0 array for both tumor and adjacent normal tissue. Gene expression profiles of tumors and adjacent normal tissue were identified with the Affymetrix GeneChip Human Exon 1.0 ST array. Partek Genomics Suite software was used for analysis and data integration. We identified recurrent (>25%) CNAs in several chromosomal regions: gain in 1q, 7p, 7q, 8q, 13q, 20p, 20q, Xp, Xq and loss in 5q, 8p, 14q, 16p, 17p, 18p, and 18q. In addition, based on the allele information from the SNP arrays, we found a subset of these recurrent CNAs were associated with LOH. Preliminary results from the integration of CNA and gene expression data in tumors indicate that a subset of these recurrent CNAs is associated with the disruption of gene expression. For example, we observed recurrent copy number gain in 8q24.21, the genomic region containing the c-MYC gene, and associated up-regulation of c-MYC gene expression in tumors compared to adjacent normal tissue. In another example, recurrent copy number loss at 5q22.2 was associated with down-regulation of APC gene expression; loss of function of this tumor suppressor is associated with both hereditary and sporadic colon cancer. This genome-wide characterization of both genomic alterations and gene expression may help identify key genes and pathways that are disrupted in this common molecular subtype of colon cancer. We will present detailed results from this integrated and comprehensive genetic profiling of MSS and CIMP-negative colon cancers. 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 2145.