Potter, John D.

Abstract Background: Both shorter and longer telomeres in peripheral blood leukocyte (PBL) DNA have been associated with cancer risk. However, associations remain inconsistent across studies of the same cancer type. This study compares DNA preparation methods to determine telomere length from patients with colorectal cancer. Methods: We examined PBL relative telomere length (RTL) measured by quantitative PCR (qPCR) in 1,033 patients with colorectal cancer and 2,952 healthy controls. DNA was extracted with phenol/chloroform, PureGene, or QIAamp. Results: We observed differences in RTL depending on DNA extraction method (P < 0.001). Phenol/chloroform-extracted DNA had a mean RTL (T/S ratio) of 0.78 (range 0.01–6.54) compared with PureGene-extracted DNA (mean RTL of 0.75; range 0.00–12.33). DNA extracted by QIAamp yielded a mean RTL of 0.38 (range 0.02–3.69). We subsequently compared RTL measured by qPCR from an independent set of 20 colorectal cancer cases and 24 normal controls in PBL DNA extracted by each of the three extraction methods. The range of RTL measured by qPCR from QIAamp-extracted DNA (0.17–0.58) was less than from either PureGene or phenol/chloroform (ranges, 0.04–2.67 and 0.32–2.81, respectively). Conclusions: RTL measured by qPCR from QIAamp-extracted DNA was less than from either PureGene or phenol/chloroform (P < 0.001). Impact: Differences in DNA extraction method may contribute to the discrepancies between studies seeking to find an association between the risk of cancer or other diseases and RTL. Cancer Epidemiol Biomarkers Prev; 22(11); 2047–54. ©2013 AACR.

Objective Genome-wide association studies have identified a large number of single nucleotide polymorphisms (SNPs) associated with a wide array of cancer sites. Several of these variants demonstrate associations with multiple cancers, suggesting pleiotropic effects and shared biological mechanisms across some cancers. We hypothesised that SNPs previously associated with other cancers may additionally be associated with colorectal cancer. In a large-scale study, we examined 171 SNPs previously associated with 18 different cancers for their associations with colorectal cancer. Design We examined 13 338 colorectal cancer cases and 40 967 controls from three consortia: Population Architecture using Genomics and Epidemiology (PAGE), Genetic Epidemiology of Colorectal Cancer (GECCO), and the Colon Cancer Family Registry (CCFR). Study-specific logistic regression results, adjusted for age, sex, principal components of genetic ancestry, and/or study specific factors (as relevant) were combined using fixed-effect meta-analyses to evaluate the association between each SNP and colorectal cancer risk. A Bonferroni-corrected p value of 2.92×10 −4 was used to determine statistical significance of the associations. Results Two correlated SNPs—rs10090154 and rs4242382—in Region 1 of chromosome 8q24, a prostate cancer susceptibility region, demonstrated statistically significant associations with colorectal cancer risk. The most significant association was observed with rs4242382 (meta-analysis OR=1.12; 95% CI 1.07 to 1.18; p=1.74×10 −5 ), which also demonstrated similar associations across racial/ethnic populations and anatomical sub-sites. Conclusions This is the first study to clearly demonstrate Region 1 of chromosome 8q24 as a susceptibility locus for colorectal cancer; thus, adding colorectal cancer to the list of cancer sites linked to this particular multicancer risk region at 8q24.

Abstract Background: Colorectal cancer (CRC) in densely affected families without Lynch Syndrome may be due to mutations in undiscovered genetic loci. Familial linkage analyses have yielded disparate results; the use of exome sequencing in coding regions may identify novel segregating variants. Methods: We completed exome sequencing on 40 affected cases from 16 multicase pedigrees to identify novel loci. Variants shared among all sequenced cases within each family were identified and filtered to exclude common variants and single-nucleotide variants (SNV) predicted to be benign. Results: We identified 32 nonsense or splice-site SNVs, 375 missense SNVs, 1,394 synonymous or noncoding SNVs, and 50 indels in the 16 families. Of particular interest are two validated and replicated missense variants in CENPE and KIF23, which are both located within previously reported CRC linkage regions, on chromosomes 1 and 15, respectively. Conclusions: Whole-exome sequencing identified DNA variants in multiple genes. Additional sequencing of these genes in additional samples will further elucidate the role of variants in these regions in CRC susceptibility. Impact: Exome sequencing of familial CRC cases can identify novel rare variants that may influence disease risk. Cancer Epidemiol Biomarkers Prev; 22(7); 1239–51. ©2013 AACR.

Abstract Background: Previous reports suggest that relatives of colorectal cancer (CRC)-affected probands carrying the BRAF p.V600E mutation are at an increased risk of CRC and extracolonic cancers (ECC). In this study, we estimated the association between a family history of either CRC or ECC and risk of CRC with a BRAF p.V600E mutation. Methods: Population-based CRC cases (probands, ages 18–59 years at diagnosis), recruited irrespective of family cancer history, were characterized for BRAF p.V600E mutation and mismatch repair (MMR) status. ORs and 95% confidence intervals (CI) were estimated using multivariable logistic regression. Results: The 690 eligible probands showed a mean age at CRC diagnosis of 46.9 ± 7.8 years, with 313 (47.9%) reporting a family history of CRC and 53 (7.7%) that were BRAF-mutated. Probands with BRAF-mutated, MMR-proficient CRCs were less likely to have a family history of CRC than probands that were BRAF wild-type (OR, 0.46; 95% CI, 0.24–0.91; P = 0.03). For probands with a BRAF-mutated CRC, the mean age at diagnosis was greater for those with a CRC-affected first- or second-degree relative (49.3 ± 6.4 years) compared with those without a family history (43.8 ± 10.2 years; P = 0.04). The older the age at diagnosis of CRC with the BRAF p.V600E mutation, the more likely these probands were to show a family history of CRC (OR, 1.09 per year of age; 95% CI, 1.00–1.18; P = 0.04). Conclusions: Probands with early-onset, BRAF-mutated, and MMR-proficient CRC were less likely to have a family history of CRC than probands that were BRAF-wild-type. Impact: These findings provide useful insights for cancer risk assessment in families and suggest that familial or inherited factors are more important in early-onset, BRAF-wild-type CRC. Cancer Epidemiol Biomarkers Prev; 22(5); 917–26. ©2013 AACR.

Abstract Background: Cigarette smoking (CS) is an established colorectal cancer (CRC) risk factor. The p53 protein, encoded by the TP53 tumor suppressor gene that is commonly mutated in CRC, is important in cell cycle arrest needed to repair DNA damage or induce apoptosis to prevent tumor propagation. In this prospective cohort study, we examined CS-associated CRC risks by p53 protein expression level in the population-based Iowa Women's Health Study (IWHS). Methods: The IWHS recruited 41,836 randomly selected Iowa women, ages 55-69 years, with a valid driver's license at study entry (1986). CS and other exposure variables were assessed at baseline, by self-report. Incident CRC cases were ascertained by annual linkage with the Iowa Cancer Registry. Archived, paraffin-embedded tissue specimens were collected from 732 CRC cases diagnosed through 12/31/2002. CS was categorized by smoking status (never, current, former), average number of cigarettes per day (1-19, 20-39, > 40) and cumulative pack-years (1-19, 20-39, > 40). P53 protein expression was determined by immunohistochemistry on tissue microarrays. Tumor cores were scored by combining both staining intensity and percent of cells stained to determine negative, low or high p53 protein expression levels. Multivariate Cox regression models were fit to estimate relative risks (RRs) and 95% confidence intervals (CIs). Results: Of the 732 available CRC cases, 486 (66%) had complete CS and p53 data. Associations between CS and CRC subtypes defined by p53 expression level are shown in the Table. Conclusions: These data demonstrate no clear heterogeneity of CS with CRC subtypes defined by p53 expression levels among older women. 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 1676. doi:1538-7445.AM2012-1676

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 Background: Colorectal cancer (CRC) is a molecularly heterogeneous disease. Three molecular pathways (traditional, serrated and alternate) have been recently proposed based on integrated combinations of microsatellite instability (MSI), CpG island methylation phenotype (CIMP), BRAF mutation, and KRAS mutation status (Gastroenterol 2010;138:2088-2100). To our knowledge, clinicopathologic associations with these CRC pathways have not been previously described. Methods: Data and tissue resources were obtained from the population-based Iowa Women's Health Study, which recruited 41,836 randomly selected women, ages 55-69 years, at study entry (1986). Incident CRC cases were ascertained by annual linkage with the Iowa Cancer Registry. Archived, paraffin-embedded tissue specimens were collected from 732 CRC cases diagnosed through 12/31/2002. Established laboratory methods were used to characterize CRC cases by MSI (MSS/MSI-L or MSI-H), CIMP (neg, low, or high), BRAF mutation (wt or mut), and KRAS mutation (wt or mut) status. Molecular pathways were defined as: traditional (MSS/MSI-L, CIMP-neg, BRAF wt, and KRAS wt); serrated (any MSI, CIMP-high, BRAF mut, and KRAS wt), alternate (MSS/MSI-L, CIMP-low, BRAF wt, and KRAS mut), or other pathway. Chi-square tests and analyses of variance were used to compare clinicopathologic features at CRC presentation (age, anatomic subsite, tumor grade, and SEER stage) across the molecular pathways. Results: Comlete and informative MSI, CIMP, BRAF and KRAS data were obtained for 491/732 (67%) CRC cases. Clinicopathologic associations are shown in the Table. Conclusions: These data suggest that distributions of age at diagnosis, subsite, and tumor grade, but not SEER stage, differ across the traditional, serrated, and alternate carcinogenic pathways, which may have implications for molecularly-targeted CRC prevention and/or therapy strategies. 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 5518. doi:1538-7445.AM2012-5518

Abstract Background: Despite recent advances in prevention and therapy, colorectal cancer (CRC) continues to account for nearly 50,000 deaths per year in the US. TNM stage is the current CRC prognostic standard, yet within-stage prognostic heterogeneity is well documented. Microsatellite instability (MSI), CpG island methylation phenotype (CIMP), and BRAF- and KRAS-mutation status represent candidate prognostic markers. However, associations between these markers and CRC survival have not been described in a population-based setting. Methods: Data and tissue resources were obtained from the Iowa Women's Health Study (IWHS), which recruited 41,836 women, ages 55-69 years, at study entry (1986). Incident CRC cases were ascertained by annual linkage with the Iowa Cancer Registry. Archived, paraffin-embedded tissue specimens were collected from 732 representative CRC cases diagnosed through 12/31/2002. Established laboratory methods were used to characterize CRC cases: MSI-H or MSS/MSI-L (by instability in β3 vs. <3 out of 10 markers, respectively); CIMP+ or CIMP− (by promoter hypermethylation in β3 vs. 0-2 out of 5 genes, respectively); BRAFwt or BRAFmut (by fluorescent allele specific PCR to detect the V600E point mutation in exon 15); and KRASwt or KRASmut (by direct sequence analysis of codons 12 and 13 in exon 2). Cox regression models were fit to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for time to death of any cause adjusted for age, stage, grade, and treatment status. Results: CRC cases with informative molecular marker data ranged from 515/732 (70%) for KRAS to 548/732 (75%) for MSI status. Mean follow-up was 4.4 years for deceased individuals and 11.2 years for women alive at last contact. After multivariable adjustment, both MSS/MSI-L (p=0.004) and KRASmut (p=0.048) tumors were associated with poorer survival. Survival did not significantly differ across levels of CIMP and BRAF status. Conclusions: MSI and KRAS status may influence CRC survival among older women. 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 4492. doi:1538-7445.AM2012-4492

Abstract Background: Folate intake (FI) appears to affect colorectal cancer (CRC) risk, but the molecular mechanism(s) involved are incompletely defined. In this study, we analyzed associations between FI and incident CRC, overall and by KRAS mutation status, in the population-based Iowa Women's Health Study (IWHS). Methods: The IWHS recruited 41,836 randomly selected Iowa women, ages 55-69 years, with a valid driver's license at study entry (1986). FI and other exposure variables were assessed at baseline, by self-report. Incident CRC cases were ascertained by annual linkage with the Iowa Cancer Registry. Archived, paraffin-embedded tissue specimens were recently collected from 732 representative CRC cases diagnosed through 12/31/2002. FI (including supplements) was categorized by quartiles. KRAS mutation status (wildtype [wt] vs. mutated [mut]) was determined by direct sequence analysis of codons 12 and 13 (exon 2). Multivariate Cox regression models were fit to estimate relative risks (RRs) and 95% confidence intervals (CIs). Results: Of the 732 available CRC cases, 470 (64%) had complete FI and KRAS data. FI was not associated with incident CRC overall (for quartile 4 vs. quartile 1, RR = 1.03; 95% CI = 0.82-1.28; p trend = 0.90), or with the KRAS-wt or KRAS-mut subtypes (see Table). Conclusions: These data do not support an association between FI and incident CRC, overall or by KRAS-defined subtypes, among older, predominately white, non-Hispanic women. 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 1892. doi:10.1158/1538-7445.AM2011-1892