CRC Susceptibility Loci and Colorectal Polyps by Type
CRC Susceptibility Loci and Colorectal Polyps by Type
Details on this study population were previously reported. Participants were enrollees in an integrated health-care delivery system in western Washington State (Group Health Cooperative, Seattle, Washington) aged 24–79 years who underwent an index colonoscopy for any indication between 1998 and 2007 and donated a buccal-cell or blood sample for genotyping analysis. Study recruitment took place in 2 phases, with phase 1 occurring in 1998–2003 and phase 2 occurring in 2004–2007. Persons who had undergone a colonoscopy less than 1 year prior to the index colonoscopy, persons with inadequate bowel preparation for the index colonoscopy, and persons with a prior or new diagnosis of colorectal cancer, a familial colorectal cancer syndrome (such as familial adenomatous polyposis), or another colorectal disease were ineligible. Patients diagnosed with adenomas or serrated polyps and persons who were polyp-free at the index colonoscopy (controls) were systematically recruited during both phases of recruitment. Approximately 75% agreed to participate and provided written informed consent. Based on medical records, persons who agreed to participate and those who refused study participation were similar with respect to age, sex, and colorectal polyp status. Study protocols were approved by the institutional review boards of the Group Health Cooperative and the Fred Hutchinson Cancer Research Center (Seattle, Washington).
Participants completed a structured questionnaire that elicited information on their demographic characteristics, including race/ethnicity, and personal risk factors for colorectal cancer. Participant sex and age at the index colonoscopy were confirmed through standardized medical record abstraction. The size of the index polyp was determined through abstraction of endoscopy and pathology reports.
Two study pathologists worked in tandem to conduct a standardized pathology review of clinical biopsy specimens, which had previously been fixed in paraffin, cut and mounted onto slides, and stained with hematoxylin and eosin. Using established protocols and criteria, these pathologists classified polyps as belonging to one of 6 types: 1) tubular adenomas; 2) tubulovillous adenomas (having ≥20% villous components); 3) hyperplastic polyps; 4) SSPs; 5) traditional serrated adenomas; and 6) other colorectal polyps. Disagreements between study pathologists were reconciled through re-review by both pathologists and by referral to a standard training set of polyp slides.
Four groups of study participants were defined on the basis of the clinical findings at endoscopy and the standardized pathology review. If a participant had at least 1 index tubular or tubulovillous adenoma and no serrated polyps, he/she was classified as an adenoma case. Participants with hyperplastic polyps, traditional serrated adenomas, or SSPs and no synchronous adenomas were classified as serrated polyp cases. Cases with both adenomas and serrated polyps were placed into a separate category. Controls were persons who had no colorectal pathology identified and no biopsies collected during the index colonoscopy.
Adenomas were classified as advanced if they 1) were ≥10 mm in diameter according to the endoscopic determination of polyp size or 2) had ≥20% villous components or high-grade dysplasia according to the standard pathology review. Among serrated polyps, SSPs were considered advanced lesions and hyperplastic polyps were considered nonadvanced lesions. Notably, traditional serrated adenomas are also a distinct type of advanced serrated polyp. Because traditional serrated adenomas and SSPs tend to exhibit different molecular markers and have differing distributions in the colon and rectum, these polyps are generally hypothesized to have distinct developmental trajectories within the serrated pathway. Ideally, we would categorize traditional serrated adenomas into their own advanced serrated case group; however, there were only 14 cases with traditional serrated adenomas, so these were excluded from analyses of lesion severity.
We selected 13 single-nucleotide polymorphisms (SNPs) that were associated with colorectal cancer in prior GWAS at a significance level of P < 5 × 10 and for which the association was replicated in a separate study population.Table 1 lists these SNPs from prior GWAS and the particulars of their associations with colorectal cancer. We extracted genomic DNA from lymphocytes during phase 1 of data collection and from buccal samples during phase 2, using the Qiagen QIAamp DNA extraction kit (Qiagen Inc., Valencia, California). Extracted DNA was stored as pellets at −80°C until the pellets were genotyped. We used the Illumina GoldenGate assay with VeraCode microbeads and the BeadXpress reader (Illumina, Inc., San Diego, California) to perform multiplex genotyping of SNPs. For quality control and to monitor for possible contamination between samples, we included wells with reagents only and genotyped a 3% random sample of study participants in duplicate. Genotyping results were similar between duplicate samples, and all SNPs were in Hardy-Weinberg equilibrium among controls (P < 0.05 for each SNP).
For each SNP, we defined the risk allele as the allele associated with an increased risk of colorectal cancer in prior GWAS. Genotypes were then coded as 0, 1, or 2, according to the number of risk alleles that were present for each SNP. This coding was done to make it easy to see whether associations were in the same direction as prior colorectal cancer GWAS or in the opposite direction. We also constructed a risk-allele score for each individual which was the sum of risk alleles present in that individual across the 13 SNPs of interest (possible values ranged from 0 to 26). Genotypes were analyzed with log-additive models, and the risk-allele score was analyzed as a continuous variable. We used polytomous logistic regression models to compare each polyp case group with the polyp-free control group and to estimate adjusted odds ratios and 95% confidence intervals for the associations between polyp subtypes and increasing numbers of risk alleles for each SNP and for the risk-allele score. These same models were used to compare case groups with one another, and we evaluated the Wald P value for the comparison of heterogeneity between case groups for each SNP and the risk-allele score. Results of all regression analyses were adjusted for study phase, age, sex, and race/ethnicity. Among eligible participants, data were complete for these variables. All statistical analyses were performed using Stata 12.0 (StataCorp LP, College Station, Texas).
Methods
Study Population
Details on this study population were previously reported. Participants were enrollees in an integrated health-care delivery system in western Washington State (Group Health Cooperative, Seattle, Washington) aged 24–79 years who underwent an index colonoscopy for any indication between 1998 and 2007 and donated a buccal-cell or blood sample for genotyping analysis. Study recruitment took place in 2 phases, with phase 1 occurring in 1998–2003 and phase 2 occurring in 2004–2007. Persons who had undergone a colonoscopy less than 1 year prior to the index colonoscopy, persons with inadequate bowel preparation for the index colonoscopy, and persons with a prior or new diagnosis of colorectal cancer, a familial colorectal cancer syndrome (such as familial adenomatous polyposis), or another colorectal disease were ineligible. Patients diagnosed with adenomas or serrated polyps and persons who were polyp-free at the index colonoscopy (controls) were systematically recruited during both phases of recruitment. Approximately 75% agreed to participate and provided written informed consent. Based on medical records, persons who agreed to participate and those who refused study participation were similar with respect to age, sex, and colorectal polyp status. Study protocols were approved by the institutional review boards of the Group Health Cooperative and the Fred Hutchinson Cancer Research Center (Seattle, Washington).
Study Questionnaire and Medical Record Abstraction
Participants completed a structured questionnaire that elicited information on their demographic characteristics, including race/ethnicity, and personal risk factors for colorectal cancer. Participant sex and age at the index colonoscopy were confirmed through standardized medical record abstraction. The size of the index polyp was determined through abstraction of endoscopy and pathology reports.
Standardized Pathology Review
Two study pathologists worked in tandem to conduct a standardized pathology review of clinical biopsy specimens, which had previously been fixed in paraffin, cut and mounted onto slides, and stained with hematoxylin and eosin. Using established protocols and criteria, these pathologists classified polyps as belonging to one of 6 types: 1) tubular adenomas; 2) tubulovillous adenomas (having ≥20% villous components); 3) hyperplastic polyps; 4) SSPs; 5) traditional serrated adenomas; and 6) other colorectal polyps. Disagreements between study pathologists were reconciled through re-review by both pathologists and by referral to a standard training set of polyp slides.
Case-control Classification
Four groups of study participants were defined on the basis of the clinical findings at endoscopy and the standardized pathology review. If a participant had at least 1 index tubular or tubulovillous adenoma and no serrated polyps, he/she was classified as an adenoma case. Participants with hyperplastic polyps, traditional serrated adenomas, or SSPs and no synchronous adenomas were classified as serrated polyp cases. Cases with both adenomas and serrated polyps were placed into a separate category. Controls were persons who had no colorectal pathology identified and no biopsies collected during the index colonoscopy.
Classification of Lesion Severity
Adenomas were classified as advanced if they 1) were ≥10 mm in diameter according to the endoscopic determination of polyp size or 2) had ≥20% villous components or high-grade dysplasia according to the standard pathology review. Among serrated polyps, SSPs were considered advanced lesions and hyperplastic polyps were considered nonadvanced lesions. Notably, traditional serrated adenomas are also a distinct type of advanced serrated polyp. Because traditional serrated adenomas and SSPs tend to exhibit different molecular markers and have differing distributions in the colon and rectum, these polyps are generally hypothesized to have distinct developmental trajectories within the serrated pathway. Ideally, we would categorize traditional serrated adenomas into their own advanced serrated case group; however, there were only 14 cases with traditional serrated adenomas, so these were excluded from analyses of lesion severity.
Selection of Single-nucleotide Polymorphisms and Genotyping
We selected 13 single-nucleotide polymorphisms (SNPs) that were associated with colorectal cancer in prior GWAS at a significance level of P < 5 × 10 and for which the association was replicated in a separate study population.Table 1 lists these SNPs from prior GWAS and the particulars of their associations with colorectal cancer. We extracted genomic DNA from lymphocytes during phase 1 of data collection and from buccal samples during phase 2, using the Qiagen QIAamp DNA extraction kit (Qiagen Inc., Valencia, California). Extracted DNA was stored as pellets at −80°C until the pellets were genotyped. We used the Illumina GoldenGate assay with VeraCode microbeads and the BeadXpress reader (Illumina, Inc., San Diego, California) to perform multiplex genotyping of SNPs. For quality control and to monitor for possible contamination between samples, we included wells with reagents only and genotyped a 3% random sample of study participants in duplicate. Genotyping results were similar between duplicate samples, and all SNPs were in Hardy-Weinberg equilibrium among controls (P < 0.05 for each SNP).
Statistical Analyses
For each SNP, we defined the risk allele as the allele associated with an increased risk of colorectal cancer in prior GWAS. Genotypes were then coded as 0, 1, or 2, according to the number of risk alleles that were present for each SNP. This coding was done to make it easy to see whether associations were in the same direction as prior colorectal cancer GWAS or in the opposite direction. We also constructed a risk-allele score for each individual which was the sum of risk alleles present in that individual across the 13 SNPs of interest (possible values ranged from 0 to 26). Genotypes were analyzed with log-additive models, and the risk-allele score was analyzed as a continuous variable. We used polytomous logistic regression models to compare each polyp case group with the polyp-free control group and to estimate adjusted odds ratios and 95% confidence intervals for the associations between polyp subtypes and increasing numbers of risk alleles for each SNP and for the risk-allele score. These same models were used to compare case groups with one another, and we evaluated the Wald P value for the comparison of heterogeneity between case groups for each SNP and the risk-allele score. Results of all regression analyses were adjusted for study phase, age, sex, and race/ethnicity. Among eligible participants, data were complete for these variables. All statistical analyses were performed using Stata 12.0 (StataCorp LP, College Station, Texas).
Source...