BRAF Mutations in Aberrant Crypt Foci and Hyperplastic Polyposis
2005; Elsevier BV; Volume: 166; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)62327-9
ISSN1525-2191
AutoresRobyn Beach, Annie On-On Chan, Tsung‐Teh Wu, Jill A. White, Jeffrey S. Morris, Simón Lunagómez, Russell R. Broaddus, Jean‐Pierre J. Issa, Stanley R. Hamilton, Asif Rashid,
Tópico(s)Colorectal Cancer Screening and Detection
ResumoPatients with hyperplastic polyposis have multiple hyperplastic polyps (HPs) and increased risk of colorectal carcinomas. Aberrant crypt foci (ACF) are postulated to be the earliest precursor lesions in colorectal carcinogenesis. We evaluated BRAF mutations by DNA sequencing in 53 ACF from patients with sporadic colorectal carcinomas and familial adenomatous polyposis, in 18 sporadic HPs from patients with resected colorectal cancer, and in 70 HPs, 4 serrated adenomas, 3 admixed hyperplastic-adenomatous polyps, 10 tubular adenomas, and 6 carcinomas from 17 patients with multiple/large HPs and/or hyperplastic polyposis. BRAF mutation status was compared with clinicopathological features and other genetic alterations by marginal logistic regression. BRAF mutation was present in only 2% of ACF and 6% of sporadic HPs. In contrast, BRAF mutation was present in 43% of HPs (P = 0.01 versus sporadic HPs), 75% of serrated adenomas, 33% of admixed hyperplastic-adenomatous polyps, 30% of tubular adenomas, and 33% of carcinomas from patients with multiple/large HPs and/or hyperplastic polyposis. BRAF mutation status in patients with multiple/large HPs and/or hyperplastic polyposis correlated with HPs from the same patient (odds ratio, 5.8; P = 0.0002) but associated with younger age (odds ratio, 0.83; P = 0.006 compared to older age), with a large HP (odds ratio, 22.5; P = 0.01 compared with patients with multiple HPs), with location of HPs in the right colon (odds ratio, 3.0; P = 0.03), and with methylation of the p16 gene and the MINT31 locus [odds ratio, 12.2 (P = 0.0001) and 4.4 (P = 0.02), respectively]. Our study shows that BRAF mutation status is heterogeneous among patients with multiple/large HPs and/or hyperplastic polyposis, suggesting differences in pathogenesis of HPs that indicate subsets within this phenotype. Patients with hyperplastic polyposis have multiple hyperplastic polyps (HPs) and increased risk of colorectal carcinomas. Aberrant crypt foci (ACF) are postulated to be the earliest precursor lesions in colorectal carcinogenesis. We evaluated BRAF mutations by DNA sequencing in 53 ACF from patients with sporadic colorectal carcinomas and familial adenomatous polyposis, in 18 sporadic HPs from patients with resected colorectal cancer, and in 70 HPs, 4 serrated adenomas, 3 admixed hyperplastic-adenomatous polyps, 10 tubular adenomas, and 6 carcinomas from 17 patients with multiple/large HPs and/or hyperplastic polyposis. BRAF mutation status was compared with clinicopathological features and other genetic alterations by marginal logistic regression. BRAF mutation was present in only 2% of ACF and 6% of sporadic HPs. In contrast, BRAF mutation was present in 43% of HPs (P = 0.01 versus sporadic HPs), 75% of serrated adenomas, 33% of admixed hyperplastic-adenomatous polyps, 30% of tubular adenomas, and 33% of carcinomas from patients with multiple/large HPs and/or hyperplastic polyposis. BRAF mutation status in patients with multiple/large HPs and/or hyperplastic polyposis correlated with HPs from the same patient (odds ratio, 5.8; P = 0.0002) but associated with younger age (odds ratio, 0.83; P = 0.006 compared to older age), with a large HP (odds ratio, 22.5; P = 0.01 compared with patients with multiple HPs), with location of HPs in the right colon (odds ratio, 3.0; P = 0.03), and with methylation of the p16 gene and the MINT31 locus [odds ratio, 12.2 (P = 0.0001) and 4.4 (P = 0.02), respectively]. Our study shows that BRAF mutation status is heterogeneous among patients with multiple/large HPs and/or hyperplastic polyposis, suggesting differences in pathogenesis of HPs that indicate subsets within this phenotype. Colorectal cancer is the second most common cause of cancer deaths in the United States. 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We compared the BRAF mutation status with polyp and patient characteristics, including correlation among multiple HPs from the same patient. All patients had given informed consent for the collection of specimens according to institutional guidelines. ACF were isolated from the grossly normal mucosa in 10 colectomy specimens from patients with sporadic colorectal cancers and from the nonpolypoid mucosa in two colectomy specimens from FAP patients with numerous polyps but no cancer. These ACF have been characterized previously.10Chan AO Broaddus RB Houlihan PS Issa J-PJ Morris JS Hamilton SR Rashid A CpG island methylation in aberrant crypt foci of the colorectum.Am J Pathol. 2002; 160: 1823-1830Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar Thirty ACF were from patients with sporadic colorectal cancers and 23 ACF from FAP patients. The ACF were classified as dysplastic, heteroplastic, or mixed (features of both dysplastic and heteroplastic ACF).10Chan AO Broaddus RB Houlihan PS Issa J-PJ Morris JS Hamilton SR Rashid A CpG island methylation in aberrant crypt foci of the colorectum.Am J Pathol. 2002; 160: 1823-1830Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar Eighteen sporadic HPs from 15 patients undergoing resection of colorectal cancer at The University of Texas MD Anderson Cancer Center, Houston, TX, and the patients and specimens from patients with multiple/large HPs and/or hyperplastic polyposis have been reported previously (Figure 1).16Rashid A Houlihan PS Booker S Peterson GM Giardiello FM Hamilton SR Phenotypic and molecular characteristics of hyperplastic polyposis.Gastroenterology. 2000; 119: 323-332Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 31Chan AO Issa J-PJ Morris JS Hamilton SR Rashid A Concordant CpG island methylation in hyperplastic polyposis.Am J Pathol. 2002; 160: 529-536Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar The patients were classified into three groups based on the number and size of HPs: large HPs (patients with HP greater than 1 cm), hyperplastic polyposis (patients with more than 20 HPs), and multiple HPs (patients with 5 to 10 HPs), as described previously.16Rashid A Houlihan PS Booker S Peterson GM Giardiello FM Hamilton SR Phenotypic and molecular characteristics of hyperplastic polyposis.Gastroenterology. 2000; 119: 323-332Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar Predominance of HPs in the right colon and predominance of HPs in the left colorectum were defined by the location of the majority of HPs in the right colon or in the left colon and rectum, respectively.31Chan AO Issa J-PJ Morris JS Hamilton SR Rashid A Concordant CpG island methylation in hyperplastic polyposis.Am J Pathol. 2002; 160: 529-536Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar We evaluated 70 HPs, 4 SAs, 3 AHAPs, 10 tubular adenomas, and 6 carcinomas from 17 patients with multiple/large HPs and/or hyperplastic polyposis. Exons 11 and 15 of the BRAF gene were amplified and sequenced as previously described.35Yuen ST Davies H Chan TL Ho JW Bignell GR Cox C Stephens P Edkins S Tsui WW Chan AS Futreal PA Stratton MR Wooster R Leung SY Similarity of the phenotypic patterns associated with BRAF and KRAS mutations in colorectal neoplasia.Cancer Res. 2002; 62: 6451-6455PubMed Google Scholar Exons 11 and 15 were amplified by genomic polymerase chain reaction using intronic primers and a commercial DNA sequencing kit according to the manufacturer's instructions (BigDye Terminator version 1.1 cycle sequencing kit; Applied Biosystems, Foster City, CA). The polymerase chain reaction products were analyzed with an Applied Biosystems 3730 automated sequencer using forward and reverse primers. All mutations were confirmed by an independent polymerase chain reaction amplification and sequencing. All BRAF mutations identified were a missense mutation at codon 599, exon 15 replacing GTG (valine) to GAG (glutamic acid). No mutations were identified in exon 11 or other codons of exon 15. Germline mutations were excluded by sequencing nonlesional DNA from these patients. KRAS mutation status of ACF, and KRAS mutations, loss of heterozygosity of chromosome 1p, MSI and CIMP status of sporadic HPs, and of HPs, SAs, AHAPs, tubular adenomas, and carcinomas from patients with multiple/large HPS and/or hyperplastic polyposis have been reported previously.10Chan AO Broaddus RB Houlihan PS Issa J-PJ Morris JS Hamilton SR Rashid A CpG island methylation in aberrant crypt foci of the colorectum.Am J Pathol. 2002; 160: 1823-1830Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 16Rashid A Houlihan PS Booker S Peterson GM Giardiello FM Hamilton SR Phenotypic and molecular characteristics of hyperplastic polyposis.Gastroenterology. 2000; 119: 323-332Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 31Chan AO Issa J-PJ Morris JS Hamilton SR Rashid A Concordant CpG island methylation in hyperplastic polyposis.Am J Pathol. 2002; 160: 529-536Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar MSI-high was defined by presence of allelic shift in comparison with control DNA in at least 30% of evaluated markers. Methylation was assessed at the p16 gene and loci methylated in tumor (MINT): MINT1, MINT2, and MINT31. MINT1 is an island associated with a cDNA transcript of unknown function. MINT2 corresponds to a CpG island that is in the 5′ region of a cDNA with an open reading frame that has no protein homology. MINT31 is 2 kb upstream of the CACNAIG, a T-type calcium channel gene (J.P. Issa, unpublished data). HPs, SAs, adenomas, and carcinomas were classified as CIMP-high if two or more (50%) of the p16 gene or MINT loci were methylated, CIMP-low if one (25%) marker was methylated, and CIMP-negative if no marker was methylated. Patients with more than one HP were represented multiple times in this data set. To model correctly the correlation among polyps coming from the same patient as well as simultaneously partition out the effects of the various factors considered, marginal logistic regression models for correlated binary data44Carey V Zeger SL Diggle P Modelling multivariate binary data with alternating logistic regression.Biometrika. 1993; 80: 517-526Crossref Scopus (399) Google Scholar were used to assess associations between BRAF mutations and the various polyp and patient characteristics. These associations were tested for association with BRAF mutations and were represented as odds ratios, in which an odds ratio of greater than one suggests positive correlation of BRAF mutations with patients or polyp characteristics, respectively. We used three models. The first model with no factors was used to estimate the correlation among the BRAF mutation status in polyps from the same patient, without adjusting for other covariates. A second model included various patient- and polyp-level factors, including the methylation status of the p16 gene and MINT1, MINT2, and MINT31 loci as potential predictors of BRAF mutation status. A third model was used with CIMP status (CIMP-high versus CIMP-low and CIMP-negative) substituted in place of the methylation status of the p16 gene and MINT1, MINT2, MINT31 loci, individually. The statistical analysis was performed using PROC GENMOD in SAS (SAS Institute, Cary, NC), using an assumption that all polyps within a patient were equally correlated. In all models, factors with P values less than 0.05 were considered statistically significant. Twenty-three ACF were from 2 FAP patients and 30 ACF from 10 patients with sporadic colorectal carcinomas. As previously reported,10Chan AO Broaddus RB Houlihan PS Issa J-PJ Morris JS Hamilton SR Rashid A CpG island methylation in aberrant crypt foci of the colorectum.Am J Pathol. 2002; 160: 1823-1830Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar 91% (21 of 23) of ACF from FAP patients were dysplastic and 9% (2 of 23) were heteroplastic. In contrast, 87% (26 of 30) of ACF from patients with sporadic colorectal cancer were heteroplastic, only 10% (3 of 10) were dysplastic, and 3% (1 of 30) were mixed. BRAF mutation was present in 0% (0 of 23) of ACF from FAP patients and only 3% (1 of 30) of ACF from patients with sporadic colorectal cancers (Figure 2A). In contrast, KRAS mutation was present in 4% (1 of 23) of ACF from FAP patients and 40% (12 of 30) of ACF from patients with sporadic colorectal cancers. BRAF mutation was present in a heteroplastic ACF and was the missense
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