Comparison of Microsatellite Instability, CpG Island Methylation Phenotype, BRAF and KRAS Status in Serrated Polyps and Traditional Adenomas Indicates Separate Pathways to Distinct Colorectal Carcinoma End Points

Abstract

The aim of this study was to compare BRAF and KRAS, CpG island methylator phenotype (CIMP), and microsatellite instability (MSI) status in each of the histologic categories, including end-point carcinomas with residual adenoma, of the serrated polyp neoplasia pathway and the traditional (nonserrated) adenoma-carcinoma sequence. Deoxyribonucleic acid (DNA) was extracted from the selected samples and assayed for BRAF, KRAS2 codon12, 13, CIMP using markers hMLH1, MGMT, MINT1, MINT2, p16, and MSI using an assay for BAT25 and BAT26. A BRAF mutation was present in 82% of serrated carcinomas (SCas), 62% of serrated adenomas (SAs), 83% of serrated polyps with abnormal proliferation (SPAPs-syn. sessile serrated adenoma [SSA]), 76% of microvesicular serrated polyps (MVSPs), and was not found in any of the histologic categories of the traditional adenoma-carcinoma sequence. KRAS2 mutations were found in 43% of the goblet cell serrated polyp (GCSP) category, 13% of MVSPs, 7% of SPAPs, and 24% of SAs; in 26% of large traditional adenoma (lTAs) compared with small traditional adenomas (sTAs) (0/30; P<0.005) and in 37.3% of traditional carcinomas (TCa). CIMP-H (>1 marker positive) was significantly more frequent in SPAP, SA, and SCa compared with MVSP (P<0.05); CIMP-H was present in 10% of sTAs but was found more frequently in lTA (44.4%; OR 7.2; P=0.007) and TCa (38.9%; OR 5.8; P=0.007). Higher CIMP levels (4 or more markers positive) were significantly more frequent in advanced categories of the serrated pathway (SAs [31%] and SCas [30%]) compared with lTAs [0%] and TCAs [3.4%] (OR 12.2; P=0.02). MSI-H was identified only in the adenocarcinoma component of SCas (9/11) or in the contiguous SAs (3/7). The findings indicate that a BRAF mutation is a specific marker for a serrated polyp pathway that has its origin in a hyperplastic polyp (MVSP) and a potential end point as MSI carcinoma. CIMP-High (CIMP-H) develops early in this sequence and MSI-H develops late. The data provided a less complete picture of a second serrated pathway, identified by a KRAS2 mutation in SAs, but showed that the progressive stages of both iterations of the serrated neoplasia pathway are separate and distinct from those of the traditional adenoma-carcinoma sequence.

Loss of heterozygosity, aberrant methylation, BRAF mutation and KRAS mutation in colorectal signet ring cell carcinoma

“…45,46 BRAF V600E mutations and CpG island methylation phenotype-positive status were more common in signet ring cell carcinoma and mucinous adenocarcinoma compared with conventional adenocarcinoma. Similar observations regarding BRAF V600E in signet ring cell carcinoma have been noted by Ogino et al 35 Expression of the gastric mucin MUC5AC, a typical feature of serrated polyps, is also more common in mucinous and signet ring cell carcinomas, but is relatively uncommon in conventional adenocarcinoma.…”

Distinct WNT/β-catenin signaling activation in the serrated neoplasia pathway and the adenoma-carcinoma sequence of the colorectum

“…Interestingly, stepwise increment of AXIN2 methylation was identified from SSA/Ps (4%) through those with high-grade dysplasia (64%) to those with submucosal carcinoma (78%), indicating that AXIN2 methylation has an important role in the serrated neoplasia pathway as well as microsatellite instability associated colorectal carcinomas, some of which has been considered to be the end point of progression in the serrated pathway. [3][4][5] Loss of APC function by gene mutation or methylation is the reason for b-catenin translocation into the nucleus in the conventional adenomacarcinoma sequence. 18,25 The majority (60-91%) of conventional tubular adenomas or adenocarcinomas have APC mutations, 15,16 but to our knowledge no mutational study of APC has been conducted in SSA/Ps, although methylation of APC has been reported to be more frequent in tubular adenomas (56-65%) than SSA/Ps (22-25%).…”

“…shown associations of SSA/P and those with dysplasia or carcinoma with methylation or loss of protein expression for DNA repair genes, ie, MLH1, 1,4,6-9 a CpG island methylator phenotype, 3,4,6,8 BRAF mutations, 3,4,[6][7][8][9][10][11][12][13][14] and a lack of genetic alterations in CTNNB1 (the gene coding for b-catenin protein). 14 This pathway is thought to be distinct from the conventional adenoma-carcinoma pathway, where adenomas progress to invasive colorectal carcinomas through the influence of a series of genetic alterations including adenomatous polyposis coli (APC) and KRAS mutations.…”

“…14 This pathway is thought to be distinct from the conventional adenoma-carcinoma pathway, where adenomas progress to invasive colorectal carcinomas through the influence of a series of genetic alterations including adenomatous polyposis coli (APC) and KRAS mutations. 4,6,10,11,15,16 The WNT signaling pathway has a vital role in embryogenesis, 17 and its deregulation is also implicated in colorectal carcinogenesis. 18 b-Catenin in the resting state is degraded by proteasomes resulting from its phosphorylation by a multiprotein complex containing APC, AXIN, and glycogen synthase kinase 3b (GSK3b).…”

Immunohistochemistry with a mutation-specific monoclonal antibody as a screening tool for the BRAFV600E mutational status in primary cutaneous malignant melanoma

“…BRAF mutations were first assayed by allele-specific PCR (codon V600E) by previously described methods. [17][18][19] Two sets of allele-specific primers (forward and reverse gene coding strand) were designed to duplex with a single pair of GAPDH primers as DNA quality internal control to amplify the mutated allele. Following allele-specific PCR, direct DNA Sanger sequencing was used to confirm the mutation positive cases and a few allele-specific PCR failed cases (as indicated by negative GAPDH PCR).…”