A subset of colorectal cancers with CpG island methylator phenotype-high (CIMP-H) is frequently associated with MSI and BRAF V600E mutation. Since limited data are available on different histological types of colorectal polyps, we compared the pattern and the frequency of promoter methylation, CIMP-H, MSI, KRAS and BRAF V600E mutations and the relationship among these molecular parameters and the clinicopathologic characteristics in 110 serrated polyps (48 hyperplastic polyps, 32 sessile serrated adenomas and 30 serrated adenomas) and 32 tubular adenomas using 7 commonly used tumor-associated gene loci. No significant difference in the frequency of overall methylation frequency (86% vs. 100%) and CIMP-H (39% vs. 28%) between serrated polyps and tubular adenomas was observed, but proximally located serrated polyps showed more frequent methylation at 5 of 7 loci examined, and were more likely to be CIMP-H (62% vs. 22%). MGMT methylation was more common in tubular adenomas while MLH1 and HIC1 were more frequently methylated in serrated polyps. BRAF mutation was frequently present in all types of serrated polyps (80%), but was absent in tubular adenomas and was not associated with CIMP or MSI status. These results show comparable frequencies of promoter methylation of tumor-associated genes and CIMP-H, but distinct differences in gene-specific or colonic site-specific methylation profiles occur in serrated polyps and tubular adenomas. BRAF mutation occurs independently of CIMP and MSI in all types of serrated polyps and may serve as a marker of serrated pathway of colorectal carcinogenesis. ' 2008 Wiley-Liss, Inc.Key words: serrated polyp; tubular adenoma; DNA methylation; CpG island methylator phenotype; BRAF mutation Hypermethylation of CpG islands in the promoter region of tumor suppressor genes and tumor-associated genes may cause transcriptional silencing, with subsequent loss of protein expression in colorectal cancers. 1-3 Moreover, a high degree of concurrent promoter methylation in multiple genes, called the CpG island methylator phenotype (CIMP), has been described in a subset of colorectal cancer. 4-6 CIMP positive colorectal cancers have been reported to have distinct clinicopathological features such as proximal location, microsatellite instability (MSI), BRAF V600E mutation and wild-type p53. [5][6][7][8][9] CpG island methylation has also been reported to occur in precursor lesions such as aberrant crypt foci and colorectal polyps. [10][11][12][13][14][15][16][17][18][19] Although a higher frequency and the level of promoter methylation and CIMP in serrated colorectal polyps such as sessile serrated adenomas (SSA) and serrated adenomas (SA) has been reported, [11][12][13][14] there are also reports that indicate that CIMP occurs in 6-30% of tubular adenomas (TA). [15][16][17] Higher frequency of methylation has been associated with large size (>1 cm) and presence of high-grade dysplasia in TA. 18,19 In addition, a higher frequency of promoter methylation of MGMT and CDKN2A has been reported in TA (49...

The relationship of molecular abnormalities with clinicopathologic features and survival in colorectal signet ring cell carcinoma, and its comparison with mucinous and conventional adenocarcinomas, has not been well studied. High-level microsatellite instability, loss of heterozygosity (LOH) at four loci, CpG island methylation phenotype based on seven loci, BRAF V600E mutation and KRAS mutation in signet ring cell carcinoma were compared with mucinous and conventional adenocarcinomas. The relationship of these molecular features in signet ring cell carcinoma with clinicopathologic features and survival was examined. LOH was observed in 93% of signet ring cell carcinomas compared with 62 and 70% of mucinous and conventional adenocarcinomas. Also, 80% of signet ring cell carcinomas with high-level microsatellite instability showed LOH compared with 14% each of mucinous and conventional adenocarcinomas. High-level microsatellite instability, CpG island methylation phenotype-positive status and BRAF V600E mutation were more often seen in signet ring cell carcinoma and mucinous adenocarcinoma compared with conventional adenocarcinoma. BRAF V600E mutation was significantly associated with CpG island methylation phenotype-positive status. Stage and BRAF V600E mutation in microsatellite-stable cases were the only variables with an affect on survival. In conclusion, chromosomal instability manifested by LOH is nearly a universal finding in signet ring cell carcinoma, including cases with highlevel microsatellite instability. This may explain the aggressive behavior of signet ring cell carcinoma irrespective of high-level microsatellite-instability status. BRAF V600E mutation and CpG island methylation phenotypepositive status are similar in signet ring cell carcinoma and mucinous adenocarcinoma but more frequent when compared with conventional adenocarcinoma. In signet ring cell carcinoma, BRAF V600E mutation adversely affects survival in microsatellite-stable tumors, but not in high-level microsatellite-unstable tumors. The high frequency of methylation and BRAF V600E mutation suggests that many signet ring cell carcinomas may be related to the serrated pathway of carcinogenesis.

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Abstract. Abundant mucin production and MUC2 expression is the key feature of mucinous colorectal cancer (CRC). Although MUC2 gene methylation has been thought to play an important role in loss of MUC2 expression, the tissues are difficult to analyze because of the cellular heterogeneity of tissue samples. In the present study, we determined the role of region-specific methylation in the MUC2 promoter in MUC2 expression in CRC. Additionally, we optimized the conditions for quantification of methylation analysis in mucinous and non-mucinous CRC tissues. We identified two regions in MUC2 promoter, region A (-289 and -274) and region C (-193 and -160), that correlated with loss of MUC2 expression by comparing the methylation status in 13 CRC cell lines with no or low MUC2 expression and those in 4 cell lines with high MUC2 expression. To prove the correlation of MUC2 methylation status and loss of expression in CRC tissues, MUC2 methylation status in tumors needs to be determined. Since the critical CpG sites have been identified in cell lines by sequencing, a more rapid and sensitive methylation specific PCR (MSP) was used. We conducted MSP at 3 CpG sites (-289, -274, -193) in 19 mucinous and 34 non-mucinous CRC tissues because this analysis worked at only these sites in the preliminary cell line experiments. Our results showed that methylation status of mucinous CRC was significantly lower than that of non-mucinous CRC at 3 sites (-289; p=0.001, -274; p=0.013, -193; p=0.001), and correlated with high level of MUC2 expression as determined by immunohistochemistry. Besides, these results indicated that MUC2 expression and mucin contents decreased in accordance with the increase of methylation status. We concluded that low methylation status of MUC2 gene plays a predominant role in high level MUC2 expression in mucinous CRC.