In the development of colorectal cancer (CRC), it is now widely accepted that some forms of genetic instability lead to the sequential accumulation of genetic alterations and consequently develop carcinomas. 1 RAS activation in the MAP kinase cascade is supposed to constitute a part of the primary events in colorectal carcinogenesis, and the KRAS gene mutations have been found in about 30 -40% cases of sporadic CRCs. [2][3][4] Recently, activating BRAF mutations have been found almost invariably in melanoma cells and sometimes in other types of carcinoma, including CRCs, 5-7 implying a function of BRAF as a protooncogene. The RAF genes are members of MAPK pathway, encoding serine/threonine kinases that integrate the upstream input signals. 8,9 Once recruited at the cell membrane by GTP-loaded RAS, RAF becomes activated and subsequently phosphorylates the downstream kinases, MEKs, which eventually induce transcriptional activation of the target genes. 9 More recently, frequent BRAF mutations and infrequent KRAS mutations have been reported in DNA-mismatch repair (MMR)-deficient CRCs. 10 Inactivation of MMR genes incurs instability of genomic microsatellite sequence (microsatellite instability, or MSI), which is found in the majority of patients with hereditary nonpolyposis colorectal cancer syndrome (HNPCC) and in 10 -15% of cases of sporadic CRCs. 11-13 Moreover, it was also reported that 70 -90% of sporadic CRCs with MSI (MSI ϩ CRCs) are associated with hypermethylation of hMLH1, one of DNA-MMR genes, and have distinct clinical and pathologic characteristics, i.e., occurrence in older females, location in the proximal colon and histopathology of mucious or poor differentiation. 14 -20 We have previously examined the methylation status of hMLH1 gene in sporadic CRCs by use of 5 sets of primer spanning the whole CpG sites within its promoter region and have classified the methylation status into 3 subtypes: full methylation, partial methylation and nonmethylation. 21,22 We reported that an extensive methylation, or full methylation, of hMLH1 promoter was found in about 80% of MSI ϩ CRC cases and was highly associated with loss of expression of its gene product. Interestingly, this type of CRC cells are rarely associated with KRAS mutations and loss of heterozygosity (LOH) of TP53 gene. 22 It is therefore possible that extensive methylation of hMLH1 promoter region may contribute to the carcinogenesis of the right-sided sporadic CRCs, independently of KRAS/p53 alterations.From these results, 2 questions may arise. First, does the activation of BRAF, instead of KRAS, take part in the carcinogenesis of CRCs with extensive hMLH1 methylation? Second, if so, does the BRAF activation have any relationship with the CRCs with partial methylation, although most of which are microsatellite stable (MSI Ϫ ), maintain MMR gene expression and show a relatively high incidence of KRAS and p53 alterations? 22 Additionally, in the melanoma cells, high frequency of mutations of -catenin and BRAF has been recognized. 23 Some resea...
It has been reported that MLH1 is silenced by promoter methylation, and that this phenomenon is associated with microsatellite instability (MSI) in sporadic colorectal cancer (CRC). To clarify the significance of MLH1 promoter methylation in sporadic CRC, we examined the correlation between methylation status over the entire promoter region and mRNA expression in cases showing high-frequency MSI (MSI-H). MLH1 promoter methylation was analyzed using the bisulfite modification sequencing in 48 MSI-H cases. We also screened for somatic mutation, loss of heterozygosity, and immunohistochemical staining of MLH1. The results showed that methylation patterns could be subdivided into three types: methylation of more than 80% of the CpG sites analyzed (type 1 methylation), methylation of less than 20% (type 2 methylation), and methylation mainly in the region 500 to 921 bases upstream from the translation start site (type 3 methylation). Of the three types, only type 1 methylation correlated with decreased mRNA expression. The frequency of type 1 methylation was significantly higher in cases involving the proximal colon (66.7%, 18/27) compared to that of the distal colon and rectum (23.8%, 5/21, P = 0.004). Immunohistochemical staining of MSI-H cases showed that decreased MLH1 was found in 77.1% (37/48). Of the cases with decreased MLH1, type 1 methylation was present in 59.5% (22/37). Overall, our data suggested that the type 1 methylation pattern may affect MLH1 mRNA expression, such that the majority of MSI-H cases in sporadic CRC, especially proximal colon cancer, exhibited type 1 methylation.
Aims-Microsatellite instability (MSI) was first observed in hereditary non-polyposis colorectal carcinoma (HNPCC) and was subsequently seen in non-familial colorectal carcinoma. The relation between MSI and cancer associated genes in nonfamilial colorectal carcinomas has yet to be evaluated. To clarify this matter, changes in cancer associated genes were examined in non-familial colorectal carcinomas. Methods-Alterations in the adenomatous polyposis coli (APC), p53, and Ki-ras genes were analysed in 24 MSI high (alterations in four to seven of seven loci), nine MSI low (alterations in one to three of seven loci), and 31 MSI negative non-familial carcinomas. The hMSH2 and hMLH1 genes were also analysed in 24 MSI high carcinomas. Results-Both the frequencies and types of alterations in the APC and p53 genes in MSI high carcinomas were the same as those in MSI low and MSI negative carcinomas; however, they were diVerent from those seen in HNPCC. The frequency of Ki-ras mutation was significantly lower in the MSI high cases (two of 24; 8%) than in the others (15 of 38; 39%). Somatic mutation of hMSH2 or hMLH1 was detected in six of 24 (25%) of the MSI high cases. Conclusions-These results suggest that APC and p53 alterations occur irrespective of microsatellite instability status in non-familial colorectal carcinomas, and that Ki-ras mutation is not involved in MSI high non-familial colorectal carcinoma. The pathogenesis of these carcinomas may diVer from both the usual adenoma-carcinoma sequence and HNPCC carcinogenesis. (J Clin Pathol 2000;53:841-845)
The JCCA are suitable for selecting cases to analyze for gene mutations, but the JCCB are not useful for the clinical setting. The authors suggest that an age at onset younger than 50 years is also important for screening. Analyzing TGF beta RII mutations and immunohistochemical staining of hMLH1 or hMSH2 for cases with MSI phenotype are useful for selecting cases who should be tested for germline mutations.
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