Kazuhisa Shitoh

Mutations of BRAF are associated with extensive hMLH1 promoter methylation in sporadic colorectal carcinomas

Activating mutations of BRAF have been frequently observed in microsatellite unstable (MSI+) colorectal carcinomas (CRCs), in which mutations of BRAF and KRAS are mutually exclusive. Previously, we reported that hypermethylation of hMLH1 might play an important role in the tumorigenesis of right‐sided sporadic CRCs with MSI showing less frequency of KRAS/TP53 alteration. Therefore, we have assumed that BRAF mutations might be highly associated with hMLH1 methylation status rather than MSI status. In this study, mutations of BRAF and KRAS and their relationship with MSI and hMLH1 methylation status were examined in 140 resected specimens of CRC. The methylation status was classified into 3 types: full methylation (FM), partial methylation (PM) and nonmethylation (NM). Only FM closely linked to reduced expression of hMLH1 protein. BRAF mutations were found in 16 cases (11%), all leading to the production of BRAFV599E. As for MSI status, BRAF mutations were found in 43% of MSI+ and 4% of MSI− cases (p < 0.0001). Among the MSI+ individuals, BRAF mutations were more frequent in cases with hMLH1 deficiency (58%) than those with hMSH2 deficiency (0%; p = 0.02). Moreover, they were found in 69% of FM, 4% of PM and 4% of NM, revealing a striking difference between FM and the other 2 groups (FM vs. PM or NM; p < 0.0001). These findings suggest that BRAF activation may participate in the carcinogenesis of sporadic CRCs with hMLH1 hypermethylation in the proximal colon, independently of KRAS activation.

Densely methylated MLH1 promoter correlates with decreased mRNA expression in sporadic colorectal cancers

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.

Frequent activation of the ?-catenin-Tcf signaling pathway in nonfamilial colorectal carcinomas with microsatellite instability

It has been reported that wild‐type APC protein forms a complex with β‐Catenin and GSK3β, inducing degradation of β‐Catenin in normal cells. Both β‐Catenin and APC gene mutations have recently been shown to activate the same signaling pathway. Frequent mutations of β‐Catenin in hereditary nonpolyposis colorectal carcinomas have also been reported. It was, however, controversial whether the mutation of the β‐Catenin gene was frequent in nonfamilial colorectal carcinomas with high‐frequency microsatellite instability (MSI‐H). We analyzed the mutations of the APC and β‐Catenin genes in 56 nonfamilial colorectal carcinomas stratified according to the presence or absence of microsatellite instability (MSI). APC mutations were identified in 11 of 22 (50%) cases of MSI‐H and 14 of 34 (41%) cases of microsatellite‐stable (MSS)/low‐frequency microsatellite instability (MSI‐L). In contrast, the frequency of β‐Catenin mutations was significantly higher in MSI‐H (6/22; 27%) than in MSS/MSI‐L (1/34; 3%) (P = 0.01). β‐Catenin mutations were not detected in carcinomas with APC mutation. APC mutation occurred irrespective of MSI status. β‐Catenin mutation, however, occurred frequently in MSI‐H carcinomas. Our data suggest that activation of the β‐Catenin‐Tcf signaling pathway, through either β‐Catenin or APC mutation, frequently contributes to MSI‐H nonfamilial colorectal carcinomas (17/22; 77%).

Microsatellite instability as a marker in predicting metachronous multiple colorectal carcinomas after surgery. A cohort-like study

AbstractPURPOSE: In case-control studies, it was reported that microsatellite instability might be helpful in predicting the development of metachronous multiple colorectal cancers. The purpose of this cohort-like study was to determine whether microsatellite instability is a novel independent marker in predicting metachronous colorectal carcinomas after colorectal cancer surgery. METHODS: Three hundred twenty-eight colorectal carcinoma patients were surveyed by periodic colonoscopy for at least three years after surgery. Among these, DNA from paraffin-embedded sections was available for 272 cases. DNA of these cases was studied for six microsatellite markers (five dinucleotide repeats, one mononucleotide repeat). Microsatellite instability phenotype was defined as alterations in one or more loci. RESULTS: Median follow-up period was 74 months, and the median number of colonoscopies was 4.6. The percentage of microsatellite instability-positive cases was 26.4 percent (72/272). Seventeen metachronous colorectal carcinomas were detected during the follow-up period. Incidences of metachronous colorectal carcinomas in microsatellite instability-positive and microsatellite instability-negative cases were 15.3 and 3 percent, respectively (P < 0.001). The cumulative five-year incidence of metachronous colorectal carcinomas was significantly higher in microsatellite instability-positive cases than in microsatellite instability-negative cases (12.5 vs. 2.5 percent, P < 0.0001). Logistic regression analysis of the relationship between incidence of metachronous colorectal carcinomas and possible risk factors (namely, coexistence of adenoma at the time of surgery, family history of colorectal carcinoma , history of extracolonic malignancy, and microsatellite instability status) showed that microsatellite instability and coexistence of adenoma were significant independent risk factors for the occurrence of metachronous colorectal carcinomas, with values of P = 0.001 and 0.02, respectively. CONCLUSION: These data indicate that microsatellite instability can be regarded as a novel independent and important marker for predicting the development of metachronous colorectal carcinoma after surgery.

Mutation of β-Catenin Does Not Coexist with K-ras Mutation in Colorectal Tumorigenesis

Alterations of the APC, K-ras, and β-catenin genes are defined as early events in colorectal tumorigenesis. These alterations are well-known as constitutents of Vogelsteins pathway, however, the relationship among them is unclear. For understanding colorectal tumorigenesis it is important to evaluate their relationship. We analyzed the relationship between β-catenin and K-ras gene mutations in clinical colorectal samples. Sixty-four cases of colorectal cancers (44 proximal, 20 distal) without a family history of colorectal cancer were used for this study. We purified genomic DNAs from fresh surgical samples and, thus, analyzed the mutations of β-catenin (exon 3) and K-ras (codons 12 and 13) by PCR direct sequencing method using Big Dye terminator cycle sequencing with AmpliTaq polymerase FS. We found 27% (17/64) K-ras mutations (proximal 25%, 11/44; distal 30%, 6/20). The frequency of β-catenin mutations was 11% (7/64; proximal 9%, 4/44; distal 15%, 3/20). All cases with β-catenin mutation had no mutation of K-ras. All sites of β-catenin mutation have been reported previously (codons 33, 34, 41, 45). In cell lines, it has been reported previously that β-catenin and K-ras play the same roles in activation of cyclin D1 transcription. Our results may support this report and suggest that some colorectal cancers with β-catenin mutation will progress without K-ras mutation. Further study may disclose a new pathway or new mechanism of colorectal tumorigenesis.