Microsatellite Instability, MLH1 Promoter Methylation, and BRAF Mutation Analysis in Sporadic Colorectal Cancers of Different Ethnic Groups in Israel

Colorectal cancer (CRC) is the second leading cause of cancer mortality in the Western countries and affects 5% to 6% of the population. In Israel, the rate of CRC differs significantly among different ethnic groups. The incidence is highest in Ashkenazi Jews (European-and American-born Jews), intermediate in Sephardic Jews (Asian- and African-born Jews), and lower in Israeli-born Jews.1 Israeli-born Arabs have the lowest incidence rate of CRC.2 The cause of these differences is not understood well and is believed to result from a combination of many genetic, epigenetic, and environmental factors, such as the I1307K adenomatous polyposis coli gene (APC) variant in Ashkenazi Jews3 and CpG island methylation that results in gene silencing.4 Microsatellite instability (MSI) is the hallmark of DNA mismatch-repair gene (MMR)-deficient CRCs. MSI has been observed in approximately 15% of sporadic colon cancers and in virtually all colorectal tumors arising in patients with hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome (LS).5 Sporadic tumors that exhibit MSI have unique clinicopathologic characteristics, such as poor differentiation, mucinous histology, Crohn-like lymphoid infiltrate, preferential proximal location, and older age.6,7 The prognosis for patients who have sporadic tumors with high MSI (MSI-H) is better than that for patients who have microsatellite-stable (MSS) tumors, but they respond poorly to chemotherapy. 8,9 In contrast to HNPCC tumors, in which loss of MMR gene expression is caused by germline mutation, MSI in sporadic colon cancers is caused by epigenetic modification of the mutL homolog 1 gene (MLH1).4,7 The MLH1 gene has a large CpG island within its promoter region. Deng et al10 compared the methylated status of the MLH1 promoter and its protein expression in 24 cancer cell lines and observed that methylation of a small proximal region closer to the transcriptional start site (defined as Region C) in the promoter invariably was correlated with loss of gene expression; methylation outside of this region did not always correlates with the absence of MLH1 expression. Those results were confirmed by others in clinical samples and suggest that partial MLH1 promoter methylation limited to the upstream region of the MLH1 promoter is not critical for gene silencing.11,12 The mechanism of epigenetic modification of tumor suppressor genes in cancer remains unclear. It has been proposed that low consumption of folic acid, vitamin B6, and vitamin B12, high alcohol intake, and smoking may affect DNA methylation,13,14 but the critical factors in human cancer remain uncertain. Recently, a v-raf murine sarcoma viral oncogene homolog B1 (BRAF) somatic mutation (valine-to-glutamic acid mutation at residue 600 [V600E]), has been identified in multiple human cancers. Davies et al15 reported a BRAF mutation in 66% of malignant melanomas and at lower frequency in other human malignancies, including CRC. BRAF is a serine/threonine kinase that is part of the Ras/Raf-extracellular signal-regulated kinase/mitogen activated protein kinase (MEK/MAPK) signaling pathway and is an essential component of intracellular signaling. In CRC, this BRAF mutation is associated strongly with sporadic MSI tumors and extensive MLH1 promoter methylation,12,16–18 but not with MMR-deficient tumors in patients with LS.19,20 The objective of this study was to investigate a possible relationship between MLH1 methylation in 2 different promoter regions, MSI status, and BRAF mutation in sporadic patients with CRC from different Israeli ethnic groups.