Microsatellite instability, MLH1 promoter methylation, and BRAF mutation analysis in sporadic colorectal cancers of different ethnic groups in Israel

Abstract

Background: The molecular mechanisms that underlie colorectal cancer (CRC) include microsatellite instability (MSI), chromosomal instability, and the CpG island methylator phenotype. There is evidence to suggest that CRC incidence varies among different ethnic populations worldwide. The authors of this report hypothesized that environmental factors and lifestyle differences among various ethnic groups may differentially influence the epigenetic regulation of tumor suppressor genes in CRC.

Methods: In the current study, microdissection and DNA extraction were performed on 128 samples of CRC from Israeli patients (85 Jews and 43 Arabs). MSI analysis, mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2) protein expression levels, and MLH1 promoter methylation were investigated by combined bisulfite restriction analysis. The v-raf murine sarcoma viral oncogene homolog B1 (BRAF) valine-to-glutamic acid mutation at residue 600 was investigated by direct DNA sequencing.

Results: High MSI (MSI-H), MLH1 methylation, and BRAF mutations were observed in 11.6%, 9.4%, and 23.5% of Jews, respectively, and in 16.2%, 17.6%, and 20.9% of Arabs, respectively (P value nonsignificant). MLH1 promoter methylation was observed in 22.6% of microsatellite-stable (MSS) tumors and in 53.8% of MSI-H tumors (P < .015). Extensive methylation (covering both 5' and 3' promoter regions) was present in all MSI-H tumors with loss of MLH1 expression. BRAF mutation was observed in 15.6% and 46.1% of MSS tumors and MSI-H tumors, respectively (P < .007). BRAF mutation was observed in 66%, 22.2%, and 14.7% of patients who had tumors with extensive MLH1 promoter methylation, methylation of the 5' region alone, or without methylation, respectively (P < .006).

Conclusions: There was no difference in molecular signatures examined between Jewish and Arab patients with CRC in Israel. Extensive promoter methylation was associated with MLH1 inactivation, MSI, and BRAF mutation.

FIGURE 1

Immunohistochemistry staining for protein expression of the mutL homolog 1 gene MLH1 and the mutS homolog 2 gene MSH2. (A,B) Immunohistochemistry results for MLH1 protein expression from a microsatellite-stable (MSS) tumor (original magnification, ×100 in A, ×200 in B). MLH1 expression can be seen as nuclear brown staining in normal and tumor tissue. (C,D) Immunohistochemistry results for MLH1 expression from a tumor with high microsatellite instability (original magnification, ×100 in C, ×200 in D) There is nuclear staining in normal mucosa and absent nuclear staining in the tumor tissue. Inflammatory cells and non-neoplastic cells (internal control) are positively stained. (E,F) MSH2 protein expression in an MSS tumor (original magnification, ×100 in E, ×200 in F). There is normal nuclear staining in the normal mucosa and the tumor tissue.

FIGURE 2

Methylation analysis of the mutL homolog 1 gene (MLH1) promoter in the 5′ and 3′ regions. Combined bisulfite restriction analysis for the MLH1 promoter region: (A) shows the 5′ region after digestion with the restrictive enzyme HhaI and (B) is the 3′ region after digestion with the restriction enzyme RsaI. The upper band is the unmethylated product and the lower band is a methylated product. Lanes 1–3, unmethylated samples; lanes 4–7, methylated samples; U, unmethylated control; M, methylated control; SM, size marker.

FIGURE 3

Direct sequencing for a v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation. Representative sequence chromatographs from (A) a wild-type BRAF gene and (B) a BRAF gene with the V600E valine-to-glutamic acid mutation at residue 600 in exon 15. G indicates guanine; T, thymine; A, adenine.