Hypermethylation of the hMLH1 gene promoter in solitary and multiple gastric cancers with microsatellite instability

Abstract

Human cancers with a high frequency microsatellite instability phenotype develop due to defects in DNA mismatch repair genes. Silencing of a DNA mismatch repair gene, hMLH1 gene, by promoter hypermethylation is a frequent cause of the microsatellite instability-H phenotype. Using methylation specific PCR we investigated the methylation status of the hMLH1 gene promoter in 17 solitary gastric cancers (12 microsatellite instability-H and five microsatellite stable tumours from 17 patients), and 13 multiple gastric cancers (eight microsatellite instability-H, one low frequency microsatellite instability-L and four microsatellite stable tumours from five patients) and also examined non-cancerous gastric mucosa both adjacent to and distant from each tumour. Expression of hMLH1 protein was evaluated by immunohistochemistry. All microsatellite instability-H tumours (20 out of 20) had evidence of methylation of hMLH1 promoter, whereas only one out of 10 microsatellite instability-L and microsatellite stable tumours did (P<0.0000005), and the methylation status correlated with hMLH1 protein expression (P<0.000003). Furthermore, methylation of the hMLH1 promoter was detected in 50% (6 out of 12) and 63% (5 out of 8) of non-cancerous gastric mucosa samples adjacent to, and in 33% (4 out of 12) and 40% (2 out of 5) of those obtained from distant portion of, solitary and multiple cancers with microsatellite instability-H. Thus both solitary and multiple gastric cancers with microsatellite instability-H have evidence of similar high levels of hMLH1 promoter hypermethylation in the surrounding non-cancerous tissue. Hypermethylation of the hMLH1 promoter occurs in non-cancerous gastric mucosa of microsatellite instability-H tumours and may increase the risk of subsequent neoplasia.

Figure 1

Representative results from methylation-specific PCR (MSP) of the hMLH1 gene promoter in multiple (A) and solitary (B) gastric cancers. The presence of PCR product in lanes marked M indicates hypermethylated hMLH1 product, lanes marked U indicate unmethylated hMLH1. T, Tumour DNA; A, normal mucosa adjacent to tumour; N, normal mucosa from the surgical margin; PC, positive control; NC, negative control; SM, size marker.

Figure 2

Immunohistochemical staining for hMLH1 protein expression in gastric cancers with unmethylated (A) or hypermethylated (B) gene promoters. (A) Nuclear staining of hMLH1 in a MSS tumour without promoter hypermethylation (intramucosal well differentiated tubular adenocarcinoma). (B) Loss of hMLH1 expression in a MSI-H tumour with promoter hypermethylation (left, intramucosal well differentiated tubular adenocarcinoma; right, intestinal metaplastic mucosa exhibiting hMLH1 expression).