High-resolution methylation analysis of the hMLH1 promoter in sporadic endometrial and colorectal carcinomas

BACKGROUND Microsatellite instability (MSI) has been reported in endometrial carcinoma (EC) and in colorectal carcinoma (CRC), primarily as a result of defective DNA mismatch repair (MMR). The MMR gene hMLH1 commonly is inactivated in both EC and CRC. In the current study, epigenetic mechanisms involved in hMLH1 inactivation have been investigated to further elucidate the role of these mechanisms in the pathogenesis of EC and CRC. METHODS Polymerase chain reaction (PCR)-based microsatellite analysis performed on paraffin-embedded tissues was used to select 42 sporadic carcinomas (21 ECs and 21 CRCs) with MSI. Immunohistochemistry (IHC), using the anti-hMLH1 antibody, and mutation analysis, using denaturing high-performance liquid chromatography and automated sequencing, were performed on unstable carcinoma samples. Methylation analysis, using modified protocols for bisulfite treatment and methylation-specific PCR (MSP), was performed on DNA from archival tissue samples. RESULTS No MSI-positive tumor samples with normal hMLH1 immunostaining (n = 7) exhibited hMLH1 promoter methylation, whereas 8 of 35 unstable cases with loss of hMLH1 expression (23%) exhibited MSP amplification. Among analyzed cases, germ-line mutations of hMLH1 were found in 4 of 20 unmethylated samples (20%) and in 0 of 8 methylated samples. Bisulfite sequencing of amplification products from methylated samples demonstrated that almost all CpG dinucleotides within the hMLH1 promoter elements underwent methylation. CONCLUSIONS Although an MMR gene other than hMLH1 may be responsible for genetic instability in MSI-positive/IHC-positive tumors, the presence of MSP amplification and allelic deletions within the hMLH1 locus in subsets of MSI-positive/IHC-negative cases strongly suggests that hMLH1 promoter methylation may contribute to the inactivation of both hMLH1 alleles. Bisulfite analysis suggests that the mechanisms of hMLH1 silencing may depend on CpG density rather than site-specific methylation. Cancer 2003;98:1540–6. © 2003 American Cancer Society. DOI 10.1002/cncr.11651