Mismatch repair gene expression defects contribute to microsatellite instability in ovarian carcinoma

BACKGROUND hMLH1, the human MutL homologue, has been linked to microsatellite instability (MSI) in gastrointestinal tumors. However, to the authors' knowledge, the role of hMLH1, the other mismatch repair genes (MMR), and MSI in ovarian carcinoma has not been well defined. The purpose of the current study was to determine the relation between MSI of ovarian carcinoma and MMR gene expression, hMLH1 and hMSH2 hypermethylation, and hMLH1 and hMSH2 null mutations. METHODS hMLH1 mRNA was detected by reverse transcriptase‐polymerase chain reaction (RT‐PCR) and amplification of cDNA using a housekeeping gene (glycerol 3‐phosphate dehydrogenase) as a control for mRNA quality and quantity. Methylation‐specific PCR (MS‐PCR) was used to correlate methylation of the hMLH1 and hMSH2 CpG islands with mRNA expression status. Similar techniques were used to evaluate the concomitant expression of five other MMR: hMSH2, hMSH3, hMSH6, PMS1, and PMS2. Microsatellite instability was studied using the National Cancer Institute consensus markers (D2S123, D5S346, D17S250, BAT25, and BAT26) and NM23 as described previously. RESULTS One hundred twenty‐five primary tumors were analyzed. High‐frequency MSI (MSI‐H) was found in 21 tumors (16.8%). hMLH1 mRNA was absent in 10 of these 21 tumors (47.6%). In each case, coordinated hypermethylation of both regions A and C of the promoter was identified. Microsatellite stable and low‐frequency MSI tumors all were found to express not only hMLH1 but the other MMR genes as well (P < 0.001). Absence of expression of hMSH2 and the four other MMRs occurred in tumors with absent hMLH1 mRNA expression because of CpG island hypermethylation. No absence of expression of hMSH2, hMSH3, hMSH6, PMS1, or PMS2 was found to occur in tumors expressing hMLH1. None of the 11 MSI‐H tumors without promoter hypermethylation demonstrated a null mutation in hMLH1 or hMSH2. CONCLUSIONS A molecular mechanism to explain > 50% of the MSI‐H phenotype in ovarian carcinoma cases was demonstrated. MSI‐H may occur because of MMR defects, especially hMLH1 promoter hypermethylation. Additional mechanisms are required to explain the balance between the cases of MSI‐H as well as the phenomenon of MSI‐L tumors. Cancer 2003. © 2003 American Cancer Society. A molecular mechanism that the authors believe explains greater than 50% of ovarian carcinoma cases with high frequency microsatellite instability is discussed in the current study. See also pages 2091–4.