Molecular characterization of MSI-H colorectal cancer by MLHI promoter methylation, immunohistochemistry, and mismatch repair germline mutation screening

Abstract

Microsatellite instability (MSI) occurs in 10% to 20% of colorectal cancers (CRC) and has been attributed to both MLH1 promoter hypermethylation and germline mutation in the mismatch repair (MMR) genes. We present results from a large population- and clinic-based study of MLH1 methylation, immunohistochemistry, and MMR germline mutations that enabled us to (a) estimate the prevalence of MMR germline mutations and MLH1 methylation among MSI-H cases and help us understand if all MSI-H CRC is explained by these mechanisms and (b) estimate the associations between MLH1 methylation and sex, age, and tumor location within the colon. MLH1 methylation was measured in 1,061 population-based and 172 clinic-based cases of CRC. Overall, we observed MLH1 methylation in 60% of population-based MSI-H cases and in 13% of clinic-based MSI-H cases. Within the population-based cases with MMR mutation screening and conclusive immunohistochemistry results, we identified a molecular event in MMR in 91% of MSI-H cases: 54% had MLH1 methylation, 14% had a germline mutation in a MMR gene, and 23% had immunohistochemistry evidence for loss of a MMR protein. We observed a striking age difference, with the prevalence of a MMR germline mutation more than 4-fold lower and the prevalence of MLH1 methylation more than 4-fold higher in cases diagnosed after the age of 50 years than in cases diagnosed before that age. We also determined that female sex is an independent predictor of MLH1 methylation within the MSI-H subgroup. These results reinforce the importance of distinguishing between the underlying causes of MSI in studies of etiology and prognosis.

Figure 1. MMR mutation status, methylation status, and IHC results for population-based CRC

Blue boxes represent the initial sample set, red boxes represent samples where an alteration in MMR function was observed, and green boxes represent no detected alteration in MMR function. 1. 50 MSI-H cases with no results from MMR testing and 40 additional MSI-H cases with incomplete results for IHC were excluded 2. 26 MSI-L cases with no results from MMR testing and 14 additional MSI-L cases with incomplete results for IHC were excluded 3. 259 MSS cases with no results from MMR testing and 34 additional MSS cases with incomplete results for IHC were excluded 4. MLH1 methylation was detected in a tumor from 1 individual with a germline mutation in MSH2 5. MMR loss = loss of MLH1, MSH2 or MSH6