The main objective of this analysis was to perform molecular characterization of the MSI-H phenotype within a large series of population-based CRC. When we restricted the population-based series to cases with MMR germline mutation testing and complete IHC 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 (MSH2, MLH1, MSH6 or PMS2), and 23% had isolated IHC evidence for loss of a MMR protein (). The prevalence of MLH1 methylation and germline MMR mutation differed greatly by age at diagnosis, with cases diagnosed after age 50 having a lower prevalence of germline mutation and a much higher prevalence of MLH1 methylation than cases diagnosed before age 50.
MLH1 methylation was observed more frequently in population-based than clinic-based MSI-H tumors. This can be explained by the higher frequency of clinic-based MSI-H cases with a MMR germline mutation (). In addition, the MSI-H cases in the population-based series were diagnosed at an older age than the MSI-H cases in the clinic-based series (median age 63, range 22−75 vs. median age 44, range 19−77, respectively). The low frequency of MLH1 methylation in the clinic-based sample has clinical implications. Our data suggest that MLH1 methylation may explain MSI-H CRC in the absence of a detected germline mutation in some, but not all, of these cases. The low frequency of MLH1 methylation in clinic-based cases with a germline mutation also suggests that germline mutation and methylation are largely independent mechanisms for inactivation of MLH1, and that the remaining wild-type allele in most Lynch syndrome cases is not typically inactivated by DNA methylation.
Previous studies have reported that MLH1
methylation is associated with older age at diagnosis and female sex (18
). In addition, MSI-H tumors in general (35
) as well as tumors with MLH1
methylation are more likely to be located within the proximal colon (23
). Our large sample of population-based MSI-H cases allowed us to evaluate independent associations between these descriptive characteristics and MLH1
methylation within the MSI-H subgroup. We observed statistically significant positive associations for female sex and older age at diagnosis in the multivariable adjusted model and a statistically significant inverse association with Amsterdam II criteria (Table 3). Although we have not measured CpG island methylator phenotype (CIMP) in these samples to date, a previous report found that most sporadic MSI-H were CIMP positive because MLH1
was methylated in these samples (30
). CIMP can occur in the context of both MSI-H and MSS CRC, with different molecular alterations distinguishing the two groups of CIMP positive tumors (21
). These data suggest that the MSI-H tumors with MLH1
methylation are likely CIMP positive, and the associations we have observed between MLH1
methylation and descriptive characteristics may also apply to CIMP positive MSI-H CRC.
Possible explanations for MSI in the group with no detected MLH1
methylation or MMR germline mutation include: 1) false negative results for either MLH1
methylation or MMR germline mutation; 2) somatic inactivation in one of the known MMR genes; or 3) some other method of inactivation of mismatch repair. False negatives for MLH1
methylation or MMR germline mutation are unlikely to explain all of these cases in this population-based series as MLH1
methylation was rarely observed in cases without loss of MLH1 and MMR germline mutations are estimated to occur in only 1−2% of CRC (2
). We detected variants of uncertain biological significance in our MMR germline mutation screening, and it is also possible that some of these variants may be functional. Among the cases without a clearly deleterious MMR germline mutation, unclassified variants were observed in 4% of cases with loss of MLH1 protein and no DNA methylation, 4% of cases with loss of one of the other MMR genes, and 24% of cases with no detected loss of MMR function (Supplementary Table 1
). Somatic mutations have been reported in MSI-H CRC in previous studies (17
), and this may explain MSI in some of these cases. Additionally, a germline polymorphism in the MLH1
promoter has been reported to be associated with risk of MSI-H CRC (40
). Such sequence variants, particularly when homozygous, may also offer an explanation for a portion of the remainder of MSI-H CRC. We did not perform screening for this polymorphism.
While the majority of MLH1
methylation was observed in MSI-H cases, we did observe MLH1
methylation in 3.1% of MSI-L and 0.7% of MSS cases in the population-based series with no resulting loss of MLH1 expression, although we did not have MLH1 IHC data for 3 MSS cases. We observed a lower PMR value in the MSS tumors with MLH1
methylation compared to the MSI-H tumors with MLH1
methylation. In addition, we observed MLH1
methylation in 4 MSI-H cases with no observed loss of MLH1 expression (). One plausible explanation for these findings is mono-allelic MLH1
methylation. MethyLight is a quantitative assay and a previous study has demonstrated that this technique is capable of distinguishing between monoallelic and bi-allelic DNA methylation (41
This study has several limitations. Because the prevalence of MLH1 methylation decreased with increasing ALU C(t) value, it is likely that we have some samples with undetected MLH1 methylation in the unmethylated category. However, there were no statistically significant differences in ALU C(t) value by age, gender, or tumor location within the MSI-H category (data not shown), suggesting that the percentage of false negatives should not differ within these groups. Undetected carriers of MMR germline mutations may exist in our study population because we did not test all individuals for MSH6 and PMS2 mutations. In addition, IHC results for MSH6 and PMS2 were not available for all cases. This study also has several strengths, including the largest sample size to date of tumors with both MSI and MLH1 methylation status, systematically collected epidemiologic data and tumor characteristics and inclusion of both population- and clinic-based families.
In summary, we observed MLH1 methylation in 60% of population-based MSI-H tumors and 13% of clinic-based MSI-H colorectal tumors. As expected, the prevalence of germline mutation in one of the MMR genes was higher in cases diagnosed before age 50 compared to cases diagnosed after age 50 (39% vs. 9%, respectively) while the prevalence of MLH1 methylation was much lower in cases diagnosed before age 50 than in cases diagnosed after age 50 (14% vs. 63%). Within population-based MSI-H colon cancer, we were able to establish that older age at diagnosis and female sex are independent predictors of MLH1 methylation, and that a great majority of MSI-H CRC could be explained by either germline mutation within one of the MMR genes or MLH1 methylation. However, there was a subset of cases where the MSI-H phenotype could apparently not be explained by either of these mechanisms. Further research will be required to better understand the MSI-H phenotype in these cases.