Aberrant methylation of MLH1 is responsible for causing approximately 90% of sporadic MSI colon cancers. Other mechanisms such as somatic MLH1 mutations may be responsible for the minority of cases where aberrant MLH1 methylation is absent. In most studies, aberrant MLH1 methylation has been detected in only a small percentage of LS colon cancers in individuals with germline mutations in MLH1.[250,251,252,253] Thus, detection of aberrantly methylated MLH1 in colon cancer is more suggestive of a sporadic MSI tumor. Since assays of methylation are complex and resource-intensive, surrogate markers of MLH1 methylation have been examined. One study found that loss of immunohistochemical staining for p16 correlated strongly with both MLH1 methylation and BRAF V600E mutations (BRAF mutations are discussed in detail in the following paragraphs). However, only 30% of sporadic tumors examined in this study exhibited loss of p16 expression, limiting the utility of this assay.
BRAF mutations have been detected predominantly in sporadic MSI tumors.[255,256,257,258] This suggests that somatic BRAF V600E mutations may be useful in excluding individuals from germline mutation testing; however, limitations of current studies preclude this conclusion. For example, none of the studies clearly define the clinical criteria used to diagnose the families with LS, limiting the general application of the results to patients seen in the clinical setting. Furthermore, at least one person with a germline mutation in MLH1 (mutation not described) had colon cancer with a BRAF mutation. Recommendations for BRAF testing to stratify individuals for subsequent germline MMR testing cannot be made until a study is performed using a population of individuals who meet borderline clinical criteria for LS and who have had germline MMR testing.
(Refer to the Diagnostic strategies for all individuals diagnosed with CRC section of this summary for more information about the clinical role of BRAF and hypermethylation testing.)
Germline MLH1 hypermethylation
Reports of patients with germline MLH1 hypermethylation should not be confused with EPCAM mutation-induced hypermethylation of MSH2, as described below. Prior paragraphs have emphasized the issues associated with the common, acquired somatic hypermethylation of the MLH1 promoter. However, examples of hypermethylation of the MLH1 promoter described in the germline have generally not been associated with a stable Mendelian inheritance.
A comprehensive review of MLH1 constitutional epigenetic alterations involving hypermethylation of one MLH1 allele has been published. Such epimutations are seen in patients with early onset LS and/or multiple tumors of the LS type. Germline sequence variations or rearrangements are not seen in these patients, although the tumors show MSI-H, loss of MLH1 protein expression, and an absence of BRAF V600E mutations. These patients commonly have no family history of LS-like tumors. Interestingly, inheritance appears to be maternal, and therefore nonMendelian. The constitutional monoallelic hypermethylation may appear as a mosaic, involving different tissues to a varying extent. In addition, the constitutional epimutation is typically reversible in the course of meiosis, such that offspring are usually unaffected. Because inheritance has been demonstrated in very few families, performing genetic counseling and genetic testing (which requires specialized research techniques) is particularly challenging.