Hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch Syndrome, is an autosomal dominant condition associated with increased risk of multiple cancers particularly colorectal and endometrial. This syndrome most commonly results from mutations of genes encoding DNA mismatch repair proteins including MLH1, MSH2, MSH6 and PMS2. These mutations lead to tumors with microsatellite instability (MSI) – a change in length of a microsatellite allele due to either insertion or deletion of repeating units. However, MSI can also be seen in sporadic colorectal and endometrial cancers that are not associated with HNPCC. The majority of cancers with sporadic MSI demonstrate hypermethylation of the MLH1 promoter resulting in silenced MLH1 protein expression. MLH1 promoter hypermethylation is extremely uncommon in tumors from patients with HNPCC. Similarly, BRAF V600E mutations are frequently observed in colorectal carcinoma with sporadic MSI and are virtually never observed in tumors associated with HNPCC. Therefore, the evaluation of neoplastic cells for MLH1 promoter methylation and/or BRAF V600E can be useful in screening colorectal cancers with MSI for suspected HNPCC. MLH1 promoter methylation is particularly useful in evaluating endometrial carcinomas with MSI since BRAF V600E mutations are extremely rare in these tumors.

In this assay, MLH1 promoter methylation is detected using methylation specific real-time PCR (MethyLight). The absence of MLH1 promoter methylation in tumors demonstrating loss of MLH1 protein expression and/or MSI may suggest a MLH1 mutation associated with HNPCC. Genetic testing for germline MLH1 mutation is recommended in this setting. Conversely, the presence of MLH1 promoter methylation in neoplastic tissue is suggestive of sporadic MSI, not associated with HNPCC. However, rare instances of co-occurring MLH1 promoter hypermethylation and MLH1 germline mutations have been described. In addition, constitutional epimutations that result in heritable MLH1 promoter hypermethylation also exist, conferring an HNPCC (Lynch Syndrome) phenotype even in the absence of primary sequence alternations in the MLH1 gene. If there is a clinical suspicion of germline MLH1 promoter methylation, performing germline MLH1 promoter methylation testing on peripheral blood is recommended. Genetic counseling may be indicated based on the clinical and laboratory findings.