Identification of constitutional MLH1 methylation in MLH1-deficient colorectal cancer

Constitutional MLH1 methylation (epimutation), a poorly recognized mechanism for Lynch syndrome (LS), is usually overlooked due to current screening strategies that rely on tumor MLH1 methylation as an indication of sporadic mismatch repair-deficient (dMMR) colorectal cancer (CRC). We aimed to assess the frequency and clinicopathological characteristics of constitutional MLH1 methylation among dMMR CRC cases with tumor MLH1 methylation.

We conducted a retrospective study on two independent cohorts: a discovery cohort of 48 MLH1-deficient CRC cases (2017-2021) and a validation cohort of 159 consecutive CRC cases (2022-2024). Real-time methylation-specific PCR (qMSP) screened for tumor MLH1 methylation, followed by constitutional MLH1 methylation testing on paired normal colorectal mucosa (NCM) DNA, confirmed by CpG pyrosequencing.

In the discovery cohort, constitutional MLH1 methylation was identified in 4 of 31 individuals (12.9 %) with tumor MLH1 methylation, including 2 with high-level and 2 with low-level mosaic methylation. These patients were generally younger (median age: 56 years) , and three of the four tested negative for the BRAF V600E mutation. When applying an age threshold of ≤55 years, the highest positivity rate for constitutional MLH1 methylation was achieved at 2 of 4 (50.0 %), and notably, both exhibited relatively high-level epimutations. A refined screening strategy specially targeting high-level constitutional MLH1 methylation was developed. In the validation cohort, 35 MLH1-deficient, BRAF V600E-negative cases were tested, including 18 individuals aged ≤55 years and 17 randomly selected controls aged >55 years. Constitutional MLH1 methylation was successfully identified in one patient from the younger group, while none was detected among the older controls. All five cases lacked a significant family history, and one patient developed three LS-associated cancers. In addition, heterogeneous MMR protein expression patterns, and early partial MLH1/PMS2 loss in precancerous lesions were observed.

Our findings highlight a subset of younger patients with constitutional MLH1 methylation among MLH1-deficient, MLH1-methylated CRC cases. Patients aged ≤55 years, whose tumors exhibit MLH1 methylation or lack the BRAF V600E mutation, are recommended to undergo additional testing for constitutional MLH1 methylation to improve LS-related clinical management.