MLH1 Promoter Methylation Could Be the Second Hit in Lynch Syndrome Carcinogenesis

Abstract

(1) Background: MLH1 hypermethylation is an epigenetic alteration in the tumorigenesis of colorectal cancer (CRC) and endometrial cancer (EC), causing gene silencing, and, as a consequence, microsatellite instability. Commonly, MLH1 hypermethylation is considered a somatic and sporadic event in cancer, and its detection is recognized as a useful tool to distinguish sporadic from inherited conditions (such as, Lynch syndrome (LS)). However, MLH1 hypermethylation has been described in rare cases of CRC and EC in LS patients. (2) Methods: A total of 61 cancers (31 CRCs, 27 ECs, 2 ovarian cancers, and 1 stomach cancer) from 56 patients referred to cancer genetic counselling were selected for loss of MLH1 protein expression and microsatellite instability. All cases were investigated for MLH1 promoter methylation and MLH1/PMS2 germline variants. (3) Results: Somatic MLH1 promoter hypermethylation was identified in 16.7% of CRC and in 40% of EC carriers of MLH1 germline pathogenic variants. In two families, primary and secondary MLH1 epimutations were demonstrated. (4) Conclusions: MLH1 hypermethylation should not be exclusively considered as a sporadic cancer mechanism, as a non-negligible number of LS-related cancers are MLH1 hypermethylated. Current flow charts for universal LS screening, which include MLH1 methylation, should be applied, paying attention to a patient's family and personal history.

Keywords: LS universal screening; Lynch syndrome; MLH1 methylation; constitutional epimutation.

Figure 1

Distribution of samples according to MLH1 germinal status (g-MLH1) and somatic MLH1 promoter methylation in CRC (blue) and EC (pink). Lighter colors indicate the MLH1 unmethylated samples, darker colors identify MLH1 hypermethylated samples. (a) Somatic MLH1 methylation status in CRC and EC associated with LS. (b) Somatic MLH1 methylation status in sporadic CRC and EC. The bars indicate 95% confidence interval.

Figure 2

LS family pedigree: patient ID, cancer site, and age of onset are reported together with presence of MLH1 germline pathogenic variants (blue dot) and somatic MLH1 hypermethylation (purple triangle). Arrow indicates the proband. (a) Family F-1: patient P-18 is affected by two CRCs that are MLH1 defective and negative for MLH1 methylation P-02, mother of P-18, is affected by EC that is MLH1 defective and showing MLH1 hypermethylation; (b) Family F-2: patient P-13 is affected by CRC and EC; both tumors are MLH1 defective but only EC revealed MLH1 hypermethylation. Legend: gMLH1: germinal MLH1 status; PV: pathogenic variant; CRC: colorectal cancer; EC: endometrial cancer; d-MLH1/PMS2: defect of MLH1 and PMS2 protein expressions; MSH-H: presence of high level of microsatellite instability; IHC: immunohistochemistry.

Figure 3

LS family pedigree: patient ID, cancer site, and age of onset are reported together with presence of MLH1 germline (gMLH1) pathogenic variant (PV, blue dot). Arrow indicates the proband. (a) Family F-3: P-05 is affected by EC that is MLH1 defective and demonstrates MLH1 hypermethylation; (b) family F-4: patient P-03 is affected by CRC showing focal MLH1 loss. In this tumor, MLH1 hypermethylation was observed only in MLH1-defective areas. Legend: gMLH1: germinal MLH1 status; PV: pathogenic variant; CRC: colorectal cancer; EC: endometrial cancer; d-MLH1/PMS2: defect of MLH1 and PMS2 protein expressions; MSH-H: presence of high level of microsatellite instability; IHC: immunohistochemistry.

Figure 4

LS family pedigree: patient ID, cancer site, and age of onset are reported together with presence of MLH1 germline variant (blue dot), PMS2 germline variant (green dot), constitutional MLH1 hypermethylation (purple dot), and somatic MLH1 hypermethylation (purple triangle). Arrow indicates the proband. (a) Family F-5: patient P-09 is affected by CRC that is MLH1 defective and MLH1 hypermethylated; P-10 is affected by CRC and EC, with both tumors being MLH1 defective and MLH1 hypermethylated; (b) family F-6: patient P-38 is affected by CRC that is MLH1 defective and MLH1 hypermethylated. Legend: gMLH1: germinal MLH1 status; gPMS2: germinal PMS2 status; PV: pathogenic variant; CRC: colorectal cancer; EC: endometrial cancer; d-MLH1/PMS2: defect of MLH1 and PMS2 protein expression; MSH-H: presence of high level of microsatellite instability; VUS: variant of uncertain significance.