High-risk constitutional MLH1 methylation as a cause of early-onset colorectal and endometrial cancers displaying mismatch repair deficiency and MLH1 methylation.
Authors: Hitchins Megan Philippa , Alvarez Rocio , Aguirre Francesca Paola , Zhou Lisa , Diniz Marcio A. , Yuan Xiaopu , Damaso Estela , Hodan Rachel , Ryan Shawnia R , Cannon Timothy Lewis , Biswas Rakesh , DeMarco Tiffani , Sathe Devika , Salo-Mullen Erin E. , Stadler Zsofia Kinga , Pearlman Rachel , Hampel Heather 
Published: June 2023

10518 Background: Mismatch repair deficiency (MMRd) occurs in ~15% of colorectal (CRC) and ~30% of endometrial (EC) cancers, of which ~20% are caused by Lynch syndrome (LS). Universal screening of all incident CRC and EC for MMRd uses MLH1 methylation (in tumors with MLH1-loss by immunohistochemistry) to omit common sporadic cases from follow-up germline testing for LS. However, this overlooks unusual cases with constitutional MLH1 methylation (epimutation), a poorly-recognized high-risk mechanism for LS-type cancers that are MLH1 methylated. We aimed to determine the role and prevalence of constitutional MLH1 methylation among CRC and EC cases with tumor MLH1 methylation from clinical and population-based series and if age limits could be used to triage cases warranting additional germline methylation testing. Methods: We screened blood DNA for constitutional MLH1 methylation by pyrosequencing and methylation-specific qPCR in patients with MLH1-loss, MLH1 methylated CRC and EC ascertained from: i) cancer clinics, referred by oncologists and genetic counsellors; ii) Ohio population-based “Columbus” and “OCCPI” cohorts. Bisulfite sequencing confirmed constitutional MLH1 methylation. Results: In clinical cases, constitutional MLH1 methylation was identified in 3/7 EC and 3/8 CRC cases < 60 years. In population-based cohorts, constitutional MLH1 methylation was identified in 4/95 (4.2%) and 4/281 (1.4%) CRC cases of all ages from Columbus and OCCPI, respectively. Highest rates of detection with the majority of cases detected were at age ≤55 years, with 3/4 (75%) in Columbus and 4/17 (23.5%) in OCCPI ≤55 years. For EC, constitutional MLH1 methylation was identified in 0/68 Columbus cases of all ages and 1/24 OCCPI cases < 60 years (age-limited testing). She was one of six (~17%) cases < 50 years in the combined cohorts. EC was the first/dual-first presenting “sentinel” cancer in three female patients with constitutional MLH1 methylation. Of the 14 cases with constitutional MLH1 methylation, nine had hemiallelic methylation (~50% alleles methylated, affecting a single parental allele) and five had mosaicism across various normal tissues. Somatic “second-hits” affecting the unmethylated allele were found in the tumors of mosaic cases, demonstrating causation. Conclusions: A correct diagnosis of constitutional MLH1 methylation at first cancer presentation is important as it will significantly alter clinical management of these high-risk patients. Although rare overall, high rates of detection were found ≤55 years for CRC and ≤50 years for EC cases with a MMRd, MLH1 methylated tumor, entailing minimal extra screening. Testing for constitutional MLH1 methylation is warranted in patients with young-onset CRC or EC, or syn/metachronous cancers at any age, with MMRd and MLH1 methylated tumor(s).