MLH1 methylation status is associated with tumor immune microenvironment and prognosis in MMRd-type endometrial cancer

To investigate the association between MLH1 promoter methylation and tumor immune microenvironment (TIME) characteristics and prognosis in mismatch repair-deficient (MMRd) endometrial cancer. MLH1 promoter methylation was assessed using multiplex fluorescence PCR targeting eight CpG dinucleotides in a retrospective cohort of 152 MMRd endometrial cancer patients from Peking University People's Hospital (PKUPH). Immune microenvironment was evaluated by bioinformatics (TIMER, CIBERSORT, TIDE, ssGSEA-based TME subtyping, and T cell-inflamed GEP scoring) in the TCGA-UCEC cohort, and by immunohistochemistry in the PKUPH cohort. Survival analysis employed Kaplan–Meier and Cox regression methods. MLH1 methylation was strongly and negatively correlated with MLH1 expression across all 30 promoter CpG sites (all P < 0.05). Higher methylation was associated with reduced anti-tumor immune infiltration (CD8 + T cells, M1 macrophages, NK cells), elevated T cell exclusion scores, lower T cell-inflamed GEP scores (Spearman ρ = − 0.442, P < 0.001), and enrichment in fibrotic and immune-desert TME subtypes (Kruskal–Wallis P < 0.0001) — collectively indicating an immunologically cold phenotype. IHC in the PKUPH cohort confirmed significantly lower CD3 + , CD4 + , and CD8 + T cell infiltration in methylated tumors. MLH1 methylation was associated with older age, lower ER expression, and reduced MELF pattern, and predicted poorer disease-free survival (HR 3.766, 95%CI 1.092–12.990), an association that remained significant after adjustment for FIGO stage in a penalized-likelihood model and was robust to adjustment for individual established prognostic factors. MLH1 promoter methylation defines an immunosuppressive, immune-excluded subtype within MMRd endometrial cancer with worse prognosis, and may serve as a clinically actionable biomarker for immunotherapy stratification.