MLH1 promoter methylation in pancreatic ductal adenocarcinoma: frequent but not prognostic.

BACKGROUND: MutL homolog 1 (MLH1) is a key component of the mismatch repair (MMR) pathway, and promoter methylation-mediated silencing is a well-established oncogenic mechanism in several tumor types. However, its prevalence and prognostic relevance in pancreatic ductal adenocarcinoma (PDAC) remain unclear. METHODS: We retrospectively analyzed 55 patients with PDAC diagnosed between 2012 and 2021 at a tertiary center. MLH1 promoter methylation (%) was quantified by pyrosequencing in formalin-fixed, paraffin-embedded tumor samples and classified using predefined (8%) and exploratory ROC-derived (2.6%) cut-offs. Associations with clinicopathological variables and disease-specific survival (DSS) were assessed using Cox regression models, with additional exploratory quartile-based and cubic spline analyses. External validation was performed using multi-omics data from The Cancer Genome Atlas Pancreatic Adenocarcinoma (TCGA-PAAD) cohort. RESULTS: MLH1 promoter methylation levels varied widely (mean 11.3%, range 2.0-42.2%) with 41.8% and 74.5% of cases classified as methylated using the predefined and exploratory cut-offs, respectively. No significant associations were observed with clinicopathological variables. In multivariable analysis, MLH1 methylation was not associated with DSS at either cut-off (HR = 1.55, 95% CI 0.85-2.85, p = 0.153; HR = 1.31, 95% CI 0.66-2.62, p = 0.442). Quartile-based and cubic spline analyses did not identify threshold-dependent or non-linear associations with DSS. In TCGA-PAAD data, neither MLH1 methylation nor gene expression correlated with overall survival, whereas low MLH1 protein expression was associated with worse outcomes. CONCLUSION: Although MLH1 promoter methylation is frequent in PDAC, it was not associated with DSS. These findings suggest limited prognostic value of MLH1 promoter methylation alone and support the integration of functional and multi-layered molecular data.