Integrated analysis of microsatellite instability, MMR deficiency, and MLH1 promoter methylation in colorectal cancer: a comprehensive Tunisian study

Abstract Background Colorectal cancer (CRC) is a major cause of cancer-related morbidity and mortality worldwide, including in Tunisia. Microsatellite instability (MSI) and mismatch repair (MMR) deficiency are key biomarkers that inform prognosis, guide therapeutic decision-making, and facilitate the identification of patients with suspected cases of Lynch syndrome. However, integrated molecular characterization combining MSI testing, MMR protein expression, and MLH1 promoter methylation remains underexplored in North African populations. Methods This retrospective study included 93 consecutive archived CRC cases. MSI status was determined using the five-locus Bethesda panel. MMR protein expression (MLH1, PMS2, MSH2, and MSH6) was assessed by immunohistochemistry (IHC). MLH1 promoter methylation was evaluated using methylation-specific PCR. Associations with clinicopathological characteristics were analyzed, and overall survival and progression-free survival were estimated using the Kaplan–Meier method. Results MSI-H was observed in 18.3% of cases, MSI-L in 12.9%, and MSS in 68.8%. Among MSI-positive tumors ( n = 29), 72.4% exhibited a deficient MMR (dMMR) profile, while 27.6% retained MMR expression, two discordant MSI-H/pMMR cases were identified. Concurrent MLH1 and PMS2 loss was the most common dMMR pattern. MLH1 promoter methylation was detected in 23.1% of MLH1-deficient tumors. MSI status was not significantly associated with clinicopathological characteristics or survival outcomes (log-rank p > 0.05). Conclusion This study provides an integrated molecular characterization of CRC in a Tunisian cohort, combining MSI testing, MMR IHC, and MLH1 promoter methylation analysis. The low frequency of MLH1 promoter methylation among MLH1-deficient tumors suggests that alternative mechanisms of MLH1 loss may be relevant in this population and warrants confirmation in larger, multicenter studies. These findings, while exploratory given the sample size, underscore the importance of combined molecular workup — rather than MSI testing alone — when evaluating patients for suspected Lynch syndrome.