Precision oncology in endometrioid endometrial carcinoma integrating genetic and epigenetic features

Endometrial carcinoma (EC) displays marked molecular heterogeneity driven by complex interactions between genetic and epigenetic mechanisms. Among these, somatic mutations, microsatellite instability (MSI) and DNA methylation play key diagnostic and biological roles, yet the molecular drivers that shape the methylation landscape and deficiency of DNA mismatch repair system (dMMR) or MSI remain partly understood. This thesis integrates two complementary studies aimed at exploring the link between genetic and epigenetic alterations; and using computational approaches to optimise Next-Generation Sequencing (NGS)-based MSI detection to advance molecular diagnostics and clinical application. The first study interrogated the interplay between driver mutations, global DNA methylation and MSI status with particular focus on MLH1 promoter methylation in endometrioid EC (EEC). We combined in-silico analysis of public datasets with an institutional cohort characterised by NGS, MMR immunohistochemistry (IHC), PCR-based MSI testing, MS-MLPA for MLH1 methylation and long interspersed nuclear element-1 (LINE-1) pyrosequencing. Our findings demonstrate that KRAS-activating mutations are significantly associated with MSI and MLH1 promoter hypermethylation. Furthermore, co-occurrence of KRAS and ARID1A mutations correlated with a hypermethylated phenotype, suggesting a cooperative role in epigenetic remodeling. LINE-1 analysis indicated that KRAS mutations contribute not only to locus-specific but also to global DNA hypermethylation, highlighting mechanistic links between oncogenic signaling and epigenetic regulation. The second study focused on optimising and validating an NGS-based MSI detection panel. Starting from 122 microsatellite loci, we identified a refined subset of 51 loci that retained discriminatory performance for MSI status through model-based clustering and threshold optimisation in colorectal carcinoma (CRC) and EC cohorts. NGS-derived MSI scores demonstrated high concordance with conventional gold standard methods, validating a streamlined workflow capable of integrating mutational and MSI assessment in a single assay. Collectively, these studies emphasize the biological and clinical significance of KRAS and ARID1A mutations in shaping MSI and methylation landscapes and establishing a robust NGS-based framework for accurate MSI detection across tumor types. The findings support the potential utility of integrated molecular profiling for improved risk stratification, therapeutic decision-making and identification of actionable subset of EC, including candidates for KRAS-targeted or immunotherapy-based interventions.