Do secondary MSH3/MSH6 alterations define an immune-modulatory subtype of MSI gastric cancer?

Gastric cancer (GC) is one of the leading causes of cancer-related morbidity and mortality worldwide. Molecular classification stratifies GC into four major subtypes: Epstein-Barr virus-positive, microsatellite instability (MSI), genomically stable, and chromosomal instability. Among these, MSI GC-representing approximately 8-25% of cases-is characterized by high mutational burden resulting from defects in the mismatch repair (MMR) system. For early-stage cancer the main therapeutic approaches include surgery, along with chemotherapy. Recently, immune checkpoint inhibitors, administered alone or in combination with other treatments, have demonstrated potent antitumoral activity in some subgroups of GC; among the responders are MSI tumors, which are associated with a better clinical prognosis compared with microsatellite-stable (MSS) ones. However, only around 50% of MSI GC obtain a benefit from immunotherapy. This study aims to elucidate the molecular mechanisms underlying the different ability of MSI CG to respond to immunotherapy, focusing on how alterations in mismatch repair genes can contribute to mutational burden, neoantigen formation, and immune activation. Taking advantage from our molecularly annotated platform including around 1000 primary tumors and 250 patient-derived xenografts (PDXs), we performed a multilevel molecular characterization to define MMR status and to functionally evaluate how specific MMR alterations influence immune responsiveness in vivo. Our preliminary data indicate that gastric cancers are characterized by intratumoral heterogeneity of the MMR, which may manifest as primary heterogeneity, defined by the coexistence of MSS and MSI regions, or as secondary heterogeneity, where MLH1 silencing is frequently accompanied by the additional loss of another MMR component, typically MSH3 or MSH6.This loss often results from frameshift mutations at mononucleotide repeat hotspots. These alterations, potentially reversible, have been linked to immune evasion. Notably, our findings indicate that secondary MSH3 and MSH6 mutations represent a frequent event in gastric MSI tumors (56% of MSI GCs display MSH3/MSH6 frameshift mutations), and we aim to explore their potential clinical implications. Future directions include dissecting the molecular pathways through which alterations in MMR genes generate subclonal neoantigenic diversity, ultimately shaping tumor-immune system interactions. A deeper understanding of these processes may inform the development and refinement of immunotherapeutic strategies for MSI gastric cancer.