Abstract A001: Reshaping the immune microenvironment after temozolomide priming in metastatic colorectal cancer patients in ARETHUSA clinical trial
Authors: Crisafulli Giovanni , Sartore-Bianchi Andrea , Lazzari Luca , Pietrantonio Filippo , Battuello Paolo , Bartolini Alice , Nicolantonio Federica Di , Marsoni Silvia , Siena Salvatore , Bardelli Alberto 
Published: December 2023

Abstract
Mismatch repair (MMR) proficient (MMRp) tumors, known for their weak immune response, often exhibit limited effectiveness in immunotherapy. In contrast, responsive mismatch repair deficient (MMRd) tumors, characterized by heightened immune activity, show a stronger response to immune checkpoint blockade (ICB). In the ARETHUSA clinical trial (NCT03519412), we revealed that treatment with temozolomide (TMZ) can pharmacologically deactivate the MMR machinery, as indicated by mutational signature analysis and the emergence of MMR gene mutations. We focused our investigation on a subset of initially MMRp CRC patients who experienced sustained disease stabilization with ICB after receiving TMZ priming treatment. This treatment resulted in the emergence of an inactivating MSH6 mutation and the TMZ mutational signature. Additionally, TMZ treatment induced diverse genomic changes, leading to the identification of three distinct subtypes through analysis of blood and tissue samples. Notably, we observed a dose-dependent accumulation of mutations with a specific molecular signature visible at the clonal level (subtype B2) in 10% of patients, at the subclonal level (subtype B1) in 71% of patients, and the absence of these mutations (subtype A) in 19% of patients within our cohort of 21 patients. In conjunction with previous genetic findings, in this new study we performed an analysis of T-cell receptors (TCRs) in the tumor microenvironment following TMZ treatment. Our results revealed expanded clonotypes in tumors that exhibited a clonal increase in mutations following priming treatment. Furthermore, the diversity of TCRs within the immune infiltrate confirmed the categorization of tumors into three distinct classes based on Tumor Mutational Burden (TMB) and mutational signatures. We also established a significant linear correlation (p-value 0.0048) between the diversity of T-cells in the microenvironment and the number of mutations induced by TMZ. Based on these clinically-oriented preliminary findings, we propose that increasing the number of TMZ-induced mutations could enhance the likelihood of TMZ-driven neoantigens triggering clonal expansion of specific T-cell clonotypes within the tumor. Interestingly, the subset of TMZ-treated tumors displaying an acquired MSH6 mutation, TMZ mutational signature, and increased TMB not only achieved temporary disease stabilization with ICB but also exhibited clonal expansion of T-cells in the tumor microenvironment. These clinically relevant findings suggest that the inactivation of MMR achieved through TMZ priming has the potential to reshape the immune microenvironment modulating the immune response in metastatic colorectal cancer.
Citation Format: Giovanni Crisafulli, Andrea Sartore-Bianchi, Luca Lazzari, Filippo Pietrantonio, Paolo Battuello, Alice Bartolini, Federica Di Nicolantonio, Silvia Marsoni, Salvatore Siena, Alberto Bardelli. Reshaping the immune microenvironment after temozolomide priming in metastatic colorectal cancer patients in ARETHUSA clinical trial [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A001.