Temozolomide treatment alters mismatch repair genes and can prime colorectal cancer tumors for immunotherapy

Most metastatic colorectal cancers (mCRC) are mismatch repair (MMR) proficient (MMRp) and unresponsive to immunotherapy, while MMR deficient tumors often respond to immune checkpoint blockade (ICB). It has been previously reported that a priming treatment of CRC preclinical models with temozolomide (TMZ) leads to MMR deficiency (MMRd), increasing tumor mutational burden (TMB) and response to immunotherapy. To clinically translate these finding, the ARETHUSA trial has been designed, in which O6-Methylguanine-DNA-methyltransferase (MGMT) methylated/MMR proficient mCRC patients received TMZ treatment, which was followed by inhibition of the immune checkpoint programmed death-1 (PD-1) by pembrolizumab only in cases showing TMB >20 mutations/MB after priming treatment. Moreover, we used mutational signature analysis of both CRC preclinical models and tissue biopsies to monitor the effect of TMZ post treatment, providing an integrative approach to TMB for assessing drug response. Robustness of signature fitting analysis was evaluated by comparing cosine similarity measurements against multiple reference datasets. Finally, the impact of genetic heterogeneity was assessed considering the effect induced by the TMZ treatment on TMB and TMZ signature at both clonal and subclonal level. Genetic alterations in genes involved in DNA mismatch repair including the MSH6 p.T1219I variant were found in 6/14 patients showing TMZ signature. Overall, we provide initial evidence that treatment of MMR proficient mCRCs with TMZ leads not only to TMB increase but also to the appearance of a specific signature (SBS11), highlighting the possible application of mutational signatures analysis for monitoring the TMZ-priming followed by ICB in a clinical setting.