Multiple alterations in the nucleotide context favored by the TMZ signature emerged in MMR genes, and the p.T1219I MSH6 variant was detected in ctDNA and tissue of 94% (16/17) of the cases….Overall, these results indicate that in colorectal cancer, the p.T1219I MSH6 mutation emerges at a very high frequency (94%) as a molecular response marker to extended exposure to TMZ....In addition, we have highlighted the MSH6 p.T1219I variant as a potential marker for TMZ molecular efficacy in colorectal cancer.