Heterogeneity and evolution of DNA mutation rates in microsatellite stable colorectal cancer

Historically, DNA sequence mutability has been considered relatively uniform and low in tumors with chromosomal instability (CIN), based on the assumption that high mutability would be detrimental in karyotypically aberrant contexts. Recent in silico analyses have challenged this view, suggesting some heterogeneity in mutation rates across CIN tumors; however, these predictions lack experimental validation. It also remains unclear how the intertumor variability of mutation rates compares to intratumor diversification and evolves along disease progression, whether mutation rates are functionally relevant in CIN cancers, and which mutational processes shape mutational accrual during CIN tumor onset and evolution. To address these gaps, we performed mutation accumulation experiments using clonal populations of patient-derived tumoroids from seven CIN, microsatellite-stable colorectal cancers (CRCs), and one microsatellite-unstable CRC. Each tumor exhibited a distinctive mutation rate footprint that was conserved among different clones from the same ancestor. In contrast, mutation rates diverged markedly across different tumors, with variations in magnitude within microsatellite-stable tumors as prominent as those distinguishing them from microsatellite-unstable tumors. New mutations reflected mutational processes associated with defective DNA replication and repair, which were not detected in normal tissues. Last, both mutation accumulation assays and high-depth whole-exome sequencing of subclonal variants showed higher mutation rates in metastatic lesions compared with matched primary tumors, suggesting positive selection for cells with increasing mutability during cancer dissemination. By providing an empirical assessment of mutation rates in human cancer, our data delineate heterogeneity, heritability, and progression-associated evolvability of DNA mutational instability as hallmarks of microsatellite-stable CRC.