Extensive subclonal mutational diversity in human colorectal cancer and its significance

Human colorectal cancers (CRCs) contain both clonal and subclonal mutations. Clonal driver mutations are positively selected, present in most cells, and drive malignant progression. Subclonal mutations are randomly dispersed throughout the genome, providing a vast reservoir of mutant cells that can expand, repopulate the tumor, and result in the rapid emergence of resistance, as well as being a major contributor to tumor heterogeneity. Here, we apply duplex sequencing (DS) methodology to quantify subclonal mutations in CRC tumor with unprecedented depth (10⁴) and accuracy (<10^-7). We measured mutation frequencies in genes encoding replicative DNA polymerases and in genes frequently mutated in CRC, and found an unexpectedly high effective mutation rate, 7.1 × 10^-7. The curve of subclonal mutation accumulation as a function of sequencing depth, using DNA obtained from 5 different tumors, is in accord with a neutral model of tumor evolution. We present a theoretical approach to model neutral evolution independent of the infinite-sites assumption (which states that a particular mutation arises only in one tumor cell at any given time). Our analysis indicates that the infinite-sites assumption is not applicable once the number of tumor cells exceeds the reciprocal of the mutation rate, a circumstance relevant to even the smallest clinically diagnosable tumor. Our methods allow accurate estimation of the total mutation burden in clinical cancers. Our results indicate that no DNA locus is wild type in every malignant cell within a tumor at the time of diagnosis (probability of all cells being wild type, 10^-308).