Abstract LB-338: Extensive subclonal mutations in human colorectal cancers detected by duplex sequencing

The accumulation of somatic mutations is a defining hallmark of cancer. Human colorectal cancers (CRC) contain thousands of mutations that are detected at frequencies greater than 1%. Some of these are “driver mutations” that have been positively selected during tumor progression. In addition, subclonal mutations—those occurring only in a fraction of malignant cells—are randomly distributed and contribute to phenotypic and morphologic heterogeneity of cancer cells within a tumor. Cells containing specific subclonal mutations may be present prior to therapy and can be positively selected and account for the rapid emergence of therapeutic resistance. The extent of subclonal mutations in cancer, however, has been difficult to quantify, as the high error rate of next-generation sequencing precludes reliable detection of mutations present in fewer than 1-5% of cells. Here, we apply the highly accurate duplex sequencing methodology to quantify the extent of subclonal mutations in CRC. We analyzed somatic mutations in genes encoding replicative DNA polymerases (POLδ and POLϵ) as well as in genes reported to be frequently mutated in CRC. Duplex sequencing was carried out at depths varying from 500X-30,000X and an accuracy of 10-8. We find that CRCs with DNA repair deficits harbor an unexpectedly large complement of unique subclonal mutations; indicating that the mutational diversity of CRCs has been greatly underestimated. The frequency of subclonal mutations in normal colonic mucosa and CRC is greater than reported for clonal mutations. The burden of tumor-associated subclonal mutations does not correlate with age and the spectrum of single-nucleotide substitutions is different from that in nonmalignant cells, indicating that different mechanisms of mutation accumulation are operative in normal and CRC. Our data are consistent with a mutation rate of 1.1 x 10 per base pair, indicating that each malignant cell and its daughter encoded genomic DNA differs by more 3,000 nucleotides. The linearity of subclonal mutation accumulation as a function of sequencing depth, using DNA obtained from five different tumors, is in accord with a neutral model of tumor evolution. We calculate that the probability of mutations at any type is so high that it is likely that every possible somatic point mutation is present by the time a tumor is clinically detectable, and this could account for the high frequency by which tumors become resistant to therapeutic agents. Citation Format: Lawrence A. Loeb, Kaitlyn J. Loubet-Senear, Brendan F. Kohrn, Michael W. Schmitt, Mary P. Bronner, Robert A. Beckman. Extensive subclonal mutations in human colorectal cancers detected by duplex sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3377.