Abstract 3770: Unexpectedly high subclonal mutational diversity in human colorectal cancer and its significance
Authors: Beckman Robert A. , Kohrn Brendan , Loubet-Senear Kaitlyn , Dunn Jasmin , OSullivan Jacintha , Bronner Mary , Loeb Lawrence A. 
Published: August 2019

Abstract
Human colorectal cancers (CRC) contain numerous positively selected clonal somatic mutations in 10% or more of the cells. In addition, subclonal mutations in a fraction of cells contribute to phenotypic heterogeneity. Several groups have shown either by analysis of the number of unique subclones as a function of sequencing depth, or by the evaluation of synonymous to nonsynonymous mutation ratios, that most subclonal mutations are not selected and evolve neutrally. Because of the branching nature of tumor evolution, the clonal mutations arise in the founder cell, or very early thereafter. Subclonal mutations appear next, the rare ones on progressively smaller branches of the evolutionary tree. Here, we apply Duplex Sequencing (DS) methodology to quantify subclonal mutations in 5 fresh human MSI-low CRC diagnostic samples with unprecedented depth (104) and accuracy (10-7), and confirm neutral evolution further forward in time than previously known. We find that CRCs without known DNA repair deficits harbor unexpectedly many subclonal mutations; indicating that the mutational diversity of CRCs has been greatly underestimated. The “effective mutation frequency”, or mutation frequency per new cell added to the tumor (taking cell death into account) is also unexpectedly high: 6 X 10-7per base. Given a genome length of 3 X 109, a new daughter cell (taking turnover into account) would have ca 2000 new private mutations compared to its parent. Further, the smallest clinically diagnosable tumor has ca 109 cells, leading to violation of the common modeling assumption that a given mutation arises uniquely in only one cell (“infinite sites assumption”), since at an effective mutation frequency of 6 X 10-7, a cell generation leading to the formation of 109 new cells would produce the same mutation in 600 of those cells. We have developed a new theoretical approach to model neutral evolution independent of the infinite sites assumption, and find that intratumoral heterogeneity in clinical tumors is also underestimated by previous theoretical approaches. Our novel experimental and theoretical methods show that every possible somatic point mutation is present when a tumor is clinically detectable, leading to pre-existing resistant cells to any therapy. We can more accurately predict emergence of cells simultaneously mutationally resistant to multiple non-cross resistant therapies with increasing cell number; targeting genetic instability itself may prevent this.
Citation Format: Robert A. Beckman, Brendan Kohrn, Kaitlyn Loubet-Senear, Jasmin Dunn, Jacintha OSullivan, Mary Bronner, Lawrence A. Loeb. Unexpectedly high subclonal mutational diversity in human colorectal cancer and its significance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3770.