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Proc Natl Acad Sci U S A. 2019 Dec 5. pii: 201910301. doi: 10.1073/pnas.1910301116. [Epub ahead of print]

Extensive subclonal mutational diversity in human colorectal cancer and its significance.

Author information

Department of Pathology, University of Washington, Seattle, WA 98195;
Department of Biochemistry, University of Washington, Seattle, WA 98195.
Department of Pathology, University of Washington, Seattle, WA 98195.
Trinity Translational Medicine Institute, Department of Surgery, Trinity College Dublin, St. James's Hospital, Dublin 8, Ireland.
Division of Medical Oncology, University of Washington, Seattle, WA 98195.
TwinStrand Biosciences, Inc., Seattle, WA 98121.
Department of Pathology, University of Utah, Salt Lake City, UT 84112.
Department of Oncology, Georgetown University Medical Center, Washington, DC 20007.
Department of Biostatistics, Bioinformatics, and Biomathematics, Georgetown University Medical Center, Washington, DC 20007.
Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20007.
Innovation Center for Biomedical Informatics, Georgetown University Medical Center, Washington, DC 20007.


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).


drug resistance; duplex sequencing; genetic instability; mathematical modeling; tumor evolution

Conflict of interest statement

Competing interest statement: L.A.L. and the University of Washington have a license agreement with TwinStrand Biosciences for the use and development of Duplex Sequencing technology. L.A.L. and J.J.S. are founding members of TwinStrand Biosciences. L.A.L. is a member of the Scientific Advisory Board of Stratos Genomics, Inc. J.J.S. is the Chief Scientific Officer of TwinStrand Biosciences. R.A.B. consults for AstraZeneca, EMD Serono, Vertex, Zymeworks, and CStone, and is the Founder and Chief Scientific Officer of Onco-Mind, LLC.

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