Abstract LB-283: Human cancers harbor extensive subclonal mutations
Authors: Schmitt Michael W. , Fox Edward J. , Prindle Marc J. , Reid –Bayliss Kate S. , Becker Pamela S. , Loeb Lawrence A. 
Published: August 2015

Abstract
Recent advances in next-generation DNA sequencing (NGS) have revealed greater than expected mutational heterogeneity, not only between tumors of similar cancer type, but also within individual tumors. This mutational heterogeneity could serve as a reservoir for the emergence of new phenotypes, including resistance to therapy. While mutational diversity has been reported in many human cancers, the high error-rate of conventional NGS limits its ability to confidently resolve mutations present in less than 1.0 to 5% of the cells that comprise a tumor. These low-level subclonal mutations likely contribute to the rapid emergence of resistance to radiation and chemotherapy and the ability of cancer cells to invade adjacent tissues and to metastasize.
In order to study subclonal mutations, we have developed a highly accurate sequencing protocol, termed Duplex Sequencing, which takes advantage of the double-stranded nature of DNA to increase the accuracy of DNA sequencing by more than 10,000-fold; Duplex Sequencing has an unprecedented background error frequency of &lt;5×10-8. Additionally, in order to analyze rare subclonal mutations in human diseases, we have also established a highly efficient, targeted capture method, which utilizes sequential rounds of hybridization to enrich targeted genes by &gt;1,000,000-fold, with up to 99.8% of resultant sequencing reads on target.
Using these new methodologies, we have investigated the subclonal makeup of several cancers, including acute myeloid leukemia (AML), colon cancers (CRC), and glioblastoma (GBM). Upon targeting genes identified by NGS as drivers of clonal proliferation, we identified multiple subclonal mutations in each of these cancers, some with the potential to elicit drug resistance and others to drive tumor cell proliferation. We also investigated the five human replicative DNA polymerases. No reports have previously implicated mutations in these polymerases in either AML or GBM, nor in sporadic CRC with the exception of mutations in the exonuclease domain of DNA polymerase epsilon. However, our approach, combining targeted gene capture with Duplex Sequencing, revealed multiple subclonal mutations in the catalytic domains of all five replicative DNA polymerases in each of these tumors.
The presence of subclonal mutations in the catalytic domains of replicative DNA polymerases supports the mutator phenotype hypothesis, which posits that an increased mutation rate is a driving force during early tumorigenesis. Furthermore, extrapolating the results on the number of subclonal mutations in target genes to the entire genome, our results indicate that sites harboring subclonal mutations are &gt;100-fold more frequent than sites with clonal mutations. Thus, by the time a tumor is clinically diagnosed, every position in the genome could be mutated in at least one cell in the tumor; these subclones could be the reservoir for the emergence of new phenotypes and resistance to therapy.
Citation Format: Michael W. Schmitt, Edward J. Fox, Marc J. Prindle, Kate S. Reid – Bayliss, Pamela S. Becker, Lawrence A. Loeb. Human cancers harbor extensive subclonal mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-283. doi:10.1158/1538-7445.AM2015-LB-283