Development and Validation of an Ultradeep Next-Generation Sequencing Assay for Testing of Plasma Cell-Free DNA from Patients with Advanced Cancer.

Abstract

<b>Purpose:</b> Tumor-derived cell-free DNA (cfDNA) in plasma can be used for molecular testing and provide an attractive alternative to tumor tissue. Commonly used PCR-based technologies can test for limited number of alterations at the time. Therefore, novel ultrasensitive technologies capable of testing for a broad spectrum of molecular alterations are needed to further personalized cancer therapy.<b>Experimental Design:</b> We developed a highly sensitive ultradeep next-generation sequencing (NGS) assay using reagents from TruSeqNano library preparation and NexteraRapid Capture target enrichment kits to generate plasma cfDNA sequencing libraries for mutational analysis in 61 cancer-related genes using common bioinformatics tools. The results were retrospectively compared with molecular testing of archival primary or metastatic tumor tissue obtained at different points of clinical care.<b>Results:</b> In a study of 55 patients with advanced cancer, the ultradeep NGS assay detected 82% (complete detection) to 87% (complete and partial detection) of the aberrations identified in discordantly collected corresponding archival tumor tissue. Patients with a low variant allele frequency (VAF) of mutant cfDNA survived longer than those with a high VAF did (<i>P</i> = 0.018). In patients undergoing systemic therapy, radiological response was positively associated with changes in cfDNA VAF (<i>P</i> = 0.02), and compared with unchanged/increased mutant cfDNA VAF, decreased cfDNA VAF was associated with longer time to treatment failure (TTF; <i>P</i> = 0.03).<b>Conclusions:</b> Ultradeep NGS assay has good sensitivity compared with conventional clinical mutation testing of archival specimens. A high VAF in mutant cfDNA corresponded with shorter survival. Changes in VAF of mutated cfDNA were associated with TTF. <i>Clin Cancer Res; 23(18); 5648-56. ©2017 AACR</i>.