Abstract LB-314: Development of reference material guaranteed copy number of DNA for liquid biopsy

Abstract

Abstract cfDNA (cell free DNA) analysis using liquid biopsy plays an important role in cancer research. In particular NGS (next-generation sequencer)-based assay has attracted wide interests, because it can not only screen many cancer genes but also can detect ultra-low-concentration mutations at the same time. However, there is no reference material to evaluate correctly its performance. Currently, most researchers use diluted human genomic DNA that is commercially available as reference, but adjusting its concentration is difficult because of the Poisson distribution. Herein, we have developed a method to dispense cells with counting a cell one by one using inkjet technology (Manabu Seo, et. Al., 2019, Anal. Chem.). By lysing cells after dispensing, the defined copy number of DNA is prepared with a single molecule level. In this study, we assessed the feasibility of our reference material prepared by inkjet as a control for rare mutation detection (e.g. minimal sampling bias and high reproducibility). Spike-in control samples with exact allele frequencies with 0.07%, 0.53%, 0.64%, and 1.0%, were prepared by accurately dispensing with inkjet the cells that contain a sequence of exons 18, 19, 20, and 21 of EGFR. The EGFR sequence has G719A, E746_A750del, T790M, C797S, L858R, and L861Q mutations. For comparison, a few samples were also prepared by manual dilution process. The EGFR mutations were analyzed by the method of high-sensitivity procedure using NGS (Yoji Kukita, et. Al., 2013, PLoS ONE.). Result of the inkjet method had less variation than the manual dilution method, and high repeatability was obtained. Especially in E746_A750del (0.53%) the inkjet samples were tested three times (n = 24 each), and their CVs (coefficient of variation) were 17%, 17%, and 12%. In contrast, the manual sample's one was 33%. In addition, linearity of T790M and L858R were R2 = 0.98 and R2 = 0.96, respectively. The reference material produced by inkjet method can be provided under the same conditions at each facility and make it possible to improve the reliability of liquid biopsy. Therefore, the reference material could be appropriate standard for external quality control for EGFR mutant detection of liquid biopsy. Citation Format: Yusuke Osaki, Yoshiharu Sato, Miho Ishii, Yuki Yonekawa, Ryo Matoba. Development of reference material guaranteed copy number of DNA for liquid biopsy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-314.