Abstract 4908: Establishment of multiplexed ultra-sensitive detection of epidermal growth factor receptor mutations using picodroplet digital PCR
Authors: Akamatsu Hiroaki , Koh Yasuhiro , Watanabe Masaru , Kikuchi Takashi , Nakanishi Masanori , Matsunaga Kazuto , Yamamoto Nobuyuki 
Published: August 2015

Abstract
BACKGROUND: The epidermal growth factor receptor (EGFR) mutations have been used as the strongest predictor of EGFR tyrosine kinase inhibitor (TKI) treatment. Three most common EGFR mutations (L858R, exon19 deletion, and T790M) are known to be major selection marker for EGFR-TKI therapy. Noninvasive techniques for tumor genotyping may be needed to fully realize the potential of genotype-directed cancer care as a “Liquid biopsy”. The low abundance of mutants and limited amount and quality of available cell-free DNA (cfDNA) in plasma samples render it difficult to reliably detect multiple mutations with current platforms and methods. Here, we develop a multiplex dPCR assay to detect three common EGFR mutations in one reaction using picodroplet digital PCR (ddPCR) technology, which enable us to provide 3-fold throughput and save the cost as well as DNA samples.
METHODS: Positive and negative control plasmids for the EGFR assay were prepared by cloning DNA fragments containing wild-type or the EGFR mutations. The appropriate concentration of plasmid DNA was determined empirically to yield a mixture in which the number of copies of mutant DNA was ca. 0.01-1.00% of the number of wild-type EGFR fragments. Genomic DNA from the lung tumor cell lines H1975, PC-9/ZD, A549 and wild-type human genomic DNA were digested with CviQ1, and then used to quantitatively assess each EGFR mutant sequence in the multiplex assay panels.
RESULTS: Serial dilutions experiments using plasmid as well as genomic DNA harboring EGFR mutations revealed a linear performance with an analytical sensitivity of approximately 0.01% for each mutation. The regression analysis between duplex and multiplex assay demonstrated a correlation coefficient (R2) of 0.9983for L858R, 0.9911 for exon19 deletion, and 0.9781 for T790M. The limit of blank (LOB) was defined by the frequency of positive droplets measured in 400 ng of wild-type DNA control samples. The number of false events of mutant droplets detected per analysis is: 8 for L858R, 6 for Exon 19 deletion, 6 for T790M from human normal gDNA, and 9 for L858R, 4 for Exon 19 deletion, 2 for T790M from A549 gDNA. Thus, the LOB was determined as a 10 events/assay.
CONCLUSIONS: Using ddPCR, we established multiplex and ultra-sensitive genotyping platform for three common EGFR mutations. Results of a proof-of-principle study using preclinical model indicated that the clinical utility of multiplex ddPCR to detect for multiple EGFR mutations. Further evaluation using clinical samples is warranted.
Citation Format: Hiroaki Akamatsu, Yasuhiro Koh, Masaru Watanabe, Takashi Kikuchi, Masanori Nakanishi, Kazuto Matsunaga, Nobuyuki Yamamoto. Establishment of multiplexed ultra-sensitive detection of epidermal growth factor receptor mutations using picodroplet digital PCR. [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 4908. doi:10.1158/1538-7445.AM2015-4908