Abstract 5248: Non-invasive EGFR T790M detection using droplet digital PCR system
Authors: Hashida Shinsuke , Ohashi Kadoaki , Matsubara Takehiro , Ohtsuka Tomoaki , Watanabe Mototsugu , Suzawa Ken , Maki Yuho , Yamamoto Hiromasa , Soh Junichi , Asano Hiroaki , Tsukuda Kazunori , Miyoshi Shinichiro , Kiura Katsuyuki , Toyooka Shinichi 
Published: August 2015

Abstract
Background: Non-small cell lung cancers (NSCLCs) harboring activating epidermal growth factor receptor (EGFR) mutations show significant response to EGFR-tyrosine kinase inhibitors (TKIs). However, most of them acquire resistance to EGFR-TKIs, and a half of the resistance is caused by EGFR T790M mutation. Recently, the third generation-TKIs which are effective to lung cancer harboring T790M mutation have been developed, thus the importance of T790M mutation detection is increasing in NSCLC treatment. Nowadays, the highly sensitive assays such as PNA-LNA PCR clamp, Scorpion ARMS have been developed to detect T790M mutation. In this study, we newly established non-invasive assay for T790M mutation detection from circulating cell free DNA (ccfDNA) using droplet digital PCR system (ddPCR).
Materials and Methods: The sensitivity of ddPCR was determined using variable proportion of mixture DNAs of human bronchial epithelial cell with wild-type EGFR and NCI-H1975 with EGFR L858R and T790M mutations. We collected ccfDNA extracted from the 39 plasma samples of 22 NSCLC patients, who had been treated with EGFR-TKI and examined EGFR T790M mutational status by re-biopsy. The T790M mutational status of the re-biopsy samples were determined using PNA-LNA PCR clamp method. The circulating T790M alleles in ccfDNAs were detected using ddPCR, and compared with the T790M mutational status of re-biopsy samples.
Results: In 8 of the 39 plasma samples, T790M alleles were detected by ddPCR. As 10 of 39 plasma samples were collected from the patient who determined T790M positive by re-biopsy after acquiring resistance to EGFR -TKI, the sensitivity of ddPCR was 80%. In 3 plasma samples, T790M alleles were detected by ddPCR although PNA-LNA PCR clamp method could not detect T790M mutation from the re-biopsy tissues of the patients who provided their plasmas. The specificity of ddPCR was determined as 89.7% although we have to consider about the difference between the timings of plasma collection and re-biopsy, and tumors’ heterogeneity.
Conclusions: The result of circulating T790M allele detection using ddPCR was highly concordant with the result of T790M detection in re-biopsy samples using PNA-LNA PCR clamp method. The ddPCR is promising method for non-invasive T790M mutation detection.
Citation Format: Shinsuke Hashida, Kadoaki Ohashi, Takehiro Matsubara, Tomoaki Ohtsuka, Mototsugu Watanabe, Ken Suzawa, Yuho Maki, Hiromasa Yamamoto, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi, Katsuyuki Kiura, Shinichi Toyooka. Non-invasive EGFR T790M detection using droplet digital PCR system. [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 5248. doi:10.1158/1538-7445.AM2015-5248