Predominance Of Egfr Mutations Responsible For Resistance To Tyrosine Kinase Inhibitors Among Tumor Dnas Assessed By Picoliter Droplet Digital Pcr Analysis Of Cell-Free Plasma Dnas

Abstract Introduction & Objectives: The new generation EGFR-tyrosine kinase inhibitors (TKIs), which suppress the kinase activity of EGFR proteins with secondary T790M substitution, are being developed to overcome resistance to therapy using conventional EGFR-TKI. Non-invasive monitoring of EGFR gene mutations conferring sensitive (i.e., L858R and various types of exon 19 deletion mutations) and T790M mutations to TKIs are inevitable for efficient therapy of lung adenocarcinoma (LADC) using conventional and/or new generation EGFR-TKIs. We aimed to evaluate the utility of picoliter droplet digital PCR (pdPCR)-based cell-free plasma DNA (cfDNA) examination to assess the tumor progression status of LADC patients against EGFR-TKI treatments. Experimental Design: cfDNAs from eight LADC patients acquiring resistance to EGFR-TKIs including gefitinib or erlotinib were subjected to pdPCR assay that enables us to assess fractions of EGFR DNAs with T790M, L858R and representative exon 19 deletion mutations among all EGFR DNAs. Each cfDNA sample was subjected to two pdPCR assays, one for T790M mutation and the other for either of TKI-sensitive exon 19 deletion or L858R mutation (determined before EGFR-TKI therapy). Mutation positivity was decided according to a threshold set based on cfDNAs from mutation-negative LADC patients, and fractions of T790M mutated EGFR DNAs among EGFR DNAs with TKI-sensitive mutations were estimated. The pdPCR assay for exon 19 deletion was designed to detect loss of the wild-type signal and therefore could detect exon 19 deletions of representative exon 19 in-flame deletion mutations while the L858R and T790M assays were designed to specifically detect DNA with corresponding single nucleotide variations. Results: All eight patients examined after acquiring resistance to EGFR-TKIs showed positivity for TKI-sensitive mutations, and five of them (62.5%) also for T790M mutations. The mutation status of cfDNAs from four patients was consistent with those of their re-biopsied tissues from the lesion acquiring resistance. T790M mutation was deduced to have occurred in a variety of fractions (7-97%) of tumor DNAs. Conclusions: pdPCR-based examination of cfDNAs will present a robust non-invasive assessment of tumor progression status against EGFR-TKI treatments by informing us of the predominance of T790M mutated DNAs among tumor DNAs. A prospective study to validate the utility of the pdPCR assay is on-going on patients with NSCLC receiving EGFR-TKI therapy. Citation Format: Yoshitaka Seki, Yutaka Fujiwara, Takashi Kohno, Erina Takai, Kuniko Sunami, Hidehito Horinouchi, Shintaro Kanda, Hiroshi Nokihara, Noboru Yamamoto, Shun-ichi Watanabe, Kazuyoshi Kuwano, Yuichiro Ohe. Predominance of EGFR mutations responsible for resistance to tyrosine kinase inhibitors among tumor DNAs assessed by picoliter droplet digital PCR analysis of cell-free plasma DNAs. [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 5247. doi:10.1158/1538-7445.AM2015-5247