Abstract 617: Ultrasensitive detection of the pretreatment EGFR T790M mutation in non-small cell lung cancer patients with an EGFR-activating mutation using picodroplet digital PCR
Authors: Koh Yasuhiro , Kawaguchi Tomoya , Watanabe Masaru , Isa Shun-ichi , Ando Masahiko , Tamiya Akihiro , Kubo Akihito , Saka Hideo , Takeo Sadanori , Adachi Hirofumi , Tagawa Tsutomu , Kakegawa Seiichi , Yamashita Motohiro , Kataoka Kazuhiko , Ichinose Yukito , Takeuchi Yukiyasu , Sakamoto Kazuhiro , Matsumura Akihide 
Published: August 2015

Abstract
Purpose: The resistance of the epidermal growth factor receptor (EGFR) to tyrosine kinase inhibitors (TKIs) is a major concern in non-small-cell lung cancer (NSCLC) treatment. T790M mutation in EGFR accounts for nearly 50% of the acquired resistance to EGFR-TKIs. Earlier studies suggested that EGFR T790M mutation was also detected in TKI-naive NSCLCs in a small cohort and the prevalence of a de novo EGFR T790M mutation in patients with EGFR-mutant NSCLC remains unclear. Here, we use an ultra-sensitive droplet digital PCR (ddPCR) technique to address the incidence and clinical significance of pretreatment EGFR T790M mutation in a larger cohort.
Experimental design: ddPCR was established as follows: EGFR wild-type or T790M mutation-containing DNA fragments were cloned into plasmids. Candidate threshold was identified using wild-type plasmid, normal human genomic DNA, and human A549 cell line DNA, which expresses wild-type EGFR. Surgically resected tumor tissues from 373 early-stage NSCLC patients with EGFR-activating mutations detected by Cycleave PCR method were then examined for the presence of pretreatment EGFR T790M mutation using ddPCR.
Results: Preclinical model data revealed a linear performance for this ddPCR method (R2 = 0.998) with an analytical sensitivity of ∼0.001%. The overall incidence of the pretreatment EGFR T790M mutation was 79.9% (298/373) and the frequency ranged from 0.009% to 26.9%, while only five patients were diagnosed positive for EGFR T790M mutation by Cycleave PCR method. The EGFR T790M mutation was detected more frequently in patients with a larger tumor size (p = 0.019) and those with common EGFR-activating mutations (p = 0.022), as compared to the others. Among the 69 patients who had co-incident mutations in EGFR and p53, 61 patients (88.4%) had the EGFR T790M mutation (p = 0.096). Moreover, all 11 patients with mutations in PIK3CA and EGFR had the EGFR T790M mutation (p = 0.097). These results, while not statistically significant, suggest that tumors with the pretreatment EGFR T790M mutation may manifest greater genomic complexity.
Conclusions: To our knowledge, this is the largest study thus far using an ultra-sensitive method for detecting the pretreatment EGFR T790M mutation that has demonstrated associations between the incidence of the pretreatment T790M mutation and clinicopathological as well as genetic features in early-stage NSCLC. The ultra-sensitive ddPCR assay revealed that pretreatment EGFR T790M mutation was found in the majority of NSCLC patients with EGFR-activating mutations. ddPCR should be utilized for detailed assessment of the impact of the low frequency pretreatment EGFR T790M mutation on treatment with EGFR-TKIs, allowing the possibility of developing strategies for personalized cancer therapies in NSCLC patients.
Citation Format: Yasuhiro Koh, Tomoya Kawaguchi, Masaru Watanabe, Shun-ichi Isa, Masahiko Ando, Akihiro Tamiya, Akihito Kubo, Hideo Saka, Sadanori Takeo, Hirofumi Adachi, Tsutomu Tagawa, Seiichi Kakegawa, Motohiro Yamashita, Kazuhiko Kataoka, Yukito Ichinose, Yukiyasu Takeuchi, Kazuhiro Sakamoto, Akihide Matsumura. Ultrasensitive detection of the pretreatment EGFR T790M mutation in non-small cell lung cancer patients with an EGFR-activating mutation 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 617. doi:10.1158/1538-7445.AM2015-617