Development, validation, and comparison of gene analysis methods for detecting EGFR mutation from non-small cell lung cancer patients-derived circulating free DNA

// Masaki Hanibuchi 1 , 2 , Akira Kanoh 3 , Takuya Kuramoto 3 , Tatsuro Saito 4 , Makoto Tobiume 1 , Atsuro Saijo 1 , Hiroyuki Kozai 1 , Mayo Kondo 1 , Shun Morizumi 1 , Hiroto Yoneda 1 , Kozo Kagawa 1 , Hirokazu Ogino 1 , Seidai Sato 1 , Hiroshi Kawano 1 , Kenji Otsuka 1 , Yuko Toyoda 1 , Hiroshi Nokihara 1 , Hisatsugu Goto 1 and Yasuhiko Nishioka 1 1 Department of Respiratory Medicine and Rheumatology, Graduate School of Biomedical Sciences, Tokushima University, Tokushima, 770-8503, Japan 2 Department of Internal Medicine, Shikoku Central Hospital of the Mutual Aid Association of Public School Teachers, Shikoku-Chuo, 799-0193, Japan 3 Biomarker Research, Early Development Strategy and Planning, Taiho Pharmaceutical Co., Ltd., Tsukuba, 300-2611, Japan 4 Riken Genesis Co., Ltd., Shinagawa-ku, Tokyo, 141-0032, Japan Correspondence to: Yasuhiko Nishioka, email: yasuhiko@tokushima-u.ac.jp Keywords: non-small cell lung cancer; circulating free DNA; epidermal growth factor receptor mutation; epidermal growth factor receptor-tyrosine kinase inhibitor Received: September 13, 2018     Accepted: May 04, 2019     Published: June 04, 2019 ABSTRACT The feasibility and required sensitivity of circulating free DNA (cfDNA)-based detection methods in second-line epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) treatment are not well elucidated. We examined T790M and other activating mutations of EGFR by cfDNA to assess the clinical usability. In 45 non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations, cfDNAs were prepared from the plasma samples. EGFR mutations in cfDNA were detected using highly sensitive methods and originally developed assays and these results were compared to tissue-based definitive diagnoses. The specificity of each cfDNA-based method ranged 96–100% whereas the sensitivity ranged 56–67%, indicating its low pseudo-positive rate. In EGFR-TKI failure cohort, 41–46% samples were positive for T790M by each cfDNA-based method, which was comparable to re-biopsy tissue-based T790M positive rates in literature. The concordance of the results for each EGFR mutation ranged from 83–95%. In eight patients, the results of the cfDNA-based assays and re-biopsy-derived tissue-based test were compared. The observed overall agreement ranged in 50–63% in T790M, and in 63–100% in activating EGFR mutations. In this study, we have newly developed three types of assay which have enough sensitivity to detect cfDNA. We also detected T790M in 44% of patients who failed prior EGFR-TKI treatment, indicating that cfDNA-based assay has clinical relevance for detecting acquired mutations of EGFR .