title: Ultra-Sensitive Detection of the Pretreatment EGFR T790M Mutation in Non-Small Cell Lung Cancer Patients with an EGFR-Activating Mutation Using Droplet Digital PCR.
abstract: OBJECTIVE
  The resistance to the EGFR tyrosine kinase inhibitors (TKI) 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 T790M mutation was also detected in TKI-naïve NSCLCs in a small cohort. Here, we use an ultra-sensitive droplet digital PCR (ddPCR) technique to address the incidence and clinical significance of pretreatment T790M in a larger cohort.
  
  
  METHODS
  ddPCR was established as follows: 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. Surgically resected tumor tissues from 373 NSCLC patients with EGFR-activating mutations were then examined for the presence of T790M using ddPCR.
  
  
  RESULTS
  Our data revealed a linear performance for this ddPCR method (R(2) = 0.998) with an analytical sensitivity of approximately 0.001%. The overall incidence of the pretreatment T790M mutation was 79.9% (298/373), and the frequency ranged from 0.009% to 26.9%. The 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 with the others.
  
  
  CONCLUSIONS
  The ultra-sensitive ddPCR assay revealed that pretreatment T790M 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 T790M mutation on treatment with EGFR-TKIs.
authors.affiliation: Department of Respiratory Medicine and Medical Oncology, National Hospital Organization Nagoya Medical Center, Aichi, Japan. Third Department of Internal Medicine, Wakayama Medical University, Kimiidera, Wakayama, Japan. Drug Discovery and Development Division, Shizuoka Cancer Center Research Institute, Shizuoka, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: National Hospital Organization Kinki-Chuo Chest Medical Center, Osaka, Japan. Department of Respiratory Medicine, Graduate School of Medicine, Osaka City University, Osaka, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Kawaguchi
authors.affiliation: Clinical Research Center, Department of Lung Cancer Research, National Hospital Organization Kinki-chuo Chest Medical Center, Osaka, Japan.
authors.forename: Shun-ichi
authors.initials: S
authors.lastname: Isa
authors.affiliation: Center for Advanced Medicine and Clinical Research, Nagoya University Hospital, Aichi, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Ando
authors.affiliation: Department of Internal Medicine, National Hospital Organization Kinki-chuo Chest Medical Center, Osaka, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Tamiya
authors.affiliation: Department of Respiratory Medicine and Medical Oncology, National Hospital Organization Nagoya Medical Center, Aichi, Japan. Division of Respiratory Medicine and Allergology, Aichi Medical University School of Medicine, Aichi, Japan.
authors.forename: Akihito
authors.initials: A
authors.lastname: Kubo
authors.affiliation: Department of Respiratory Medicine and Medical Oncology, National Hospital Organization Nagoya Medical Center, Aichi, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Saka
authors.affiliation: Department of Thoracic Surgery, Clinical Research Institute, National Hospital Organization Kyushu Medical Center, Fukuoka, Japan.
authors.forename: Sadanori
authors.initials: S
authors.lastname: Takeo
authors.affiliation: Department of Thoracic Surgery, National Hospital Organization Hokkaido Cancer Center, Hokkaido, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Adachi
authors.affiliation: Department of Thoracic Surgery, National Hospital Organization Nagasaki Medical Center, Nagasaki, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Tagawa
authors.affiliation: Department of Thoracic Surgery, National Hospital Organization Nishigunma National Hospital, Gunma, Japan.
authors.forename: Seiichi
authors.initials: S
authors.lastname: Kakegawa
authors.affiliation: Department of Thoracic Surgery, National Hospital Organization Shikoku Cancer Center, Ehime, Japan.
authors.forename: Motohiro
authors.initials: M
authors.lastname: Yamashita
authors.affiliation: Department of Thoracic Surgery, National Hospital Organization Iwakuni Clinical Center, Yamaguchi, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Kataoka
authors.affiliation: Clinical Research Institute, National Kyushu Cancer Center, Fukuoka, Japan.
authors.forename: Yukito
authors.initials: Y
authors.lastname: Ichinose
authors.affiliation: Department of General Thoracic Surgery, National Hospital Organization Toneyama National Hospital, Osaka, Japan.
authors.forename: Yukiyasu
authors.initials: Y
authors.lastname: Takeuchi
authors.affiliation: Department of Respiratory Surgery, National Hospital Organization Yokohama Medical Center, Kanagawa, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Sakamoto
authors.affiliation: Department of Surgery, National Hospital Organization Kinki-chuo Chest Medical Center, Osaka, Japan.
authors.forename: Akihide
authors.initials: A
authors.lastname: Matsumura
authors.affiliation: Department of Respiratory Medicine and Medical Oncology, National Hospital Organization Nagoya Medical Center, Aichi, Japan. Third Department of Internal Medicine, Wakayama Medical University, Kimiidera, Wakayama, Japan. Drug Discovery and Development Division, Shizuoka Cancer Center Research Institute, Shizuoka, Japan. ykoh@wakayama-med.ac.jp.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Koh
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D066246:ErbB Receptors / Q000235:genetics* / Q000302:isolation & purification; D005260:Female; D005787:Gene Frequency; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D057285:Precision Medicine; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use
chemical_list: D047428:Protein Kinase Inhibitors; C512478:EGFR protein, human; D066246:ErbB Receptors