title: Picoliter-Droplet Digital Polymerase Chain Reaction-Based Analysis of Cell-Free Plasma DNA to Assess EGFR Mutations in Lung Adenocarcinoma That Confer Resistance to Tyrosine-Kinase Inhibitors.
abstract: OBJECTIVE
  The objective of this study was to evaluate the utility of analyzing cell-free plasma DNA (cfDNA) by picoliter-droplet digital polymerase chain reaction (ddPCR) to detect EGFR mutations that confer resistance to tyrosine-kinase inhibitors (TKIs) used for treatment of lung adenocarcinoma (LADC).
  
  
  METHODS
  Thirty-five LADC patients who received epidermal growth factor receptor (EGFR)-TKI therapy, including ten who received tumor rebiopsy after development of resistance, were subjected to picoliter-ddPCR-cfDNA analysis to determine the fraction of cfDNA with TKI-sensitive (L858R and inflame exon 19 deletions) and -resistant (i.e., T790M) mutations, as well as their concordance with mutation status in rebiopsied tumor tissues.
  
  
  RESULTS
  cfDNA samples from 15 (94%) of 16 patients who acquired resistance were positive for TKI-sensitive mutations. Also, 7 (44%) were positive for the T790M mutation, with fractions of T790M (+) cfDNA ranging from 7.4% to 97%. T790M positivity in cfDNA was consistent in eight of ten patients for whom rebiopsied tumor tissues were analyzed, whereas the remaining cases were negative in cfDNA and positive in rebiopsied tumors. Prior to EGFR-TKI therapy, cfDNAs from 9 (38%) and 0 of 24 patients were positive for TKI-sensitive and T790M mutations, respectively. Next-generation sequencing of cfDNA from one patient who exhibited innate resistance to TKI despite a high fraction of TKI-sensitive mutations and the absence of the T790M mutation in his cfDNA revealed the presence of the L747P mutation, a known driver of TKI resistance.
  
  
  CONCLUSIONS
  Picoliter-ddPCR examination of cfDNA, supported by next-generation sequencing analysis, enables noninvasive assessment of EGFR mutations that confer resistance to TKIs.
  
  
  CONCLUSIONS
  Noninvasive monitoring of the predominance of tumors harboring the secondary T790M mutation in the activating mutation in EGFR gene is necessary for precise and effective treatment of lung adenocarcinoma. Because cells harboring the T790M mutation are resistant to epidermal growth factor receptor-tyrosine-kinase inhibitors (TKIs), the predominance of tumor cells harboring the T790M mutations influences the choice of whether to use conventional or next-generation TKIs. Digital polymerase chain reaction-based examination of cfDNA is a promising method; however, its feasibility, including its consistency with examination of rebiopsied tumor tissue, has not been fully proven. Here, picoliter-droplet digital polymerase chain reaction technology is presented as a candidate method for testing cfDNA and assessing the predominance of T790M-mutant tumors.
authors.affiliation: Division of Genome Biology, National Cancer Center Research Institute, Tokyo, Japan Division of Respiratory Diseases, Department of Internal Medicine, Jikei University School of Medicine, Tokyo, Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Seki
authors.affiliation: Department of Thoracic Oncology, National Cancer Center Hospital, Tokyo, Japan yutakafu@ncc.go.jp.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Fujiwara
authors.affiliation: Division of Genome Biology, National Cancer Center Research Institute, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kohno
authors.affiliation: Division of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan.
authors.forename: Erina
authors.initials: E
authors.lastname: Takai
authors.affiliation: Department of Thoracic Oncology, National Cancer Center Hospital, Tokyo, Japan.
authors.forename: Kuniko
authors.initials: K
authors.lastname: Sunami
authors.affiliation: Department of Thoracic Oncology, National Cancer Center Hospital, Tokyo, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Goto
authors.affiliation: Department of Thoracic Oncology, National Cancer Center Hospital, Tokyo, Japan.
authors.forename: Hidehito
authors.initials: H
authors.lastname: Horinouchi
authors.affiliation: Department of Thoracic Oncology, National Cancer Center Hospital, Tokyo, Japan.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Kanda
authors.affiliation: Department of Thoracic Oncology, National Cancer Center Hospital, Tokyo, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nokihara
authors.affiliation: Department of Thoracic Surgery, National Cancer Center Hospital, Tokyo, Japan.
authors.forename: Shun-ichi
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Division of Genetics, National Cancer Center Research Institute, Tokyo, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Ichikawa
authors.affiliation: Department of Thoracic Oncology, National Cancer Center Hospital, Tokyo, Japan.
authors.forename: Noboru
authors.initials: N
authors.lastname: Yamamoto
authors.affiliation: Division of Respiratory Diseases, Department of Internal Medicine, Jikei University School of Medicine, Tokyo, Japan.
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Kuwano
authors.affiliation: Department of Thoracic Oncology, National Cancer Center Hospital, Tokyo, Japan.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Ohe
mesh_terms: D000230:Adenocarcinoma / Q000097:blood / Q000235:genetics* / Q000473:pathology; D000077192:Adenocarcinoma of Lung; D000368:Aged; D004273:DNA, Neoplasm / Q000097:blood* / Q000302:isolation & purification; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000097:blood / Q000235:genetics*; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D047428:Protein Kinase Inhibitors / Q000008:administration & dosage; D016896:Treatment Outcome
chemical_list: D004273:DNA, Neoplasm; D047428:Protein Kinase Inhibitors; C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: Acquired resistance; Cell-free DNA; Digital PCR; EGFR tyrosine-kinase inhibitor; Lung cancer; Next-generation sequencing