title: Optimal method for quantitative detection of plasma EGFR T790M mutation using droplet digital PCR system.
abstract: Though patients with EGFR mutations are initially responsive to EGFR-tyrosine kinase inhibitors (TKIs), most tumors ultimately acquire resistance to EGFR-TKIs. The most frequently reported mechanism is EGFR T790M mutation. In this study, using a droplet digital PCR (ddPCR) system, we assessed optimal conditions for a mutation detection assay for EGFR T790M obtained from circulating cell-free DNA (cfDNA) in plasma. The advantages of locked nucleic acids (LNA) probe, short amplicon size, and blocking oligo using peptide nucleic acids (PNA) were assessed using control DNAs from cell lines to improve the sensitivity of mutation detection. T790M alleles were then analyzed using ddPCR in 59 plasma samples from 24 NSCLC patients with EGFR mutations, and compared to the T790M status which were determined thorough re-biopsies. The assessment of the optimal assay method revealed that the assay using the short amplicon can efficiently detect more fragmented-DNA. The LNA probe and PNA clamp contributed better separation between positive and negative droplets. This PNA-LNA-ddPCR clamp method can detect mutant alleles in the sample with a mutant allele content of 0.01%. In clinical plasma samples, T790M alleles were detected via ddPCR with a sensitivity of 42.8% and specificity of 97.3%. We established a highly-sensitive detection assay for the T790M allele using the PNA-LNA-ddPCR clamp method. ddPCR is a promising method for detecting non-invasive T790M mutation.
authors.affiliation: Department of Thoracic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Suzawa
authors.affiliation: Department of Thoracic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Yamamoto
authors.affiliation: Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Kadoaki
authors.initials: K
authors.lastname: Ohashi
authors.affiliation: Department of Thoracic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Hashida
authors.affiliation: Biobank, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Shuta
authors.initials: S
authors.lastname: Tomida
authors.affiliation: Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Kubo
authors.affiliation: Department of Thoracic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Yuho
authors.initials: Y
authors.lastname: Maki
authors.affiliation: Department of Thoracic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Junichi
authors.initials: J
authors.lastname: Soh
authors.affiliation: Department of Thoracic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Tsukuda
authors.affiliation: Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Kiura
authors.affiliation: Department of Thoracic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Miyoshi
authors.affiliation: Department of Thoracic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Toyooka
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis / Q000379:methods*; D066246:ErbB Receptors / Q000097:blood*; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: C512478:EGFR protein, human; D066246:ErbB Receptors