Optimal method for quantitative detection of plasma EGFR T790M mutation using droplet digital PCR system

Ken Suzawa, Hiromasa Yamamoto, Kadoaki Ohashi, Shinsuke Hashida, Shuta Tomida, Toshio Kubo, Yuho Maki, Junichi Soh, Kazunori Tsukuda, Katsuyuki Kiura, Shinichiro Miyoshi, Shinichi Toyooka

Research output: Contribution to journalArticle

10 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)3100-3106
Number of pages7
JournalOncology reports
Volume37
Issue number5
DOIs
Publication statusPublished - May 2017

Keywords

  • Circulating DNA
  • Digital PCR
  • Locked nucleic acid
  • T790M mutation

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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