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T. Hanami

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    P1.03 - Poster Session 1 - Technology and Novel Development (ID 150)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.03-003 - Novel detection method for EGFR T790M mutation by Eprobe mediated PCR and melting curve analysis (ID 2006)

      09:30 - 16:30  |  Author(s): T. Hanami

      • Abstract

      Figure 1Epidermal growth factor receptor (EGFR) mutation status is the primary issue on the appropriate therapy of EGFR-tyrosine kinase inhibitor (EGFR-TKI) in non-small lung cancer. The point mutation of EGFR T790M (C→T) is known to be an acquired and the most common resistance against EGFR-TKI. Highly sensitive mutation detection system has been desired considering the difficulty in obtaining tissue specimens during disease progression. Eprobe is new fluorescence labeled probe with high affinity toward target ssDNA and works as competitive probe (Figure 1). Here we describe a novel method to identify T790M mutation using Eprobe on real-time monitoring of PCR and melting curve analysis.

      Figure 1Eprobe was designed to bind wild type allele including T790M region with competing primer, which enriches mutant allele amplification (Figure 2). The T790M mutation was detected by melting curve analysis following real-time monitoring of PCR. We verified detection ability by genomic DNA containing wild type and mutated EGFR T790M (cell lines H1975) gene. For clinical evaluation, 338 tumor tissues from the patients with lung adenocarcinoma, of which EGFR gene mutation status had been revealed by the nucleic acid-locked nucleic acid PCR clamp (PNA-LNA PCR clamp), were assayed by Eprobe method. We compared the two methods on T790M mutation assay.

      The T790M mutant genome could be detected when it accounted for as little as 0.5% of a mixture of wild type genome by enrichment of mutation amplicon. The activating EGFR mutation (exon19 deletion, L858R, and L861Q) had been detected in 143 out of 338 samples (42.3%) but no T790M mutation was identified by PNA-LNA PCR clamp. Among 143 samples harboring activating EGFR mutation, T790M mutation was identified in 2 samples (1.4%) by Eprobe method.

      The Eprobe method is sensitive for detecting EGFR T790M. Since Eprobe works in simple manner, current method is expected to apply to other gene detections.