Author of

• 18973

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  P2.01 - Advanced NSCLC (ID 618)

  • Event: WCLC 2017
  • Type: Poster Session with Presenters Present
  • Track: Advanced NSCLC
  • Presentations: 2
  • Moderators:
  • Coordinates: 10/17/2017, 09:00 - 16:00, Exhibit Hall (Hall B + C)

  • 24663

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    P2.01-075f - Detection of EML4-ALK and ROS1 Fusion Gene in NSCLC by PNA-Assisted Real-Time RT-PCR Using Fluorescent Meting Curve Analysis (ID 9912)

    09:00 - 16:00  |  Presenting Author(s): Myosun Kim
    • Abstract

    Background:
    In patients with non-small cell lung cancer, the target therapy varies depending on the gene alteration. Recently, it is progressed therapy using ALK inhibitor for patients with resistance against EGFR TKI. The therapy is targeted patients with ALK or ROS1 fusion gene. In generally, two-step RT-PCR is used as a method for detecting EML4-ALK and ROS1.
    
    Method:
    Our method can be easily and quickly used by performing RT-PCR and real-time PCR in one step using PNA probe. In addition, a PNA probe is designed at the ALK and ROS1 kinase sites and the fusion target is detected as a melting curve to screen EML4-ALK and ROS1 gene. Using 43 of FFPE clinical samples, we compared our screening method with direct sequencing method.
    
    Result:
    This real-time PCR-based PNA probe-assisted fluorescent melting curve analysis assay(EML4-ALK and ROS1 screening) can detect a total of EML4-ALk 28 and ROS1 20 different Fusion gene. A total of 43 FFPE clinical samples and 3 cell lines(H2228, H3122 and HCC78) were used for EML4-ALK and ROS1 tests and results was good concordance. In case of discordance samples, we confirmed using direct sequencing.
    
    Conclusion:
    We have a simple and rapid method for detecting EML4-ALk or ROS1 Fusion genes within four hours. Therefore, it can be accomplished preventing experimental error, thus generating significant cost savings reductions.
    
    

  • 24664

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    P2.01-075g - Genotyping of EML4-ALK Fusion Gene by PNA-Assisted Real-Time RT-PCR Using Fluorescent Meting Curve Analysis (ID 9931)

    09:00 - 16:00  |  Presenting Author(s): Myosun Kim
    • Abstract

    Background:
    Fusion partner of echinoderm microtubule-associated protein-like 4 (EML4) is frequently found in non-small-cell lung cancer (NSCLC). The EML4-ALK fusion gene has various variants depending on the exon region of the EML4 gene, and the tests are required to establish a new strategy for the treatment and prevention of disease according to the genotype.
    
    Method:
    EML4-ALK gene was identified by PNA assisted one step RT-real-time PCR using fluorescent melting curve analysis. This method is run cDNA synthesis, target amplification at a time. Genotyping technology confirmed concordance using existing screening method and direct sequencing method. Genotyping Probe can detect accurately break point between EML4 gene and ALK gene. Test was performed using 12 of standard RNA and 2 of EML4-ALK gene cell line(H2228 and H3122).
    
    Result:
    We have developed a simple and rapid RT-real time PCR method for detecting EML4-ALk genes within four hours using PNA. This method is able to detect 12 of standard RNA with detection limit as low as 1x10^3 copies and in H2228 and H3122 cell line concentration confirmed 10ng, 1ng, 0.1ng.
    
    Conclusion:
    This method can be easily and quickly detected EML4-ALK genes and have a high sensitivity and accuracy for genotyping. Therefore, it can be accomplished preventing experimental error, thus generating significant cost savings reductions.