Author of

• 14490

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  P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)

  • Event: WCLC 2015
  • Type: Poster
  • Track: Biology, Pathology, and Molecular Testing
  • Presentations: 1
  • Moderators:
  • Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)

  • 14512

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    P2.04-022 - Development of Microfluidic Devices for Rapid, Low-Cost Detection of EGFR Mutations in Cytological Samples from Patients with Lung Cancer (ID 999)

    09:30 - 09:30  |  Author(s): M. Tokeshi
    • Abstract

    Background:
    Epidermal Growth Factor Receptor (EGFR) mutation testing plays an important role in selecting patients for targeted therapy with EGFR-tyrosine kinase inhibitors (TKIs). However, a currently available PCR-based sequencing is time-consuming and expensive. In order to overcome these problems, we have developed microfluidic devices which enable rapid and specific detection of mutant EGFR proteins at low-cost in cytological samples from patients with non-small cell lung cancer (NSCLC).
    
    Methods:
    The diagnostic device consisted of the capture antibody against EGFR and photo-reactive polymer. The antibody-immobilized photo-reactive polymer wall (40 μm width, 40 μm height and 4 mm length) was constructed at the center of a microchannel (1 mm width, 40 μm height and 8.5 mm length) by ultraviolet light irradiation. The substrate was made of cyclic olefin polymer by using injection molding. The inner wall of the microchannel was blocked with bovine serum albumin. By using the diagnosis devices, the sandwich-type fluorescence immunoassay procedure was conducted. The sample, detection antibody reagent (mutation specific monoclonal antibody against EGFR with the E746_A750 deletion in exon 19 or the L858R point mutation and control EGFR antibody), and fluorescence-labeled anti-IgG antibody reagent were injected in turn. Between each injection, we performed a washing procedure, in which the microchannel was filled with the washing buffer for 1 minute followed by flushing with 5 μL of the same washing buffer. The amount of the sample and reagents to fill the microchannel was 1 μL. Incubation times were 15 minutes, 30 seconds, and 30 seconds for capture antibody-antigen reaction, antigen-detection antibody reaction, and detection antibody-fluorescence-labeled antibody reaction, respectively. After the immunoassay, fluorescence images were captured by using a digital CCD camera. Malignant pleural effusions (MPEs) were obtained from patients with NSCLC with written informed consent. After centrifugation, the cell pellets of MPEs were lysed in 200μl of lysis buffer.
    
    Results:
    First, we performed a pilot study by using cell lysates of 3 lung cancer lines expressing wild-type (H358) or E746_A750del mutant (HCC827) or L858R mutant (H3255) EGFR. Using our newly developed diagnostic device, we were able to specifically distinguish EGFR mutant proteins from that of wild-type EGFR in all of these cell lines. Next, we tested the device for detecting EGFR mutations in cytological samples of MPEs. Results of the mutation testing of the lysates using this device were consistent with those obtained by commercially available techniques in Japan although the number of samples assessed in this experiment was limited. The cost was less than a few dollars per assay.
    
    Conclusion:
    These results suggest that our device may be possible candidates for the next generation companion diagnostics devices for EGFR-TKI. Further investigation will be needed to elucidate the most appropriate detection method of EGFR mutation as a companion diagnostics.