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D. Kuo

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    ORAL 38 - Liquid Biopsies (ID 147)

    • Event: WCLC 2015
    • Type: Oral Session
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      ORAL38.03 - Assessing the Feasibility of Detecting ALK Fusions with qRT-PCR Assays in Cell-Free Plasma RNA (ID 1437)

      16:45 - 18:15  |  Author(s): D. Kuo

      • Abstract
      • Presentation
      • Slides

      Chromosomal rearrangements that result in transcript fusions have been a focus of attention in cancer as they provide attractive therapeutic targets. Identifying tumors that harbor chromosomal rearrangements by in situ hybridization assays has been a challenge in the clinic because these assays demand large quantities of tissue specimens. Cell-free nucleic acids from patient plasma may provide a non-invasive, alternative tool for detecting transcript fusions. Here, we demonstrate the feasibility of detecting ALK fusions with a qRT-PCR assay using cell-free plasma RNA (cfRNA).

      We designed a one-tube, four-channel multiplex ALK qRT-PCR assay that incorporates two strategies to detect ALK fusions. One channel employs variant-specific primers to detect >90% of the reported ALK fusions. The remaining three channels measure the expression of the 5’ and 3’ ends of the ALK gene relative to an internal reference and to each other, in theory, permitting the detection of all ALK fusions including those without knowledge of the fusion partner. To assess the performance of the multiplex ALK prototype assay, we made contrived samples blending mutant and wildtype cell line RNAs. In addition, we tested the multiplex ALK assay on NSCLC FFPET specimens (n=209). Moreover, to mimic plasma cfRNA, we made contrived samples by blending mutant cell line conditioned media with normal plasma.

      Data from the cell line RNA blends demonstrate that both the variant-specific and the 5’ and 3’ differential expression successfully detect the EML4-ALK fusion-positive RNA. The variant-specific component of the assay is sensitive enough to detect at least 25pg of fusion-positive cell line RNA at a 1:4000 dilution with wildtype cell line RNA. From the NSCLC FFPET specimens, we identified 4 samples positive for the ALK fusion. These results were validated by a reference method that uses anchored-PCR to enrich ALK targets followed by NGS that employs a novel algorithm to identify potential fusion products. In addition, the multiplex ALK qRT-PCR assay detected transcript fusions in blends composed of plasma and EML4-ALK positive conditioned media at a dilution of 3:1. Lastly, we tested the multiplex ALK assay on confirmed ALK-fusion positive NSCLC plasma specimens, and were able to detect ALK fusions (7 out of 8) from as little as 750 ul of plasma.

      In summary, we have developed a one-tube, multiplex ALK qRT-PCR assay that exhibits performance characteristics suitable for transcript fusion detection in plasma cfRNA. Efforts are underway to further test and optomize the performance of this assay in clinical samples and to apply this multiplex qRT-PCR design concept to other transcript fusions including RET and ROS1.

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