Author of

• 15382

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  P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)

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

  • 15446

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    P3.04-064 - A 24h-Single Highthroughput Assay to Identify ALK or ROS-1 Gene Fusions and EGFR Mutations in DNA from FFPE Tumor Samples or Free Circulating DNA (ID 2811)

    09:30 - 09:30  |  Author(s): P. Innominato
    • Abstract

    Background:
    The diagnosis of metastatic lung adenocarcinoma to decide tyrosine kinase inhibitors (TKI) targeting either EGFR mutations or ALK or ROS translocations requires the combination of several techniques and different biological or pathological expertises. These are DNA sequence analysis, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) that are performed independently and require time and DNA materials. Importantly, to our knowledge no diagnostic can be performed on extracted DNA from FFPE tumors for the identification of ALK or ROS translocations except FISH. FISH is considered as the gold standard technique for gene translocations but time-consumable and not applicable to highthoughput diagnosis. Some unsuccessful attempts have been made using RNA extracted from FFPE.
    
    Methods:
    We have developed and patented an assay using the i-plex technology and mass spectrometry detection (Sequenom-Agena Bioscience, CA, USA) allowing the concomitant identification of 20 targeted EGFR exon 18-21 gene sequence abnormalities as well as variants of EML4-ALK (variants 1-2-3a-3b) or ROS1-SLC34A2/EZR/CD74 gene fusions on extracted DNA samples in a single 24h experiment. DNA has been extracted eitherfrom either FFPE tumor samples or plasma free circulating DNA.
    
    Results:
    DNA samples from 6 different patient can be analyzed on the same 96 wells-assay (more if a 384 well-assay). As low as 16ng DNA per sample from FFPE biopsies (10 slices) or plama can be used. We have applied this new panel to a cohort of 90 lung adenocarcinoma samples positive for EGFR mutations (n=30), ALK (n=30) or ROS (n=30) translocations; one third being extracted DNA from circulating plasma samples. The limit of detection of the assay is as low as 1 to 5% depending on the gene abnormality. When compared to IHC (EML4-ALK 5A4 clone; ROS1 D4D6 clone, Cell Signalling) / FISH techniques (Vysis LSI ALK Break Apart Rearrangement Probe Kit, Abbott; ROS1 Split FISH Probe, Abnova), the specificity of the identification of ALK or ROS gene rearrangements is 97%.
    
    Conclusion:
    In conclusion, we have developed a promising and performant assay based on an innovative methodology that we have patented for the identification in a single experiment of both gene mutations and gene translocations using very low amounts of DNA extracted from FFPE tumor biopsies or plasma samples.