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C. Paweletz

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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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      ORAL38.01 - A Prospective Study of Rapid Plasma Genotyping Utilizing Sequential ddPCR and NGS in Newly Diagnosed Advanced NSCLC Patients (ID 935)

      16:45 - 18:15  |  Author(s): C. Paweletz

      • Abstract
      • Presentation
      • Slides

      Plasma genotyping of cell-free DNA (cfDNA) has the potential to allow for noninvasive genotyping while avoiding the inherent shortcomings of tissue genotyping and repeat biopsies. We have developed a quantitative droplet digital PCR (ddPCR)-based plasma genotyping assay capable of detecting common EGFR and KRAS mutations in NSCLC (Oxnard et al., CCR 2014). Although rapid and highly specific, this assay lacks the ability to both multiplex and detect complex genomic alterations such as rearrangements. In this prospective study, we evaluate the test characteristics of ddPCR combined with plasma next-generation gene sequencing (NGS) as a new paradigm for plasma genotyping.

      Patients with newly diagnosed advanced NSCLC were eligible. All patients were required to have a biopsy available or planned for tissue genotyping which was used for gold standard comparison. Patients underwent an initial blood draw and immediate plasma ddPCR for EGFR exon 19 del/L858R and KRAS G12X. A subset of patients additionally underwent plasma NGS using a unique probe set designed by our group to detect rearrangements and mutations in 12 genes (EGFR, KRAS, ALK, ROS1, BRAF, RET, NRAS, ERBB2, MET, MEK1, PIK3CA and p53). This plasma NGS assay utilized a novel bias corrected NGS which minimizes off-target reads (Resolution Bio) performed on a desktop MiSeq platform. Test turnaround time (TAT) was measured in business days from date of blood draw until test reporting.

      120 patients with newly diagnosed advanced NSCLC have been enrolled and 94 have completed tissue and plasma genotyping. Tumor genotype included 25 EGFR exon 19/L858R mutants, 17 KRAS G12X mutants, 24 rare genotypes and 15 others. Median TAT for plasma ddPCR was 3 days (range 1-5). Specificity of plasma ddPCR was 99% for EGFR exon 19 del/L858R (68/69) and 100% for KRAS (77/77). Sensitivity of plasma ddPCR was 76% for EGFR exon 19 del/L858R (19/25) and 71% for KRAS (12/17). Plasma NGS is ongoing with testing completed on 11 patients with a known tumor genotype. 8 had a genotype detected on plasma NGS: 2 ALK rearrangements, 1 ROS1 rearrangement, 1 RET rearrangement, an EGFR G719A mutation, a KRAS G12C and a combined KRAS G12C/PIK3CA mutation - all matched the tumor genotype. Preliminary plasma NGS turnaround time ranged from 5-10 business days.

      Rapid plasma genotyping using sequential plasma ddPCR (1-5 day TAT) followed by plasma NGS (5-10 day TAT) represents a new paradigm for noninvasive plasma genotyping. This approach capitalizes on the use of rapid ddPCR for common targetable mutations and the ability of plasma NGS using an augmented MiSeq platform to multiplex and detect complex alterations. This new model for plasma genotyping uses testing platforms that can readily be employed in most molecular pathology laboratories allowing for widespread adoption.

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