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  P3.06 - Poster Session 3 - Prognostic and Predictive Biomarkers (ID 178)

  • Event: WCLC 2013
  • Type: Poster Session
  • Track: Biology
  • Presentations: 1
  • Moderators:
  • Coordinates: 10/30/2013, 09:30 - 16:30, Exhibit Hall, Ground Level

  • 12494

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    P3.06-054 - Development of a clinical-grade quantitative assay for non-invasive measurement of tumor genotype in cell-free plasma DNA (cfDNA) using next-generation quantitative genotyping (ID 741)

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

    Background
    Non-invasive genotyping of cfDNA has been shown to be feasible using highly sensitive assays. However, for detection of uncommon genomic events, specificity must approach 100% or false positive results impair clinical utility. Digital droplet PCR (ddPCR) is a quantitative genotyping technology that emulsifies input DNA into ~20,000 droplets which are PCR amplified, fluorescently labeled, and read as mutant or wildtype in a droplet flow cytometer. Using this quantitative technology, we aimed to develop a clinical-grade assay for non-invasive plasma genotyping and serial disease monitoring.

    Methods
    Patients with advanced NSCLC known to harbor EGFR or KRAS mutations were studied in an IRB-approved fashion. Plasma was collected in 10cc EDTA-tubes. Extracted DNA was quantified with a PCR for LINE1 and genotyped using ddPCR. Specificity of EGFR genotyping was determined using patients with KRAS-mutant lung cancer as gold standard negative cases. Serial assessment was piloted on EGFR-mutant cases receiving first-line erlotinib.

    Results
    To minimize risk of false positive results, we identified the “normal range” for EGFR L858R and exon 19 deletions in specimens from KRAS-mutant lung cancers as 0-1 and 0-8 copies/mL of plasma, respectively. Using this threshold for positive, ddPCR for EGFR sensitizing mutations had 67% sensitivity and 100% positive predictive value (Figure 1). Sensitivity was 100% with LINE-1 levels between 60-60000 pg/mcL but was poor with higher or lower cfDNA concentrations. Serial assessment on erlotinib (Figure 2) demonstrated pretreatment detection of EGFR mutations with ddPCR, complete plasma response on erlotinib, and subsequent reemergence of plasma EGFR up to 16 weeks prior to objective progression. Figure 1 Figure 2

    Conclusion
    Plasma genotyping of cfDNA using ddPCR has 100% specificity when using a rigorously defined threshold for a positive result. Sensitivity is highest in specimens with optimal cfDNA concentration. Clinical development is underway to use this non-invasive assay to guide genotype-directed therapy.