Predictive Biomarkers and Personalized Medicine NoninvasiveDetection of Response andResistance inEGFR- Mutant Lung Cancer Using Quantitative Next-Generation Genotyping of Cell-Free Plasma DNA

Purpose: Tumor genotyping using cell-free plasmaDNA (cfDNA) has the potential to allow noninvasive assessment of tumor biology, yet many existing assays are cumbersome and vulnerable to false-positive results. We sought to determine whether droplet digital PCR (ddPCR) of cfDNAwould allow highly specific and quantitative assessment of tumor genotype. ExperimentalDesign:ddPCRassays for EGFR,KRAS, andBRAFmutationswere developed using plasma collected frompatientswith advanced lung cancer ormelanomaof a known tumor genotype. Sensitivity and specificity were determined using cancers with nonoverlapping genotypes as positive and negative controls. Serial assessment of response and resistance was studied in patients with EGFR-mutant lung cancer on a prospective trial of erlotinib. Results:We identified a reference range for EGFR L858R and exon 19 deletions in specimens from KRASmutant lung cancer, allowing identification of candidate thresholds with high sensitivity and 100% specificity. Received operative characteristic curve analysis of four assays demonstrated an area under the curve in the range of 0.80 to 0.94. Sensitivity improved in specimens with optimal cfDNA concentrations. Serial plasma genotyping of EGFR-mutant lung cancer on erlotinib demonstrated pretreatment detection of EGFRmutations, complete plasma response in most cases, and increasing levels of EGFR T790M emerging before objective progression. Conclusions: Noninvasive genotyping of cfDNA using ddPCR demonstrates assay qualities that could allow effective translation into a clinical diagnostic. Serial quantification of plasma genotype allows noninvasive assessment of response and resistance, including detection of resistance mutations up to 16 weeks before radiographic progression. Clin Cancer Res; 20(6); 1698–705. 2014 AACR.