The detection of primary and secondary EGFR mutations using droplet digital PCR in patients with nonsmall cell lung cancer

BACKGROUND: We share our experience of using droplet digital polymerase chain reaction (DdPCR) in liquid biopsy specimens for detecting primary and secondary epidermal growth factor receptor (EGFR) mutations among patients with nonsmall-cell lung cancer who had tissue biopsy initially analyzed for del19, L858R and T790M. MATERIALS AND METHODS: Three groups of patients were chosen: Group 1: patients positive for EGFR mutation (del 19 or L858R) by conventional tissue biopsy that were treatment naïve, Group 2: patients positive for EGFR mutation (del 19 or L858R) by conventional tissue biopsy with acquired resistance to tyrosine kinase inhibitor (TKI) therapy, documented by radiology, and Group 3: no known EGFR mutation detected on primary tissue biopsy and treatment naive. RESULTS: One hundred and thirty-three patients were included in the study. Group 1 had 40 cases, of which 21 (52.5%) and 19 (47.5%) were positive for del19 and L858R mutations, respectively, by tissue biopsy. DdPCR detected primary mutation in all but 5 cases. DdPCR additionally found four patients to have T790M mutation. Group 2 had 73 cases and DdPCR detected T790M mutation in 39 (53.4%) cases. Liquid biopsy also picked the original primary mutation in 56/73 cases. Secondary tissue biopsy for T790M mutation status was performed in 11 patients and while it detected mutation in 2 out of 11 cases, DdPCR detected the same in 7 cases, thus providing significantly superior yield (46% difference, McNemar's test, P value 0.063). Tissue biopsy additionally detected c-MET amplification in a patient who had T790M mutation on liquid biopsy. Group 3 had 20 patients and none were falsely positive for EGFR mutation on liquid biopsy. Overall, DdPCR had a Cohen's kappa of 0.82 (standard error 0.074, 95% CI 0.68–0.97) indicating “very good agreement” with conventional tissue biopsy. CONCLUSION: DdPCR demonstrated 87.5% sensitivity and 100% specificity in detecting primary EGFR mutations in patients who were treatment naïve with overall positive and negative predictive value of 100% and 80%, respectively. DdPCR demonstrated T790M mutation postprogression on TKI therapy in 53.4% patients.