A Noninvasive System for Monitoring Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors with Plasma DNA

Introduction: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are widely used to treat lung adenocarcinomas with EGFR-activating mutations. However, half of the patients acquire resistance because of the gatekeeper T790M mutation. Noninvasive mutation detection system is desired considering the difficulty in obtaining tissue specimens during disease progression. Methods: Sixty-seven plasma DNA samples from 49 patients with lung adenocarcinoma and 30 healthy volunteers were evaluated. T790M in plasma DNA was determined using the mutation-biased polymerase chain reaction (PCR) quenching probe (MBP-QP) method. The method combines MBP and genotyping, the latter based on analysis of the melting curve of the probe DNA binding the target mutated site using a fluorescence QP system. Results: The detection limit was two copies of control plasmid and 0.2 ng of genomic DNA. The mutant plasmid could be detected when it accounted for as little as 0.3% of a mixture of plasmids carrying EGFR exon 20 with or without T790M. The T790M mutation was detected in plasma DNA from 10 of 19 patients (53%) who acquired resistance, but not in nonresponders, patients responding to treatment, or those not treated with EGFR tyrosine kinase inhibitor. Other mutation detection systems, such as the nucleic acid-locked nucleic acid PCR clamp, the cycleave PCR technique, and allele-specific oligonucleotide PCR, detected T790M in three, four, and six patients, respectively, among 10 in which T790M was detected by the MBP-QP method. Conclusions: The MBP-QP method is simple, sensitive, and-intriguingly-reflective of clinical course, compared with the other three mutation-detection systems. Thus, the MBP-QP method is an ideal noninvasive monitoring system for detecting T790M in plasma samples.