Identification and characterization of preexisting resistant subclone in lung cancer with EGFR mutation sensitive to EGFR tyrosine kinase inhibitor

Abstract

EGFR tyrosine kinase inhibitor (EGFR-TKI) is the first molecularly targeted drug to change the paradigm in the management of lung cancer. These drugs are specifically effective in lung cancers with activating EGFR mutations. However, most cancer that initially responds to EGFR-TKI eventually acquires drug resistance. Several mechanisms are responsible for acquired resistance to EGFR-TKI, and the most common is the emergence of the T790M mutation in EGFR. This study aimed to evaluate whether the tumor cells carrying the T790M mutation exists before drug exposure and expands under the selective drug pressure. We collected pretreatment tumor tissues from 124 advanced non-small cell lung cancer patients with activating EGFR mutations that were detected by direct sequencing. Genotyping for T790M by matrix-assisted laser desorption/ionization-time of flight/mass spectrometry identified 31 (25.0%) tumors with pretreatment T790M. Furthermore, 68 cases which additionally underwent droplet digital PCR showed 27 (39.7%) tumors had pretreatment T790M mutation. We also observed clonal expansion of preexisting T790M clones during EGFR-TKI treatment both in in vivo model and in the paired tissue samples. In co-culture study mixing drug-sensitive cell and luciferase-tagging drug-resistant cell, we showed this growth advantage of resistant cell in regressing tumors was caused by dying sensitive cells. The T790M mutation frequency at which the risk of progression to EGFR-TKI begins to increase was estimated to be 3.2%. The patients with T790M-positive tumor had shorter time to progression (TTP) after EGFR-TKI (median 6.3 months vs. 11.5 months; P < 0.001) and overall survival (OS) (median 16.1 months vs. 26.5 months; P = 0.065) than those with T790M-negative tumor. Among the T790M-positive patients, the patients with high T790M frequency (n= 9) had shorter TTP (median 2.4 months vs. 6.7 months; P = 0.009) and OS (median 9.1 months vs. 18.7 months; P = 0.018) than those with low T790M frequency (n= 22). In conclusions, EGFR T790M mutation preexisted substantially in EGFR-mutant lung cancer. The T790M clones may expand during EGFR-TKI treatment by dying sensitive cell and finally be responsible for the patients’ resistance and progression. Thus, the patients with high T790M mutation frequency had worse clinical outcomes to EGFR-TKI.