The impact of intermittent versus continuous exposure to EGFR tyrosine kinase inhibitor on selection of EGFR T790M-mutant drug-resistant clones in a lung cancer cell line carrying activating EGFR mutation

Drug-resistant cell lines are essential tools for investigating the mechanisms of resistance to molecular-targeted anti-cancer drugs. However, little is known about how to establish clinically relevant drug-resistant cell lines. Our study examined the impact of a drug-free period on the establishment of a cell line with clinically relevant resistance to molecular-targeted drugs. We used PC9 cells, a lung cancer cell line carrying EGFR mutation, because this is a validated target for EGFR tyrosine kinase inhibitors (TKI). PC9 cells were intermittently or continuously exposed to increasing concentrations of gefitinib (0.01 mu M to 1.0 mu M) and the emergence of the most common acquired resistance mutation in EGFR, T790M, was determined. T790M was detected at a 25-fold lower drug concentration in cells continuously exposed to gefitinib (PC9/GRc) than in cells intermittently exposed to gefitinib (PC9/GRi) (0.04 mu M vs 1.0 mu M, respectively). The mutation frequencies at those drug concentrations were 19.8% and 8.0% in PC9/GRc and PC9/GRi cells, respectively. After drug-free culture for 8 weeks, resistance to gefitinib decreased in the PC9/GRi cells but not in the PC9/GRc cells. In the PC9/GRc cells, the frequency of the T790M mutation was consistently about 20% from 0.04 mu M to 1.0 mu M of gefitinib. In the PC9/GRc cells, the T790M mutation was detected in all single-cell clones, at frequencies ranging from 7.0% to 37.0%, with a median of 19.5% (95% confidence interval, 17.3%-20.9%). In conclusion, compared with intermittent drug exposure, continuous exposure might select better minor drug-resistant clones when creating cell lines resistant to molecular-targeted drugs.