Gefitinib resistance caused by a secondary mutation of the epidermal growth factor receptor

2637 Mutations of the epidermal growth factor receptor gene conferring sensitivity to anilinoquinazoline EGFR inhibitors have recently been identified. Despite the typical dramatic tumor responses to such inhibitors, patients will eventually relapse with time and the mechanism of relapse has not been identified. A patient with advanced lung adenocarcinoma with a del747-752 EGFR mutation treated with gefitinib had a durable remission, but relapsed after 24 months of treatment. The DNA sequencing of exons 18-21 of his original specimen as well as his biopsy on relapse demonstrated persistence of the 747-752 deletion. The DNA sequence of his tumor biopsy from his relapse revealed a new, secondary C-to-T base pair change leading to a Thr-Met amino acid substitution at position 790. This amino acid change corresponds to one of the common bcr-abl kinase mutations conferring STI-571 resistance. Structural modeling suggested that this change would lead to the introduction of a bulkier amino acid side chain into a hydrophobic pocket of the ATP-binding site of the EGFR molecule. Further modeling also suggested that this substitution would lead to likely steric hindrance of either erlotinib or gefitinib binding due to the juxtaposition of the methionine side chain and the acetylene or chloride side chain of the anilino group of the respective inhibitor. Plasmid constructs were generated introducing this amino acid change into wild-type, L858R mutant, del747-753insS as well as del747-752 constructs. In transient transfection experiments, equal expression as well as phosphoprotein generation upon EGF stimulation between all the corresponding construct pairs (primary versus resistance mutant) was seen. While gefitinib completely inhibited EGF-stimulated phospho-EGFR generation of the non-resistance mutant constructs at concentrations of 2-20 nM, all four constructs carrying the Thr-Met substitution showed high level resistance to gefitinib with sustained EGFR phosphorylation at concentrations as high as 2 μM. A screen of commercially available EGFR inhibitors demonstrated that one of the screened inhibitors, CL-387,785, inhibited both the del747-752 as well as the del747-752 plus resistance mutant constructs with nearly the same potency with full inhibition observed at a concentration as low as 30nM. CL-387,785 is an irreversible, anilinoquinazoline inhibitor of EGFR. In summary, we have identified a novel, in vivo occurring mutation of the EGFR conferring gefitinib resistance and thereby describe the first resistance mutation in an oncogenic tyrosine kinase in a common epithelial cancer. We also demonstrate an alternative compound which can inhibit this mutant receptor, suggesting that drugs of this class might be useful in patients who relapse while on treatment with gefitinib.