Double EGFR mutants containing rare EGFR mutant types show reduced response to in-vitro gefitinib compared to common activating missense mutations

Abstract

Epidermal growth factor receptor (EGFR) mutations are common in lung adenocarcinomas (AD) especially from non-smoking women of Asian descent. Tumors with EGFR mutations respond to tyrosine kinase inhibitor (TKI) treatment with improved patient survival and/or reduction of tumor size. We have previously shown that EGFR mutations occur in >70% of lung cancers from non-smokers with a complex mutational profile that includes 13% of EGFR double mutations in our population. In this study, we investigated the in vitro TKI response patterns of four novel EGFR double mutants previously identified in untreated patients with AD, including Q787R+L858R, E709A+G719C, T790M+L858R and H870R+L858R. For the first three pairs, previous cloning studies had shown that the component mutants were in-cis to each other. The respective single mutants were transfected into the H358 human lung cancer cell line with low endogenous EGFR expression and subjected to increasing dosages of the TKI gefitinib. The tyrosine phosphorylation profiles of EGFR and AKT, STAT3/5 and ERK1/2 downstream effectors were compared by immunoblot analyses to those of the corresponding double mutant transfectants. Results demonstrated that the mutants responded to in-vitro TKI treatment with different sensitivities. The G719C and L858R single mutants showed ligand-independent activation and the highest gefitinib sensitivity compared to the corresponding coexisting single mutants E709A, Q787R, H870R and T790M. The double mutants E709A+G719C, Q787R+L858R and H870R+L858R showed attenuated responses to gefitinib in both the EGFR and downstream effectors phosphorylation profiles compared to G719C or L858R alone. T790M+L858R showed strong resistance to gefitinib. Clinically, the patient whose tumor contained H870R+L858R showed tumor stabilization by daily treatment of 250mg gefitinib but leptomeningeal tumor metastasis developed 6 months later. Correlation with the in-vitro phosphorylation profile of H870R+L858R suggested that treatment failure was probably due to drug resistance at the level usually attainable in the cerebral spinal fluid by the given oral gefitinib dosage. Overall, the findings suggested that rare types of EGFR substitution mutations could induce relative gefitinib resistance compared to the common activating mutants alone. The response of EGFR double mutants cannot be predicted from the response of the individual single mutant partners. Information of the mutation types and signaling changes are important factors for designing treatment protocols for lung cancers with EGFR mutations.