P3.02-039 Acquired Resistance to EGFR-TKI in the Uncommon EGFR Mutation, G719S

Abstract

Acquired resistance (AR) to EGFR-TKI is a common event and several mechanisms including T790M, MET amplification, PTEN down regulation have been reported for the common EGFR mutations, Deletion 19 and L858R. An EGFR G719X mutation is an uncommon mutation and is known to show sensitivity to EGFR-TKIs in a series of clinical reports and in experiments using transformed cultured cells. However, there is neither established lung cancer cell line nor resistant cell line reported in the literature, which harbors the EGFR G719X mutation. Here we established a lung adenocarcinoma cell line (G719S-GR) from malignant pleural effusion of a patient whose tumor developed acquired resistance from initial gefitinib treatment. G719S-GR cells were established and maintained in RPMI1640 medium supplemented with 10%FBS and 10μM ROCK inhibitor (Y-27632, Wako). The ROCK inhibitor was removed from the medium for the following experiments. Cell growth inhibition was examined with gefitinib, afatinib and osimertinib using CellTiter-Glo (Promega), and a comprehensive genomic analysis was performed using hybrid capture based NGS (NCC oncopanel, Agilent; MiSeq, Illumina) for G719S-GR and MLPA (Salsa, MRC-holland) for clinical samples. Western blot analyzes were also performed to identify the mechanism of resistance to gefitinib. Cell growth inhibition test revealed EGFR-TKI resistance in G719S-GR cells with LC50 of approximately 20μM for either gefitinib or afatinib, and 2μM for osimertinib, indicating the G719S-GR cells are also resistant to EGFR-TKIs in vitro. From the NGS analysis, G719S-GR cells are proven to harbor EGFR mutations (G719S and E709A) as well as amplification of EGFR, IL7R, MYC and FGFR1 locus. Homozygous deletion of CDKN2A and loss of PTEN, TSC1 were also detected. In order to estimate the mechanism of EGFR-TKI resistance, copy number analyzes of several tumor suppressor genes were performed by MLPA using genomic DNA from G719S-GR and tumor biopsy sample (obtained before gefitinib treatment). Losses of CDKN2A, PTEN and TSC1 were confirmed in G719S-GR cells, whereas the loss of PTEN was not observed in gefitinib-naïve tumor sample. Finally, an AKT inhibitor, LY294002 could inhibit the AKT pathway, when it was combined with gefitinib. EGFR-TKI resistant mechanisms have not been investigated for uncommon mutations so far. The newly established G719S-GR cell line could be a useful tool for AR mechanism of the G719X mutation, and PTEN loss could be one of the AR mechanisms. Further experiments are warranted.