Background: Acquired resistance (AR) to EGFR-TKI is a common event and several underlying mechanisms, including T790M, MET amplification and PTEN downregulation have been reported for the common EGFR mutations: Deletion 19 and L858R. An EGFR G719X mutation is an uncommon mutation that was reported to show sensitivity to EGFR-TKIs in a series of clinical reports and experiments using transformed cultured cells. However, no established lung cancer or resistant cell lines harboring the EGFR G719X mutation have been reported in the literature. We established a lung adenocarcinoma cell line (G719S-GR) from the malignant pleural effusion of a patient whose tumor developed acquired resistance from initial treatment with gefitinib.

Materials and methods: G719S-GR cells were established and maintained in RPMI1640 medium supplemented with 10%FBS and 10 μM ROCK inhibitor (Y-27632, Wako). The ROCK inhibitor was removed from the medium for the following experiments. Cell growth inhibition was examined with gefitinib and afatinib 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) was used for the analysis of clinical tumor samples.

Results: A cell growth inhibition test revealed EGFR-TKI resistance in G719S-GR cells with an LC50 of more than 100 μM for either gefitinib or afatinib, indicating that the G719S-GR cells are also resistant to EGFR-TKI in vitro. The NGS analysis showed that G719S-GR cells harbor EGFR mutations (G719S and E709A) as well as the amplification of EGFR, IL7R, MYC and the FGFR1 locus. The homozygous deletion of CDKN2A and the loss of PTEN and TSC1 were also detected. In order to estimate the mechanism underlying the development of EGFR-TKI resistance, copy number analyses of several tumor suppressor genes were performed by an MLPA using genomic DNA from G719S-GR and a tumor biopsy sample (obtained before gefitinib treatment). Losses of CDKN2A, PTEN and TSC1 were confirmed in G719S-GR cells. Interestingly, the loss of PTEN was not observed in the gefitinib-naïve tumor sample.

Discussion: Thus far, the mechanisms underlying the development of EGFR-TKI resistance in uncommon mutations have not been investigated. The newly established G719S-GR cell line could be a useful tool for investigating the mechanism underlying the development of AR in the G719X mutation; the loss PTEN could be one such mechanism. Further experiments are warranted.

Citation Format: Atsushi Osoegawa, Tomonori Yamada, Takafumi Hashimoto, Yohei Takumi, Ryoji Kobayashi, Shuji Suehiro, Michiyo Miyawaki, Hideya Takeuchi, Tatsuro Okamoto, Kenji Sugio. Acquired resistance to EGFR-TKI in an uncommon G719S EGFR mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4107. doi:10.1158/1538-7445.AM2017-4107