Abstract 3164: A comprehensive analysis of autopsied specimens and patient-derived cell lines in ALK-positive lung cancers with rapid acquired resistance to alectinib
Authors: Go Makimoto, Kadoaki Ohashi, Kazuya Nishii, Shuta Tomida, Hiroe Kayatani, Tomoki Tamura, Hisao Higo, Kiichiro Ninomiya, Takashi Ninomiya, Toshio Kubo, Eiki Ichihara, Akiko Sato, Katsuyuki Hotta, Masahiro Tabata, Katsuyuki Kiura
Journal: Cancer Research (2017)

Abstract Background Alectinib, a highly selective anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI), demonstrated a stunning disease control rate (95.7%) and unprecedented median progression-free survival (not reached: 95% CI: 20.3-) (Lancet Oncology 2013, ASCO 2016, Abstract #9008). However, acquired resistance to alectinib is inevitable; some ALK-positive lung tumors rapidly acquire alectinib resistance. Therefore, we sought to investigate the mechanism of the rapid resistance to alectinib using clinical samples and patient-derived ALK-positive cell lines. Materials and Methods Autopsied samples (primary lung tumors and metastatic tumors from the esophagus, liver, and bilateral kidney) were obtained from a 51-year-old male with advanced ALK-positive lung cancer. The lung tumors acquired resistance to first-line treatment of alectinib in three months, and subsequent treatment with ceritinib or crizotinib showed only a moderate and temporary effect. We established novel ALK-positive cell lines; ABC-14 was established from a pleural effusion after alectinib treatment, and ABC-17 was established from patient-derived xenograft (PDX) tumors of autopsied liver metastases. Western blot analysis, immunostaining, a receptor tyrosine kinase assay, fluorescence in situ hybridization (FISH), and next-generation sequencing were performed. Results ALK overexpression was confirmed in all autopsied samples by immunostaining. PCR revealed that ABC-14 harbored the EML-4/ALK fusion variant 3. An in vitro cell proliferation assay showed that ABC-14 exhibited resistance to alectinib (IC50 &amp;gt; 1 µM), but was sensitive to crizotinib (IC50 = 98 nM). The receptor tyrosine kinase assay revealed the activation of MET and EGFR in ABC-14. Quantitative RT-PCR and FISH confirmed MET gene amplification. Quantitative RT-PCR also indicated the overexpression of an EGFR ligand, EGF, TGF-α but no EGFR mRNA overexpression. The combination of crizotinib (dual ALK/MET-TKI) and an EGFR-TKI, gefitinib, showed an additive inhibitory effect on cell growth compared with each drug alone in vitro. ABC-17 showed resistance to both alectinib and crizotinib; consistently showed no activation of MET and no MET gene amplification. Interestingly, ABC-17 showed metastatic ability in the lung in NOG mouse PDX models. Conclusion The mechanism of rapid resistance to alectinib may be complicated and heterogeneous. Crizotinib combined with gefitinib, which inhibits the ALK, EGFR, and MET pathways, may represent one potent strategy against alectinib resistance. Further next-generation sequencing of clinically relevant samples should provide deeper insights into its resistance. (This work was supported by KAKEN 16K19454 and KAKEN 15H04830.) Citation Format: Go Makimoto, Kadoaki Ohashi, Kazuya Nishii, Shuta Tomida, Hiroe Kayatani, Tomoki Tamura, Hisao Higo, Kiichiro Ninomiya, Takashi Ninomiya, Toshio Kubo, Eiki Ichihara, Akiko Sato, Katsuyuki Hotta, Masahiro Tabata, Katsuyuki Kiura. A comprehensive analysis of autopsied specimens and patient-derived cell lines in ALK-positive lung cancers with rapid acquired resistance to alectinib [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 3164. doi:10.1158/1538-7445.AM2017-3164