Molecular landscape of osimertinib resistance revealed by targeted panel sequencing and patient-derived cancer models in non-small cell lung cancer patients

Background: Recent studies demonstrated profound clinical activity of osimertinib in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) patients. However, the emergence of resistance limits the clinical benefit inevitably, demanding dissection of its underlying mechanisms. Here, we provide the molecular landscape of osimertinib resistance investigated by clinical sample sequencing and patient-derived cancer models. Methods: The paired tumor tissue (n = 10) and plasma samples (n = 8) were collected from 12 EGFR-mutant NSCLC patients before and after osimertinib treatment in the ASTRIS trial (NCT02474355). The tissue and plasma DNAs were analyzed by targeted next-generation sequencing (NGS) of 112 cancer-related genes (AZ100 panel). The patient-derived cancer cell lines (PDC) and tumor xenografts (PDTX) were established from osimertinib-treated patients. Osimertinib resistant cell line (PC9-GR/AR) was also established by chronic drug administration. The preclinical resistance models were investigated by whole-exome sequencing (WES) and RNA-seq. Results: The paired baseline and progression sample analysis identified the emergence of acquired mutations, EGFR C797S (n = 1), KRAS G12D (n = 1), and PIK3CA E545K mutations (n = 2), in the progression samples. One of these progression tissue samples with an acquired PIK3CA mutation also lost EGFR. A preexisting KRAS G12D mutation and PTEN loss were also identified in two patients who showed primary resistance to osimertinib. The WES of osimertinib-resistant PDC revealed amplification of GLI1, CDK4, and CCND1. The osimertinib-resistant PDTXs harboring PIK3CA H1047R mutation and MET amplification were established, and PI3K inhibitors and MET inhibitors will be tested. The RNA-seq analysis showed upregulation of epithelial-mesenchymal transition signatures in PC9-GR/AR cells compared to PC9-GR cells that are potentially related to epigenetic resistance mechanisms. Conclusions: Our multi-layered molecular analysis of osimertinib-resistant patients' clinical samples and patient-derived cancer models demonstrates a diverse spectrum of osimertinib resistance mechanisms. Legal entity responsible for the study: Byoung Chul Cho. Funding: AstraZeneca. Disclosure: J.C. Barrett, D. Stetson, J. Chmielecki, A. Markovets: Employee: AstraZeneca. All other authors have declared no conflicts of interest.