Abstract 3260: Enhanced lineage plasticity in RTK-independent TKI-resistant EGFR-mutant NSCLC

Abstract Background: Resistance to targeted tyrosine kinase inhibitors (TKI) inevitably develops in metastatic EGFR-mutant non-small cell lung cancer (NSCLC). Resistance mechanisms are diverse, and mechanisms beyond receptor tyrosine kinase (RTK) pathway mutations are poorly understood. We hypothesized that the use of osimertinib as first-line therapy is increasing the prevalence of RTK-independent resistance mechanisms, and that RTK-independent resistant tumors undergo enhanced tumor cell lineage plasticity as an escape mechanism to EGFR TKI therapy. Methods: We identified patients who developed osimertinib resistance (OR) after first line (1L, n=54) and second line (2L, n=42) treatments and determined the resistance mechanisms based on clinical sequencing and histopathology. We also performed single-cell RNA-seq of 24 samples from 13 patients with EGFRm NSCLC at TKI treatment-naïve (TN, n=2), residual disease (RD, n=4), and progression disease (PD, n=7) stages. Results: Compared to 2L OR tumors, 1L OR tumors had increased RTK-independent mechanisms of resistance (76% vs. 46%, p=0.002), including 8% with small cell transformation (n=4), 2% with squamous transformation (n=1) and 66% with unknown mechanisms (n=34). To understand inter- and intra-tumor heterogeneity, we analyzed transcriptomic profiles of 76,266 single cells. Lung developmental lineages were assigned to 10,250 EpCAM+ cells, including 4,735 cells classified as malignant cells by inferCNV and RTK signaling analysis. In the two EGFRm TN tumors, the malignant cells demonstrated bronchoalveolar lineage and moderate EGFR expression. In the TKI resistant cases (PD, n=7), both RTK-dependent and RTK-independent resistance were observed. The RTK-dependent tumors (EGFR T790M n=1; ERBB2 amplification n=1) demonstrated preserved bronchoalveolar lineage identity. In the RTK-independent resistant tumors (n=5), one had complete lineage switch from epithelial to small cell neuroendocrine and very low expression level of EGFR. The remaining 4 PD tumors displayed varying expression of epithelial-to-mesenchymal transformation (EMT) features. One tumor had sarcomatoid histology and a high proportion of cells having positive VIM expression (84%) and 92% of cells having complete loss of NAPSA expression; 3 tumors had partial EMT demonstrated by heterogeneous proportion of cells having VIM expression (18-56%) and loss of NAPSA (26-67%). Interestingly, some of the cells with EMT and partial-EMT had moderate levels of EGFR expression, similar to the levels in the TN tumors. Conclusion: With osimertinib use at 1L, the incidence of RTK-independent resistance has increased to become the dominant mechanism, whereas RTK-dependent resistance has decreased. Increased lineage plasticity (small cell neuroendocrine, squamous and EMT) potentially serves as an RTK-independent TKI-resistance mechanism in EGFRm NSCLC. Citation Format: Xiuning Le, Ruiping Wang, Natalie Vokes, Yasir Elamin, Neda Kalhor, Daniel McGrail, Yuanxin Xi, Santiago Treviño III, Lingzhi Hong, Robyn Du, George Blumenschein, Carl Gay, Marcelo Negrao, Mehmet Altan, Hai Tran, Limei Hu, Jing Wang, Simon Heeke, Monique Nilsson, Jacqulyne Robichaux, Minghao Dang, Guangchun Han, Lauren Byers, Anne Tsao, Boris Sepesi, Chantale Bernatchez, Jianjun Zhang, Linghua Wang, John Heymach. Enhanced lineage plasticity in RTK-independent TKI-resistant EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3260.