Abstract 7036: EcDNA-mediated oncogene amplification underlies EGFR TKI resistance in NSCLC.

Abstract Extrachromosomal DNA (ecDNA) drives oncogene amplification and therapeutic resistance in multiple cancers, but its contribution to acquired resistance to EGFR inhibitors (EGFRi) in EGFR-mutant non-small cell lung cancer (NSCLC) is not well defined. While EGFR TKI resistance frequently arises through acquisition of the EGFR T790M mutation, many cases remain unexplained. We therefore aimed to determine whether ecDNA represents an additional mechanism associated with EGFR TKI resistance. We analyzed whole-genome sequencing datasets from TCGA, PCAWG, and the Hartwig Medical Foundation, encompassing 536 NSCLC tumors, and detected ecDNA structures using AmpliconArchitect. Across these public cohorts, ecDNA prevalence was markedly elevated in EGFR TKI-treated tumors (50%, 10/20) compared with primary tumors (14%, 20/147; OR≈6.35, p0.001) and advanced untreated tumors (25%, 92/369; OR≈3.01, p=0.02), indicating a significant association between EGFR TKI exposure and increased ecDNA prevalence. Notably, all ecDNAs detected in the EGFR TKI-treated group harbored oncogenic drivers, underscoring their potential functional relevance. To investigate whether this enrichment reflects resistance-associated events, we established an isogenic PC9 resistance model by generating WGS and RNA-seq data from resistant subclones derived from a single EGFR TKI-sensitive clone following long-term erlotinib exposure. Analysis of 25 resistant PC9 samples revealed ecDNA acquisition in approximately 20% of clones (5/25), all of which contained oncogenes. These ecDNA-positive clones were mutually exclusive with EGFR T790M-positive samples (also 20%, 5/25), suggesting that ecDNA emergence represents an alternative resistance route rather than a secondary event downstream of T790M. Functional analyses demonstrated activation of EGFR downstream signaling pathways in ecDNA-positive resistant cells. For example, in one resistant subclone harboring a newly formed RAF1 ecDNA (copy number ∼24), we observed over 100-fold RAF1 overexpression and marked MAPK/ERK pathway activation, as supported by GSEA (NES = 1.39, p 0.01). Functional RAF1-inhibition experiments further confirmed that suppressing RAF1-driven signaling restored erlotinib sensitivity, providing strong evidence that ecDNA-mediated RAF1 amplification leads to EGFR-independent MAPK pathway activation. EcDNA may promote the development of EGFR TKI resistance by enabling alternative signaling pathways that bypass EGFR activity. EcDNA-mediated oncogene amplification represents an important feature of EGFR TKI-resistant NSCLC and a potential therapeutic vulnerability, providing a foundation for strategies aimed at overcoming ecDNA-driven resistance. Citation Format: Boyoon Kim, Sujin Kim, Jeonghee Cho, Hoon Kim. EcDNA-mediated oncogene amplification underlies EGFR TKI resistance in NSCLC abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7036.