ctDNA features of acquired resistance to immunotherapy in advanced NSCLC.

2563 Background: Acquired resistance to systemic therapies, including immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKIs), is a major clinical challenge in advanced non–small cell lung cancer (NSCLC). While mechanisms of resistance to targeted therapies are well-documented, the genomic alterations associated with resistance to immunotherapy remain poorly understood. Circulating tumor DNA (ctDNA) profiling offers a non-invasive approach to identify real-time genomic changes driving resistance, providing novel insights into the underlying biology of immunotherapy failure. Methods: We performed a prospective ctDNA sequencing study in 57 advanced NSCLC patients from two cohorts: STING (n = 30, NCT04932525) and COPE (n = 27, NCT04258137). Plasma samples were collected before treatment initiation and at disease progression following objective response to anti–PD-1 therapy or TKIs. ctDNA analysis was conducted to detect emergent genomic alterations, evaluate tumor mutation burden (TMB), and quantify circulating tumor fraction (TF). Results: The study included 57 patients, with a median age of 62 years [IQR: 54.5–70], and 54.4% (31/57) were male. At disease progression, 64.9% (37/57) of patients exhibited emergent ctDNA alterations associated with secondary resistance, independent of therapy type. Among these, 70% (26/37) harbored multiple newly arising mutations. In the non–oncogene-addicted NSCLC cohort receiving anti–PD-1 therapy (n = 30), 56.6% (17/30) exhibited emergent resistance alterations, with 76.5% (13/17) harboring multiple mutations. Frequently observed aberrations included mutations in NOTCH1/3 (n = 3), KEAP1 (n = 3), KMT2B (n = 2), POLE (n = 2), SETD2 (n = 2), TYRO3 (n = 2), STK11 (n = 2), TSC2 (n = 1), TGFBR2 (n = 1), PTPN11 (n = 1), SPEN (n = 1), STAG (n = 1), CDH1 (n = 1), and CTNNB1 (n = 1). The median progression-free survival (mPFS) was 7.0 months [95% CI: 5.0–10.3]. In the anti-EGFR TKI cohort (n = 13), 84.6% (11/13) displayed emergent ctDNA alterations, including mutations in EGFR (n = 2), PIK3CA (n = 2), and MET (n = 1). The mPFS was 8.6 months [95% CI: 6.7–11.2]. TMB and circulating TF did not significantly change between baseline and progression in both cohorts. Conclusions: This study highlights the high prevalence of emergent genomic alterations detected by ctDNA in advanced NSCLC patients developing resistance to ICIs and TKIs. In the ICI-treated cohort, mutations in NOTCH1/3, KEAP1, and STK11 emerged as significant resistance drivers, consistent with their roles in immune evasion, oxidative stress regulation, and impaired immune cell infiltration, as supported by prior studies. Notably, this is the first study investigating features of ctDNA at acquired resistance to immunotherapy using an FDA-approved assay. These findings underscore the heterogeneous and polyclonal nature of resistance to ICIs.