Baseline ctDNA profiling identifies molecular predictors of resistance to second-generation ALK inhibitors in EML4-ALK-positive NSCLC

Introduction: Lung cancer remains the leading cause of cancer-related death worldwide. Although tyrosine kinase inhibitors (TKIs) have improved outcomes for EML4-ALK-positive non-small cell lung cancer (NSCLC), most patients experience disease progression. Understanding the clinical and molecular factors that drive resistance is essential for guiding more effective treatment strategies. Aim: To identify molecular features associated with treatment resistance and survival in patients receiving first-line alectinib or brigatinib. Patients and Methods: Seventy-two patients with EML4-ALK-positive NSCLC treated with first-line alectinib or brigatinib were included. Plasma samples were obtained at baseline, during treatment, and at disease progression. Circulating tumor DNA (ctDNA) was analyzed using cancer personalized profiling by deep sequencing (CAPP-seq) and the R/Bioconductor package, DNAfusion , recently developed in our lab. ctDNA features at progression and at baseline were correlated to time to progression (TTP) and overall survival (OS). Results: During a median follow-up of 27 months, 54% (39/72) of patients developed disease progression. ALK on-target resistance mutations at progression were observed exclusively in short EML4-ALK variants (40% vs 0% in long variants, p<0.05). Patients with short variants also had numerically shorter TTP (14 vs 35 months, p=0.12) and OS (38 months vs NE, p=0.16). Baseline ctDNA profiles were analyzed to assess associations with clinical outcomes. Patients with a high plasma mutation load (>1 mutation) had significantly shorter TTP (13 vs 51 months, p<0.05) and OS (36 months vs NE, p<0.05) compared with those with a low load (0-1 mutation). In addition, TP53 mutations were strongly associated with poor outcomes (TTP: 10 vs 45 months, p<0.05; OS: 12 months vs NE, p<0.05). Conclusion: Development of ALK on-target resistance mutations at progression demonstrates a significant association with short EML4-ALK variants. A high mutation load and TP53 mutations at baseline identify patients at risk of early progression and poor survival under second-generation ALK TKI treatment. These findings suggest that ctDNA-based molecular profiling may help guide personalized treatment strategies and improve survival outcomes for EML4-ALK-positive NSCLC.