Introduction: By implementing dynamic circulating tumor DNA (ctDNA) analysis, we explored the impact of TP53 mutations on tumor evolution and resistance mechanisms to ensartinib in patients with ALK-positive NSCLC. Methods: In a multicenter phase 2 trial, patients with ALK-positive NSCLC who progressed on crizotinib were treated with ensartinib. Blood samples for ctDNA analysis were collected at baseline, cycle 3 day 1, and progression disease (PD) and analyzed with a 212-gene panel. Results: A total of 440 samples were collected from 168 patients. Baseline TP53 mutations (20.2%) significantly correlated with inferior progression-free survival (4.2 mo versus 11.7 mo, p < 0.0001). Patients with TP53 mutations had higher mutation load than those without TP53 mutations at baseline (13.79 +/- 3.72 versus 4.67 +/- 0.39, p < 0.001). Although there was no significant difference in mutation load between these groups at cycle 3 day 1 (5.89 +/- 2.25 versus 3.72 +/- 0.62, p = 0.425), patients with mutated TP53 developed more mutations at PD (7.07 +/- 1.25 versus 3.20 +/- 0.33, p = 0.003). Frequency and abundance of secondary ALK mutations G1269A, G1202R, and E1210K increased markedly at PD than baseline. In patients without secondary ALK mutations, we identified ALK-independent resistance mechanisms including bypass signaling activation, downstream effector protein reactivation, epithelial-mesenchymal transformation, and epigenetic dysregulation. Conclusions: Our study highlighted the advantage of ctDNA analysis for monitoring tumor evolution. TP53 mutations promoted genetic evolution and accelerated occurrence of resistance. We also unveiled ALK-dependent resistance mechanisms, mainly by G1269A, G1202R, and E1210K mutations, and ALK-independent resistance mechanisms to ensartinib. (C) 2021 International Association for the Study of Lung Cancer. Published by Elsevier Inc.