Background: Lorlatinib, a third-generation ALK tyrosine kinase inhibitor, has shown overall and intracranial activity in ALK+ advanced NSCLC. In the randomized, multicenter, phase 3 study in pts with previously untreated ALK+ advanced NSCLC (CROWN; NCT03052608), lorlatinib showed a statistically significant and clinically meaningful improvement in progression-free survival (PFS) vs crizotinib (Shaw AT, et al. N Engl J Med. 2020;383:2018-2029). Comprehensive molecular profiling of circulating tumor DNA (ctDNA) and tumor tissue was performed to identify molecular correlates of response. Methods: At baseline (BL), plasma samples were available from 134 and 129 pts in the lorlatinib and crizotinib arms, respectively. Analyses returned results for tumor tissue (archived or new biopsy) from 147 pts across both arms. Plasma and tumor DNA were analyzed by next-generation sequencing (NGS; Guardant360 and TissueNext, respectively, Guardant Health, Inc.). Objective response rate (ORR), duration of response, and PFS based on the September 20, 2021, cutoff, all assessed by blinded independent central review, were summarized according to mutation and tumor mutation burden (TMB) status. Results: At BL, 22% of pts had no detectable ctDNA. ALK missense mutations (n=19) or deletion (n=1) were detected in plasma of 12 pts (n=5 and 7 in the lorlatinib and crizotinib arms, respectively). Most pts harbored 1 mutation, but 3 pts harbored ≥3 mutations. In tumor samples, no somatic ALK mutation was detected. ALK fusions were detected in plasma of 48% of pts and in tumor of 80%. EML4-ALK variant (v) subtypes were highly concordant between ctDNA and tumor tissue. Based on ctDNA, ORRs were generally higher in the lorlatinib vs crizotinib arm, reaching 80% and 72% for EML4-ALK v1 and v3, respectively, in the lorlatinib arm, and 50% and 74% in the crizotinib arm. Median PFS was not reached for v1 in the lorlatinib arm and was 7.4 mo in the crizotinib arm; for v3, mPFS was 33.3 and 5.5 mo, respectively. TP53 mutations were found in 42% of pts with detectable ctDNA, and their presence did not seem to influence lorlatinib activity. In the crizotinib arm, absence of TP53 mutations led to longer PFS. These findings are being verified in tumor tissue. A pt treated with lorlatinib with an ongoing partial response in tumor lesions at the data cutoff date was found to have a KRAS G12V mutation and the presence of ALK fusion in tumor tissue but had no ctDNA detected at BL. Conclusions: Pts with untreated ALK+ advanced NSCLC had higher ORRs and potentially longer PFS across predefined biomarker subgroups when treated with lorlatinib compared with crizotinib in the phase 3 CROWN study. Based on pretreatment ctDNA and tumor tissue analyses, lorlatinib led to strong clinical benefit regardless of the type of ALK rearrangement or presence of potential driver co-mutation. Clinical trial information: NCT03052608.