Response and outcomes for the 50 patients with a molecular response evaluable cohort (baseline Max-VAF 0.3%) compared to the 16 patients in the unevaluable cohort (baseline Max-VAF 0.3%). patients, 51 (76.1%) had 1 variant detected at a variant allele fraction 0.3% and thus were eligible for calculation of molecular response from paired baseline and 9-week samples. Molecular response values were significantly lower in patients with an objective radiologic response (log mean 1.25% 27.7%, .001). Patients achieving a DCB had significantly lower molecular response values compared to patients with no durable benefit (log mean 3.5% 49.4%, .001). Molecular responders had significantly longer progression-free survival (hazard ratio, 0.25; 95% CI, 0.13 to 0.50) and overall survival (hazard ratio, 0.27; 95% CI, 0.12 to 0.64) compared with molecular nonresponders. CONCLUSION Molecular response assessment using circulating tumor DNA may serve as a noninvasive, on-therapy predictor of response to pembrolizumab-based therapy in addition to standard of care imaging in mNSCLC. This strategy requires validation in impartial prospective studies. INTRODUCTION Over the past Firategrast (SB 683699) decade, treatment for metastatic nonCsmall-cell lung cancer (mNSCLC) has undergone a paradigm shift. For patients with NSCLC not harboring an actionable mutation, immunotherapy with or without concurrent chemotherapy has become the standard first-line therapeutic approach. Pembrolizumab is currently approved for the treatment of patients with mNSCLC with a programmed death ligand 1 (PD-L1) Tumor Proportion Score (TPS) 1%, and in combination with chemotherapy regardless of PD-L1 TPS.1,2 In practice, pembrolizumab monotherapy is given to relatively asymptomatic patients with a low overall disease burden and a PD-L1 TPS 50%.3 By contrast, patients with PD-L1 50% are usually treated p85 with histology-specific platinum-doublet therapy in Firategrast (SB 683699) combination with pembrolizumab.2,4 CONTEXT Key Objective For patients with metastatic nonCsmall-cell lung cancer without a targetable mutation, programmed death 1 or programmed death ligand 1 antibody therapy alone, or in combination with chemotherapy, has become the standard first-line therapeutic approach. However, only a subset of patients respond to these therapies. Predicting which patients will experience a durable clinical benefit remains a clinical challenge. Knowledge Generated We performed plasma next-generation sequencing on circulating tumor DNA (ctDNA) from paired blood samples obtained at baseline and at 9 weeks in a prospective cohort of patients with metastatic nonCsmall-cell lung cancer treated with pembrolizumab-based therapy. We demonstrate that a reduction in on-treatment ctDNA level is associated with improved response rates at 9 weeks and 6 months, as well as improved progression-free survival and overall survival. Relevance These results highlight the potential role of assessing on-treatment changes in ctDNA as Firategrast (SB 683699) a noninvasive means to predict long-term efficacy from pembrolizumab-based therapy in advanced nonCsmall-cell lung cancer (NSCLC). Despite significant advances, many patients do not benefit from immunotherapy, and currently available biomarkers are unable to adequately predict which patients are most likely to respond.3,5,6 Current management and therapeutic decisions continue to be based on clinical symptoms and radiographic evaluation. Plasma-based approaches using circulating tumor DNA (ctDNA) and next-generation sequencing (NGS) are being increasingly used for noninvasive detection of mutations in mNSCLC. Our group recently evaluated plasma NGS as a potential biomarker for selection of immunotherapy for treatment-naive patients with mNSCLC. Using a 500-gene plasma NGS assay, we demonstrated that patients with a higher blood tumor mutational burden at baseline were more likely to benefit from pembrolizumab-based therapy.7 With the integration of plasma-based NGS testing into clinical practice, emerging reports have correlated changes in ctDNA on-treatment compared to baseline with response to targeted and immunotherapy in various cancer types.8-10 There are still very limited data, however, regarding changes in ctDNA during the course of immunotherapy and how these changes correlate with response and outcome in patients with mNSCLC.11-15 Recently, Zhang et al14 demonstrated that early on-treatment decreases in ctDNA levels were associated with improved progression-free survival (PFS) and overall survival (OS) across three clinical trial cohorts treated with durvalumab. Similarly, on-treatment ctDNA kinetics were recently associated with improved patient outcomes in five clinical trial cohorts of various solid tumors treated with pembrolizumab.15 However, additional data validating the role of a clinically available ctDNA NGS assay to predict response and outcomes to standard of care pembrolizumab-based therapy in mNSCLC are needed. We hypothesized that serial ctDNA molecular response assessment could serve as a biomarker of therapeutic response and outcome in patients with mNSCLC who receive pembrolizumab either alone or in combination with platinum-based chemotherapy. MATERIALS AND METHODS.