BackgroundPD-1/PD-L1 inhibitors have improved survival for patients with non-small cell lung cancer (NSCLC). We evaluated natural killer cell activity (NKA) and methylatedHOXA9circulating tumor DNA (ctDNA) as prognostic biomarkers in NSCLC patients treated with PD-1/PD-L1 inhibitors.MethodsPlasma was prospectively collected from 71 NSCLC patients before treatment with PD-1/PD-L1 inhibitors and before cycles 2–4. We used the NK Vue®assay to measure the level of interferon gamma (IFNγ) as a surrogate for NKA. MethylatedHOXA9was measured by droplet digital PCR.ResultsA score combining NKA and ctDNA status measured after one treatment cycle had a strong prognostic impact. Group 1 had IFNγ < 250 pg/ml and detectable ctDNA (n= 27), group 2 consisted of patients with either low levels of IFNγ and undetectable ctDNA or high levels of IFNγ and detectable ctDNA (n= 29), group 3 had IFNγ ≥250 pg/ml and undetectable ctDNA (n= 15). Median OS was 221 days (95% CI 121–539 days), 419 days (95% CI 235–650 days), and 1158 days (95% CI 250 days—not reached), respectively (P= 0.002). Group 1 had a poor prognosis with a hazard ratio of 5.560 (95% CI 2.359–13.101,n= 71,P< 0.001) adjusting for PD-L1 status, histology, and performance status.ConclusionsCombining NKA and ctDNA status after one cycle of treatment was prognostic in patients with NSCLC treated with PD-1/PD-L1 inhibitors.