Background: In advanced NSCLC (aNSCLC) with PD-L1 < 50% chemo-immunotherapy is the standard of care. Although the activity of single agent pembrolizumab was reported, no biomarkers have been identified able to select patients who mostly benefit. The trial aimed to identify immune biomarkers associated with PFS in aNSCLC patients with PD-L1 levels < 50% treated with first-line pembrolizumab. Here we reported for the first time the clinical outcomes of the trial and circulating immune profiling (CIP) biomarkers correlated to PFS. Methods: This phase II trial was conducted at Fondazione IRCCS Istituto Nazionale dei Tumori of Milan. Eligible patients were previously untreated stage IIIB-IV NSCLC, EGFR and ALK wild type with PD-L1 < 50%, PS 0-2. PD-L1 was assessed with 22-C3 antibody (Dako). Patients received pembrolizumab 200 mg every 3 weeks until 35 cycles, disease progression or unacceptable toxicity. The primary endpoint was the association of immune biomarkers with PFS. OS, ORR, DCR, DoR and safety were secondary endpoints. CIP was performed by determination of absolute cell counts for 36 innate and adaptive immunity subsets through multiparametric flow cytometry on freshly isolated whole blood samples at baseline. An orthoblique principal components-based clustering approach and multivariable Cox regression model adjusted for clinical variables were used to analyse CIP variables and their correlation with clinical endpoints. Results: From May 2018 to October 2020, of 87 screened, 65 patients were enrolled. The median age was 70.9 years, most patients were male (67.7%), smoker (87.7%), non-squamous (83.1%), PDL1 + (70.8%). 12 patients (18.5%) had PS 2. During a median follow-up of 26.4 months (mo), 51 radiological progressions, 46 deaths and 60 PFS events were observed. The median PFS was 2.9 mo (95%CI 1.8 - 5.6) and the median OS was 12.1 mo (95%CI 8.7 - 17.1). The ORR was 24.1% (2 complete and 12 partial responses), DCR was 53.4% and median DoR was 14.5 mo (95%CI 6.4 - 24.9). Drug related G3-G4 adverse events were: 2 hyponatremia, 2 lipase increased, 1 pneumonitis. Out of 7 CIP clusters identified, 2 were statistically significant at multivariable analysis on 57 patients. Higher baseline counts of 8 subsets within these two clusters were associated with better PFS (HR values range: 0.88-0.98; p values range: 0.001- 0.016). The significant subsets included lymphocytes (cells expressing CD3, or CD19 or CD56, but lacking granulocyte and monocyte markers) and main NK subsets (including CD56dim CD16+, CD56dim CD16- and HLA-DR+ NK cells). Conclusions: This trial confirmed that pembrolizumab as first-line single agent is safe and active also in a subgroup of aNSCLC patients with PD-L1 < 50%. CIP biomarkers can be useful to identify patients with a favourable outcome, thus avoiding the adding toxicity of chemotherapy. Clinical trial information: NTC03447678.