Background: Chemo-immunotherapy is the standard of care for patients with advanced NSCLC and PD-L1 < 50%. Efficacy has been reported in this setting with single agent pembrolizumab, but no reliable biomarkers yet exist for selecting patients likely to respond to single agent immunotherapy. The aim of this trial was to identify potential new immune biomarkers associated with PFS in NSCLC patients with PD-L1 < 50% treated with first line pembrolizumab. Methods: Advanced EGFR and ALK wild type treatment-naïve NSCLC patients with PD-L1 < 50% were enrolled. Gene expression profile was performed using nCounter PanCancer IO 360 Panel (Nanostring) on baseline tissue. Circulating immune profiling was performed by determination of absolute cell counts with multiparametric flow cytometry on freshly isolated whole blood samples at baseline and at first radiological evaluation. Gut bacterial taxonomic abundance was obtained by shotgun metagenomic sequencing of stool sample at baseline. Pembrolizumab was administered at 200 mg flat dose every 3 weeks until 35 cycles, disease progression or unacceptable toxicity. Omics data was analyzed with sequential univariate Cox Proportional Hazards regression predicting PFS, with Benjamini-Hochberg multiple comparisons correction. Biological features significant with univariate analysis were analyzed with multivariate Least Absolute Shrinkage and Selection Operator (LASSO). Results: From May 2018 to October 2020 65 patients were enrolled. Main characteristics were: male 67.6%, smokers 87.7%, PD-L1 positive 70.8%. Median follow up and PFS were 26.4 and 2.9 months, respectively. Gene expression profile was performed in 48 patients, microbiome in 54 patients and circulating immune profiling in 56 patients at baseline and in 46 patients at first radiologic evaluation. LASSO multivariate analysis with optimal lambda of 0.28 showed high expression levels of IRF9 and COMP genes was associated with an unfavorable PFS (HR 3.03, 1.52 – 6.02, p = 0.08 and HR 1.22, 1.08 – 1.37, corrected p = 0.06, respectively). High expression levels of CD244 (HR 0.74, 0.62- 0.67, p = 0.05), PTPRC (HR 0.55, 0.38 – 0.81, p = 0.098), KLRB1 (HR 0.76, 0.66 – 0.89, p = 0.05) in tissue samples and NK cells/CD56dimCD16+ (HR 0.56, 0.41 – 0.76, p = 0.006) in peripheral blood at baseline and non classical CD14dim CD16+ monocytes (HR 0.52, 0.36 – 0.75, p = 0.004), eosinophils (CD 15+CD16-) (HR 0.62, 0.44 – 0.89, p = 0.003) lymphocytes (HR 0.28, 0.15 – 0.5, p < 0.001) in peripheral blood after first radiologic evaluation were associated to a favorable PFS. No microbiome features were selected by LASSO. Conclusions: To the best of our knowledge, this is the first prospective trial in NSCLC with PD-L1 < 50% performed with a multi-omic approach able to identify immune cell subsets and expression levels of genes associated to PFS under first line treatment with pembrolizumab. Clinical trial information: NTC03447678.