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9033 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.

9051 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.