Asaf Dan ^1,2* Ozan Aricak ^3,4 Konstantinos Rounis ^1,2 M.Angeles Montero-Fernandez ⁵ Ricardo Guijarro ⁶ Simon Ekman ^1,2 Cristian Ortiz-Villalón ² Luigi De Petris ^1,2*

• ¹ Thoracic Oncology Center, Department of Oncology, Karolinska University Hospital, Stockholm, Stockholm, Sweden
• ² Department of Oncology-Pathology, Karolinska Institutet (KI), Stockholm, Stockholm, Sweden
• ³ Division of Pathology, Department of Laboratory Medicine, Karolinska Institutet (KI), Huddinge, Stockholm, Sweden
• ⁴ Department of Clinical Pathology and Cytology, Karolinska University Hospital, Huddinge, Sweden
• ⁵ Department of Cellular Pathology, Royal Liverpool University Hospital, Liverpool, United Kingdom
• ⁶ Department of Thoracic Surgery, University of Valencia, Valencia, Valencian Community, Spain

Introduction: Programmed death ligand -1 (PD-L1) expression is a well-established predictive biomarker for immunotherapy in non-small cell lung cancer (NSCLC). Programmed death -1 (PD-1) serves as the target protein to PD-L1 and their interaction serves as a crucial pathway for immune evasion. This study aimed to investigate the expression pattern of PD-1 on Tumor-infiltrating lymphocytes (TILs) in early-stage NSCLC, and its potential role as prognostic biomarker.Materials & Methods: PD-1 was evaluated in 474 surgical resected early-stage NSCLC specimens, using Tissue microarray and immunohistochemical staining. Expression was scored as negative (<1%) or positive. Positive PD-1 expression was further divided into low (<10%) and high (≥10%). None of the patients had received treatment with PD-1/PD-L1 inhibitors.Results: PD-1 expression ≥1% in TILs was observed in 83.5% of cases and was associated with pT stage (p=0.02), grade 3 (p=0.004), and adenocarcinoma subtype (p=0.05). Individuals with high PD-1 expression (≥10%) experienced reduced 10-year overall survival (Log-Rank test = 0.005). In addition, high PD-1 expression emerged as an independent factor associated with reduced survival on multivariate analysis (HR: 1.328 (95% CI: 1.074-1.641). Conclusions: Patients with early-stage NSCLC who exhibited PD-1 expression of ≥10% on TILs had an unfavorable 10-year OS rate. These findings indicate that elevated PD-1 expression on TILs can be associated with immune evasion during the early stages of malignancy evolution in the NSCLC setting and further research is required to further delineate the role of PD-1/PD-L1 pathway on tumor immune senescence. These results underline the potential role of PD-1/PD-L1 inhibitors in the treatment of early-stage NSCLC.