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Juliana Machado-Rugolo,1,2 Tabatha Gutierrez Prieto,1 Alexandre Todorovic Fabro,3 Edwin Roger Parra Cuentas,4 Vanessa Karen Sá,5 Camila Machado Baldavira,1 Claudia Aparecida Rainho,6 Erick C Castelli,7,8 Cecilia Farhat,1 Teresa Yae Takagaki,9 Maria Aparecida Nagai,10,11 Vera Luiza Capelozzi1 1Laboratory of Genomics and Histomorphometry, Department of Pathology, University of São Paulo Medical School (USP), São Paulo, Brazil; 2Health Technology Assessment Center, Clinical Hospital (HCFMB), Medical School of São Paulo State University (UNESP), Botucatu, São Paulo, Brazil; 3Department of Pathology and Legal Medicine, Ribeirão Preto School of Medicine, University of São Paulo (FMRP-USP), Ribeirão Preto, Brazil; 4Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA; 5Laboratory of Genomics and Molecular Biology, Centro Internacional De Pesquisa (CIPE), AC Camargo Cancer Center, São Paulo, SP, Brazil; 6Department of Chemical and Biological Sciences, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil; 7Molecular Genetics and Bioinformatics Laboratory, Experimental Research Unit (UNIPEX), Medical School of São Paulo State University (UNESP), Botucatu, São Paulo, Brazil; 8Department of Pathology, Medical School of São Paulo State University (UNESP), Botucatu, São Paulo, Brazil; 9Division of Pneumology, Heart Institute (Incor), Clinical Hospital, University of São Paulo Medical School (USP), São Paulo, São Paulo, Brazil; 10Department of Radiology and Oncology, University of São Paulo Medical School (USP), São Paulo, Brazil; 11Laboratory of Molecular Genetics, Center for Translational Research in Oncology, Cancer Institute of São Paulo (ICESP), São Paulo, BrazilCorrespondence: Vera Luiza CapelozziDepartment of Pathology, University of São Paulo Medical School (USP), Av Dr Arnaldo 455, Room 1143, São Paulo, São Paulo State, 01246-903, BrazilTel +55 11 3061 7427Fax +55 11 3064-2744Email vera.capelozzi@fm.usp.brPurpose: Although non-small cell lung cancer (NSCLC) remains a deadly disease, new predictive biomarkers have emerged to assist in managing the disease, of which one of the most promising is the programmed death‐ligand 1 (PD-L1). Each, PD-L1 variant seem to modulate the function of immune checkpoints differently and affect response to adjuvant treatment and outcome in NSCLC patients. We thus investigated the influence of these PD-L1 genetic variations in genetically admixed NSCLC tissue samples, and correlated these values with clinicopathological characteristics, including prognosis.Materials and Methods: We evaluated PD-L1 non-coding genetic variants and protein expression in lung adenocarcinomas (ADC), squamous cell carcinomas (SqCC), and large cell carcinomas (LCC) in silico. Microarray paraffin blocks from 70 samples of ADC (N=33), SqCC (N=24), and LCC (N=13) were used to create PD-L1 multiplex immunofluorescence assays with a Cell Signaling E1L3N clone. Fifteen polymorphisms of the PD-L1 gene were investigated by targeted sequencing and evaluated in silico using dedicated tools.Results: Although PD-L1 polymorphisms seemed not to interfere with protein expression, PD-L1 expression varied among different histological subtypes, as did clinical outcomes, with the rs4742098A>G, rs4143815G>C, and rs7041009G>A variants being associated with relapse (P=0.01; P=0.05; P=0.02, respectively). The rs7041009 GG genotype showed a significant correlation with younger and alive patients compared to carriers of the A allele (P=0.02 and P< 0.01, respectively). The Cox regression model showed that the rs7041009 GG genotype may influence OS (P< 0.01) as a co-dependent factor associated with radiotherapy and recurrence in NSCLC patients. Furthermore, the Kaplan–Meier survival curves showed that rs7041009 and rs4742098 might impact PPS in relapsed patients. In silico approaches identified the variants as benign.Conclusion: PD-L1 non-coding variants play an important role in modulating immune checkpoint function and may be explored as immunotherapy biomarkers. We highlight the rs7041009 variant, which impacts OS and PPS in NSCLC patients.Keywords: PD-L1, non-small cell lung cancer, single nucleotide polymorphisms, next-generation sequencing