Yosra Messai1, Sophie Gad2, Muhammad Zaeem Noman1, Gwenael Le Teuff3, Sophie Couve2, Bassam Janji4, Solenne Florence Kammerer5, Nathalie Rioux-Leclerc5, Meriem Hasmim1, Sophie Ferlicot6, Véronique Baud7, Arnaud Mejean8, David Robert Mole9, Stéphane Richard2, Alexander M M Eggermont10, Laurence Albiges11, Fathia Mami-Chouaib1, Bernard Escudier11, Salem Chouaib12. 1. INSERM (Institut National de la Santé et de la Recherche Médicale) UMR1186, Laboratory Integrative Tumor Immunology and Genetic Oncology, Villejuif, France; INSERM, Gustave Roussy, Univ. Paris-Sud, Université Paris-Saclay, Villejuif, F-94805, France. 2. INSERM (Institut National de la Santé et de la Recherche Médicale) UMR1186, Laboratory Integrative Tumor Immunology and Genetic Oncology, Villejuif, France; INSERM, Gustave Roussy, Univ. Paris-Sud, Université Paris-Saclay, Villejuif, F-94805, France; Laboratoire de Génétique Oncologique de l'Ecole Pratique des Hautes Etudes, Paris, France. 3. Department of Biostatistics and Epidemiology, Gustave Roussy, Villejuif, France; Institut National de la Santé et de la Recherche Médicale, CESP, Université Paris-Sud, Villejuif, France. 4. Laboratory of Experimental Cancer Research, Department of Oncology, Luxembourg Institute of Health, Luxembourg City, Luxembourg. 5. Department of Pathology, University Hospital of Rennes, Rennes, France; The French National Centre for Scientific Research, IGDR Biosit, Rennes 1 University, Rennes, France. 6. INSERM (Institut National de la Santé et de la Recherche Médicale) UMR1186, Laboratory Integrative Tumor Immunology and Genetic Oncology, Villejuif, France; INSERM, Gustave Roussy, Univ. Paris-Sud, Université Paris-Saclay, Villejuif, F-94805, France; Université Paris-Sud, Assistance Publique-Hôpitaux de Paris, Service d'Anatomo-Pathologie, Hôpital Bicêtre, Le Kremlin-Bicêtre, France. 7. Institut National de la Santé et de la Recherche Médicale, Institut Cochin, Paris, France; The French National Center for Scientific Research, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France. 8. Service d'Urologie, AP-HP Hôpital Européen Georges Pompidou, Paris, France. 9. The Henry Wellcome Building for Molecular Physiology, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom. 10. Cancer Institute, Gustave Roussy Cancer Campus, Grand Paris, Villejuif, France. 11. INSERM (Institut National de la Santé et de la Recherche Médicale) UMR1186, Laboratory Integrative Tumor Immunology and Genetic Oncology, Villejuif, France; INSERM, Gustave Roussy, Univ. Paris-Sud, Université Paris-Saclay, Villejuif, F-94805, France; Medical Oncology Department, Gustave Roussy Cancer Campus, Villejuif, France. 12. INSERM (Institut National de la Santé et de la Recherche Médicale) UMR1186, Laboratory Integrative Tumor Immunology and Genetic Oncology, Villejuif, France; INSERM, Gustave Roussy, Univ. Paris-Sud, Université Paris-Saclay, Villejuif, F-94805, France. Electronic address: salem.chouaib@gustaveroussy.fr.
Abstract
BACKGROUND: Clear cell renal cell carcinomas (ccRCC) frequently display a loss of function of the von Hippel-Lindau (VHL) gene. OBJECTIVE: To elucidate the putative relationship between VHL mutation status and immune checkpoint ligand programmed death-ligand 1 (PD-L1) expression. DESIGN, SETTING, AND PARTICIPANTS: A series of 32 renal tumors composed of 11 VHL tumor-associated and 21 sporadic RCCs were used to evaluate PD-L1 expression levels after sequencing of the three exons and exon-intron junctions of the VHL gene. The 786-O, A498, and RCC4 cell lines were used to investigate the mechanisms of PD-L1 regulation. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Fisher's exact test was used for VHL mutation and Kruskal-Wallis test for PD-L1 expression. If no covariate accounted for the association of VHL and PD-L1, then a Kruskal-Wallis test was used; otherwise Cochran-Mantel-Haenzsel test was used. We also used the Fligner-Policello test to compare two medians when the distributions had different dispersions. RESULTS AND LIMITATIONS: We demonstrated that tumors from ccRCC patients with VHL biallelic inactivation (ie, loss of function) display a significant increase in PD-L1 expression compared with ccRCC tumors carrying one VHL wild-type allele. Using the inducible VHL 786-O-derived cell lines with varying hypoxia-inducible factor-2 alpha (HIF-2α) stabilization levels, we showed that PD-L1 expression levels positively correlate with VHL mutation and HIF-2α expression. Targeting HIF-2α decreased PD-L1, while HIF-2α overexpression increased PD-L1 mRNA and protein levels in ccRCC cells. Interestingly, chromatin immunoprecipitation and luciferase assays revealed a direct binding of HIF-2α to a transcriptionally active hypoxia-response element in the human PD-L1 proximal promoter in 786-O cells. CONCLUSIONS: Our work provides the first evidence that VHL mutations positively correlate with PD-L1 expression in ccRCC and may influence the response to ccRCC anti-PD-L1/PD-1 immunotherapy. PATIENT SUMMARY: We investigated the relationship between von Hippel-Lindau mutations and programmed death-ligand 1 expression. We demonstrated that von Hippel-Lindau mutation status significantly correlated with programmed death-ligand 1 expression in clear cell renal cell carcinomas.
BACKGROUND:Clear cell renal cell carcinomas (ccRCC) frequently display a loss of function of the von Hippel-Lindau (VHL) gene. OBJECTIVE: To elucidate the putative relationship between VHL mutation status and immune checkpoint ligand programmed death-ligand 1 (PD-L1) expression. DESIGN, SETTING, AND PARTICIPANTS: A series of 32 renal tumors composed of 11 VHL tumor-associated and 21 sporadic RCCs were used to evaluate PD-L1 expression levels after sequencing of the three exons and exon-intron junctions of the VHL gene. The 786-O, A498, and RCC4 cell lines were used to investigate the mechanisms of PD-L1 regulation. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Fisher's exact test was used for VHL mutation and Kruskal-Wallis test for PD-L1 expression. If no covariate accounted for the association of VHL and PD-L1, then a Kruskal-Wallis test was used; otherwise Cochran-Mantel-Haenzsel test was used. We also used the Fligner-Policello test to compare two medians when the distributions had different dispersions. RESULTS AND LIMITATIONS: We demonstrated that tumors from ccRCC patients with VHL biallelic inactivation (ie, loss of function) display a significant increase in PD-L1 expression compared with ccRCC tumors carrying one VHL wild-type allele. Using the inducible VHL 786-O-derived cell lines with varying hypoxia-inducible factor-2 alpha (HIF-2α) stabilization levels, we showed that PD-L1 expression levels positively correlate with VHL mutation and HIF-2α expression. Targeting HIF-2α decreased PD-L1, while HIF-2α overexpression increased PD-L1 mRNA and protein levels in ccRCC cells. Interestingly, chromatin immunoprecipitation and luciferase assays revealed a direct binding of HIF-2α to a transcriptionally active hypoxia-response element in the humanPD-L1 proximal promoter in 786-O cells. CONCLUSIONS: Our work provides the first evidence that VHL mutations positively correlate with PD-L1 expression in ccRCC and may influence the response to ccRCC anti-PD-L1/PD-1 immunotherapy. PATIENT SUMMARY: We investigated the relationship between von Hippel-Lindau mutations and programmed death-ligand 1 expression. We demonstrated that von Hippel-Lindau mutation status significantly correlated with programmed death-ligand 1 expression in clear cell renal cell carcinomas.
Authors: Jing-Ping Zhang; Zhihui Song; Hong-Bo Wang; Lang Lang; Yuan-Zhong Yang; Wenming Xiao; Daniel E Webster; Wei Wei; Stefan K Barta; Marshall E Kadin; Louis M Staudt; Masao Nakagawa; Yibin Yang Journal: Blood Date: 2019-05-31 Impact factor: 22.113