The Clinical Significance And Regulation Of Programmed Death-ligand 1 In Nasopharyngeal Carcinoma

Part Ⅰ PD-L1 predicts poor prognosis for nasopharyngeal carcinoma irrespective of PD-1 and EBV-DNA loadObjective Programmed death-1(PD-1)is an immunosuppressive receptor functionally bound with programmed death-ligand 1(PD-L1),which has been reported in various malignancies.However,only a few studies are available for the clinical significance of PD-1/PD-L1 in nasopharyngeal carcinoma(NPC).The aim of this study was then designed to evaluate the clinical significance of PD-1/PD-L1 expression and related factors in NPC patients.Methods Immunohistochemistry analysis was performed in a cohort of first diagnosed NPC patients(n = 99).Clinical data were collected for further statistical analysis.Chi-square test was used to assess the correlation of the expression of PD-1 and PD-L1 with various clinical parameters such as age,clinical stage and EBV-DNA status.T-test was used to compare the differences between the Body Mass Index(BMI),hemoglobin(HB)level in patients with different PD-1/PD-L1 immunoreactivity.Univariate analyses and multivariate analyses were performed with a Log-rank test and Cox regression analysis.Results PD-1-positive immune cells were present in 44 of the total 99 tumours(44.4%),and PD-L1 staining was detectable in 96 patients(97.0%).Lower HB and BMI levels were associated with high levels of PD-L1 staining in NPC patients.In contrast,a correlation between the PD-1/PD-L1 level and EBV load was not identified.PD-L1 might be a negative indicator for NPC patients,while PD-1 positivity was suggested to not be significantly correlated with clinical outcomes.Conclusion PD-L1 might be a potential prognostic biomarker for NPC patients.However,further studies are needed to clarify the underlying mechanism of EBV status in the immunosuppression process induced by the PD-1/PD-L1 axis.Part Ⅱ PD-1/PD-L1 expression in recurrent nasopharyngeal carcinoma:the correlation with anemia and outcomesObjective To investigate the clinical significance and related factors of programmed death-1(PD-1)/programmed death-ligand 1(PD-L1)expression in recurrent NPC patients.Methods Immunohistochemistry analysis were performed to detect the alterations in of PD-1/PD-L1 in 132 recurrent NPC patients.The clinical data and hematologic biomarkers were collected for further statistical analysis.Chi-square test was used to assess the correlation of the expression of PD-1 and PD-L1 with various clinical parameters such as age,clinical stage and anemia.T-test was used to compare the differences between the HB level in patients with different PD-1/PD-L1 immunoreactivity.Univariate analyses and multivariate analyses were performed with a log-rank test and Cox regression analysis.To further explore the association between PD-L1 and anemia,immunofluorescence analysis was performed to investigate the correlation of PD-L1 with hypoxia inducible factor-1α(HIF-1α).Results PD-1 positive immune cells were presented in 50 of the total 132(37.9%)biopsy samples.PD-L1 expression was detect in 128(97.0%)recurrent tumors.Advanced rT classification and anemia status before salvage treatment was associated with high level of PD-L1 in recurrent NPC patients,and PD-L1 and was co-located with HIF-1α in recurrent tumors by immunofluorescence analysis.Moreover,our result suggested that PD-L1 might be a negative indicator for recurrent NPC patients as well as age,rT classification,anemia and tumor necrosis at diagnose of recurrence.Conclusion PD-L1 might be a potential prognostic biomarker for recurrent NPC patients,and advanced re-stage,anemia might represent as candidate biomarkers for evaluating patients’ response to anti-PD-1/PD-L1-treatment.However,further studies are needed to clarify the underlying mechanism of hypoxia in immunosuppression process induced by PD-1/PD-L1 axis.Part Ⅲ Hypoxia induces PD-L1 expression in nasopharyngeal carcinoma cellsObjective To investigate the effect of hypoxia on the expression of PD-L1 in nasopharyngeal carcinoma cells and to explore the potential mechanism.Methods CNE2 cells were transfected with siRNAs targeting HIF-1α and RT-qPCR were performed to confirm the transfection efficiency.CNE2 cells with or without HIF-1α-siRNA transfection were cutured at 1-2%O2(hypoxia)or 20%(normoxia)for 24 hours,siRNA of β-actin were used as a control for HIF-1α-siRNA(NC-siRNA),CNE2 cells were then divided into 4 groups according to different treatments:cells under normoxia,cells under hypoxia,cells with HIF-1α-siRNA transfection under hypoxia,cells with NC-siRNA transfection under hypoxia.The mRNA and protein expression levels of PD-L1,HIF-1α,STAT3 and VEGF in CNE2 cells under different conditions were evaluated by realtime-quantitivePCR(RT-qPCR)and western blot(WB),respectively.The phosphorylation level of STAT3(pSTAT3)was also determined by WB.MTT and flow cytometry were applied to explore cell activity and apoptosis of CNE2 cells.Results After CNE2 cells was cultured under hypoxia for 24 hour,the mRNA and protein expression levels of PD-L1,HIF-1α,STAT3 and VEGF were significantly increased,the phosphorylation level of STAT3(pSTAT3)showed a similar siginificant trend.After HIF-la-siRNA transfection,the mRNA and protein expression levels of PD-L1,HIF-1α,STAT3 and VEGF in CNE2 cells under hypoxia were significantly decreased,the phosphorylation level of STAT3(pSTAT3)was down-regulated simultaneously.Bivariate correlation analysis suggested that the protein level of HIF-1α,STAT3,pSTAT3 and VEGF significantly correlated with PD-L1 expression.The cell proliferation rate of CNE2 cells with HIF-1α-siRNA transfection were significantly down-regulated,whereas the apoptosis rate of CNE2 cells with HIF-1α-siRNA transfection were significantly elevated compared with cells under normoxia,hypoxia or cells transfected with NC-siRNA.Conclusion Hypoxia might enhanced the mRNA and protein expression of PD-L1,HIF-1α,STAT3 and VEGF as well as the phosphorylation level of STAT3 in CNE2 cells,the enhancement could be inhibited by HIF-1α-siRNA transfection meanwhile.HIF-1α-siRNA transfection might promote opoptosis and interfere the proliferation of CNE2 cells.These findings indicated that hypoxia might promote overexpression of PD-L1 in CNE2 via HIF-1α related pathway including VEGF and STAT3.Downregulation of HIF-1α could attenuate the immunosuppression effect mediated by PD-L1.