Association Of PD-1,PD-L1/L2 Gene Polymorphism And Protein Expression With Prognosis In Gastric Cancer

Gastric cancer(GC)is a major cancer-related threat to global health,GC prognosis remains poor because typical symptoms are usually absent in the early stages,resulting in delayed diagnosis and treatment.There is also a lack of reliable biomarkers available for screening targeted therapies and predicting prognosis.Immunotherapy has been considered as an anticancer treatment.PD-1 and its ligand PD-L1/PD-L2 are a group of negative co-stimulatory molecules that can suppress T cell proliferation in carcinoma.The clinical efficacy of PD-1/PD-L1 inhibition has been observed in many cancers.single nucleotide polymorphisms are the most commen variations in human genetics.These variations could affect not only with the progress of GC,but also with the treatment response and long time survival of GC.We tested the functional SNPs in PD-1,PD-L1/L2 genes and analysis the association between the genotypes of these SNPs and GC prognosis.Meanwhile,We evaluated PD-1 and PD-L1/L2 expression in tumor cells(TCs)and tumor-infiltrating immune cells(TIICs).We determined the Helicobacter pylori(Hp)and Epstein-Barr virus(EBV)infection status in a GC cohort,then analyzed the relationship between the expression of PD-1,PD-L1/L2 and GC prognosis.Objective: We tested the functional SNPs in PD-1,PD-L1/L2 genes and evaluated PD-1 and PD-L1/L2 protein expressions in tumor cells and tumor-infiltrating immune cells of GC,meanwhile we also assess the m RNA expression levels in patients' tumor tissues and paired adjacent non-tumorous tissues.The comprehensive evaluation could help in predicting the prognosis of gastric cancers and selecting patients who might benefit from targeted treatment.Methods:756 cases those underwent a radical operation,without distant metastasis and with negative surgical margins were included in the survival analysis to evaluate the association of the functional SNPs(rs2227982,rs10204525,rs36084323,rs822336,rs866066,rs822338,rs2297136,rs7854413 and rs16923198)in PD-1,PD-L1/L2 with gastric cancer prognosis using Mass ARRAY method.Then we determined the Helicobacter pylori(Hp)and Epstein-Barr virus(EBV)infection status in a GC cohort(n=340),and analyzed the relationship between the protein expression of PD-1,PD-L1/L2 in TCs,TIICs and GC prognosis using IHC method.Total m RNA was extracted from 21 patients' tumor tissues and paired adjacent non-tumorous tissues.The m RNA levels of these three genes were analyzed with the 2-??Ct method.The log-rank test was used to compare Kaplan-Meier survival curves.Univariate and multivariate Cox regressions were performed to assess the hazard ratios(HRs)and 95% CIs of the possible prognostic factors.Result: PD-L1rs822336 CC genotype was associated with better prognosis of GC.Compared with the patients carrying TT+TC genotypes,the HR of patients with CC genotype was decreased 49.2%(P=0.020).TT genotype of PD-L1 rs822338 seemed to be correlated with worse survival,but this association did not reach statistical significance,(P=0.051,HR=1.366,95%CI: 0.999-1.868).In patients without chemotherapy,carrying GG genotype of PD-L1rs2297136 may increase the risk of death in GC patients(HR=2.872,95%CI:1.03-7.99,P=0.043).Carrying PD-L1822336 CC genotype also associated with better survival of GC(HR=0.385,95%CI:0.19-0.79,P=0.009).PD-1 protein expression in TIICs was observed in 22.6% of GC patients.The PD-L1 and PD-L2 positivity rates were 40.3% and 53.8% in TCs,respectively,and 60.0% and 60.9% in TIICs,respectively.PD-L1 was up-regulated in EBV-infected GC patients in both TCs(P=0.009)and TIICs(P=0.003).Hp status was not associated with PD-1 or PD-L1/PD-L2 expression.In TIICs,PD-L1 expression was independently associated with better GC prognosis(HR=0.72,95%CI: 0.53-0.99).Co-expression of PD-1 and PD-L1,but not PD-L2,was a favorable prognostic marker that indicated a dose effect on the mortality risk of GC patients(P-value for trend=0.005).PD-L1 rs2297136 AA+AG genotype was associated with a higher PD-L1 protein expression.We found that PD-1,PD-L1 and PD-L2 m RNA levels were up-regulated in GC tissues,and were positively correlated with one another(P=0.043,P=0.008 and P=0.035).After the bioinformatic analysis,we predicted that JAK2-STAT1 signal pathway may regulated the expression of GC in gastric cancer.Conclusion:PD-L1rs822336 CC genotype was associated with better prognosis of GC.In patients without chemotherapy,carrying GG genotype of PD-L1rs2297136 and GC+GG genotypes of PD-L1822336 were Independent risk factors with wrose survival of GC.PD-L1 was over-expressed in EBV-infected GC.The PD-1,PD-L1,and PD-L2 m RNA levels were up-regulated in GC tissues and positively correlated with one another.Co-expression of PD-1 and PD-L1,but not PD-L2,was a favorable prognostic marker in gastric cancer.PD-L1 rs2297136 AA+AG genotype was associated with a higher PD-L1 protein expression.The comprehensive evaluation of tumor cells and tumor-infiltrating immune cells could help in predicting the prognosis of gastric cancers and selecting patients who might benefit from targeted treatment.