| Objective:Inflammation and immune signatures are known to play important roles in tumorigenesis,tumor progression and tumor prognosis and they have always been hot issues which are widely studied in both basic and clinical researches.Urothelial carcinoma of bladder(UCB)is a typical inflammation-related and immune infiltration-related tumor among all kinds of malignancies.This study aimed to investigate the prognostic value of systemic inflammation related markers in bladder cancer patients who underwent radical cystectomy;besides,we aimed to establish a prognostic model based on these screened markers to estimate the overall survival(OS)for bladder cancer patients after radical cystectomy.Moreover,we aimed to investigate the prognostic value of tumor-infiltrating lymphocyte(TIL)in UCB patients using meta-analysis.We also aimed to mine TCGA bladder cancer database by bioinformatic methods to identify tumor immune-related differentially expressed hub genes.We aimed to analyze the characteristics of transcription level,translation level and mutation burden of immune-related hub genes(IRHGs).Additionally,the association of IRHGs and tumor-infiltrated immune cells,and the prognostic value of IRHGs were explored.Methods:1.434 patients obtained from West China Hospital were randomly split into a training cohort(n=218)and a validation cohort(n=216).Univariate Cox regression analysis,Kaplan-Meier analysis and Least Absolute Shrinkage and Selection Operator(LASSO)regression analysis were performed in the training cohort to identify prognostic factors.A nomogram predicting OS was constructed based on multivariate Cox regression model.The prognostic model’s performance was assessed by calibration curves,the receiver operating characteristic(ROC)curves,concordance index(C-index),decision curve analysis(DCA),the net reclassification improvement(NRI)analysis and the integrated discrimination improvement(IDI)analysis.2.We used Pubmed,Embase and Cochrane Library to conduct this systematic review and meta-analysis.Studies were eligible if they investigated the prognostic value of CD3+,CD4+,CD8+,Foxp3+ lymphocytes or TILs in UCB patients,by time-to-event survival analysis.All studies were appraised for risk of bias using the Quality and Prognosis Studies(QUIPS)criteria.Hazard ratios(HRs)with their 95%confidence interval(CIs)from each study were used to generate pooled HRs and obtain forest plots.3.Bladder cancer gene expression profiles were obtained from the TCGA database.Differentially expressed genes between bladder cancer and normal bladder tissue were displayed in the volcano map and the heat map.Functional enrichment analysis was performed through KEGG and GO.The immune-related gene set was obtained from the Imm Port database,and the intersection between the two databased were immune-related differentially expressed genes of bladder cancer.The protein-protein interaction network was constructed by Cytoscape,and IRHGs were screened out.Box plots were used to analyze the expression levels of IRHGs.Protein translation levels of IRHGs in bladder cancer and normal bladder tissues were obtained from the HPA database.Oncoplot was used to analyze the mutation burden of IRHGs.CIBERSORT and TIMER were used to analyze the correlation between IRHGs and tumor-infiltrating immune cells and the level of immune infiltration.Finally,combined with TCGA clinicopathological information,univariate and multivariate Cox regression methods were used to analyze the prognosis of IRHGs in bladder cancer,and a nomogram prognostic model was constructed.Results:1.Out of all systemic inflammation related markers,systemic immune inflammation index(SIRI),prognostic nutritional index(PNI)and lymphocyte-eosinophil ratio(LER)were independently associated with OS.The prognostic nomogram model incorporated age,tumor recurrence,T stage,lymph node metastasis,SIRI,LER and PNI.In detail,age ≥60 was a hazardous factor(HR=3.1,95% CI:1.4-6.7,p=0.005);recurrent tumor was a hazardous factor(HR=2.0,95% CI:1.2-3.4,p=0.007);positive lymph node metastasis was a hazardous factor(HR=4.0,95% CI:1.9-8.2,p<0.001);SIRI ≥0.6 was a hazardous factor(HR=1.3,95% CI:1.1-3.1,p=0.029);LER ≥5.42 was a protective factor(HR=0.6,95% CI:0.4-0.9,p=0.043);PNI ≥5.42 was a protective factor(HR=0.6,95% CI:0.3-0.9,p=0.031).The C-index of the prognostic nomogram was 0.783(95%CI: 0.730-0.834)in validation cohort.AUC of the nomogram was 0.825 for 3-year OS and 0.784 for 5-year OS in the validation cohort.Calibration plots for comparing predicted outcome and actual outcome of 3-and 5-year OS displayed good concordance.DCA curves of the nomogram showed larger benefits than other established models.The NRI and IDI indicated the nomogram outperformed other prognostic models.A webtool based on this prognostic model demonstrated acceptable applicability and convenience.2.A total of 2120 studies were retrieved and 14 studies assessing the impact of TILs on prognostic outcomes in UCB patients were included in final analysis through inclusion-exclusion criteria.We concluded features such as population baseline characteristics,specimen processing methods and immune infiltration estimation methods from these studies.QUIPS quality assessment of literature revealed that included studies were all at low of moderate levels of bias.Due to high heterogeneity among the studies,all meta-analyses adopted random-effect models.The pooled analysis indicated a favorable role of higher CD3+ TILs(HR=0.74,95%CI: 0.62-0.88,p<0.05)for overall survival and higher CD8+ TILs(HR=0.46,95%CI: 0.28-0.74,p<0.05)for overall survival in the clinical outcomes of UCB,while higher Foxp3+ TILs were associated with worse survival(HR=2.21,95% CI:1.47-3.32,p<0.05)for recurrence-free survival.3.A total of 3135 differentially expressed genes were obtained from the TCGA database,2498 immune-related genes were identified by Imm Port database,and 610immune-related differentially expressed genes were obtained from the intersection of the two.Functional enrichment analysis showed that these genes were involved in the occurrence and development of bladder cancer.Cytoscape was used to screen out the most important IRHGs,including BDKRB2,CXCL12,GNG11,GPR183 and S1PR1.IRHGs were differentially down-regulated genes in bladder cancer,and pan-cancer analysis showed that the expression is generally down-regulated in 33 cancers.The structure of IRHGs was relatively stable,and there was no or very low degree of mutation(mutation rate=0 or <2%).TIMER showed that the expression level of IRHGs had a significant correlation with the infiltration levels of CD4+T cells,CD8+T cells,B cells,neutrophils,macrophages and dendritic cells(p<0.05).Univariate and multivariate Cox regression analysis revealed that for overall survival in bladder cancer,higher expression level of BDKRB2 was an independent hazardous factor(HR=1.19,95% CI:1.02-1.39,p=0.026),older age was an independent hazardous factor(HR=1.02,95% CI:1.01-1.04,p=<0.0001),and higher p TNM stage was an independent hazardous factor(HR=1.62,95% CI:1.33-1.98,p<0.0001).These factors were incorporated to construct a prognostic nomogram(C index=0.727,95% CI: 0.683-1,p<0.001).Conclusion:1.SIRI,LER and PNI were independently associated with OS of bladder cancer patients.We have established a prognostic nomogram with improved discriminative ability and clinical benefits for bladder cancer patients after radical cystectomy.The nomogram alongside an easy-access webtool might assist clinicians in optimizing postoperative management.2.This systematic review and meta-analysis confirmed the favorable prognostic impact of CD3+ and CD8+ tumor-infiltrating lymphocytes in UCB patients and demonstrated that higher Foxp3+ TILs was associated with worse survival.Future studies using large cohorts and standardized methodology with regard to tumor subsites,stages and treatment modalities are needed to incorporate TILs with clinical practice.3.We found that BDKRB2,CXCL12,GNG11,GPR183 and S1PR1 were immune-related hub genes of bladder cancer.Their expression levels were significantly associated with the levels of tumor-infiltrated immune cells.Immune-related hub genes of bladder cancer are potential prognostic markers,which may provide value in predicting patients’ survival lengths. |
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