CD8+ Cytotoxic Immune Infiltrate in Non-Muscle Invasive Bladder Cancer: A Standardized Methodology to Study Association with Clinico-Pathological Features and Prognosis

BACKGROUND: Major interest lies in the evaluation of immune infiltrate in bladder cancer. CD8+ cytotoxic lymphocytes are key effectors of adaptive immune response. OBJECTIVES: The aims of the study were to set up a standardized methodology for CD8+ lymphocytes estimation in NMIBC and investigate how intra-tumoral heterogeneity influences CD8+ immune infiltrate. METHODS: We considered 995 NMIBC included in the Spanish Bladder Cancer (SBC)/EPICURO Study. Duplicate 0.6mm TMA spots and paired full sections (FS) for 50 selected cases were double stained with anti-pan cytokeratin antibody and anti-CD8 antibody. Slides were digitalized and CD8+ cells were automatically counted after tissue recognition (tumor vs stroma). Spatial heterogeneity was assessed and a resampling strategy was applied to estimate the proper number of 0.6mm TMA spots providing an adequate CD8+ cell estimate. Association between CD8+ count and expression of urothelial differentiation markers was estimated. Cox regression models were performed to assess association between CD8+ cell count and risk of recurrence and progression. RESULTS: Microscopic examination of full sections showed spatial heterogeneity for CD8+ infiltrates. Simulation analyses demonstrated that 5 TMA regions provided a correct sampling of tumor and stromal compartments in Ta while 2 and 6 TMA regions were necessary in T1, respectively. CD8+ cells infiltration was associated with stage, regardless of the histological compartment analyzed (median CD8+ /mm(2) were 25/mm(2) and 129/mm(2) in tumor and stroma respectively in Ta and 111/mm(2) and 344/mm(2) in T1; p-value = 0.006). CD8+ infiltration in tumor compartment was significantly associated with low FGFR3 expression. CD8+/mm(2) count in the tumor compartment was not associated with prognosis. CONCLUSION: Differences identified between Ta and T1 tumours supported the hypothesis that rigorous efforts should be placed in proper study design. These results provide a new framework to investigate microenvironment complexity in bladder cancer.