LAUSR

Background: Tumor protein 63 (p63) is a transcription factor of the p53 gene family which is regularly expressed in the normal urothelium. Recently proposed RNA expression based molecular classifiers of bladder cancer identified high p63 expression as a component of a basal/squamous subtype linked to poor patient prognosis. The interplay between p63 expression status and the anti-tumor immunity in bladder cancer is unknown. Design: To assess the prognostic impact of p63 expression and the relationship between p63 and the immune tumor microenvironment we have stained tissue microarrays containing more than 2300 urothelial bladder carcinomas with 22 antibodies (i.e., p63, CD3, CD8, CD4, FOXP3, CD20, CD68, CD163, CD11c, TIM3, PD-L1, PD-1, CTLA-4, panCK, Ki-67, CD31, Vimentin, HLA-DRa, Myosin-11, Desmoglein 3, PAX-8, CDH16) using conventional brightfield and multiplex fluorescence immunohistochemistry (BLEACH&STAIN). A framework of several neuronal networks for image analysis were used. Spatial immune parameters were compared with histopathological parameters and overall survival data. The area under (tAUC) time-dependent receiver operating characteristic curves was used to compare the prognostic relevance of different prognostic markers. Results: Nuclear p63 staining was seen in all cells of normal urothelium and in all pTaG2 tumors, mostly at high levels. The rate of p63 positive cases and the staining intensity was lower in pTaG3 tumors (93.2%, p<0.0001 for pTaG3 vs pTaG2) and markedly lower in pT2-4 carcinomas (83.5%, p=0.0120 for pT2-4 vs. pTaG3). A low p63 expression was linked to a low density of T-helper cells (p=0.044) and regulatory T-cells (p=0.0053) localized in the intraepithelial tumor component as well as in the stroma, while all other analyzed T-cells and macrophages subsets where unrelated to p63 expression. Within pT2-4 carcinomas, low p63 expression was linked to nodal metastasis (p=0.0028) and overall survival (p=0.0005). The association of p63 loss with survival was independent of pT and pN (p=0.0109). The predictive performance of intraepithelial CD8+ cytotoxic T-cells (tAUC: 0.70) was even higher than the predictive performance of p63 expression (tAUC: 0.57, p=0.0017). Conclusion: In summary, our data show that p63 is downregulated in a fraction of urothelial neoplasms that are associated with a particularly poor prognosis and a low density of T-helper and regulatory T-cells. The even higher predictive performance of intraepithelial CD8+ cytotoxic T-cells underlines the strong prognostic role of the immune tumor microenvironment in muscle invasive bladder cancer. Citation Format: Henning Plage, Sebastian Hofbauer, Kira Kornienko, Paul G. Bruch, Sarah Weinberger, Florian Roßner, Simon Schallenberg, Sefer Elezkurtaj, Martina Kluth, Maximilian Lennartz, Tim Mandelkow, Elena Bady, Niclas C. Blessin, Andreas H. Marx, Henrik Samtleben, Margit Fisch, Michael Rink, Marcin Slojewski, Krystian Kaczmarek, Thorsten Ecke, Steffen Hallmann, Stefan Koch, Nico Adamini, Sarah Minner, Ronald Simon, Guido Sauter, Tobias Klatte, David Horst, Thorsten Schlomm, Henrik Zecha. Reduced p63 expression is linked to a low density of regulatory T-cells and unfavorable prognosis in muscle-invasive urothelial carcinoma of the bladder [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3436.