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Background: Most inactivating p53 mutations result in a nuclear accumulation of the defective p53 protein. However, p53 alterations that result in a complete lack of cellular p53 and complete absence of p53 immunostaining do also occur. As p16 is upregulated in p53 inactivated cells, p16 immunohistochemistry may be a surrogate marker for p53 inactivation. Design: In this study, we investigated p53 and p16 immunostaining on more than 2,500 urothelial bladder carcinomas in a tissue microarray format to better understand their impact in relation to clinicopathological parameters of disease progression and patient outcome. Results: p16 immunostaining was not observed in normal urothelium but occurred in 1,576 (63.5%) of cancers including 755 (30.4%) with a strong staining. The fraction of p16 positive cases increased markedly from pTaG2 low grade (9.6%) to pTaG3 high grade tumors (46.5% strongly positive, p<0.0001 for pTaG2 low vs. pTaG3) but continuously decreased from pTaG3 to pT2 (41.3% strongly positive), pT3 (36.5%) and pT4 (33.3%; p=0.0030). Within pT2-4 carcinomas, p16 positivity was also linked to high grade (p=0.0005) but unrelated to overall survival. p53 staining has been recorded as negative in 203 (8.4%), very weak in 373 (15.4%), weak in 1,341 (55.3%), strong in 115 (4.7%), and very strong in 393 (16.2%) cancers. The fraction of tumors with negative (potentially p53 null phenotype), strong, and very strong p53 positivity increased markedly from pTaG2 low grade to pTaG3 high grade tumors (p<0.0001) and from pTaG3 to muscle-invasive pT2-4 cancers (p=0.0007). p53 staining pattern was unrelated to histopathological parameters of malignancy or patient prognosis within pT2-4 carcinomas, however. There was a significant overall association between p53 and p16 expression but strong p16 expression predominated in tumors with very strong, strong, and negative p53 staining. Subset analyses showed that the combination of p53 negative/p16 strongly positive cancers was particularly linked to features of tumor aggressiveness. Conclusion: Our data show that altered function of p53 and p16 immunostaining increases during grade and stage progression although these alterations lack prognostic significance in pT2-4 carcinomas. That high level p16 expression is limited to neoplastic urothelium and that the p53 null phenotype is largely limited to grade 3 and invasive urothelial carcinomas are features with potential diagnostic utility. Citation Format: Simon Schallenberg, Henning Plage, Sebastian Hofbauer, Kira Kornienko, Sarah Weinberger, Paul G. Bruch, Florian Roßner, 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, David Horst, Tobias Klatte, Thorsten Schlomm, Henrik Zecha. Altered p53/p16 expression is linked to urothelial carcinoma progression but is unrelated to prognosis in muscle-invasive tumors. [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 5555.

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.