The urothelial transcriptomic response to interferon gamma predicts T1 recurrence-free and basal/squamous muscle-invasive bladder cancer survival and better targeted strategies for immune checkpoint blocking.

Intravesical Bacillus Calmette-Guerin vaccine (BCG) is an established immunotherapeutic in bladder cancer (BlCa), provoking inflammation leading to tumour-specific immunity. Immune checkpoint blockers such as anti-PD-L1 have potential for enhancing tumour-specific lymphocyte-mediated cytotoxicity in BCG-refractive or advanced disease. In both cases, Interferon-gamma (IFN{gamma}) plays a central role. We investigated the transcriptomic response of normal human urothelium to IFN{gamma} to disentangle mechanisms of BCG and anti-PD-L1 therapy failure. Exposure of differentiated human urothelium to IFN{gamma} resulted in upregulated MHC class I and class II and de novo expression of CXCL9-11 chemokine genes. Normal urothelium expressed only immuno-inhibitory B7 family members: PD-L1 expression was induced by IFN{gamma}, whereas VISTA was expressed constitutively. A urothelial IFN{gamma} response gene set was derived and used for unsupervised clustering of tumours, which predicted longer recurrence-free survival in non-muscle invasive bladder cancer (NMIBC). In muscle invasive bladder cancer (MIBC), the IFN{gamma}-signature split the basal/squamous consensus subtype, with significantly worse overall survival when weak/absent. Normal urothelium has few resident lymphocytes. Tumour cell killing requires recruitment and activation of IFN{gamma}-secreting pro-inflammatory/cytotoxic lymphocytes while surmounting both innate (VISTA) and upregulated (PD-L1) inhibitory mechanisms. This study offers supportive evidence for strategies to enhance immunotherapy via the IFN{gamma} and VISTA/PD-L1 nexus. Patient SummaryImmunotherapy brings promise of harnessing a patients own immune system to seek and destroy malignant cells, but it has yet to deliver widespread clinical benefit. We exposed human urothelium to interferon gamma, a key messenger of the immune system and identified a novel signature of 33 genes that predicted cancers with better outcomes. Our study revealed alternative strategies for targeting checkpoint proteins to improve immunotherapy in the future.