Background. Intravenous immune checkpoint inhibition achieves a 40% three-month response in BCG-unresponsive non-muscle invasive bladder cancer (NMIBC) with carcinoma in situ (CIS). Yet only half of early responders will continue to be disease free by 12 months, and resistance mechanisms are poorly defined. Objective: We assessed the molecular features associated with response to immunotherapy in BCG unresponsive non-muscle invasive bladder cancers treated with pembrolizumab. Design, Setting, and Participants: We performed digital spatial profiling (DSP) of BCG unresponsive NMIBC tumors before and after IV pembrolizumab therapy. Intervention: Pembrolizumab was administered intravenously in patients with NMIBC at the time of recurrence after BCG therapy. Biopsies were obtained before starting IV pembrolizumab and three months post-treatment. Outcomes and Statistical Analysis: Spatial gene expression profiling of the tumor niche pre- and post IV pembrolizumab. Results and Limitations: We evaluated 119 regions of interest (ROIs) from five patients, which included 60 epithelial (PanCK+) and 59 stromal segments (PanCK-). ROIs from responders had distinct expression signatures from non-responders for both the tumor and TME. Responders were more likely to have a dynamic change in expression after pembrolizumab than non-responders. A major limitation of this study was the number of patients evaluated. Conclusion: For the first time, we have identified distinct expression signatures associated with response and resistance to IV pembrolizumab in NMIBCs. Further research with more patients and alternative checkpoint inhibitors is essential to validate our findings. Patient Summary: We identify the molecular features of tumors associated with response to pembrolizumab for patients with BCG unresponsive NMIBCs.

KM: None NF: Research Funding from the AUA Foundation JM advisory board/consulting: Merck, AstraZeneca, Incyte, Janssen, BMS, UroGen, Prokarium, Imvax, Pfizer, Seagen/Astellas, Ferring; Research Funding: VHA, NIH, DoD, Compensation for talks/educational courses: AUA, OncLive, Olympus, UroToday; Clinical Trials: SWOG, Genentech, Merck, AstraZeneca

This study was funded by the Merck Investigator Studies Program (MISP) and the Robert H. Lurie Cancer Center. JM is supported by grants from the VHA BX005599 and BX003692. This work was supported by the Northwestern University RHLCCC Flow Cytometry Facility and the Cancer Center Support Grant (NCI CA060553).

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