Background: The functional status of immune cells within the tumor microenvironment and tumor characteristics may explain Bacillus Calmette Guérin (BCG)-failure in high-risk non-muscle invasive bladder cancer (NMIBC). Objective: To characterize molecular correlates of BCG-failure using a multiomics approach. Design, Setting, and Participants: BCG-treated NMIBC patients (n=156) were included. Metachronous tumors were analyzed using RNA-sequencing (n=170) and whole exome sequencing (n=198). Urine samples were analyzed for immune-oncology related proteins (n=190), and tumor-derived DNA (tdDNA; n=192). Outcome Measurement and Statistical Analysis: Primary endpoint was BCG-failure. Cox regression, Wilcoxon Rank Sum test, t-test or Fisher,s exact test were used. Results and Limitations: BCG caused activation of the immune system regardless of clinical response; however, immune-inhibitory proteins were observed in the urine of BCG-unresponsive patients post-treatment (CD70, PD1, CD5). BCG-failure was associated with post-BCG T-cell exhaustion (p=0.0021). Pre-BCG tumors from patients with post-BCG T-cell exhaustion were characterized by high expression of cell division and immune-related genes. A high post-BCG exhaustion prediction score in pre-BCG tumors was associated with worse post-BCG high-grade recurrence free survival (HGRFS), reflecting BCG-failure (p=0.0084). Pre-BCG tumors of class 2a and 2b were likewise associated with worse post-BCG HGRFS(p=0.0023). Post-BCG exhaustion was observed in patients with high pre-BCG neoantigen load (p=0.023) and mutations in MUC4 (p=0.0007). Finally, absence of post-BCG tdDNA clearance identified patients at high risk of recurrence (p=0.028). The retrospective design, lack of maintenance BCG, and partial overlap in analyses are limitations to the study. Conclusions: BCG failure may be caused by T-cell exhaustion. Tumor subtype and Pre-BCG tumor characteristics may identify patients at high risk of BCG-failure prior to treatment. Urinary measurements have the potential to be used as a real-time assessment of treatment response. Patient Summary: A dysfunctional immune response to BCG therapy may explain lack of response to the treatment.

Lars Dyrskjøt has sponsored research agreements with C2i, AstraZeneca, Natera, Photocure, and Ferring; has an advisory/consulting role at Ferring; and is Chairman of the Board in BioXpedia A/S. Jørgen Bjerggaard Jensen is proctor for Intuitive Surgery; is a member of advisory board for Olympus Europe, Cephaid, and Ferring; and has sponsored research agreements with Medac, Photocure ASA, Cephaid, and Ferring.

This work was funded by Ferring Pharmaceuticals, the Danish Cancer Society, Aarhus University, Dagmar Marshalls Fond, Christian Larsen og Dommer Ellen Larsens Legat, Fabrikant Einar Willumsens Mindelegat, the Danish Medical Association, Danish Cancer Biobank, and Dansk Kraeftforskningsfond.

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The Danish National Committees on Health Research Ethics gave ethical approval for this work (#1708266).

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