Elevated T-cell Exhaustion and Urinary Tumor DNA Levels Are Associated with Bacillus Calmette-Guérin Failure in Patients with Non–muscle-invasive Bladder Cancer

Trine Strandgaard, Sia Viborg Lindskrog, Iver Nordentoft, Emil Christensen, Karin Birkenkamp-Demtröder, Tine Ginnerup Andreasen, Philippe Lamy, Asbjørn Kjær, Daniel Ranti, Yuanshuo Alice Wang, Christine Bieber, Frederik Prip, Julie Rasmussen, Torben Steiniche, Nicolai Birkbak, John Sfakianos, Amir Horowitz, Jørgen Bjerggaard Jensen, Lars Dyrskjøt

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


Background: The functional status of immune cells in 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 post-BCG high-grade (HG) recurrence using multiomics analysis. Design, setting, and participants: Patients with BCG-treated NMIBC (n = 156) were included in the study. Metachronous tumors were analyzed using RNA sequencing (n = 170) and whole-exome sequencing (n = 195). Urine samples were analyzed for immuno-oncology–related proteins (n = 190) and tumor-derived DNA (tdDNA; n = 187). Outcome measurements and statistical analysis: The primary endpoint was post-BCG HG recurrence. Cox regression and Wilcoxon rank-sum, t, and Fisher's exact tests were used for analyses. Results and limitations: BCG induced activation of the immune system regardless of clinical response; however, immunoinhibitory proteins were observed in the urine of patients with post-BCG HG recurrence (CD70, PD1, CD5). Post-BCG HG recurrence was associated with post-BCG T-cell exhaustion (p = 0.002). Pre-BCG tumors from patients with post-BCG T-cell exhaustion had high expression of genes related to cell division and immune function. A high predicted post-BCG exhaustion score for pre-BCG tumors was associated with worse post-BCG HG recurrence–free survival (HGRFS; p = 0.002). This was validated in independent cohorts. Pre-BCG class 2a and 2b tumors (UROMOL2021 scheme) were associated with worse post-BCG HGRFS (p = 0.015). Post-BCG exhaustion was observed in patients with high pre-BCG neoantigen load (p = 0.017) and MUC4 mutations (p = 0.002). Finally, the absence of post-BCG tdDNA clearance identified patients at high risk of recurrence (p = 0.018). The retrospective design and partial overlap for analyses are study limitations. Conclusions: Post-BCG HG recurrence may be caused by T-cell exhaustion. Tumor subtype and pre-BCG tumor characteristics may identify patients at high risk of post-BCG HG recurrence. Urinary measurements have potential for real-time assessment of treatment response. Patient summary: A dysfunctional immune response to bacillus Calmette-Guérin (BCG) therapy may explain high-grade recurrences of bladder cancer.

Original languageEnglish
Pages (from-to)646-656
Number of pages11
JournalEuropean Urology
Issue number6
StatePublished - Dec 2022


  • Bacillus Calmette-Guérin
  • Biomarkers
  • Bladder cancer
  • Response
  • T-cell dysfunction


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