Abstract 1282: Elevated T cell exhaustion and immune cell infiltration is associated with BCG failure in patients with non-muscle invasive bladder cancer
Authors: Strandgaard Trine , Nordentoft Iver , Christensen Emil , Lindskrog Sia , Lamy Philippe , Birkenkamp-Demtröder Karin , Steiniche Torben , Jensen Jørgen Bjerggaard , Dyrskjøt Lars 
Published: June 2022

Abstract
Introduction: Gold standard treatment in patients with high-risk non-muscle invasive bladder cancer (NMIBC) includes Bacillus Calmette-Guérin (BCG), which activates the immune cells to kill remaining cancerous cells after surgical removal of the tumors. However, 40% of patients do not have a clinical benefit of BCG. The presence of immune cells, their functional state, as well as genomic alterations in the tumor and the normal appearing urothelial tissue, defined as field effect, may have significant impact on therapeutic outcome.
Materials and methods: To investigate this, we analyzed samples from 156 patients diagnosed with NMIBC, including 237 tumors, 569 urine samples, and 305 biopsies of adjacent normal appearing urothelium. Patients had a median follow-up time of 8 years. 70 patients (45%) progressed to muscle-invasive bladder cancer or experienced early high grade recurrence within two years after ended BCG treatment, defined as BCG failure. Urinary levels of 92 immune-oncology related proteins were measured in pre- and post-treatment samples using the Olink proteomics platform. Total RNA- and whole exome sequencing (WES) data was generated from tumors before and after BCG. Clonal patient-specific mutations were selected for deep-targeted sequencing of the adjacent normal appearing biopsies. The level of field effect was defined as the mean of the variant allele frequency for mutations observed in normal biopsies.
Results: We found that treatment with BCG activated the immune system regardless of clinical outcome. However, patients differed in urinary protein profile after BCG depending on their clinical response. Transcriptomic analysis of tumors showed that UROMOL2021 subtypes were significantly associated with outcome and clinical response (p=0.018). Paired tumors showed a shift in subtype to an immune infiltrated subtype (class 2b) after treatment in 36% of cases. Patients with BCG failure showed signs of immune exhaustion after treatment indicated by higher expression of the T cell exhaustion marker genes CTLA4 (p=0.0067), LAG3 (p=0.00012), TIM-3 (p=0.022), KLRG1 (p=0.028), and PD-1 (p=0.047), and higher immune cell infiltration (p=0.011). Genomic features such as mutational signatures and mutational load were not predictive of clinical response. Interestingly, BCG-responsive patients had a high level of field effect before BCG (p=0.0026), suggesting that field effect could lead to increased BCG efficacy.
Conclusion: BCG treatment was associated with immune system activation reflected in both urine and tissue samples. In patients with BCG failure, we observed an immune exhausted phenotype after treatment, and we observed a high level of field effect before treatment in responsive patients. Collectively, this could indicate that increased immunogenicity and prolonged immune activation are key factors in BCG treatment responsiveness.
Citation Format: Trine Strandgaard, Iver Nordentoft, Emil Christensen, Sia Lindskrog, Philippe Lamy, Karin Birkenkamp-Demtröder, Torben Steiniche, Jørgen Bjerggaard Jensen, Lars Dyrskjøt. Elevated T cell exhaustion and immune cell infiltration is associated with BCG failure in patients with non-muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1282.