Introduction: Immunotherapy with live-attenuated Bacillus Calmette-Guérin (BCG) is the standard treatment for non-muscle invasive bladder cancer (NMIBC). Unfortunately, 40% of patients do not benefit clinically from this treatment. It was recently shown that the success to various immunotherapies rely on a healthy gut microbiome, which is negatively altered by antibiotic (ATB) intake. However, the impact of ATB use on BCG treatment is unclear.

Methods: This retrospective study involved NMIBC patients who started a first BCG immunotherapy at CHU de Québec from 2009-2019. We reviewed ATB in prescriptions and medical records. Patients were considered exposed when ATB use lasted at least 3 days within 3 months before BCG initiation. The impact of ATB on BCG response was assessed using recurrence-free survival (RFS) and progression-free survival (PFS) using Kaplan-Meier analyses. To test the impact of ATB on BCG efficacy in vivo, C3H mice were randomized to either receive a broad-spectrum antibiotic regimen for one week, or continue this ATB treatment throughout the experiment. Mice were s.c injected with MBT-2 bladder cancer cells and received weekly intratumoral BCG treatments.

Results: Of 622 NMIBC patients, 77 (12%) were exposed to ATB before BCG treatment, while 545 (88%) were not. Compared to unexposed patients, ATB-exposed patients receiving BCG had a decreased RFS (HR:0.68, CI95%:1.38-2.84, p=0.0002) and a trend for decreased PFS (HR:0.68, CI95%:0.95-4.1, p=0.06). In the preclinical in vivo model, the prolonged use of ATB reduced the anti-tumor activity of BCG immunotherapy compared to controls (p<0.01).

Conclusions: In this first large single-site retrospective analysis of ATB use for at least 3 days before BCG treatment in NMIBC patients, ATB use within 3 months before BCG reduces treatment response. Prolonged ATB in vivo reduces BCG’s antitumor activity. Our findings suggest that the gut microbiota may play a crucial role in the efficacy of BCG immunotherapy.