Tertiary lymphoid structures (TLSs) are emerging indicators of prognosis and therapeutic efficacy specifically in the context of immunomodulatory therapies. TLSs form at mucosal sites as a result of biological aging, following exposure to normal commensal and pathogenic microbes, chronic inflammation, and cancer, and generally evolve to provide local immune protection. Therapy induced TLSs have also been shown as predictive biomarkers in muscle invasive bladder cancer. Given the widespread presence of TLSs in bladder mucosa as a result of biological aging or urinary tract infections or cancer, we hypothesized that tumor associated pre-treatment TLS may inform response to locally administered Bacillus Calmette-Guérin (BCG) in non-muscle invasive bladder (NMIBC) cancer. The goal of this study was to characterize lymphoid aggregates/TLSs in tumors from patients with NMIBC who either responded to or failed BCG immunotherapy.

Using a multiplexed immunofluorescence (IF) assay, we characterized the organizational and developmental status of lymphoid aggregates/TLSs in tumors from NMIBC patients who either responded (recurrence free survival over 2 years) to or failed BCG therapy (recurrence within 1 year of BCG therapy). Formalin fixed paraffin embedded whole tumor sections from patients (n=31) who underwent adequate BCG immunotherapy were subjected to multiplexed immunofluorescence assay using markers specific TLS (CD79a+ B cells, PNAd+ high endothelial venules, CD3+ T cells, CD8+ cytotoxic T cells, CD208+ and CD21+ dendritic cells).

Various stages of pre-existing TLSs were present in tumors from both groups of patients. Higher number of mature TLSs were located in peri-tumoral regions in tumors from patients deemed as BCG non-responders.

Findings from this study demonstrate the significance of pre-treatment TLSs and their spatial organization, as prognostic indicators in NMIBC and will potentially help in the early identification of BCG non-responsive patients.