Systemic and tumor intrinsic expansion of FCRL5 expressing B cells associates with poor response to Bacillus Calmette-Guerin immunotherapy in patients with non-muscle invasive bladder cancer

Intravesical Bacillus Calmette-Guerin (BCG) immunotherapy remains the standard treatment for intermediate and high-risk non-muscle invasive bladder cancer (NMIBC), yet more than half of the patients do not respond and experience recurrence or progression. BCG induced humoral immune responses remain poorly defined in patients with NMIBC. Building upon our previous findings showing the pathogenic role of atypical B cells (ABCs) in cancer progression, and following repeated intravesical treatment with BCG, in an aging murine model of bladder cancer, we conducted a longitudinal study to characterize B cell associated local and systemic responses in 45 patients (37 males and 8 females) with high-risk NMIBC who underwent treatment with BCG. Peripheral B cell immune phenotyping, B cell single-cell transcriptomics, spatial multi-omics, and systemic proteomics were performed. We identified expansion of circulating ABCs following the 4th BCG instillation, as a defining feature of patients who experienced early recurrence following BCG therapy. Spatial mapping of corresponding pre- and post-BCG recurrent tumors, at single cell transcriptomic and proteomic levels, revealed preferential enrichment of ABCs within tumor-adjacent stroma and tertiary lymphoid structures, where they co-localized with PD-1 B cells, regulatory T cells, and CD163 macrophages, forming immunosuppressive niches. BCG non-responders exhibited IgG skewed antibody responses at both local and systemic levels with expanded IgG autoantibody repertoires, progressive IgG reactivity against BCG antigens, and higher IgG deposition within the tumor microenvironment. Independent validation in tumors from two independent cohorts (total n = 409) of patients treated with BCG immunotherapy, revealed a significant association between high expression of the ABC specific transcript, FCRL5, and shorter recurrence and progression free survival. Findings from this study demonstrate that a BCG unresponsive state arises within a pre-existing ABC-dominated immune landscape that is further amplified during repeated BCG instillations. Our study identifies a novel role of ABCs as key regulators of local and systemic humoral immune dysfunction in high-risk NMIBC, and highlights ABC signatures as a potential predictive biomarker of response to BCG.