Data from Atypical B cells promote cancer progression and poor response to Bacillus Calmette Guérin in non-muscle invasive bladder cancer

Abstract Formation of tertiary lymphoid structures (TLS) within the bladder microenvironment because of chronic mucosal inflammation has been associated with variable clinical outcomes. While the immune cell composition and functional states of TLS have been characterized in both non-invasive and muscle-invasive bladder tumors, the TLS-adjacent tumor epithelial compartments remain poorly characterized. Evaluation of a 16-gene TLS signature in treatment-naïve tumor bulk RNA sequencing profiles from 283 non-muscle invasive bladder tumors, from patients treated with Bacillus Calmette-Guérin (BCG) immunotherapy, and 348 muscle-invasive bladder tumors from patients treated with immune checkpoint inhibitor therapy revealed overlapping enrichment of immune exhaustion pathways. High TLS gene expression scores correlated with upregulation of immune exhaustion, hypoxia, and epithelial-to-mesenchymal transition (EMT) pathways in tumors from both cohorts. Spatial whole transcriptomic analysis of tumor sections with high TLS density, revealed enrichment of genes associated with EMT, angiogenesis, extracellular matrix remodeling, and B cell receptor signaling pathways in tumor epithelial regions adjacent to TLS, whereas those distant from TLS exhibited enrichment of IFN-γ, TNF-α/NF-κB, p53, and metabolic pathways. Multiplex immunofluorescence further identified co-localization of exhausted immune cell populations within the core and periphery of peri-tumoral TLS. These findings indicate that a pro-tumorigenic microenvironment associated with disease progression in bladder cancer exists within peri-tumoral TLS and potentially a factor underlying contrasting therapeutic associations potentially driven by live microbial versus targeted immunomodulatory therapy in NMIBC and MIBC.

BBN exposure and BCG treatment alters plasma immunoglobulin profiles in a sex differential manner.

Effect of transient B‐cell depletion and BBN exposure on urothelium of BBN exposed female and male mice.

Splenic ABC expansion is enhanced by a combination of BBN exposure and BCG treatment.

Tumor-adjacent TLS characterized using multiplex IF and NanoString GeoMx DSP.

Gating strategy for identifying different subsets of T cells.

Abstract The majority of patients treated with Bacillus Calmette–Guérin (BCG) immunotherapy, for non-muscle invasive bladder cancer (NMIBC), experience early recurrence due to pre-existing mucosal immune dysfunction. Since B cell are mucosal immune sentinels, we characterized the systemic and local B cell responses in 45 patients with NMIBC. Expansion of circulating atypical B cells (ABCs) following repeated BCG instillation, expanded IgG autoantibody repertoire, progressive IgG reactivity against BCG antigens, and higher tumor IgG deposition, were features of patients who recurred early. Integrated spatial immunophenotyping and single cell spatial transcriptomic analysis of corresponding tumors revealed increased ABCs within tertiary lymphoid structures, and co-localization with PD-1⁺ B cells, regulatory T cells, and CD163⁺ macrophages. Independent validation in two patient cohorts (total n = 409), revealed a significant association between high expression of the ABC specific, FCRL5 , and poor outcomes. Our study identifies ABCs as key mediators of poor response to BCG in patients with high-risk NMIBC.

Expansion of ABCs in the systemic and local bladder microenvironment.

Contour plots showing myeloid and T cell populations.

B‐cell differentiation to ABCs following in vitro treatment with IFN-γ, IL-21 and BCG is sex-dependent.

Gating strategy for identifying atypical B cells (ABCs) and myeloid cell population.

Immune infiltration in the bladder microenvironment after repeated BCG treatment and B‐cell depletion in BBN exposed mice.

B‐cell depletion alters expression profiles of immune regulatory genes in the bladder microenvironment.

Intravesical Bacillus Calmette-Guérin (BCG) immunotherapy is the gold standard for high-risk NMIBC, however over half of patients experience early recurrence or disease progression, highlighting the need for reliable predictive biomarkers. Our previous research identified that B cell dominant peri-tumoral tertiary lymphoid structures (TLSs) evolve during chronic carcinogenesis in a subset of patients who experience early recurrence or progression. TLSs generally correlate with favorable outcomes following treatment with immune checkpoint inhibitors, chemotherapy or oncolytic viruses. In contrast, their abundance in peri-tumoral regions in BCG-naïve NMIBC tumors associates with poor prognosis. This highlights the importance of their in-depth characterization and further exploring their biomarker potential in NMIBC. Peri-tumoral TLSs and tumor epithelial regions were profiled using the NanoString GeoMx Digital Spatial Profiler based whole transcriptome analysis. Multiplex Immunofluorescence was performed to infer spatial profiles of B, T and myeloid cell functional states as well as immune checkpoint proteins. Spatial whole transcriptome analysis displayed increased expression of genes associated with antigen presentation, complement activation, B cell recruiting chemokines, and immunoglobulins in the TLSs regions. Tumor epithelial regions located adjacent to TLSs showed elevated expression of genes reflective of a luminal subtype. Increased PD-1+ cells in the peri-tumoral regions and enrichment of luminal subtype genes in the epithelial compartment indicated cancer cell intrinsic aggressive features associated with immune exclusion. The novel findings from this study will help establish peri-tumoral TLSs as biomarkers for predicting BCG therapy response, enabling early identification of patients who may benefit from alternative immunomodulatory treatments like oncolytic viruses, gemcitabine-docetaxel chemotherapy, or immune checkpoint inhibitors. Kartik Sachdeva. Characterizing peri-tumoral tertiary lymphoid structures in non-muscle invasive bladder cancer using a spatial whole transcriptomics approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6475.

Poor response to Bacillus Calmette-Guérin (BCG) immunotherapy remains a major barrier in the management of patients with non-muscle-invasive bladder cancer (NMIBC). Among the multiple factors contributing to poor outcomes, a B cell infiltrated pre-treatment immune microenvironment of NMIBC tumors has emerged as a key determinant of response to BCG. The mechanisms underlying the paradoxical roles of B cells in NMIBC are poorly understood. Here, we show that B cell dominant tertiary lymphoid structures (TLSs), a hallmark feature of chronic mucosal immune response, are abundant and located close to the epithelial compartment in pre-treatment tumors from BCG non-responders. Digital spatial proteomic profiling of whole tumor sections revealed higher expression of immune exhaustion-associated proteins within the TLSs from both responders and non-responders. Chronic local inflammation, induced by the N-butyl- N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen, led to TLS formation with recruitment and differentiation of the immunosuppressive atypical B cell (ABCs) subset within the bladder microenvironment, predominantly in aging female mice compared to their male counterparts. Depletion of ABCs simultaneous to BCG treatment delayed cancer progression in female mice. Our findings provide the first evidence indicating the role of ABCs in BCG response and will inform future development of therapies targeting the B cell exhaustion axis.

Non-muscle invasive bladder cancer (NMIBC) constitutes a significant clinical challenge, with over 50% of patients experiencing poor clinical outcomes in the form of early recurrence or progression following treatment with Bacillus Calmette-Guerin (BCG) immunotherapy. The pre-treatment tumor immune microenvironment (TIME) is an established determinant of response to BCG. This study explores the spatial profiles of CD79a+ B cells, CD163+ M2-like macrophages, proliferating and tissue-resident phenotypes of T cells, along with PD-1/PD-L1 checkpoint expression in pre-BCG treatment tumors of 173 patients (139 males, 34 females). Multiplex immunofluorescence staining of a tumor tissue microarray, revealed elevated infiltration of CD79a+ B cells, CD163+ M2-like macrophages, CD103+ cells, and CD8+ T cells at the tumor invasive margins. Increased epithelial PD-L1 immune-checkpoint expression in tumors was observed in female and male patients who exhibited significantly shorter recurrence-free survival (RFS). Importantly, high CD79a+ B cell density in BCG-treated females in both stromal and epithelial compartments exhibited significantly shorter RFS and progression-free survival compared to males. Stromal CD79a+ B cell density was positively correlated with M2-like macrophages, CD8+ T cells, CD103+ cells and PD-1 expressing cells. CD79a+ B cells, CD103+ cells, and M2-like macrophage density were associated with higher grade and enriched in basal subtype tumor. This study highlights the significance of an understudied role of B cells and their cellular neighborhoods in the pre-treatment TIME and BCG-therapy response. Overall, findings from this study underscore the importance of considering sex-related immunobiological differences in the stromal compartments of bladder tumors towards the development of optimal therapeutic targeting strategies.