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.

The standard-of-care for patients with higher-risk non-muscle invasive bladder cancer (NMIBC) after tumour resection is intravesical administration of Bacillus Calmette-Guérin (BCG). While this form of adjuvant immunotherapy has improved recurrence-free and progression-free survival, a large proportion of patients experience recurrences within a year of diagnosis. The reasons for this high rate of early recurrence following BCG therapy remain unclear; however, inadequate activation of systemic immunity may be a contributing factor. To address this, we analysed the transcriptomic and chromatin accessibility profiles of peripheral blood mononuclear cells obtained from patients with NMIBC at single-cell resolution before BCG immunotherapy and after five induction doses of BCG. Monocytes from patients who experienced disease recurrence within a year of initiation of BCG therapy (BCG non-responders) exhibited a pro-inflammatory phenotype consistent with age-related immunosenescence prior to BCG immunotherapy. Moreover, inflammation-associated pathways that were active before initiation of BCG therapy in the BCG non-responders were down-regulated after five instillations of BCG. In contrast, these pathways were quiescent before BCG therapy in patients who remained disease-free for at least a year but were markedly up-regulated after five doses of BCG. Genomic regions with accessible chromatin were enriched in activator protein 1 (AP-1) binding sequences in monocytes from BCG-non-responders prior to BCG therapy. AP-1 is a central regulator of the inflammatory phenotype associated with immunosenescence. Our findings indicate that a pre-existing state of innate immunosenescence underlies early disease recurrence following BCG. Patients unlikely to benefit from BCG may be offered alternative therapies early in their disease journey.

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.

BACKGROUND:This study was conducted to evaluate the population-level healthcare resource utilization (HCRU) and costs for men diagnosed with low-risk prostate cancer (PCa) either on active surveillance (AS) or not on AS, in which AS was defined as receiving no treatment within 1 year of diagnosis and two biopsies. METHODS:AS men aged 40 to 105 years, diagnosed with stage I or II PCa, had a prostate-specific antigen (PSA) level < 20 ng/mL, a Gleason score between 5 and 7, and were matched (1:1) with men not receiving AS. The index date is defined as the date 1 year after PCa diagnosis. HCRU and costs were assessed using a macro-based costing methodology and costs standardized to 2023 CAD. Means (SD) and medians (interquartile ranges) per person-year values were reported annually. RESULTS:During the year leading up to the index date, the mean number of HCRU per patient-year (PPY) was significantly lower for the AS cases versus non-AS in terms of cancer clinic visits (1.7 vs. 26.7), hospital outpatient clinic visits (3.6 vs. 4.9), all physician visits (16.3 vs. 17.5), and specialist visits (10.7 vs. 11.6). The mean overall cost PPY was $6100 ± $12,400 for AS cases and $10,400 ± $17,800 for non-AS men (median overall cost PPY= $3500 [IQR: $2100-$5700] vs. $3700 [IQR: $2100-$7200] (p = 0.0001, respectively). CONCLUSIONS:HCRU and costs calculated for AS and non-AS low-risk PCa men indicate the cost savings potential for AS.

Background - Non-muscle invasive bladder cancer (NMIBC) comprises 75% of bladder cancer diagnoses and is characterized by frequent recurrence. Clinical management is guided by risk stratification, which incorporates subjectively assessed tumour grade. Digital pathology enables objective quantification of features from whole slide images (WSIs), however the minimum cancer tissue needed for reliable grading is undefined, and the prognostic significance of small high-grade components in otherwise low-grade cancers remains uncertain. We hypothesize that there exists a minimum number of cancer cells sufficient to capture clinically relevant histopathologic features such as recurrence-free survival. Methods - The cohort included a single digitized WSI from each of 163 patients with stage Ta NMIBC and clinical follow-up data. Quantitative measurements of 11 nuclear features were extracted from each WSI using digital pathology software (Visiopharm, Hørsholm, Denmark). Each WSI was sampled into progressively smaller tissue subsets. At each sampling threshold, Jensen-Shannon (JS) divergence assessed similarity in feature distributions relative to the full WSI. Nuclear features were then summarized at each sampling threshold and used to stratify patients for Kaplan-Meier analysis of their association with recurrence-free survival. Results - We found feature distributions showed the greatest improvement in similarity to the full slide between 5% and 25% tissue sampling, with only smaller gains as more tissue was added. Lesser nuclear diameter and eccentricity retained prognostic significance with limited sampling, whereas ellipticalness, solidity, and convexity were relatively more sensitive to limited sampling. Survival analyses found higher sampling thresholds recovered more significant prognostic features, with thresholds of 20-25% showing feature recovery most comparable to full-slide analysis. Conclusions - This study begins to establish quantitative tissue thresholds for digital pathology in NMIBC. These findings may improve the efficiency and standardization of computational pathology and help define specimen adequacy thresholds.Faculty Supervisors: Dr. Steven Smith, Dr. Nicholas Held

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.

Working Group 1 at ISUP's Cancer Precursors meeting (September 2024) evaluated 5 putative precursors of invasive prostate cancer: high-grade prostatic intraepithelial neoplasia (HGPIN), intraductal carcinoma (IDC), atypical intraductal proliferation (AIP), atypical adenomatous hyperplasia (AAH)/adenosis, and proliferative inflammatory atrophy (PIA). Objectives were to compile recent evidence, interrogate current practices, and vote on recommendations, with 67% approval defined as consensus. Consensus was reached against the reporting of the low-grade form of PIN. HGPIN need not be reported when concomitant cancer or atypical small acinar proliferation suspicious for cancer exists adjacent to it, for biopsy or prostatectomy specimens. Finally, while the clinical significance of unifocal HGPIN in biopsies remains uncertain, there is stronger evidence for multifocal isolated HGPIN as a predictor of subsequent cancer detection. By consensus, multifocal HGPIN should continue being reported. Slight refinement was achieved regarding IDC criteria. The consensus opinion was that a dense cribriform to solid proliferation need not demonstrate marked nuclear atypia/ pleomorphism to qualify as IDC. The inverse scenario of marked atypia without dense cribriform/solid proliferation fell just short (65%) of consensus for IDC. Redesignating cribriform HGPIN as AIP achieved consensus. AIP found alone or with grade group 1 cancer warrants an explanatory comment. However, agreement was not attained to report AIP in the presence of invasive cancer, in either needle biopsy or prostatectomy. Finally, the optional reporting of PIA or AAH/adenosis in biopsies as pertinent negatives both fell short of consensus. This guidance should help pathologists standardize reporting, staying focused on the clinically actionable aspects of these lesions.