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

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.

Intermediate-risk prostate cancer (PCa) poses a challenge for treatment decisions, made more complex by its heterogeneous nature. Growing evidence highlights the critical role of the tumor microenvironment (TME), in cancer initiation, progression, and response to treatment, underscoring the importance of profiling cancer cells but also the TME within their spatial context. Here, we present single-cell in situ transcriptomic and spatial proteomics results from 300 intermediate-risk PCa prostatectomy samples. Multiple regions of the prostate, including regions of the index lesion, secondary lesions, regions of benign and normal were sampled then analyzed using Bruker’s CosMx SMI platform for a 6000-plex RNA assay in a subset of patients; in addition to proteomic profiling using the GeoMx DSP platform. Clustering assigned cells into 15 cell types based on their transcriptional profiles. Each cell type was tested for significantly different proportions in tumor versus non-tumor samples. The cell type with the greatest bias had a 9-fold greater proportion in tumor samples (adjusted P < 0.01). ERG, largely associated with the TMPRSS2-ERG fusion in PCa, was most specifically expressed in cells of this type. Conversely, the cell type with the greatest bias towards non-tumor samples (3-fold greater proportion, adjusted P < 0.01) is characterized most specifically by the expression of MSMB, which encodes an immunoglobulin binding factor previously found to have decreased expression in PCa. Other evidence of intratumoral heterogeneity was found in samples that are not dominated by these stereotypical cell types. For example, a relatively higher-grade sample from an index tumor possessed high proportion of the tumor-dominant cell type whereas a relatively lower-grade sample from the same tumor did not. Similarly, the tumor and non-tumor samples arising from the same patient showed similar cell type composition, highlighting inter-patient heterogeneity. Moreover, we identified proteins with significantly different expression in tumor versus non-tumor samples. In the TME, EpCAM and CD56 were significantly overexpressed and underexpressed in tumor versus non-tumor regions, respectively (adjusted P < 0.01). In the epithelium, cleaved caspase-9, which initiates the caspase cascade leading to apoptosis, was the most significantly underexpressed protein in tumor samples (adjusted P < 0.01). However, the expression of the mRNA encoding caspase-9 is not highly correlated with the expression of cleaved caspase-9, highlighting the added benefit of protein expression profiling. Taken together, this multi-modal spatial analysis of multiple samples from the same PCa patients facilitates a detailed understanding of early PCa for biomarker discovery and pathways of therapeutic intervention. Anna Y. Lee, Megan Hopkins, Linda Liao, Vida Talebian, Tamara Jamaspishvili, David M. Berman, Melanie Spears, Jane Bayani. Revealing the transcriptional and proteomic spatial neighborhoods of early prostate cancer and the tumor microenvironment [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 777.