Bacillus Calmette-Guérin Treatment Research Articles

Bacillus Calmette-Guérin immunotherapy induces an efficient antitumor response to control murine melanoma depending on MyD88 signaling

Bacillus Calmette-Guérin (BCG) is the first line treatment for bladder cancer and it is also proposed for melanoma immunotherapy. BCG modulates the tumor microenvironment (TME) inducing an antitumor effective response, but the immune mechanisms involved still poorly understood. The immune profile of B16-F10 murine melanoma cells was assessed by infecting these cells with BCG or stimulating them with agonists for different innate immune pathways such as TLRs, inflammasome, cGAS-STING and type I IFN. B16-F10 did not respond to any of those stimuli, except for type I IFN agonists, contrasting with bone marrow-derived macrophages (BMDMs) that showed high production of proinflammatory cytokines. Additionally, we confirmed that BCG is able to infect B16-F10, which in turn can activate macrophages and spleen cells from mice in co-culture experiments. Furthermore, we established a subcutaneous B16-F10 melanoma model for intratumoral BCG treatment and compared wild type mice to TLR2-/-, TLR3-/-, TLR4-/-, TLR7-/-, TLR3/7/9-/-, caspase 1-/-, caspase 11-/-, IL-1R-/-, cGAS-/-, STING-/-, IFNAR-/-, MyD88-/-deficient animals. These results in vivo demonstrate that MyD88 signaling is important for BCG immunotherapy to control melanoma in mice. Also, BCG fails to induce cytokine production in the co-culture experiments using B16-F10 and BMDMs or spleen cells derived from MyD88-/- compared to wild-type (WT) animals. Immunotherapy with BCG was not able to induce the recruitment of inflammatory cells in the TME from MyD88-/- mice, impairing tumor control and IFN-γ production by T cells. In conclusion, MyD88 impacts on both innate and adaptive responses to BCG leading to an efficient antitumor response against melanoma.

Abstract B008: High expression of the exhaustion markers PD1 and PD-L1 in non-muscle invasive bladder cancer is associated with poor outcome following Bacillus Calmette-Guérin immunotherapy

Abstract Introduction: The recommended treatment for high-risk non-muscle invasive bladder cancer (NMIBC) is intravesical instillations of Bacillus Calmette-Guérin (BCG). However, 40 % experience recurrence within 5 years despite completing BCG treatment. T cell exhaustion has been associated with poor outcome following treatment with BCG. Here we investigated if T cell exhaustion, characterized by protein expression of PD1 and PD-L1 measured in paired samples obtained before and after BCG treatment could further explain BCG response and help predict outcome in patients with NMIBC. Methods: We included 111 patients with NMIBC, from which we had 183 TURB-T samples obtained before and after BCG treatment. Using immunohistochemistry (IHC) staining of sections from tissue microarrays (TMAs) with triplicate core biopsies, we investigated the protein expression of the exhaustion markers PD1 (clone EP239) and PD-L1 (clone 22c3). Data was analyzed using digital pathology software (Visiopharm®), where tissue areas were defined as either carcinoma or stroma based on PanCK staining. Cells were scored as positive when detection of a nucleus was combined with a positive PD1 or PD-L1 stain, which was based on visually defined thresholds to differentiate between positive and unspecific staining. In total, 167 samples from 105 patients were considered suitable for further analysis (cell count > 200 in carcinoma and stromal areas) with 2-3 cores per sample, resulting in a total of 441 tissue cores. The average cell count was 5732 per core (defined by positive nuclei stain). Stratification of samples into high or low expression was based on a median split of the positive cell fraction. Results: When investigating the number of PD1 and PD-L1 positive cells in the tumors (carcinoma and stroma combined), we observed that PD-1 expression significantly increased with tumor stage in pre- (p=0.002) and post-BCG (p=0.006) tumor samples. PD1 expression was also increased in high-grade tumors compared to low-grade tumors in pre-BCG samples (p = 0.001). Furthermore, we observed a significant association between tumors of higher stage and high PD-L1 expression in the pre-BCG samples (p = 0.007). Patients with low expression of PD1 and PD-L1 in the pre-BCG tumor samples had a superior high-grade recurrence-free survival (HG-RFS) compared to patients with high PD1 (p=0.002) and PD-L1 (p=0.021) expression. In addition, low expression of PD-L1 in pre-BCG tumors was correlated with a better progression-free survival compared to the patients with high PD-L1 expression (p = 0.013). Conclusion: Protein expression of PD1 and PD-L1 in pre-BCG tumor samples were correlated to higher stage and grade as well as worse HGFRS, indicating that T cell exhaustion plays an important role in resistance to BCG treatment. Citation Format: Tine G. Andreasen, Trine Strandgaard, Jørgen B. Jensen, Lars Dyrskjøt. High expression of the exhaustion markers PD1 and PD-L1 in non-muscle invasive bladder cancer is associated with poor outcome following Bacillus Calmette-Guérin immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B008.

Abstract B004: Deep learning unveils molecular footprints in histology: predicting molecular subtypes from bladder cancer histology slides

Abstract Introduction: Transurethral resection followed by intravesical bacillus Calmette-Guerin (BCG) is the standard of care for high-risk non-muscle-invasive bladder cancer (HR-NMIBC) patients. However, ≈20% of HR-NMIBC patients progress to muscle-invasive or metastatic disease, which is associated with a high-risk of death. Early identification of patients who are unlikely to benefit from BCG treatment could benefit from an early radical cystectomy or bladder-sparing alternatives. Previously, we showed that molecular subtyping or HR-NMIBC identified BCG response subtypes (BRS 1/2/3). BRS3 tumors were associated with a poor response to BCG and a high-risk of progression. However, molecular subtyping relies on resource-intensive and costly sequencing methods such as next-generation sequencing, and microarray analysis. We employed deep learning (DL) to predict BRS3 vs BRS1/2 molecular subtypes from digitized histology slides. Methods: Haematoxylin and eosin (H&E)-stained slides were digitized, and quality control was performed. The areas where the RNA was extracted were annotated pixel-wise by a trained pathology assistant, using QuPath. Image tiles of 512 × 512 pixels at 10X, 20X, and 40X magnifications were extracted with a 25% overlap. For classification, BRS1 and BRS2 images were grouped, while BRS3 images were categorized separately. After applying Macenko normalization, tiles were split based on individual patients into a 75/25 training/validation ratio via stratified 4-fold cross-validation. Four DL models (DenseNet, Inception, ShuffleNet, ResNet) were trained to classify tiles into BRS3 and BRS1/2, with tile labels based on the patient’s subtype. Patient-level classification was performed using majority voting from tile predictions. Model efficacy was gauged using the area under the ROC curve (AUC). Results: Out of 245 H&E slides, 45 were discarded after quality control. From 200 patients – with a 70:30 distribution between BRS1&2 and BRS3 The DenseNet model at 10x magnification was the best-performing model, achieving the highest AUC compared to all other models and magnifications. On the image tile level, the best DenseNet achieved an average AUC of 0.65 with a standard deviation of 0.03 across the four folds of the validation set. At the patient-level prediction, the best DenseNet reached an average AUC of 0.80 and a standard deviation of 0.05 on the validation set. Conclusion: Our study demonstrates that deep learning classifies BSR3 from BSR1 and 2 subtypes from H&E slides. This method holds potential to identify BRS3 HR-NMIBC patients who may benefit from alternative treatments than BCG. With further refinement, this deep learning strategy can become a key tool in histology, aiding in swift, affordable, and efficient subtype identification. Hence, this method has the potential to bridge the gap between molecular techniques and clinical practice. Future research should prioritize evaluating the model on an external test cohort and consider predicting additional molecular markers to fine-tune prediction outcomes. Citation Format: Farbod Khoraminia, Flouros C de Jong, Farhan Akram, Geert Litjens, Maarten D.J. Jansen, Alberto Nakuama Gonzalez, Danique Lichtenburg, Andrew Stubbs, Nadieh Khalili, Tahlita C.M. Zuiverloon. Deep learning unveils molecular footprints in histology: predicting molecular subtypes from bladder cancer histology slides [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B004.

Abstract PR004: Spatial proteomics and transcriptomics reveal an altered immune cell landscape in bladder cancer patients unresponsive to BCG treatment

Abstract Introduction: Patients with high-risk non-muscle invasive bladder cancer (NMIBC) are recommended treatment with Bacillus Calmette-Guérin (BCG). The therapeutic effect of BCG is highly dependent on the host immune system and the tumor microenvironment (TME). The cellular composition and the functional status of the TME have been shown to play crucial roles for treatment efficacy. However, much is still unknown regarding the cellular interactions and mechanisms of BCG. Using spatial proteomics and transcriptomics, we investigated the immune cell landscape in a cohort of BCG treated patients. Materials and methods: A total of 183 tumors from 111 patients with NMIBC treated with BCG were included on a tissue microarray (TMA). The TMA included paired pre- and post-BCG tumors. We analyzed the tumor tissue using Imaging Mass Cytometry (IMC; StandardBioTools) for simultaneous detection of 35 immune and tumor-related proteins at single cell resolution. Ilastik was used to generate probability maps and DeepCell Mesmer was used for cell segmentation. Markers were quantified for the mean intensity per cell. GeoMX Digital Spatial Profiling (DSP) was used for spatial proteomics and transcriptomics profiling of tumor and stromal areas, targeting 63 protein targets and whole transcriptome amplification, respectively. Results: We identified a total of 364,504 cells in the tumor tissue using IMC. After computing a neighborhood graph using the log normalised and z-scaled signal and embedding it using UMAP, leiden clustering was performed to detect communities of unique cell types by interrogating the presence of phenotypic markers. We identified major lineages such as macrophages, CD8 T cells, dendritic cells (DCs), NK cells and neutrophils in the TME of analyzed tumors. Specifically, we observed more CD8 T cells after BCG (p=0.015), especially in females (p=0.048). Females also had higher levels of DCs and macrophages after treatment compared to males (p=0.024 and p=0.048). Patients who progressed to MIBC after BCG had higher numbers of CD8 T cells and NK cells prior to BCG (p=0.039 and p=0.021). Prolonged exposure to interferons has been associated with immune evasion and tumor cell proliferation. In concordance, we found high pretreatment protein expression of IFNα and IFNγ in tumor regions from patients with a pronounced signature of CD8 T cell exhaustion after BCG treatment using GeoMX DSP proteomic analysis. Data analysis is currently ongoing, and additional results will be presented at the conference. Conclusion: The composition and functional status of the TME is associated with clinical and biological features such as immune cell abundance, CD8 T cell exhaustion, sex and progression. A greater understanding of the TME may help identify patients unresponsive to BCG earlier and improve the understanding of biological differences in tumor development and aggressiveness. This may ultimately improve patient outcomes. Citation Format: Trine Strandgaard, Tine Ginnerup Andreasen, Tessa Jane Divita, Liina Salminen, Sean Houghton, Kasper Thorsen, Iver Nordentoft, Iyinyeoluwa Okulate, Alexander Schmitz, Søren Riis Paludan, John Sfakianos, Amir Horowitz, Jørgen Bjerggaard Jensen, Lars Dyrskjøt. Spatial proteomics and transcriptomics reveal an altered immune cell landscape in bladder cancer patients unresponsive to BCG treatment [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR004.

Induction and maintenance of sequential intravesical gemcitabine/docetaxel for intermediate and high-risk non-muscle invasive bladder cancer with different dosage protocols.

The combination of sequential intravesical gemcitabine and docetaxel (Gem/Doce) chemotherapy has been considered a feasible option for BCG(Bacillus Calmette-Guérin) treatment in non-muscleinvasive bladder cancer (NMIBC), gaining popularity during BCG shortage period. We seek to determine the efficacy of the treatment by comparing Gem/Doce induction alone vs induction with maintenance, and to evaluate the treatment outcomes of two different dosage protocols. A bi-center retrospective analysis of consecutive patients treated with Gem/Doce for NMIBC between 2018 and 2023 was performed. Baselinecharacteristics, riskgroup stratification (AUA 2020 guidelines), pathological, and surveillance reports were collected. Kaplan-Meier survival analysis was performed to detect Recurrence-free survival (RFS). Overall, 83 patients (68 males, 15 females) with a median age of 73 (IQR 66-79), and a median follow-up time of 18months (IQR 9-25), were included. Forty-one hadan intermediate-risk disease (49%) and 42 hada high-risk disease (51%). Thirty-sevenpatients (45%) had a recurrence; 19 (23%) had a high-grade recurrence. RFS of Gem/Doce induction-only vs induction + maintenance was at 6months 88% vs 100%, at 12months 71% vs 97%, at 18months 57% vs 91%, and at 24months 31% vs 87%, respectively (log-rank, p < 0.0001). Patients who received 2g Gemcitabine with Docetaxel had better RFS for all-graderecurrences (log-rank, p = 0.017). However, no difference was found for high-grade recurrences. Gem/Doce induction with maintenance resulted in significantly better RFS than induction-only. Combining 2g gemcitabine with docetaxel resulted in better RFS for all-grade but not for high-grade recurrences. Further prospective trials are necessary to validate our results.

The role of multiparametric magnetic resonance ımaging in the diagnosis of granulomatous prostatitis mimicking prostate cancer.

Aimed to investigate the role of multiparametric magnetic resonance imaging (mp-MRI) in the diagnosis of granulomatous prostatitis caused by intravesical Bacillus Calmette-Guérin (BCG). In this prospective, single-center study, 10 male patients who were given intravesical BCG due to intermediate- and high-risk bladder cancer were included. Before transurethral resection of bladder tumors (TURB), all patients were evaluated by mp-MRI, serum prostate-specific antigen (PSA), and digital rectal examination (DRE). Serum PSA levels and DRE findings were evaluated before and after intravesical BCG treatment. Prostate mp-MRI was performed for patients with elevated levels of serum PSA and/or with abnormal DRE findings. Then, MRI fusion + systematic prostate biopsy was performed. Demographic data of the patients before and after intravesical BCG were compared. The average age of the patients was 66.9years (55-87years). While PSA was 1.7ng/ml before intravesical BCG treatment, it was 4.3ng/ml after intravesical BCG treatment (p = 0.005). PSA density (PSAD) was 0.04 and 0.10 before and after the treatment, respectively (p = 0.012). DRE findings of all patients were normal before the treatment. However, abnormal findings were detected in 80% of them after the treatment (p = 0.008). PI-RADS ≥ 3 lesions were found to be significantly higher in all patients after intravesical BCG (p = 0.004). Granulomatous prostatitis is a rare complication of intravesical BCG. High PSA, abnormal DRE, and PI-RADS ≥ 3 lesions detected after intravesical BCG should suggest granulomatous prostatitis and unnecessary biopsies may be avoided.

Integrating the PD-L1 Prognostic Biomarker in Non-Muscle Invasive Bladder Cancer in Clinical Practice-A Comprehensive Review on State-of-the-Art Advances and Critical Issues.

Bladder cancer (BC) is one of the most prevalent cancers worldwide. Non-muscle invasive bladder cancer (NMIBC), comprising the majority of initial BC presentations, requires accurate risk stratification for optimal management. This review explores the evolving role of programmed cell death ligand 1 (PD-L1) as a prognostic biomarker in NMIBC, with a particular focus on its implications in the context of Bacillus Calmette-Guérin (BCG) immunotherapy. The literature suggests a potential association between elevated PD-L1 status and adverse outcomes, resistance to BCG treatment, and disease progression. However, conflicting findings and methodological issues highlight the heterogeneity of PD-L1 assessment in NMIBC, probably due to the complex biological mechanisms that regulate the interaction between PD-L1 and the tumor microenvironment. The identification of PD-L1 as a prognostic biomarker provides ground for tailored therapeutic interventions, including immune checkpoint inhibitors (ICIs). Nevertheless, challenges such as intratumoral heterogeneity and technical issues underscore the need for standardized protocols and larger, homogeneous trials. This review contributes to the ongoing debate on the personalized management of NMIBC patients, focusing on the advances and perspectives of incorporating PD-L1 as a biomarker in this setting.

Silencing the Mitochondrial Gatekeeper VDAC1 as a Potential Treatment for Bladder Cancer.

The strategy for treating bladder cancer (BC) depends on whether there is muscle invasion or not, with the latter mostly treated with intravesical therapy, such as with bacillus Calmette-Guérin (BCG). However, BCG treatment is unsuccessful in 70% of patients, who are then subjected to radical cystectomy. Although immune-checkpoint inhibitors have been approved as a second-line therapy for a subset of BC patients, these have failed to meet primary endpoints in clinical trials. Thus, it is crucial to find a new treatment. The mitochondrial gatekeeper protein, the voltage-dependent anion channel 1 (VDAC1), mediates metabolic crosstalk between the mitochondria and cytosol and is involved in apoptosis. It is overexpressed in many cancer types, as shown here for BC, pointing to its significance in high-energy-demanding cancer cells. The BC cell lines UM-UC3 and HTB-5 express high VDAC1 levels compared to other cancer cell lines. VDAC1 silencing in these cells using siRNA that recognizes both human and mouse VDAC1 (si-m/hVDAC1-B) reduces cell viability, mitochondria membrane potential, and cellular ATP levels. Here, we used two BC mouse models: subcutaneous UM-UC3 cells and chemically induced BC using the carcinogen N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Subcutaneous UM-UC3-derived tumors treated with si-m/hVDAC1 showed inhibited tumor growth and reprogrammed metabolism, as reflected in the reduced expression of metabolism-related proteins, including Glut1, hexokinase, citrate synthase, complex-IV, and ATP synthase, suggesting reduced metabolic activity. Furthermore, si-m/hVDAC1-B reduced the expression levels of cancer-stem-cell-related proteins (cytokeratin-14, ALDH1a), modifying the tumor microenvironment, including decreased angiogenesis, extracellular matrix, tumor-associated macrophages, and inhibited epithelial-mesenchymal transition. The BBN-induced BC mouse model showed a clear carcinoma, with damaged bladder morphology and muscle-invasive tumors. Treatment with si-m/hVDAC1-B encapsulated in PLGA-PEI nanoparticles that were administered intravesically directly to the bladder showed a decreased tumor area and less bladder morphology destruction and muscle invasion. Overall, the obtained results point to the potential of si-m/hVDAC1-B as a possible therapeutic tool for treating bladder cancer.

PD-L1 Expression in High-Risk Non-Muscle-Invasive Bladder Cancer Is Influenced by Intravesical Bacillus Calmette-Guérin (BCG) Therapy.

In the expanding landscape of immune checkpoint inhibitors (CPI) in high-risk (HR) non-muscle-invasive bladder cancer (NMIBC), the role of programmed death ligand 1 (PD-L1) as prognostic and predictive is increasingly significant. However, data evaluating its variability and susceptibility to Bacillus Calmette-Guérin (BCG) therapy in HR NMIBC patients is scarce. This retrospective study analyzed 126 HR NMIBC tissue samples from 63 patients (38× BCG-treated, 25× BCG-naïve) at two time points to assess PD-L1 expression using the 'combined positivity score' (CPS) with the 22C3 DAKO antibody method and correlated it with clinicopathological parameters. A CPS > 10 defined PD-L1 positivity. The impact of initial PD-L1 status and its change over time on time-to-recurrence, progression-free survival, and overall survival (TTR, PFS, OS) was analyzed using Kaplan-Meier and Cox proportional hazard models. BCG treatment significantly increased PD-L1 expression (5.31 vs. 0.22, p = 0.0423), with PD-L1 positive cases rising post-treatment in the BCG group and remaining unchanged in BCG-naïve patients. Multivariate analysis including T-stage, CIS, grading, tumor size, multifocality, age, and sex revealed a significant correlation between PD-L1 status change to positivity and improved TTR (p = 0.03). Our findings demonstrate a potential modulation of the PD-L1 status by an intravesical BCG therapy. However, its prognostic value appears limited.

Abstract 2462: Live attenuated Salmonella ZH9 as novel microbial immunotherapy for the treatment of bladder cancer

Abstract Microbial immunotherapy with Bacillus Calmette-Guérin (BCG) has been the standard-of-care for non-muscle invasive bladder cancer (NMIBC) for over 30 years. Today, global demand for BCG exceeds supply, leaving many patients untreated. BCG is associated with high disease recurrence and presents high treatment burden for patients. There remains therefore significant unmet need for novel therapeutic approaches for NMIBC and we sought to establish the utility of the live-attenuated Salmonella enterica Typhi ZH9 as a more effective microbial immunotherapy. Efficacy of ZH9 was established in orthotopic, syngeneic, mouse MB49 tumor models in which animals were treated in the bladder via catheterization, with or without prior subcutaneous immune priming. Immune responses were measured by flow cytometry and Nanostring transcriptomic analyses of bladder tissues. An intravesical dose of ZH9 after orthotopic MB49 tumour inoculation demonstrated significant survival benefit compared to both vehicle control and equivalent colony forming unit (CFU) BCG treatments. Repeat, weekly dosing studies showed a single administration of ZH9 was sufficient for maximum efficacy. To explore the durability of protection, surviving intravesical ZH9 treated animals were rechallenged, demonstrating protection from both bladder and subcutaneous MB49 tumors suggestive of lasting local and systemic anti-tumor immunity. Flow cytometry analysis of bladders showed that intravesical ZH9 treatment resulted in cellular immune response characterised by recruitment of NK cells, CD4+ and CD8+ T cells, and dendritic cells with an activated, cross-presenting phenotype, in all cases of greater magnitude than after treatment with an equivalent dose of BCG. Based on observations that pre-existing immunity to BCG has been associated with improved outcomes to intravesical BCG therapy in NMIBC, we explored whether systemic priming with ZH9 benefits local bladder therapy. Systemic prime with ZH9 followed by a single intravesical dose of ZH9 showed a trend for improved survival and will be explored in the clinical setting. This systemic immune priming effect was associated with enhanced local bladder immune cell infiltrates and the upregulation of more than 150 immune response genes in the bladder compared to intravesical therapy alone. These data form the basis of preclinical package accepted by the FDA for a Phase 1/1b study evaluating the safety, pharmacology, and clinical effect of ZH9 treatment in patients with NMIBC, currently recruiting patients. The investigation includes thorough translational and biomarker studies to delve into the mechanism of action of ZH9 and explore patient stratification approaches. By easing the treatment burden on patients, lowering healthcare costs, and featuring a scalable manufacturing process, ZH9 has the potential to revolutionize the treatment paradigm in NMIBC and reshape the patient journey. Citation Format: Nicholas Glanville, Oluwatobiloba Oke, Claudia Prevosto, Alexandra Sevko, Andrew Scott, Sonia Domingos-Pereira, Lenka Polak, Denise Nardelli-Haefliger, Livija Deban. Live attenuated Salmonella ZH9 as novel microbial immunotherapy for the treatment of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2462.

Systemic versus localized Bacillus Calmette Guérin immunotherapy of bladder cancer promotes an anti-tumoral microenvironment: Novel role of trained immunity.

Treatment for higher-risk non-muscle invasive bladder cancer (NMIBC) involves intravesical immunotherapy with Bacillus Calmette Guérin (BCG); however, disease recurrence and progression occur frequently. Systemic immunity is critical for successful cancer immunotherapy; thus, recurrence of NMIBC may be due to suboptimal systemic activation of anti-tumor immunity after local immunotherapy. We previously reported that systemically acquired trained immunity (a form of innate immune memory) in circulating monocytes is associated with increased time-to-recurrence in patients with NMIBC treated with BCG. Herein, we used a mouse model of NMIBC to compare the effects of intravesical versus intravenous (systemic) BCG immunotherapy on the local and peripheral immune microenvironments. We also assessed whether BCG-induced trained immunity modulates anti-tumor immune responses. Compared with intravesical BCG, which led to a tumor-promoting immune microenvironment, intravenous BCG resulted in an anti-tumoral bladder microenvironment characterized by increased proportions of cytotoxic T lymphocytes (CTLs), and decreased proportions of myeloid-derived suppressor cells. Polarization toward anti-tumoral immunity occurred in draining lymph nodes, spleen, and bone marrow following intravenous versus intravesical BCG treatment. Pre-treatment with intravesical BCG was associated with increased rate of tumor growth compared with intravenous BCG pre-treatment. Trained immunity contributed to remodeling of the tumor immune microenvironment, as co-instillation of BCG-trained macrophages with ovalbumin-expressing bladder tumor cells increased the proportion of tumor-specific CTLs. Furthermore, BCG-trained dendritic cells exhibited enhanced antigen uptake and presentation and promoted CTL proliferation. Our data support the concept that systemic immune activation promotes anti-tumor responses, and that BCG-induced trained immunity is important in driving anti-tumor adaptive immunity.

Urinary bother, Urinalysis, and Two-Year Efficacy Follow-Up Results of Phase I Trial of Intravesical Bacillus Calmette-Guérin Combined with Intravenous Pembrolizumab in Recurrent or Persistent High-Grade Non-Muscle-Invasive Bladder Cancer after Previous Bacillus Calmette-Guérin Treatment

ObjectiveTo report urinary bother, urinalysis changes, disease-free survival (DFS), and overall survival (OS) over 2 years for subjects enrolled in a phase I dose-escalation trial (NCT02324582) of intravesical Bacillus Calmette-Guérin (BCG) in combination with systemic pembrolizumab for recurrent or persistent high-grade non-muscle invasive bladder cancer (HGNMIBC). MethodsEighteen patients consented to the study. Five were screen failures. Clinical activity was determined using cystoscopy and cytology with a biopsy of suspicious lesions. Urinalysis and International Prostate symptom score were assessed at pre-treatment, Week 10 (during combined BCG and pembrolizumab treatment), and 3 and 6 months from treatment completion. IPSS was analyzed using a mixed-model repeated measures analysis. A Chi-square test was used to compare urinalysis results at each interval. ResultsThe pathologic disease stage after restaging transurethral resection and before treatment was pTa in 6 (46.2%), CIS in 6 (46.2%), and pT1 in 1 (7.7%). There was no increase in reported urinary bother throughout treatment. Quality of life measurements demonstrated no change in subjective burden. On urinalysis, we did not observe significant differences at 3 months compared to baseline evaluation. At 12 months, the DFS and OS were 69.23% and 92.31%, respectively. At 24 months, the DFS and OS were 38.46% and 92.31%, respectively. ConclusionsTreatment with BCG combined with intravenous pembrolizumab is not showing increased urinary bother or adverse urinalysis changes. Two-year response data is promising and await confirmation in the phase III study (Keynote 676)

BCG as an Innovative Option for HCC Treatment: Repurposing and Mechanistic Insights.

This study investigates Bacillus Calmette-Guérin (BCG) as a potential treatment for hepatocellular carcinoma (HCC), a condition often associated with unfavorable treatment outcomes. Exploiting BCG's recognized immune-boosting properties, preclinical trials are conducted using HCC mice, with a single subcutaneous dose of BCG administered post-tumor formation. Results indicate that BCG treatment effectively diminishes tumor burden and extends survival in both male and female HCC mice. Positive influences on hepatic fibrosis and metabolism are observed, leading to a reduction in lipid levels. Spatial analysis underscores BCG's tumor-specific effects, inducing the enrichment of metabolic pathways and inhibiting various cancer-related pathways. Furthermore, BCG promotes immune cell infiltration, including CD4+, CD8+ T cells, and M1 macrophages, in both v-akt murine thymoma viral oncogene homolog 1(AKT)/neutoblastoma RAS viral oncogene homolog (RAS) and β-catenin positive HCC models. Interestingly, blocking T cells, trained immunity, and Interferon-γ (IFN-γ) function reverses BCG's anti-HCC effects. In conclusion, BCG emerges as a promising treatment option for HCC, characterized by a favorable safety profile and efficacy in inhibiting fibrosis, improving metabolism, and engaging both trained immunity and T cells in therapeutic mechanisms.

Prostate Biopsy May Not Be Indicated Early after Bacillus Calmette Guérin Treatment.

Bacillus Calmette-Guérin (BCG) treatment for non-muscle-invasive bladder cancer frequently causes an intraprostatic BCG granuloma. We investigated the optimal timing for a prostate biopsy after BCG treatment by retrospectively analyzing the cases of 22 patients with non-muscle-invasive bladder cancer who underwent a prostate biopsy after BCG treatment at our institute (2013-2017). Biopsies were indicated for a rising prostate-specific antigen (PSA) level, positive digital rectal examination findings, or the appearance of de novo low apparent diffusion coefficient lesions on MRI. The control group was comprised of 28 age- and PSA-matched patients. The relationships among the cancer detection rate and the patients' PSA levels and MRI findings were analyzed. Prostate cancer was detected by biopsy in only 13.9% (3/22) of the patients in the BCG group but in 78.5% (22/28) of the control patients (p=0.0001). The three patients in the BCG group in whom prostate cancer was detected had all undergone the biopsy > 1 year after their BCG treatment. The remaining biopsies were performed within 1 year after BCG treatment and resulted in no diagnoses of prostate cancer. We suggest that performing a prostate biopsy early after BCG treatment is not informative or useful.

BladderGATE: Atezolizumab + intravesical BCG (bacillus Calmette-Guerin) upfront combination in patients with high risk non-muscle invasive bladder cancer (NMIBC)—Phase I-II ONCOSUR study.

595 Background: Intravesical Bacillus Calmette-Guerin (BCG) induction + BCG maintenance after transurethral resection is the current standard of care for patients (pts) with high-risk non-muscle invasive bladder cancer (NMIBC). Recurrence rate at 2 years and 5 years are around 30-40% and 70-80% respectively. Atezolizumab is a IgG1 monoclonal antibody targeting PD-L1 and is associated with long-term durable remissions in pts with metastatic urothelial cancer (MS. van der Heijden . Eur Urol 2021) and for pts with unresponsive NMIBC (PC. Black . Eur Urol 2023) with excellent results. Atezolizumab in combination with standard BCG could provide synergistic benefit for pts with NMIBC. BladderGATE (NCT04134000) is a phase Ib-II study which evaluates the safety and efficacy of upfront atezolizumab + intravesical BCG in pts with high-risk NMIBC. Methods: Pt had confirmed histopathology of high-risk NMIBC, BCG naïve or stopped &gt;2 years ago, WHO PS 0-2 and adequate hematologic and organ function. Dose-escalation design to identify DLT and MTD (9 pts) and to evaluate safety and efficacy in an expansion cohort (30 pts) were programed. Pt received six weekly induction instillations, and maintenance instillations at 3, 6 and 12 months + intravenously atezolizumab 1200 mg every 3 weeks, up to 1 year. Urine/blood and tissue samples were collected for translational and biomarker correlative study. Primary objective is disease free survival. Secondary objectives include safety profile and quality of life. Results: Finally 36 pt were included, median age 70 years, 86% men, 61% of pt were former smokers, 22% smokers and 17% had never smoked, and tumor size was ≥3 cm in 44% of pt. With a 22 months median follow-up, 56% of pt had completed BCG treatment and 89% had received adequate BCG treatment (5 induction plus at least 2 maintenance instillations). Thirteen pts discontinued atezolizumab due to immune-related AEs (7 pts) (g2 dermatitis, g3 hepatitis, encephalitis, pneumonitis, myocarditis, adrenal insufficiency and psoriasis), due to early-relapse disease (3 pts) and progressive-disease (3 pts). All irAEs were solved and no toxic death were reported. Of the 36 pts analyzed, 6 pts showed local recurrence (17%), 5 pts with high-risk NMIBC (14%) and 1 pt with low-risk NMIBC, 1 pt with UTUC, and 3 pts with local muscle invasive bladder cancer progressive-disease (8 %). Preliminary 2-year disease free survival is 72.8% (95% CI: 56.1% - 89.5%). Conclusions: The combination strategy of atezolizumab + intravesical BCG upfront in high-risk NMIBC pts appears feasible and safe. A 14% of 2-years local recurrence-rate and 8% of local progressive-disease are promising results, pending to randomized Ph3 ALBAN study data (GETUG). Supported by Roche-Spain. Clinical trial information: NCT03425201 .

Phase 3 KEYNOTE-676 cohort A: Bacillus Calmette-Guérin (BCG) with or without pembrolizumab for high-risk (HR) non–muscle-invasive bladder cancer (NMIBC) that persists/recurs after BCG induction.

TPS722 Background: Intravesical instillation of BCG is a standard-of-care treatment for patients with HR NMIBC, but up to 50% of patients later experience disease recurrence or progression to muscle-invasive bladder cancer (MIBC) despite BCG treatment. In the phase 2 KEYNOTE-057 study (NCT02625961), pembrolizumab monotherapy was shown to have antitumor activity in patients with HR BCG–unresponsive NMIBC who had carcinoma in situ (CIS) with or without papillary tumors and in patients with only papillary tumors. In cohort A of the randomized, phase 3 KEYNOTE-676 trial (NCT03711032), pembrolizumab plus BCG versus BCG alone is being evaluated in patients with persistent/recurrent HR NMIBC after first BCG induction. Methods: Key eligibility criteria include adults with histologically confirmed HR NMIBC (T1, high-grade Ta, and/or CIS) that is persistent/recurrent following 1 adequate course of BCG induction therapy, have no concurrent extravesical disease or history of extravesical disease that recurred within 2 years, have an ECOG PS score of 0 to 2, and have undergone cystoscopy/TURBT ≤12 weeks before randomization. Approximately 430 patients will be randomly assigned 1:1 to receive pembrolizumab 200 mg intravenously every 3 weeks plus BCG or BCG alone. BCG (50 mg) will be administered by intravesical instillation as induction therapy once weekly for 6 weeks and then as maintenance therapy once weekly for 3 weeks at weeks 13 and 25, then every 24 weeks (Q24W) thereafter. Treatment will continue for approximately 2 years (pembrolizumab) or 3 years (BCG), or until confirmed persistent/recurrent HR NMIBC or disease progression to MIBC or metastatic bladder cancer, unacceptable toxicity, or withdrawal. Randomization to treatment will be stratified by PD-L1 combined positive score (≥10 or ˂10), histology (CIS or non-CIS), and NMIBC disease history (persistence/recurrence 0 to ≤6 months or recurrence ˃6 to ≤12 months or recurrence ˃12 to ≤24 months). Response will be assessed by local cystoscopy and blinded independent central review of urine cytology and biopsy (as applicable) every 12 weeks for years 1 to 2 and Q24W for years 3 to 5. Computed tomography urography will occur every 72 weeks through year 5, then every 104 weeks thereafter. The primary end point is complete response rate in patients with CIS. Secondary end points are duration of response in patients with CIS, event-free survival, recurrence-free survival, time to cystectomy, overall survival, disease-specific survival, safety and tolerability, and patient-reported outcomes. Enrollment incohort A of KEYNOTE-676 is ongoing in Asia, Australia, Europe, and North America. Clinical trial information: NCT03711032 .

BCG immunotherapy promotes tumor-derived T-cell activation through the FLT3/FLT3LG pathway in bladder cancer.

Bladder instillation therapy is a common treatment for superficial or nonmuscle invasive bladder cancer. After surgery or reresection, chemotherapy drugs (epirubicin) or medications such as Bacillus Calmette-Guérin (BCG) are used for bladder instillation therapy, which can reduce the risk of bladder cancer recurrence and progression. However, the specific mechanism by which BCG stimulates the antitumor response has not been thoroughly elucidated. Additionally, although BCG immunotherapy is effective, it is difficult to predict which patients will have a positive response. In this study, we explored the BCG-induced immune response and found that high levels of Fms-related receptor tyrosine kinase 3 ligand (FLT3LG) were expressed after BCG treatment. This FLT3LG can directly act on CD8+ T cells and promote their proliferation and activation. The use of FLT3 inhibitors can neutralize the antitumor effects of BCG. In vitro experiments showed that FLT3LG can synergize with T-cell receptor activators to promote the activation of tumor-derived T cells. This study partially elucidates the mechanism of CD8+ T-cell activation in BCG immunotherapy and provides a theoretical basis for optimizing BCG instillation therapy in bladder cancer.

Longitudinal Transcription Profiling of Bladder Cancers Dictate the Response to BCG Treatment and Disease Progression.

Although the intravesical instillation of Bacillus Calmette-Guerin (BCG) is widely used as adjuvant treatment for nonmuscle-invasive bladder cancers, the clinical benefit is variable across patients, and the molecular mechanisms underlying the sensitivity to BCG administration and disease progression are poorly understood. To establish the molecular signatures that predict the responsiveness and disease progression of bladder cancers treated with BCG, we performed transcriptome sequencing (RNA-seq) for 13 treatment-naïve and 22 post-treatment specimens obtained from 14 bladder cancer patients. To overcome disease heterogeneity, we used non-negative matrix factorization to identify the latent molecular features associated with drug responsiveness and disease progression. At least 12 molecular features were present, among which the immune-related feature was associated with drug responsiveness, indicating that pre-treatment anti-cancer immunity might dictate BCG responsiveness. We also identified disease progression-associated molecular features indicative of elevated cellular proliferation in post-treatment specimens. The progression-associated molecular features were validated in an extended cohort of BCG-treated bladder cancers. Our study advances understanding of the molecular mechanisms of BCG activity in bladder cancers and provides clinically relevant gene markers for evaluating and monitoring patients.

The association between BCG treatment in patients with bladder cancer and subsequent risk of developing Alzheimer and other dementia.—A Swedish nationwide cohort study from 1997 to 2019

Background Alzheimer’s disease (AD) affects 50 million people worldwide. The immune system plays a major role in the pathogenesis of AD. Several retrospective analyses have reported a decreased risk of AD and other dementias in bladder cancer patients treated with immunotherapy in the form of Bacillus Calmette-Guerin (BCG) bladder instillations. We tested this hypothesis in a Swedish population-based prospective cohort of patients with non-muscle invasive bladder cancer (NMIBC). Methods and findings We utilized the BladderBaSe 2.0 database, which contains tumor-specific, health-related, and socio-demographic information for patients diagnosed with NMIBC between 1997 and 2019. The database also includes a matched comparison cohort sampled from the general population, consisting of individuals free from urinary tract cancer at the time of the index case’s diagnosis. Five controls were randomly selected for each index case without replacement on the date of the index case’s diagnosis. Our inclusion criteria identified participants diagnosed with NMIBC who had received BCG as primary treatment, along with their corresponding comparison cohort. We excluded those diagnosed with dementia before or within 6 months of NMIBC diagnosis. To compare the NMIBC cohort with their matched comparison cohort, we used a stratified Cox model, treating each case with its controls as a stratum. We identified 38,934 patients in the NMIBC cohort, with 6,496 receiving BCG after primary diagnosis (cases). AD/dementia was diagnosed during follow-up in 6.1% of cases and 7.4% of controls. Cases had a slightly lower risk of dementia than controls, with a hazard ratio (HR) of 0.88 (95% confidence interval [CI] 0.780–0.991), and a HR of 0.89 (CI 0.703–1.119) for AD. Subgroup analysis for dementia showed that age over 75 years had an HR of 0.73 (CI 0.616–0.863), and female gender had an HR of 0.73 (CI 0.552–0.971). The associations were similar for AD specifically, but not statistically significant. Similar to previous studies, we analyzed bladder cancer patients treated with and without BCG therapy. Multivariate Cox analysis indicated that those treated with BCG had a lower risk of dementia (HR 0.81, 95% CI 0.71–0.92), and an HR of 0.98 (95% CI 0.75–1.27) for AD specifically. High age was a significant risk modifier; the HR was 3.8 (CI 3.44–4.11) for dementia and 3.1 (CI 2.59–3.73) for AD. Even patients not receiving BCG had a significantly lower risk for AD than controls (HR 0.86, CI 0.77–0.96). Conclusions This study observed a marginally decreased risk of developing AD/dementia associated with earlier intravesical BCG treatment in NMIBC patients. This small benefit was most pronounced in those with high age and female gender. The disparity from previous highly positive studies underscores the importance of using an appropriate control cohort.

Comparative Analysis of Very Reduced vs Full Dose BCG Treatment for High-Risk Non-Muscle Invasive Bladder Cancer: A Contemporary Experience from Chile

BACKGROUND: Adjuvant bacillus Calmette-Guérin (BCG) is recommended for high-risk (HR) non-muscle invasive bladder cancer (NMIBC), but BCG shortages have led to exploration of reduced-dose regimens and shortened maintenance durations out of necessity, with limited data on treatment efficacy in Latin America. OBJECTIVE: Oncological outcomes of HR-NMIBC patients treated with reduced (RD,1/4th dose) vs full dose (FD) BCG instillations of Danish Strain 1331 BCG. METHODS: We performed a retrospective study of HR-NMIBC patients treated with BCG between 2003 and 2022 at our center in Santiago Chile. We stratified patients according to either RD (1/4th dose) or FD BCG. Univariate and multivariable Cox regression models were used to predict recurrence. Kaplan-Meier method was used to calculate survival estimates. RESULTS: Of a total of 200 patients, 116 (58%) had RD and 84 (42%) FD BCG. Median follow-up was 57 months (IQR: 29–100). Patients who received FD BCG had a lower risk of recurrence (HR: 0.41, 95% CI 0.22–0.74) and high-grade (HG)-recurrence (HR: 0.30, 95% CI 0.15–0.61; p = 0.001). More patients in the RD vs FD group progressed to MIBC (10/84 vs 2/116; p = 0.18). Additionally, patients were less likely to stop BCG treatment in the RD group compared to the FD group due to toxicity (5% vs 11%, p = 0.14). CONCLUSIONS: A 1/4th dose of Danish Strain 1331 BCG treatment was associated with worse recurrence free rate and HG-recurrence rate in our cohort. Patients with RD had lower discontinuation treatment rates due to a reduced toxicity profile. These findings would suggest that RD BCG would compromise oncological outcomes in HR-NMIBC patients.