Assessing the Causal Relationship between Waterpipe Smoking and Bladder Cancer.

Rates of waterpipe use increase with very little data reporting any potential health consequences. The current study, a large case-control study, of 4,194 patients from Iran denotes an elevated risk of bladder cancer in exclusive waterpipe smokers compared with non-users. Additional studies are needed to further understand the risk waterpipe smoking has on bladder cancer. See related article by Hadji et al., p. 509.

Bladder cancer risk stratification with the Oncuria 10-plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms.

BACKGROUND: No single marker of bladder cancer (BC) exists in urine samples with sufficient accuracy for disease diagnosis and treatment monitoring. The multiplex Oncuria BC assay noninvasively quantifies the concentration of 10 protein analytes in voided urine samples to quickly generate a unique molecular profile with proven BC diagnostic and treatment-tracking utility. Test adoption by diagnostic and research laboratories mandates reliably reproducible assay performance across a variety of instrumentation platforms used in different laboratories. METHODS: We compared the performance of the clinically validated Oncuria BC multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96-well microtiter plates on Day 1, and consecutively evaluated on the LED/image-based MagPix, and laser/flow-based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX) on Day 2. The BC assay uses magnetic bead-based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. All three analyzers quantify fluorescence signals generated by the Oncuria assay. RESULTS: All three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically < 5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥ 1 analyte above the highest standard concentration, i.e., A1AT (n = 7/18), IL-8 (n = 5), and/or ANG (n = 2), while only one control sample registered an analyte (A1AT) above the highest standard concentration. CONCLUSION: Multiplex BC assays generate detailed molecular signatures useful for identifying BC, predicting treatment responsiveness, and tracking disease progression and recurrence. The similar performance of the Oncuria assay across three different analyzer systems supports test adaptation by clinical and research laboratories using existing xMAP platforms. TRIAL REGISTRATION: This study was registered at ClinicalTrials.gov as NCT04564781, NCT03193528, NCT03193541, and NCT03193515.

Bladder cancer risk stratification with the Oncuria 10-plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms.

Background: No single marker of bladder cancer (BC) exists in urine samples with sufficient accuracy for disease diagnosis and treatment monitoring. The multiplex Oncuria BC assay noninvasively quantifies the concentration of 10 protein analytes in voided urine samples to quickly generate a unique molecular profile with proven BC diagnostic and treatment-tracking utility. Test adoption by diagnostic and research laboratories mandates reliably reproducible assay performance across a variety of instrumentation platforms used in different laboratories. Methods: We compared the performance of the clinically validated Oncuria BC multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96-well microtiter plates on Day 1, and consecutively evaluated on the LED/image-based MagPix, and laser/flow based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX) on Day 2. The BC assay uses magnetic bead-based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. Results: All three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically <5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥1 analyte above the highest standard concentration, i.e., A1AT (n=7/18), IL-8 (n=5), and/or ANG (n=2). In Controls, A1AT was higher in one sample. Conclusion: Multiplex BC assays generate detailed molecular signatures useful for identifying BC, predicting treatment esponsiveness, and tracking disease progression and recurrence. The similar performance of the Oncuria assay across three different analyzer systems supports test adaptation by clinical and research laboratories using existing xMAP platforms. Trial Registration: This study was registered at ClinicalTrials.gov as NCT04564781, NCT03193528, NCT03193541, and NCT03193515.

Identifying novel biomarkers associated with bladder cancer treatment outcomes.

Bladder cancer is a complex disease with variable prognosis. Recent investigations into the molecular landscape of bladder cancer have revealed frequent genetic alterations and molecular subtypes with therapeutic implications. Consequently, a shift toward personalized treatment of bladder cancer is underway. To this end, several biomarkers have been developed and tested in their ability to predict response to treatment in patients with bladder cancer and potentially help direct therapy. We performed a search of recently published PubMed articles using terms "biomarker," "bladder cancer," and the respective treatment discussed (i.e., "neoadjuvant" or "BCG"). In this review, we summarize the latest studies on novel biomarkers in bladder cancer with a focus on those intended to improve risk stratification and treatment selection.

Association of SNPs in the PAI1 Gene with Disease Recurrence and Clinical Outcome in Bladder Cancer.

PURPOSE: Bladder cancer (BCa) is one of the most common cancer types worldwide and is characterized by a high rate of recurrence. In previous studies, we and others have described the functional influence of plasminogen activator inhibitor-1 (PAI1) in bladder cancer development. While polymorphisms in PAI1 have been associated with increased risk and worsened prognosis in some cancers, the mutational status of PAI1 in human bladder tumors has not been well defined. METHODS: In this study, we evaluated the mutational status of PAI1 in a series of independent cohorts, comprised of a total of 660 subjects. RESULTS: Sequencing analyses identified two clinically relevant 3' untranslated region (UTR) single nucleotide polymorphisms (SNPs) in PAI1 (rs7242; rs1050813). Somatic SNP rs7242 was present in human BCa cohorts (overall incidence of 72%; 62% in Caucasians and 72% in Asians). In contrast, the overall incidence of germline SNP rs1050813 was 18% (39% in Caucasians and 6% in Asians). Furthermore, Caucasian patients with at least one of the described SNPs had worse recurrence-free survival and overall survival (p = 0.03 and p = 0.03, respectively). In vitro functional studies demonstrated that SNP rs7242 increased the anti-apoptotic effect of PAI1, and SNP rs1050813 was related to a loss of contact inhibition associated with cellular proliferation when compared to wild type. CONCLUSION: Further investigation of the prevalence and potential downstream influence of these SNPs in bladder cancer is warranted.

Clinical and Preclinical Therapies for Bladder Cancer Following Bacillus Calmette-Guérin Failure.

PURPOSE: Intravesical bacillus Calmette-Guérin is the current first-line treatment for high-grade nonmuscle-invasive bladder cancer; however, a substantial proportion of patients are unresponsive to bacillus Calmette-Guérin treatment. While cystectomy is often recommended in bladder cancer following bacillus Calmette-Guérin failure, there are numerous established therapeutic agents and pre-commercialized trials describing treatments for nonmuscle-invasive bladder cancer following failed bacillus Calmette-Guérin treatment. Our objective in this systematic review is to characterize the efficacy of these therapeutic agents by reporting their corresponding complete response rates and toxicity profiles. MATERIALS AND METHODS: We conducted a systematic review of all available clinical trials evaluating therapies to treat recurring nonmuscle-invasive bladder cancer after previous intravesical bacillus Calmette-Guérin. Bacillus Calmette-Guérin failure patients who had previously failed 1 or more courses of prior bacillus Calmette-Guérin therapy were included. Studies that were not in the English language, included muscle-invasive bladder cancer patient populations, or lacked a post-treatment evaluation of response were excluded. We used PubMed/Medline, the Cochrane Library, and Embase to search for relevant studies. No formal risk of bias assessment was conducted. Complete response rates for 3, 6, 12, and 24 months post-treatment evaluation, progression rates, cystectomy rates, and 12 complications are reported. RESULTS: A total of 70 studies with 73 reports evaluating 27 treatment options were retained for final analysis. These treatments were reported in 5 categories including intravesical chemotherapy, combination therapy, hyperthermia paired with intravesical chemotherapy, immunotherapy, and novel agents, with published years ranging from 1998 to 2021. Single intravesical chemotherapy and the combination of multiple intravesical chemotherapy agents demonstrate varied complete response rates of 10%-83% at 12 months. Limited clinical data evaluating hyperthermia paired with chemotherapy demonstrate 12-month complete response rates of 50%-85%. Despite these reported response rates, progression rates ranged from 0%-18%. Moreover, immunotherapeutic agents demonstrate progression rates of 7% to 22% at a median of 12 months of follow-up. Novel agents displayed a wide range of complete response rates (6% to 91%) at 12 months based on the treatment used. Total grade 3 toxicity rates range from 0%-55% for intravesical chemotherapy and combination intravesical chemotherapy agents, 0%-15% for hyperthermia paired with chemotherapy agents, 12%-13% for immunotherapy agents, and 0%-17% for novel agents. CONCLUSIONS: Bladder-preserving treatments accomplish moderate success in nonmuscle-invasive bladder cancer following bacillus Calmette-Guérin failure. As the majority of available clinical trials are single-armed uncontrolled cohorts and contain a limited number of patients, strength and comparability of the data are limited. In general, intravesical chemotherapy and hyperthermia paired with mitomycin C demonstrate some of the highest complete response rates at 12 and 24 months. Similarly, among the pre-commercialized novel agents, N-803 and gene therapy display promising results and may serve as potential future treatment for nonmuscle-invasive bladder cancer following failed bacillus Calmette-Guérin treatment. In terms of toxicity/complication rates, both commercially available and unavailable treatments showcase low toxicity profiles for bladder cancer following bacillus Calmette-Guérin failure. The comprehensive analysis provided by this systematic review can serve as a reference for treatment decisions and clinical trial design in the bacillus Calmette-Guérin-unresponsive domain.

MRI/RNA-Seq-Based Radiogenomics and Artificial Intelligence for More Accurate Staging of Muscle-Invasive Bladder Cancer.

Accurate staging of bladder cancer assists in identifying optimal treatment (e.g., transurethral resection vs. radical cystectomy vs. bladder preservation). However, currently, about one-third of patients are over-staged and one-third are under-staged. There is a pressing need for a more accurate staging modality to evaluate patients with bladder cancer to assist clinical decision-making. We hypothesize that MRI/RNA-seq-based radiogenomics and artificial intelligence can more accurately stage bladder cancer. A total of 40 magnetic resonance imaging (MRI) and matched formalin-fixed paraffin-embedded (FFPE) tissues were available for analysis. Twenty-eight (28) MRI and their matched FFPE tissues were available for training analysis, and 12 matched MRI and FFPE tissues were used for validation. FFPE samples were subjected to bulk RNA-seq, followed by bioinformatics analysis. In the radiomics, several hundred image-based features from bladder tumors in MRI were extracted and analyzed. Overall, the model obtained mean sensitivity, specificity, and accuracy of 94%, 88%, and 92%, respectively, in differentiating intra- vs. extra-bladder cancer. The proposed model demonstrated improvement in the three matrices by 17%, 33%, and 25% and 17%, 16%, and 17% as compared to the genetic- and radiomic-based models alone, respectively. The radiogenomics of bladder cancer provides insight into discriminative features capable of more accurately staging bladder cancer. Additional studies are underway.

A Diagnostic Gene Expression Signature for Bladder Cancer Can Stratify Cases into Prescribed Molecular Subtypes and Predict Outcome.

Bladder cancer is a biologically heterogeneous disease with variable clinical presentations, outcomes and responses to therapy. Thus, the clinical utility of single biomarkers for the detection and prediction of biological behavior of bladder cancer is limited. We have previously identified and validated a bladder cancer diagnostic signature composed of 10 biomarkers, which has been incorporated into a multiplex immunoassay bladder cancer test, Oncuria™. In this study, we evaluate whether these 10 biomarkers can assist in the prediction of bladder cancer clinical outcomes. Tumor gene expression and patient survival data from bladder cancer cases from The Cancer Genome Atlas (TCGA) were analyzed. Alignment between the mRNA expression of 10 biomarkers and the TCGA 2017 subtype classification was assessed. Kaplan-Meier analysis of multiple gene expression datasets indicated that high expression of the combined 10 biomarkers correlated with a significant reduction in overall survival. The analysis of three independent, publicly available gene expression datasets confirmed that multiplex prognostic models outperformed single biomarkers. In total, 8 of the 10 biomarkers from the Oncuria™ test were significantly associated with either luminal or basal molecular subtypes, and thus, the test has the potential to assist in the prediction of clinical outcome.

PAI-1 is a potential transcriptional silencer that supports bladder cancer cell activity.

The extracellular activity of Plasminogen activator inhibitor-1 (PAI-1) is well described, acting as an inhibitor of tissue plasminogen activator and urokinase-type plasminogen activator, impacting fibrinolysis. Recent studies have revealed a pro-tumorigenic role of PAI-1 in human cancers, via the regulation of angiogenesis and tumor cell survival. In this study, immunohistochemical staining of 939 human bladder cancer specimens showed that PAI-1 expression levels correlated with tumor grade, tumor stage and overall survival. The typical subcellular localization of PAI-1 is cytoplasmic, but in approximately a quarter of the cases, PAI-1 was observed to be localized to both the tumor cell cytoplasm and the nucleus. To investigate the potential function of nuclear PAI-1 in tumor biology we applied chromatin immunoprecipitation (ChIP)-sequencing, gene expression profiling, and rapid immunoprecipitation mass spectrometry to a pair of bladder cancer cell lines. ChIP-sequencing revealed that PAI-1 can bind DNA at distal intergenic regions, suggesting a role as a transcriptional coregulator. The downregulation of PAI-1 in bladder cancer cell lines caused the upregulation of numerous genes, and the integration of ChIP-sequence and RNA-sequence data identified 57 candidate genes subject to PAI-1 regulation. Taken together, the data suggest that nuclear PAI-1 can influence gene expression programs and support malignancy.

The dynamic roles of the bladder tumour microenvironment.

Bladder cancer is a prevalent but currently understudied cancer type and patient outcomes are poor when it progresses to the muscle-invasive stage. Current research in bladder cancer focuses on the genetic and epigenetic alterations occurring within the urothelial cell compartment; however, the stromal compartment receives less attention. Dynamic changes and intercellular communications occur in the tumour microenvironment (TME) of the bladder - a new concept and niche that we designate as the bladder TME (bTME) - during tumour evolution, metastatic progression and in the context of therapeutic response. Collagens and their cognate receptors, the discoidin domain receptors, have a role in various steps of the metastatic cascade and in immune checkpoint resistance. Furthermore, the presence of another TME niche, the metastatic TME (met-TME), is a novel concept that could support divergent progression of metastatic colonization in different organs, resulting in distant metastases with distinct characteristics and genetics from the primary tumour. The stroma has divergent roles in mediating therapeutic response to BCG immunotherapy and immune checkpoint inhibitors, as well as conventional chemotherapy or trimodality therapy (that is, maximal transurethral resection of bladder tumour, chemotherapy and radiotherapy). The local bTME and distant met-TME are currently conceptually and therapeutically unexploited niches that should be actively investigated. New biological insights from these TMEs will enable rational design of strategies that co-target the tumour and stroma, which are expected to improve the outcomes of patients with advanced bladder cancer.

Cell death-induced immunogenicity enhances chemoimmunotherapeutic response by converting immune-excluded into T-cell inflamed bladder tumors.

Chemoimmunotherapy has recently failed to demonstrate significant clinical benefit in advanced bladder cancer patients; and the mechanism(s) underlying such suboptimal response remain elusive. To date, most studies have focused on tumor-intrinsic properties that render them "immune-excluded". Here, we explore an alternative, drug-induced mechanism that impedes therapeutic response via disrupting the onset of immunogenic cell death. Using two immune-excluded syngeneic mouse models of muscle-invasive bladder cancer (MIBC), we show that platinum-based chemotherapy diminishes CD8+ T cell tumor infiltration and constraines their antitumoral activity, despite expression of activation markers IFNγ and granzyme B. Mechanistically, chemotherapy induces the release of prostaglandin E2 (PGE2) from dying cancer cells, which is an inhibitory damage-associated molecular pattern (iDAMP) that hinderes dendritic cell maturation. Upon pharmaceutical blockade of PGE2 release, CD8+ T cells become tumoricidal and display an intraepithelial-infiltrating (or inflamed) pattern. This "iDAMP blockade" approach synergizes with chemotherapy and sensitizes bladder tumors towards anti-PD1 immune checkpoint inhibitor therapy. These findings provide a compelling rationale to evaluate this drug combination in future clinical trials.

Clinical Utility of Oncuria™, a Multiplexed Liquid Biopsy for the Non-Invasive Detection of Bladder Cancer-A Pilot Study.

Oncuria™ is a validated quantitative multiplex immunoassay capable of detecting bladder cancer from a voided urine sample. Herein, we sought to determine whether Oncuria™ affects physicians' use of non-invasive and invasive diagnostic tests for microhematuria, gross hematuria, and bladder cancer surveillance. We conducted a survey-based study to assess physician management of nine clinical scenarios involving real-world data from patients with gross hematuria, microhematuria, and bladder cancer on surveillance. We randomly sampled 15 practicing urologists and generated data including 135 patient-by-urologist interactions and 2160 decision points. Urologists recommended a selection of diagnostic tests and procedures before and after Oncuria™ results were provided. We assessed changes in provider use of non-invasive and invasive diagnostic tests after Oncuria™ results were provided. Over 90% of all urologists changed their diagnostic behavior in at least one patient case with the addition of Oncuria™ results. The total number of diagnostic procedures was reduced by 31% following the disclosure of a negative Oncuria™ test and 27% following the disclosure of a positive Oncuria™ test. This is pilot study has the potential to shed light on the analysis of our four large multicenter international studies deploying OncuriaTM. The Oncuria™ urine-based test, a molecular diagnostic capable of ruling out the presence of bladder cancer, reduces both unnecessary invasive and non-invasive diagnostics and has the potential to reduce costs and improve patient outcomes.

Application of a multiplex urinalysis test for the prediction of intravesical BCG treatment response: A pilot study.

BACKGROUND: Intravesical Bacillus Calmette-Guerin (BCG), a live attenuated tuberculosis vaccine that acts as a non-specific immune system stimulant, is the most effective adjuvant treatment for patients with intermediate or high-risk non-muscle-invasive bladder cancer (NMIBC). However, to date, there are no reliable tests that are predictive of BCG treatment response. In this study, we evaluated the performance of OncuriaTM, a bladder cancer detection test, to predict response to intravesical BCG. METHODS: OncuriaTM data was evaluated in voided urine samples obtained from a prospectively collected cohort of 64 subjects with intermediate or high risk NMIBC prior to treatment with intravesical BCG. The OncuriaTM test, which measures 10 cancer-associated biomarkers was performed in an independent clinical laboratory. The ability of the test to identify those patients in whom BCG is ineffective against tumor recurrence was tested. Predictive models were derived using supervised learning and cross-validation analyses. Model performance was assessed using ROC curves. RESULTS: Pre-treatment urinary concentrations of MMP9, VEGFA, CA9, SDC1, PAI1, APOE, A1AT, ANG and MMP10 were increased in patients who developed disease recurrence. A combinatorial predictive model of treatment outcome achieved an AUROC 0.89 [95% CI: 0.80-0.99], outperforming any single biomarker, with a test sensitivity of 81.8% and a specificity of 84.9%. Hazard ratio analysis revealed that patients with higher urinary levels of ANG, CA9 and MMP10 had a significantly higher risk of disease recurrence. CONCLUSIONS: Monitoring the urinary levels of a cancer-associated biomarker panel enabled the discrimination of patients who did not respond to intravesical BCG therapy. With further study, the multiplex OncuriaTM test may be applicable for the clinical evaluation of bladder cancer patients considering intravesical BCG treatment.

Case Study of Noni Extract in Men with Very Low-Risk or Low-Risk Prostate Cancer.

The optimal treatment strategy for patients with early prostate cancer (PCa) is unknown. We explored the feasibility of administering noni supplementation to modify gene expression of a relevant clinical signature in the prostate of men on active surveillance for PCa. A total of 6 participants with low-risk (n=5) to very low-risk (n=1) PCa who were candidates for active surveillance received 6200 mg/day of noni in capsule form for 1 year; median age was 65.5 years (range, 58-75 years). Participants were tested for serum prostate-specific antigen (PSA) levels every 3 months. At 12 months, they underwent a repeat transrectal ultrasound-guided prostate biopsy. These biopsy samples were queried for expressing 12 key genes and rates of apoptosis, angiogenesis, and proliferation. The primary outcome was the change in expression of the 12 genes that comprise the Oncotype DX prostate cancer test from baseline to 12 months of noni supplementation. Noni was well tolerated, with only 1 participant reporting side effects of grade 2 diarrhea, requiring a drug holiday of 7 days. Median serum PSA slightly increased from 7.1 ng/mL (4.4-9.7 ng/ mL) prior to therapy to 7.9 ng/mL (5.7-10.2 ng/mL) on therapy. Changes were observed in the expression levels of several genes, including FAM13C, KLK2 (associated with the androgen pathway), and GSTM2 (associated with cellular organization) at 12 months. Noni supplementation was associated with favorable clinical parameters, including stable serum PSA among most patients and no evidence of tumor on repeat biopsy, and correlated with modulation of numerous genes and proteins.

Phase Ib study of patients with metastatic castrate-resistant prostate cancer treated with different sequencing regimens of atezolizumab and sipuleucel-T.

BACKGROUND: Combining an immune checkpoint inhibitor with a tumor vaccine may modulate the immune system to leverage complementary mechanisms of action that lead to sustained T-cell activation and a potent prolonged immunotherapeutic response in metastatic castration resistant prostate cancer (mCRPC). METHODS: Subjects with asymptomatic or minimally symptomatic mCRPC were randomly assigned in a 1:1 ratio to receive either atezolizumab followed by sipuleucel-T (Arm 1) or sipuleucel-T followed by atezolizumab (Arm 2). The primary endpoint was safety, while secondary endpoints included preliminary clinical activity such as objective tumor response and systemic immune responses that could identify key molecular and immunological changes associated with sequential administration of atezolizumab and sipuleucel-T. RESULTS: A total of 37 subjects were enrolled. The median age was 75.0 years, median prostate specific antigen (PSA) was 21.9 ng/mL, and subjects had a median number of three prior treatments. Most subjects (83.8%) had at least one treatment-related adverse event. There were no grade 4 or 5 toxicities attributed to either study drug. Immune-related adverse events and infusion reactions occurred in 13.5% of subjects, and all of which were grade 1 or 2. Of 23 subjects with Response Evaluation Criteria in Solid Tumors measurable disease, only one subject in Arm 2 had a partial response (PR) and four subjects overall had stable disease (SD) at 6 months reflecting an objective response rate of 4.3% and a disease control rate of 21.7%. T-cell receptor diversity was higher in subjects with a response, including SD. Immune response to three novel putative antigens (SIK3, KDM1A/LSD1, and PIK3R6) appeared to increase with treatment. CONCLUSIONS: Overall, regardless of the order in which they were administered, the combination of atezolizumab with sipuleucel-T appears to be safe and well tolerated with a comparable safety profile to each agent administered as monotherapy. Correlative immune studies may suggest the combination to be beneficial; however, further studies are needed. TRIAL REGISTRATION NUMBER: NCT03024216.

An N-Cadherin 2 expressing epithelial cell subpopulation predicts response to surgery, chemotherapy and immunotherapy in bladder cancer.

Neoadjuvant chemotherapy (NAC) prior to surgery and immune checkpoint therapy (ICT) have revolutionized bladder cancer management. However, stratification of patients that would benefit most from these modalities remains a major clinical challenge. Here, we combine single nuclei RNA sequencing with spatial transcriptomics and single-cell resolution spatial proteomic analysis of human bladder cancer to identify an epithelial subpopulation with therapeutic response prediction ability. These cells express Cadherin 12 (CDH12, N-Cadherin 2), catenins, and other epithelial markers. CDH12-enriched tumors define patients with poor outcome following surgery with or without NAC. In contrast, CDH12-enriched tumors exhibit superior response to ICT. In all settings, patient stratification by tumor CDH12 enrichment offers better prediction of outcome than currently established bladder cancer subtypes. Molecularly, the CDH12 population resembles an undifferentiated state with inherently aggressive biology including chemoresistance, likely mediated through progenitor-like gene expression and fibroblast activation. CDH12-enriched cells express PD-L1 and PD-L2 and co-localize with exhausted T-cells, possibly mediated through CD49a (ITGA1), providing one explanation for ICT efficacy in these tumors. Altogether, this study describes a cancer cell population with an intriguing diametric response to major bladder cancer therapeutics. Importantly, it also provides a compelling framework for designing biomarker-guided clinical trials.

Influencing Factors on the Oncuria™ Urinalysis Assay: An Experimental Model.

BACKGROUND: The Oncuria™ urine test for the detection of bladder cancer measures a multiplex protein signature. In this study, we investigated the influence of urinary cellularity, protein, and hematuria on the performance of the Oncuria™ test in an ex vivo experimental model. MATERIALS AND METHODS: Pooled urine from healthy subjects was spiked with cultured benign (UROtsa) or malignant cells (T24), cellular proteins, or whole blood. The resulting samples were analyzed using the Oncuria™ test following the manufacturer's instructions. RESULTS: Urine samples obtained from healthy subjects were negative for bladder cancer by Oncuria™ test criteria. The majority of the manipulated conditions did not result in a false-positive test. The addition of whole blood (high concentration) did result in a false-positive result, but this was abrogated by sample centrifugation prior to analysis. The addition of cellular proteins (high concentration) resulted in a positive Oncuria™ test, and this was unaffected by pre-analysis sample centrifugation. CONCLUSIONS: The Oncuria™ multiplex test performed well in the ex vivo experimental model and shows promise for clinical application. The identification of patients who require additional clinical evaluation could reduce the need to subject patients who do not have bladder cancer to frequent, uncomfortable and expensive cystoscopic examinations, thus benefiting both patients and the healthcare system.

Safety, Tolerability, and Long-Term Clinical Outcomes of an IL-15 analogue (N-803) Admixed with Bacillus Calmette-Guérin (BCG) for the Treatment of Bladder Cancer.

Intravesical BCG is active against non-muscle invasive bladder cancer (NMIBC), but bladder cancer will recur and even progress in a significant number of patients. To improve the response rate, N-803, an IL-15 superagonist was administered in combination with BCG. To evaluate the safety and efficacy associated with the use of intravesical N-803 and BCG in patients with BCG-naïve NMIBC. This phase 1b clinical trial used a 3 + 3 dose-escalation design. Participants were enrolled from July 2014 and July 2015, with follow-up and analyses through January 15, 2021. Eligibility criteria included histologically confirmed non-muscle invasive urothelial carcinoma of intermediate or high risk who had not received prior treatment with intravesical BCG (ie, BCG-naïve). All 9 participants met the eligibility criteria, received treatment according to the protocol, and were included in all analyses. Treatment was done once weekly for 6 consecutive weeks with bladder infusion of the standard dose of BCG, 50 mg/instillation, in combination with increasing doses of N-803 (100, 200, or 400 µg N-803 per instillation). No DLTs were noted in any of the dose cohorts. All adverse events (AEs) were manageable and less than grade 3. During the 2-year follow-up, all 9 participants were disease free. Furthermore, 6 y after treatment, all 9 participants (100%) were disease free with no evidence of disease progression and an intact bladder. This phase 1b trial found the combination of intravesical N-803 and BCG to be associated with modest toxic effects, low immunogenicity, and substantial prolonged antitumoral activity; phase 2 trials are in progress.

Diagnostic performance of Oncuria™, a urinalysis test for bladder cancer.

BACKGROUND: Due to insufficient accuracy, urine-based assays currently have a limited role in the management of patients with bladder cancer. The identification of multiplex molecular signatures associated with disease has the potential to address this deficiency and to assist with accurate, non-invasive diagnosis and monitoring. METHODS: To evaluate the performance of Oncuria™, a multiplex immunoassay for bladder detection in voided urine samples. The test was evaluated in a multi-institutional cohort of 362 prospectively collected subjects presenting for bladder cancer evaluation. The parallel measurement of 10 biomarkers (A1AT, APOE, ANG, CA9, IL8, MMP9, MMP10, PAI1, SDC1 and VEGFA) was performed in an independent clinical laboratory. The ability of the test to identify patients harboring bladder cancer was assessed. Bladder cancer status was confirmed by cystoscopy and tissue biopsy. The association of biomarkers and demographic factors was evaluated using linear discriminant analysis (LDA) and predictive models were derived using supervised learning and cross-validation analyses. Diagnostic performance was assessed using ROC curves. RESULTS: The combination of the 10 biomarkers provided an AUROC 0.93 [95% CI 0.87-0.98], outperforming any single biomarker. The addition of demographic data (age, sex, and race) into a hybrid signature improved the diagnostic performance AUROC 0.95 [95% CI 0.90-1.00]. The hybrid signature achieved an overall sensitivity of 0.93, specificity of 0.93, PPV of 0.65 and NPV of 0.99 for bladder cancer classification. Sensitivity values of the diagnostic panel for high-grade bladder cancer, low-grade bladder cancer, MIBC and NMIBC were 0.94, 0.89, 0.97 and 0.93, respectively. CONCLUSIONS: Urinary levels of a biomarker panel enabled the accurate discrimination of bladder cancer patients and controls. The multiplex Oncuria™ test can achieve the efficient and accurate detection and monitoring of bladder cancer in a non-invasive patient setting.