A pilot clinical study of developing an External Assist Targeting Device for rapid and precise renal calyx access during percutaneous nephrolithotomy.

Objective: To design a device to increase the accuracy of the targeting process and reduce the radiation exposure to both the patients and the medical staff. Methods: We analyzed the inherent problem and designed the External Assist Targeting Device (EATD) to assist in the alignment of needle targeting on the desired renal calyx under fluoroscopic guidance. The EATD was designed to allow rapid and precise access to calyces at all angles, with a simple two-step puncture protocol developed for puncturing a target renal calyx. We then tested the device in a pilot human trial with four patients. Results: In experiments with phantom models, the time for successful targeting was reduced by 31% using the device. The mean fluoroscopic time was reduced by 40%. In initial human trial, the puncture time was shortened by 66% and the radiation dose was decreased by 65% compared to free-hand technique. No complication was observed during the trial. Conclusion: The EATD was found to be cost effective, portable, simple to set up, and safe to operate for assisting in the percutaneous nephrolithotomy procedures. Our preliminary tests showed high degree of accuracy in gaining precise access to a targeted renal calyx with much shorter time and lesser radiation dose. The EATD also has the potential to be used to access other organs with precision under fluoroscopic guidance.

UBC®Rapid Is Sensitive in Detecting High-Grade Bladder Urothelial Carcinoma and Carcinoma in situ in Asian Population.

OBJECTIVES: Bladder cancer is a common type of malignancy. UBC®Rapid is a novel immunoassay detecting urine cytokeratin 8/18 protein. Studies have shown good accuracy of UBC®Rapid in detecting bladder cancer. UBC®Rapid has however to date not been evaluated in Asian patients. We evaluated UBC®Rapid in detecting Asian bladder cancer, together with urine cytology. METHODS: In total, 112 patients were recruited from National University Hospital Singapore and 103 patients were included in this study, comprising 49 patients with bladder urothelial carcinoma (UC), 21 patients with bladder benign lesions, and 33 patients with normal bladder. All the bladder cancer and benign lesions were confirmed by histology. Voided urine was collected for UBC®Rapid test. The results were compared with urine cytology. RESULTS: The bladder UC group had remarkably higher UBC®Rapid value than the control groups. The sensitivity, specificity, positive, and negative predictive value of UBC®Rapid in detecting bladder UC were 53%, 85.5%, 76.5%, and 66.8%, respectively. Those of urine cytology were 40.8%, 96.3%, 90.9%, and 64.2%, respectively. Adding UBC®Rapid to urine cytology increased sensitivity to 57.1% but decreased specificity to 81.8%. UBC®Rapid was sensitive in detecting high-grade bladder UC (61.1%) and carcinoma in situ (CIS) (72.7%), as compared to urine cytology for bladder UC (55.6%) and CIS (54.5%), respectively. CONCLUSION: UBC®Rapid is sensitive in detecting high-grade bladder UC and CIS in Asian population. It may be useful as an adjunct test to achieve better detection of bladder cancer.

Wirelessly Activated Nanotherapeutics for In Vivo Programmable Photodynamic-Chemotherapy of Orthotopic Bladder Cancer.

Photochemical internalization (PCI) is a promising intervention using photodynamic therapy (PDT) to enhance the activity of chemotherapeutic drugs. However, current bladder cancer treatments involve high-dose chemotherapy and high-irradiance PDT which cause debilitating side effects. Moreover, low penetration of light and drugs in target tissues and cumbersome light delivery procedures hinder the clinical utility of PDT and chemotherapy combination for PCI. To circumvent these challenges, a photodynamic-chemotherapy approach is developed comprising tumor-targeting glycosylated nanocarriers, coloaded with chlorin e6 (Ce6) and gemcitabine elaidate (GemE), and a miniaturized implantable wirelessly powered light-emitting diode (LED) as a light source. The device successfully delivers four weekly light doses to the bladder while the nanocarrier promoted the specific accumulation of drugs in tumors. This approach facilitates the combination of low-irradiance PDT (1 mW cm-2 ) and low-dose chemotherapy (≈1500× lower than clinical dose) which significantly cures and controls orthotopic disease burden (90% treated vs control, 35%) in mice, demonstrating a potential new bladder cancer treatment option.

Hospital readmissions for patients with prostate cancer are higher after radiotherapy than after prostatectomy.

PURPOSE: To compare hospital readmissions, biochemical recurrence rates, incidence of metastasis, and cancer-specific and overall mortality for prostate cancer patients undergoing radiotherapy vs. radical prostatectomy. The secondary outcome was to identify patient and disease characteristics affecting physician's choice of either therapy. MATERIALS AND METHODS: A total of 297 patients diagnosed with prostate cancer between 2008 and 2014 were identified from a single academic center's cancer database. Clinical information including age, ethnicity, comorbidities, prostate-specific antigen, Gleason score, stage, National Comprehensive Cancer Network (NCCN) risk group, biochemical recurrence, hospital readmissions, and survival outcomes were gathered and analyzed from ambulatory medical records until 2018. RESULTS: Patients selected for radiotherapy were older and had more comorbidities and NCCN high-risk disease. Biochemical recurrence was higher after radical prostatectomy for locally advanced disease, 59.3% vs. 20.0% (p<0.001), favoring radiotherapy. Hospital readmission was higher for patients with locally advanced disease undergoing radiotherapy, 48.6% vs. 18.5% (p=0.002), and 35.2% vs. 19.7% (p=0.044) for those with localized disease, with most of these readmissions occurring 24 months after the initial therapy. Radiation proctitis and colitis were the most common complications after radiotherapy and accounted for 46.3% of readmissions. CONCLUSIONS: Selection of patients for radiotherapy instead of surgery was influenced by age, significant comorbidities, and NCCN high-risk disease. The incidence of treatment- or cancer-related hospital readmissions was significantly higher for patients undergoing radiotherapy compared with radical prostatectomy, especially for those with locally advanced prostate cancer. This information may be useful in guiding a patient's choice of therapy.

Nomograms including the UBC® Rapid test to detect primary bladder cancer based on a multicentre dataset.

OBJECTIVES: To evaluate the clinical utility of the urinary bladder cancer antigen test UBC® Rapid for the diagnosis of bladder cancer (BC) and to develop and validate nomograms to identify patients at high risk of primary BC. PATIENTS AND METHODS: Data from 1787 patients from 13 participating centres, who were tested between 2012 and 2020, including 763 patients with BC, were analysed. Urine samples were analysed with the UBC® Rapid test. The nomograms were developed using data from 320 patients and externally validated using data from 274 patients. The diagnostic accuracy of the UBC® Rapid test was evaluated using receiver-operating characteristic curve analysis. Brier scores and calibration curves were chosen for the validation. Biopsy-proven BC was predicted using multivariate logistic regression. RESULTS: The sensitivity, specificity, and area under the curve for the UBC® Rapid test were 46.4%, 75.5% and 0.61 (95% confidence interval [CI] 0.58-0.64) for low-grade (LG) BC, and 70.5%, 75.5% and 0.73 (95% CI 0.70-0.76) for high-grade (HG) BC, respectively. Age, UBC® Rapid test results, smoking status and haematuria were identified as independent predictors of primary BC. After external validation, nomograms based on these predictors resulted in areas under the curve of 0.79 (95% CI 0.72-0.87) and 0.95 (95% CI: 0.92-0.98) for predicting LG-BC and HG-BC, respectively, showing excellent calibration associated with a higher net benefit than the UBC® Rapid test alone for low and medium risk levels in decision curve analysis. The R Shiny app allows the results to be explored interactively and can be accessed at www.blucab-index.net. CONCLUSION: The UBC® Rapid test alone has limited clinical utility for predicting the presence of BC. However, its combined use with BC risk factors including age, smoking status and haematuria provides a fast, highly accurate and non-invasive tool for screening patients for primary LG-BC and especially primary HG-BC.

Beyond diabetes mellitus: role of metformin in non-muscle-invasive bladder cancer

INTRODUCTION@#Usage of metformin is associated with improved survival in lung, breast and prostate cancer, and metformin has been shown to inhibit cancer cell growth and proliferation in in vitro studies. Given the lack of clinical data on metformin use in patients with bladder cancer, we aimed to evaluate the role of metformin in their oncological outcomes.@*METHODS@#Medication use data from a prospectively maintained database of 122 patients with non-muscle-invasive bladder cancer treated with intravesical Bacille Calmette-Guerin (BCG), who were recruited under a randomised, double-blinded, controlled clinical trial, was collected and analysed. Kaplan-Meier curves were used to assess overall survival (OS) and disease-specific survival (DSS).@*RESULTS@#At a median follow-up duration of 102 (range 3-357) months, 53 (43.4%) patients experienced disease recurrence and 21 (17.2%) experienced disease progression. There was no significant difference in mortality between patients with and without diabetes mellitus. There was significant difference in OS between patients without diabetes mellitus, patients with diabetes mellitus on metformin and patients with diabetes mellitus but not on metformin (p = 0.033); patients with diabetes mellitus on metformin had the best prognosis. Metformin use was associated with significantly lower DSS (p = 0.042). Other oral hypoglycaemic agents, insulin or statins were not associated with disease recurrence or progression.@*CONCLUSION@#Metformin use was associated with improved oncological outcomes in patients with non-muscle-invasive bladder cancer treated with intravesical BCG. Prospective studies with larger patient populations are needed to validate the role of metformin as potential therapy for bladder cancer.

Intravesical High Dose BCG Tokyo and Low Dose BCG Tokyo with GMCSF+IFN α Induce Systemic Immunity in a Murine Orthotopic Bladder Cancer Model.

This study evaluates a short therapy schedule for bladder cancer using BCG Tokyo. BCG Tokyo was evaluated in vitro using bone marrow derived dendritic cells, neutrophils, RAW macrophages and the murine bladder cancer cell line, MB49PSA, and compared to other BCG strains. BCG Tokyo > BCG TICE at inducing cytokine production. In vivo, high dose (1 × 107 colony forming units (cfu)) and low dose (1 × 106 cfu) BCG Tokyo with and without cytokine genes (GMCSF + IFNα) were evaluated in C57BL/6J mice (n = 12-16 per group) with orthotopically implanted MB49PSA cells. Mice were treated with four instillations of cytokine gene therapy and BCG therapy. Both high dose BCG alone and low dose BCG combined with cytokine gene therapy were similarly effective. In the second part the responsive groups, mice (n = 27) were monitored by urinary PSA analysis for a further 7 weeks after therapy cessation. More mice were cured at day 84 than at day 42 confirming activation of the immune system. Cured mice resisted the re-challenge with subcutaneous tumors unlike naïve, age matched mice. Antigen specific T cells recognizing BCG, HY and PSA were identified. Thus, fewer intravesical instillations, with high dose BCG Tokyo or low dose BCG Tokyo with GMCSF + IFNα gene therapy, can induce effective systemic immunity.

Tryptophan-kynurenine ratio as a biomarker of bladder cancer.

OBJECTIVES: To investigate plasma and urinary kynurenine (KYN)-tryptophan (TRP) ratios in bladder cancer, expression of indoleamine 2,3-dioxygenase 1 (IDO1) in relation to tryptophan 2,3-dioxygenase (TDO2) in bladder tumour, and the correlation of KYN-TRP ratio with bladder tumour burden. METHODS: Metabotyping of the TRP-KYN metabolic axis was performed via a clinical case-control study. Expression of IDO1 and TDO2 was measured in human biopsied tissues. Correlational experiments between KYN-TRP ratio and bladder tumour were performed using a murine orthotopic prostate-specific antigen (PSA)-secreting MB49 bladder cancer model. RESULTS: We established for the first time that plasma TRP level was significantly decreased, while both plasma and urinary KYN-TRP ratios were significantly higher in bladder cancer patients, and expression level of IDO1 but not TDO2 was increased in human bladder tumour. We reported the positive correlation between IDO1 expression, KYN-TRP ratio, normalized PSA to creatinine, and bladder tumour burden in the murine model. CONCLUSION: Kynurenine-tryptophan ratio is a promising surveillance biomarker for bladder cancer, but would require further validation before clinical translation.

Polymorphism in autophagy gene ATG2B is not associated with bladder cancer recurrence after intravesical Bacillus Calmette-Guerin (BCG) immunotherapy in Asian patients.

OBJECTIVES: Optimal patient stratification is critical in the era of personalized medicine. Germline polymorphisms play an important role in the treatment response of various human diseases, including bladder cancer. Intravesical BCG therapy is widely-used for bladder cancer. However, tumor recurrence and progression are very common. Stratification based on germline polymorphisms may contribute to circumvent this clinical challenge. Autophagy pathway plays an important role in the nonspecific protective effects of BCG. Patients that carry C allele of rs3759601 in autophagy gene ATG2B showed increased risk of recurrence and progression in European population. We thus sought to analyze rs3759601 and its relevance in BCG response in Asian NMIBC patients. METHOD: Functional impact of rs3759601 ATG2B (p.Gln1383Glu) was analyzed by bioinformatics programs including NCBI Conserved Domain Search, Clustal Omega, Polyphen and SIFT. NMIBC patients who received intravesical BCG at multiple hospitals in Singapore from 1995 to 2016 were included. These patients were genotyped for rs3759601 using high resolution melt analysis. The rs3759601 polymorphism was studied in correlation with the bladder cancer recurrence rate and disease progression rate in our cohort. Statistical analysis was conducted using Kaplan-Meier plots and the Chi-squared analysis. RESULTS: In total, 307 individules were included in the study including 161 NMIBC patients and 146 healthy controls, predominately Chinese. The rs3759601 genotype distributions in our NMIBC patients were (GG 72.1%; GC 27.9%; CC 0%), which were distinct from the Dutch report (GG 32.8%; GC 47.4% and CC 19.8%, Buffen K et al, 2014). Consistently, the C allele frequencies of rs3759601 are 0.171 in our controls and 0.177 in East Asians from 1,000 Genome, but 0.406 in Europeans from 1,000 Genome. In silico analysis suggested rs3759601 ATG2B (p.Gln1383Glu) alteration is unlikely to be functionally deleterious. Statistical analysis revealed no significant association between ATG2B rs3759601 C allele and risk of bladder cancer recurrence (P= 0.353, GC vs. GG: hazard ratio [HR]= 1.324), or cancer progression (P= 0.454, GC vs. GG: HR = 0.658). CONCLUSION: In contrast to European NMIBC patients, ATG2B rs3759601 C allele is much less common in Asians and it not associated with BCG response in Asian NMIBC patients.

Slow-, Tight-Binding Inhibition of CYP17A1 by Abiraterone Redefines Its Kinetic Selectivity and Dosing Regimen.

Substantial evidence underscores the clinical efficacy of inhibiting CYP17A1-mediated androgen biosynthesis by abiraterone for treatment of prostate oncology. Previous structural analysis and in vitro assays revealed inconsistencies surrounding the nature and potency of CYP17A1 inhibition by abiraterone. Here, we establish that abiraterone is a slow-, tight-binding inhibitor of CYP17A1, with initial weak binding preceding the subsequent slow isomerization to a high-affinity CYP17A1-abiraterone complex. The in vitro inhibition constant of the final high-affinity CYP17A1-abiraterone complex ( ( K i * = 0.39 nM )yielded a binding free energy of -12.8 kcal/mol that was quantitatively consistent with the in silico prediction of -14.5 kcal/mol. Prolonged suppression of dehydroepiandrosterone (DHEA) concentrations observed in VCaP cells after abiraterone washout corroborated its protracted CYP17A1 engagement. Molecular dynamics simulations illuminated potential structural determinants underlying the rapid reversible binding characterizing the two-step induced-fit model. Given the extended residence time (42 hours) of abiraterone within the CYP17A1 active site, in silico simulations demonstrated sustained target engagement even when most abiraterone has been eliminated systemically. Subsequent pharmacokinetic-pharmacodynamic (PK-PD) modeling linking time-dependent CYP17A1 occupancy to in vitro steroidogenic dynamics predicted comparable suppression of downstream DHEA-sulfate at both 1000- and 500-mg doses of abiraterone acetate. This enabled mechanistic rationalization of a clinically reported PK-PD disconnect, in which equipotent reduction of downstream plasma DHEA-sulfate levels was achieved despite a lower systemic exposure of abiraterone. Our novel findings provide the impetus for re-evaluating the current dosing paradigm of abiraterone with the aim of preserving PD efficacy while mitigating its dose-dependent adverse effects and financial burden. SIGNIFICANCE STATEMENT: With the advent of novel molecularly targeted anticancer modalities, it is becoming increasingly evident that optimal dose selection must necessarily be predicated on mechanistic characterization of the relationships between target exposure, drug-target interactions, and pharmacodynamic endpoints. Nevertheless, efficacy has always been perceived as being exclusively synonymous with affinity-based measurements of drug-target binding. This work demonstrates how elucidating the slow-, tight-binding inhibition of CYP17A1 by abiraterone via in vitro and in silico analyses was pivotal in establishing the role of kinetic selectivity in mediating time-dependent CYP17A1 engagement and eventually downstream efficacy outcomes.

Gmcsf and Ifnα gene therapy improves the response to BCG immunotherapy in a murine model of bladder cancer.

Aim: To develop a strategy to improve response to bacillus Calmette-Gueri (BCG) using cytokine gene therapy (Gmcsf + Ifnα). Materials & methods: MB49-PSA tumor-bearing C57BL/6N mice were assigned into four groups: control; Gmcsf + Ifnα therapy; BCG therapy or combined therapy (Gmcsf + Ifnα and BCG). In schedule 1, cytokine gene therapy was delivered before BCG therapy (eight instillations). In schedule 2, cytokine gene and BCG therapy were instilled alternatively (eight instillations). Tumors were analyzed by immunohistochemistry and mRNA analysis and urinary immune cells by flow cytometry. Results: Combined therapy in schedule 2 reduced tumor growth, increased immune cell recruitment and was associated with reduced inflammation when compared with BCG therapy. Conclusion: Alternating cytokine gene delivery with BCG therapy modulates the tumor environment increasing receptivity to BCG.

The Role of Vitamin D Receptor Polymorphisms in Predicting the Response to Therapy for Nonmuscle Invasive Bladder Carcinoma.

PURPOSE: Clinical and pathological predictors of bladder carcinoma recurrence and progression are relatively well defined. However, there is a paucity of genetic data specifically on the association of single nucleotide polymorphisms in specific genes for predicting recurrence and progression following immunotherapy. The VDR gene was found to regulate the immunomodulatory effects of vitamin D and it enhances the innate immunity system. We evaluated 3 VDR single nucleotide polymorphisms and their predictive role on the response to immunotherapy. MATERIALS AND METHODS: Patients with bladder cancer at intermediate-high risk who underwent post-transurethral resection intravesical bacillus Calmette-Guérin in Singapore and Hong Kong from 1995 to 2014 were recruited for analysis. We evaluated 3 VDR single nucleotide polymorphisms using polymerase chain reaction. Kaplan-Meier survival curves and relationships with outcomes were analyzed by multivariable Cox regression. RESULTS: A total of 338 predominantly Chinese patients were included in study. Individuals carrying the VDR genotype Bsm A/G were significantly associated with lower time to recurrence after bacillus Calmette-Guérin therapy (p <0.001). On multivariable analysis the HR of recurrence in patients with the Bsm A allele was 3.95 times that in patients without the allele (p = 0.037). Patients with the VDR GATC subhaplotype were 3.05 times more likely than patients with other subhaplotypes to experience recurrences (p = 0.003). Study limitations include the small sample size and the lack of information on previous bacillus Calmette-Guérin vaccine exposure and on vitamin D levels. CONCLUSIONS: Our findings in this study suggest that various VDR single nucleotide polymorphisms are associated with recurrences after bacillus Calmette-Guérin immunotherapy. Further functional studies should be performed to elucidate the significance of the VDR gene in the management of bladder cancer and the potential therapy implications.

Bacillus Calmette-Guérin induces rapid gene expression changes in human bladder cancer cell lines that may modulate its survival.

Bacillus Calmette-Guérin (BCG) immunotherapy is the standard therapy for non-muscle invasive bladder cancer. The aim of the present study was to identify genes that are induced in response to BCG immunotherapy, as these may be potential biomarkers for the response to clinical therapy. To model clinical therapy, human bladder cancer cell lines were incubated with BCG (live or lyophilized BCG Connaught) for 2 h. RNA was extracted and evaluated by Representational Differential Analysis (RDA) and oligo arrays. Gene expression was confirmed by reverse transcription polymerase chain reaction on fresh cell lines with differential abilities to internalize BCG. The effect of 2 major BCG soluble proteins, antigen 85B (Ag85B) and Mycobacterium protein tyrosine phosphatase A (MptpA) and BCG Tice® on gene expression was also determined. GAPDH and ß-actin, which are normally used as control genes, were upregulated by BCG. Therefore, the ribosomal RNA gene ribosomal protein S27a was used to normalize gene expression. The genes likely to be induced by BCG internalization and soluble factors were: GSTT2, MGST2, CCL20, TNFα, CCNE1 and IL10RB. Those induced by BCG membrane interactions and/or soluble factors were: MGST1, CXCL6, IL12A, CSF2, IL1ß and TOLLIP. MptpA decreased GSTT2 expression, and Ag85B increased TNFα expression. The two BCG strains significantly increased GSTT2, TNFα and TOLLIP levels in MGH cells. However, in J82 cells there was a BCG strain-dependent difference in TNFα expression. An important outcome of the present study was the determination that neither GAPDH nor ß-actin were suitable control genes for the analysis of BCG-induced gene expression. BCG Connaught and Tice® induced similar expression levels of genes in bladder cancer cell lines. BCG soluble proteins modulated gene expression and therefore may affect therapeutic outcomes. The genes identified may be novel biomarkers of the response to BCG therapy.

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 µL of poly-L-lysine (PLL) is slowly instilled at a rate of 10 µL/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 µL) at a rate of 10 µL/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation. This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

Local Immune Stimulation by Intravesical Instillation of Baculovirus to Enable Bladder Cancer Therapy.

Intravesical instillation of Bacillus Calmette-Guérin is currently used as adjuvant therapy for superficial, non-muscle invasive bladder cancer (NMIBC). However, nearly 40% of patients with NMIBC will fail Bacillus Calmette-Guérin therapy. In an attempt to investigate the feasibility of using insect baculovirus-based vectors for bladder cancer therapy, we observed that intravesical instillation of baculoviruses without transgene up-regulated a set of Th1-type of cytokines and increased the survival rate of mice bearing established orthotopic bladder tumors. When baculoviral vectors were used to co-deliver the mouse CD40 ligand and IL-15 genes through intravesical instillation, the immunogene therapy triggered significantly increased bladder infiltrations of inflammatory monocytes, CD4(+), CD8(+) and γδ T lymphocytes. All treated animals survived beyond 12 months whereas control animals died around 2 months after tumor inoculation. We conclude that direct intravesical instillation of baculoviral gene transfer vectors holds the potential to be a novel therapeutic modality for NMIBC.