Urinary Bladder Cancer Patients Research Articles

Pilot study for bladder cancer detection with volatile organic compounds using ion mobility spectrometry: a novel urine-based approach

PurposeDespite many efforts, no reliable urinary marker system has so far shown the potential to substitute cystoscopy. Measuring volatile organic compounds (VOCs) from urine is a promising alternative. VOCs are metabolic products which can be measured from the headspace of urine samples. Previous studies confirmed that the urine of bladder tumor patients has a different VOC profile than healthy controls. In this pilot study, the feasibility of discriminating VOCs from urine of bladder cancer patients from that of healthy control subjects was investigated. Aim of this study was to investigate whether VOC-based diagnosis of bladder cancer from urine samples is feasible using multicapillary column ion mobility spectrometry (MCC/IMS) and to identify potential molecular correlates to the relevant analytes.MethodsHeadspace measurements of urine samples of 30 patients with confirmed transitional cell carcinoma (TCC) and 30 healthy controls were performed using MCC/IMS. In the results of the measurements, peaks showing significant differences between both groups were identified and implemented into a decision tree with respect to achieve group separation. Molecular correlates were predicted using a pre-defined dataset.ResultsEight peaks with significantly differing intensity were identified, 5 of which were highly significant. Using a six-step decision tree, MCC/IMS showed a sensitivity of 90% and specificity of 100% in group separation.ConclusionVOC-based detection of bladder cancer is feasible. MCC/IMS is a suitable method for urine-based diagnosis and should be further validated. The molecular characteristics and metabolic background of the analytes require further workup.

Abstract 1473: Exposure of arsenic promotes malignant stemness and induces bladder malignancy in preclinical models

Abstract Numerous epidemiological and scientific studies have consistently demonstrated that populations exposed to arsenic (As) concentrations ranging from >10 ppb to 2500 ppb in their drinking water face an elevated risk of urogenital cancers, with a particular emphasis on bladder cancer (BCa). Hence, the goal of the study is to investigate the underlying mechanisms that trigger malignant transformation in the bladder using physiologically relevant arsenic concentrations observed in BCa patients. In our results, we observed significantly elevated levels of arsenic in the urine of BCa patients, ranging from 20 to 50 mg/L, in stark contrast to the levels observed in healthy controls (>10 mg/L) and the recommended guidelines set by the Environmental Protection Agency (EPA) and Centers for Disease Control and Prevention (CDC). We used a median physiological concentration of arsenic (30 PPM) in drinking water-induced bladder hyperplasia in male and female mice over six months. Likewise, chronic exposure of healthy bladder epithelial cells (TRT-HU1) to similar arsenic concentrations (250nM:30 PPM) for 12 months resulted in malignant transformation, and arsenic-transformed cells (AsT) were capable of inducing tumors in xenografted and orthotopic models. Nonetheless, the molecular mechanism responsible for this malignancy remains to be elucidated. Our molecular analysis has pointed towards the activation of stem cells, notably ALDH1A1 (with a 50,000-fold increase), as a pivotal factor driving this transformation. Furthermore, our research has demonstrated that the activation of ALDH1A1 is contingent on the induction of Metal Response Element-Binding Transcription Factor-1 (MTF1). Consequently, silencing MTF1 inhibited the expression of ALDH1A1 and its associated malignant properties in As-transformed cells, strongly suggesting that MTF1 functions as the master regulator in As-induced bladder carcinogenesis. We have also observed similar molecular signatures (MTF1 and ALDH1A1 expression) in arsenic-exposed versus non-exposed mice and bladder cancer patients. In conclusion, our studies suggest that activation of stem cells is a critical step in arsenic-induced bladder carcinogenesis. Citation Format: Bhawna Tyagi, Mohit Vashishta, Neha Tyagi, Balaji Chandrasekaran, Vaibhav Shukla, Ashish Tyagi, Chendil Damodaran. Exposure of arsenic promotes malignant stemness and induces bladder malignancy in preclinical models [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 1473.

Plasma Levels of Pentraxin 3: A Potential Prognostic Biomarker in Urinary Bladder Cancer Patients.

Urinary bladder cancer (BC) represents a major health issue, and identifying novel biomarkers for early disease detection and outcome prediction is paramount. It has already been established that the immune system plays a role in tumour initiation and progression in which the inflammatory marker pentraxin 3 (PTX3) might be involved, presenting a variety of functions in different cancers. The aim of this study was to investigate whether plasma levels of PTX3 could be used as a biomarker for patients with BC. Plasma levels of PTX3 were determined in 118 BC patients and 50 controls by ELISA. Patients with BC had significantly higher PTX3 levels compared to controls. The value as a diagnostic biomarker is probably limited, however, since no significant difference in PTX3 levels was seen between patients with non-muscle-invasive BC and controls; they were seen only between patients with muscle-invasive disease and controls. However, the potential value of PTX3 as a prognostic biomarker was indicated by significantly higher PTX3 levels in patients who developed metastatic disease during follow-up compared to patients who did not develop metastatic disease. The conclusions from this study are that plasma levels of PTX3 have limited value as a diagnostic biomarker, although they have potential as a prognostic biomarker for patients with BC.

Vagococcus fluvialis isolation from the urine of a bladder cancer patient: a case report

Vagococcus fluvialis infection is rare in humans, and there is limited research on the clinical manifestations and antimicrobial susceptibility testing of Vagococcus fluvialis infection. Here, We isolated Vagococcus fluvialis from the urine samples of bladder cancer patients at Hunan Provincial People’s Hospital, and it is the first reported case of Vagococcus fluvialis isolated from the urine. The fully automated microbial identification system and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identified the bacterium as Vagococcus fluvialis with a confidence level of 99.9%. The VITEK-2Compact fully automated microbial susceptibility analysis system indicated that it was most sensitive to tigecycline, vancomycin, quinupristin/dalfopristin, linezolid, and showed moderate sensitivity to erythromycin, levofloxacin, ciprofloxacin, ampicillin/sulbactam, and tetracycline. Additionally, it exhibited synergy when combined with high-level gentamicin and vancomycin, showing sensitivity. However, it displayed poor activity against penicillin and furanth. According to our knowledge, this is the first study to isolate and identify Vagococcus fluvialis from the urine of bladder cancer patients and the systematically reviewed other reported Vagococcus infections on human, which provide an experimental basis for guiding the rational use of drugs in the clinical treatment and diagnose of Vagococcus fluvialis infection and related pathogenic mechanism research. Meanwhile, we have systematically reviewed other reported.

Relationship Between Availability of Urologists and Primary Care Providers and Stage of Diagnosis for Invasive Urinary Bladder Cancer.

With no recommended screening approach, urinary bladder cancer patients rely on referral to urologists to ensure timely diagnosis of bladder cancer. This requires coordination between primary and specialty care. We provide estimates of the relative association between primary care physician and urologist density on stage of urinary bladder cancer diagnosis. We used 2010 to 2016 Pennsylvania Cancer Registry data to identify all adult patients diagnosed with bladder cancer. Our primary outcome was locoregional stage of diagnosis, since treatment modality changes and prognosis worsens beyond this stage. Based on patient's residential location at the time of diagnosis we defined both density of urologists and number of primary care providers (defined as providers per population) within the patient's county. We used univariate and multivariate logistic regression to estimate the association between provider density and likelihood of locoregional stage of diagnosis. We also controlled for age, sex, race/ethnicity, insurance type, and year. Our sample included 11,771 urinary bladder cancer patients with 10,607 diagnosed at locoregional stage and 1164 at distant stage. Multivariate regression results show primary care density was associated with significantly higher odds of locoregional stage of diagnosis (odds ratio of 1.05 [95% CI: 1.02-1.08]) while urologist density was associated with significantly lower odds of locoregional stage (odds ratio of 0.65 [95% CI: 0.48-0.89]). We found primary care density but not urologist density was associated with earlier stage of diagnosis, highlighting the importance of access to primary care and need for timely referral to urologic care.

Real-time analysis of the cancer genome and fragmentome from plasma and urine cell-free DNA using nanopore sequencing.

Cell-free DNA (cfDNA) can be isolated and sequenced from blood and/or urine of cancer patients. Conventional short-read sequencing lacks deployability and speed and can be biased for short cfDNA fragments. Here, we demonstrate that with Oxford Nanopore Technologies (ONT) sequencing we can achieve delivery of genomic and fragmentomic data from liquid biopsies. Copy number aberrations and cfDNA fragmentation patterns can be determined in less than 24 h from sample collection. The tumor-derived cfDNA fraction calculated from plasma of lung cancer patients and urine of bladder cancer patients was highly correlated (R = 0.98) with the tumor fraction calculated from short-read sequencing of the same samples. cfDNA size profile, fragmentation patterns, fragment-end composition, and nucleosome profiling near transcription start sites in plasma and urine exhibited the typical cfDNA features. Additionally, a high proportion of long tumor-derived cfDNA fragments (> 300 bp) are recovered in plasma and urine using ONT sequencing. ONT sequencing is a cost-effective, fast, and deployable approach for obtaining genomic and fragmentomic results from liquid biopsies, allowing the analysis of previously understudied cfDNA populations.

A Protocol for Organoids from the Urine of Bladder Cancer Patients.

This study investigates the feasibility of establishing urine-derived tumor organoids from bladder cancer (BC) patients as an alternative to tissue-derived organoids. BC is one of the most common cancers worldwide and current diagnostic methods involve invasive procedures. Here, we investigated the potential of using urine samples, which contain exfoliated tumor cells, to generate urine-derived BC organoids (uBCOs). Urine samples from 29 BC patients were collected and cells were isolated and cultured in a three-dimensional matrix. The establishment and primary expansion of uBCOs were successful in 83% of the specimens investigated. The culturing efficiency of uBCOs was comparable to cancer tissue-derived organoids. Immunohistochemistry and immunofluorescence to characterize the uBCOs exhibited similar expressions of BC markers compared to the parental tumor. These findings suggest that urine-derived BC organoids hold promise as a non-invasive tool for studying BC and evaluating therapeutic responses. This approach could potentially minimize the need for invasive procedures and provide a platform for personalized drug screening. Further research in this area may lead to improved diagnostic and treatment strategies for BC patients.

A Study of DNA Methylation of Bladder Cancer Biomarkers in the Urine of Patients with Neurogenic Lower Urinary Tract Dysfunction.

Background: Bladder cancer (BCa) in patients suffering from neurogenic lower urinary tract dysfunction (NLUTD) is a significant concern due to its advanced stage at diagnosis and high mortality rate. Currently, there is a scarcity of specific guidelines for BCa screening in these patients. The development of urine biomarkers for BCa seems to be an attractive non-invasive method of screening or risk stratification in this patient population. DNA methylation is an epigenetic modification, resulting in the transcriptional silencing of tumor suppression genes, that is frequently detected in the urine of BCa patients. Objectives: We aimed to investigate DNA hypermethylation in five gene promoters, previously associated with BCa, in the urine of NLUTD patients, and in comparison with healthy controls. Design, setting and participants: This was a prospective case-control study that recruited neurourology outpatients from a public teaching hospital who had suffered from NLUTD for at least 5 years. They all underwent cystoscopy combined with biopsy for BCa screening following written informed consent. DNA was extracted and DNA methylation was assessed for the RASSF1, RARβ, DAPK, TERT and APC gene promoters via quantitative methylation-specific PCR in urine specimens from the patients and controls. Results: Forty-one patients of mixed NLUTD etiology and 35 controls were enrolled. DNA was detected in 36 patients' urine specimens and in those of 22 controls. In the urine specimens, DNA was hypermethylated in at least one of five gene promoters in 17/36 patients and in 3/22 controls (47.22% vs. 13.64%, respectively, p = 0.009). RASSF1 was hypermethylated in 10/17 (58.82%) specimens with detected methylation, APC in 7/17 (41.18%), DAPK in 4/17 (23.53%), RAR-β2 in 3/17 (17.56%) and TERT in none. According to a multivariate logistic regression analysis, NLUTD and male gender were significantly associated with hypermethylation (OR = 7.43, p = 0.007 and OR = 4.21; p = 0.04, respectively). In the tissue specimens, histology revealed TaLG BCa in two patients and urothelial squamous metaplasia in five patients. Chronic bladder inflammation was present in 35/41 bladder biopsies. Conclusions: DNA hypermethylation in a panel of five BCa-associated genes in the urine was significantly more frequent in NLUTD patients than in the controls. Our results warrant further evaluation in longitudinal studies assessing the clinical implications and possible associations between DNA hypermethylation, chronic inflammation and BCa in the NLUTD population.

BTA stat®, NMP22® BladderChek®, UBC® Rapid Test, and CancerCheck® UBC® rapid VISUAL as urinary marker for bladder cancer: Final results of a German multicenter study

Introduction and objectiveBTA stat®, NMP22® BladderChek®, UBC® Rapid Test, and CancerCheck® UBC® rapid VISUAL are urinary-based rapid tests. This multicenter study is the first study comparing all available rapid tests on a large cohort of bladder cancer patients and healthy controls in one setting. MethodsIn total 732 urine samples (second morning urine) in a real-world assessment have been analyzed. We evaluated clinical samples from 464 patients with histologically confirmed urothelial tumors of the urinary bladder (17 solitary CIS, 189 low-grade, 187 high-grade nonmuscle invasive, 71 high-grade muscle invasive), 77 patients with No Evidence of Disease (NED), and from 191 healthy controls. Urine samples were analyzed by the BTA stat®, NMP22® BladderChek®, UBC® Rapid Test point-of-care (POC) system using the concile Omega 100 POC reader, and CancerCheck® UBC® rapid VISUAL. Sensitivities and specificities were calculated by contingency analyses. ResultsAll investigated urinary markers detected more pathological concentrations in urine of bladder cancer patients compared to tumor-free patients. The calculated diagnostic sensitivities for BTA stat®, NMP22® BladderChek®, UBC® Rapid Test, CancerCheck® UBC® rapid VISUAL, and cytology were 62.4%, 13.4%, 58.2%, 28.6%, 36.2% for low-grade, 83.4%, 49.5%, 84.5%, 63.1%, 71.2% for high-grade nonmuscle invasive, and 95.8%, 35.2%, 76.1%, 50.7%, 67.7% for high-grade muscle-invasive bladder cancer. The specificity was 67.9%, 95.5%, 79.4%, 94.4%, and 83.7%, respectively. The area under the curve (AUC) after receiver operating characteristics (ROC) analysis for high-grade non–muscle-invasive tumors was 0.757, 0.725, 0.819, 0.787, and 0.774, respectively. ConclusionsThe analysis of more than 700 urine samples offers an objective view on urine-based rapid diagnostics. Elevated pathological concentrations of markers in urine of bladder cancer patients were detected in all investigated tests. The highest sensitivities for high-grade non–muscle-invasive tumors were calculated for BTA stat® and UBC® Rapid Test, whereas NMP22® BladderChek®, and cytology showed the highest specificities. BTA stat® and UBC® Rapid Test have the potential to be used as a clinical valuable urinary protein biomarker for the detection of high-grade non–muscle-invasive bladder cancer patients and could be included in the management of these tumors.

Cytomorphologic visualization of circulating tumor cells in urinary bladder cancer patients using ScreenCell™ technology: Potential as a simple cytology test.

Circulating tumor cells (CTC) are a recent technique which is a potentially important prognostic factor in many solid tumors. There are many techniques of detecting CTCs, but they usually implement costly techniques like EpCAM targeted detection, fluorescence-based diagnosis, or magnetic bead based positive or negative selection. The diagnostic utility of simple cytomorphological diagnosis after routine staining of CTCs have been rarely studied. We aimed to detect CTCs in 24 patients clinically suspected to have Urinary Bladder Cancer using a simple but efficient patented filtration technology (ScreenCell™), followed by optical microscopic visualization after routine May-Grunwald-Giemsa (MGG) staining. The detected CTCs were then tested for association with the histologic type, lamina propria invasion, deep muscle invasion and the T-stage. Out of the 24 patients tested, one was found to have papilloma, nine had low grade urothelial carcinoma, 13 had high grade urothelial carcinoma and one had poorly differentiated adenocarcinoma. Of these, two LGUC, eight HGUC and one adenocarcinoma had detectable CTC. Presence of CTCs had a statistically significant association with Lamina propria invasion (P = .006) and T-stage (P = .02), and a trend toward significance for differentiating LGUC from HGUC (P = .10). These results suggest that cytomorphological detection of CTC is likely to be clinically useful in diagnosis and prognostication of urinary blader cancers. These findings need to be confirmed on studies with larger sample sizes.

Global Meta-analysis of Urine Microbiome: Colonization of Polycyclic Aromatic Hydrocarbon–degrading Bacteria Among Bladder Cancer Patients

BackgroundThe application of next-generation sequencing techniques has enabled characterization of urinary tract microbiome. Although many studies have demonstrated associations between the human microbiome and bladder cancer (BC), these have not always reported consistent results, thereby necessitating cross-study comparisons. Thus, the fundamental questions remain how we can utilize this knowledge. ObjectiveThe aim of our study was to examine the disease-associated changes in urine microbiome communities globally utilizing a machine learning algorithm. Design, setting, and participantsRaw FASTQ files were downloaded for the three published studies in urinary microbiome in BC patients, in addition to our own prospectively collected cohort. Outcome measurements and statistical analysisDemultiplexing and classification were performed using the QIIME 2020.8 platform. De novo operational taxonomic units were clustered using the uCLUST algorithm and defined by 97% sequence similarity and classified at the phylum level against the Silva RNA sequence database. The metadata available from the three studies included were used to evaluate the differential abundance between BC patients and controls via a random-effect meta-analysis using the metagen R function. A machine learning analysis was performed using the SIAMCAT R package. Results and limitationsOur study includes 129 BC urine and 60 healthy control samples across four different countries. We identified a total of 97/548 genera to be differentially abundant in the BC urine microbiome compared with that of healthy patients. Overall, while the differences in diversity metrics were clustered around the country of origin (Kruskal-Wallis, p < 0.001), collection methodology was a driver of microbiome composition. When assessing dataset from China, Hungary, and Croatia, data demonstrated no discrimination capacity to distinguish between BC patients and healthy adults (area under the curve [AUC] 0.577). However, inclusion of samples with catheterized urine improved the diagnostic accuracy of prediction for BC to AUC 0.995, with precision-recall AUC = 0.994. Through elimination of contaminants associated with the collection methodology among all cohorts, our study identified increased abundance of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria Sphingomonas, Acinetobacter, Micrococcus, Pseudomonas, and Ralstonia to be consistently present in BC patients. ConclusionsThe microbiota of the BC population may be a reflection of PAH exposure from smoking, environmental pollutants, and ingestion. Presence of PAHs in the urine of BC patients may allow for a unique metabolic niche and provide necessary metabolic resources where other bacteria are not able to flourish. Furthermore, we found that while compositional differences are associated with geography more than with disease, many are driven by the collection methodology. Patient summaryThe goal of our study was to compare the urine microbiome of bladder cancer patients with that of healthy controls and evaluate any potential bacteria that may be more likely to be found in patients with bladder cancer. Our study is unique as it evaluates this across multiple countries, to find a common pattern. After we removed some of the contamination, we were able to localize several key bacteria that are more likely to be found in the urine of bladder cancer patients. These bacteria all share their ability to break down tobacco carcinogens.

ECOG score as a barrier for clinical trial eligibility in the patient population with genitourinary cancer: Is it time to adapt this criterion to real-world data?

78 Background: ECOG score is a common eligibility criteria used in clinical trials (CT). Many CT recruit only patients in good physical condition (ie. minimal comorbidities and good performance status), which may misrepresent the real-world cancer population.This gap may result in safety and effectiveness data that may not be applicable to the real-world patient population. Furthermore, applying strict eligibility criteria augment a barrier for patient accrual to CT. In this study we analyzed the impact of ECOG performance status on Genitourinary (GU) oncology trials eligibility of patients who used the Trialjectory web-based platform. Methods: Trialjectory is an AI- based platform that matches cancer patients to CT based on a patient reported questionnaire that translates to an individual clinical profile. The profile includes disease status and stage, biomarkers, treatment history and comorbidities and demographics. Data from patients with prostate and urinary bladder cancer was analyzed. The number of matched trials per patient was determined based on the patient’s profile, location and travel preference. To assess the impact of ECOG score, we edited the data set to reflect a better performance status per patient. We used a paired samples Wilcoxon test to assess the association between decreasing the ECOG threshold and the number of matched CT. Results: Between May 2021 and August 2022 we analyzed 153 bladder cancer profiles with ECOG=2 and 71 with ECOG=3, and 90 and 27 prostate cancer profiles with ECOG=2 and ECOG=3, respectively. In a multiple regression analysis, high ECOG score was inversely and significantly associated with the number of matched trials per patient in prostate cancer (p=0.009), independent of cancer stage, patient ethnicity and gender; and in urinary bladder cancer (p=0.003), after adjustment for cancer stage, gender and ethnicity. Editing ECOG score from 2 to 1 resulted in a profound increase (233% for prostate cancer patients and 300% for urinary bladder cancer patients respectively) in the number of matched trials per patient, from a median of 3 trials to 7 and from 2 to 6 trials per patient respectively (V = 426, p-value &lt;0.0001 and V = 14, p-value = 0.0002 respectively). Among Bladder Cancer patients, editing the ECOG score from 3 to 2 resulted in an increase of 200%, from a median 1 to 2 matched CT (V = 291, p-value = 0.0002). Conclusions: The results reflect a high magnitude reduction in eligibility for patients with higher ECOG scores. Excluding these patients may result in skewed reporting on effectiveness-toxicity of drugs that may not be applicable for the real-world patient population augmenting a significant barrier for CT accrual amongst the proactive population who seek it.

Properties of non-coding mutation hotspots as urinary biomarkers for bladder cancer detection

Mutations at specific hotspots in non-coding regions of ADGRG6, PLEKHS1, WDR74, TBC1D12 and LEPROTL1 frequently occur in bladder cancer (BC). These mutations could function as biomarkers for the non-invasive detection of BC but this remains largely unexplored. Massively-parallel sequencing of non-coding hotspots was applied to 884 urine cell pellet DNAs: 591 from haematuria clinic patients (165 BCs, 426 non-BCs) and 293 from non-muscle invasive BC surveillance patients (29 with recurrence). Urine samples from 142 non-BC haematuria clinic patients were used to optimise variant calling. Non-coding mutations are readily detectable in the urine of BC patients and undetectable, or present at much lower frequencies, in the absence of BC. The mutations can be used to detect incident BC with 66% sensitivity (95% CI 58–75) at 92% specificity (95% CI 88–95) and recurrent disease with 55% sensitivity (95% CI 36–74) at 85% specificity (95% CI 80–89%) using a 2% variant allele frequency threshold. In the NMIBC surveillance setting, the detection of non-coding mutations in urine in the absence of clinically detectable disease was associated with an increased relative risk of future recurrence (RR = 4.62 (95% CI 3.75–5.48)). As urinary biomarkers, non-coding hotspot mutations behave similarly to driver mutations in BC-associated genes and could be included in biomarker panels for BC detection.

Toll-like receptor 4 promotes bladder cancer progression upon S100A8/A9 binding, which requires TIRAP-mediated TPL2 activation

Bladder cancer is an often widely disseminated and deadly cancer. To block the malignant outgrowth of bladder cancer, we must elucidate the molecular-level characteristics of not only bladder cancer cells but also their surrounding milieu. As part of this effort, we have long been studying extracellular S100A8/A9, which is elevated by the inflammation associated with certain cancers. Extracellularly enriched S100A8/A9 can hasten a shift to metastatic transition in multiple types of cancer cells. Intriguingly, high-level S100A8/A9 has been detected in the urine of bladder-cancer patients, and the level increases with the stage of malignancy. Nonetheless, S100A8/A9 has been investigated mainly as a potential biomarker of bladder cancers, and there have been no investigations of its role in bladder-cancer growth and metastasis. We herein report that extracellular S100A8/A9 induces upregulation of growth, migration and invasion in bladder cancer cells through its binding with cell-surface Toll-like receptor 4 (TLR4). Our molecular analysis revealed the TLR4 downstream signal that accelerates such cancer cell events. Tumor progression locus 2 (TPL2) was a key factor facilitating the aggressiveness of cancer cells. Upon binding of S100A8/A9 with TLR4, TPL2 activation was enhanced by an action with a TLR4 adaptor molecule, TIR domain-containing adaptor protein (TIRAP), which in turn led to activation of the mitogen-activated protein kinase (MAPK) cascade of TPL2. Finally, we showed that sustained inhibition of TLR4 in cancer cells effectively dampened cancer survival in vivo. Collectively, our results indicate that the S100A8/A9-TLR4-TPL2 axis influences the growth, survival, and invasive motility of bladder cancer cells.

“Seeing” invisible volatile organic compound (VOC) marker of urinary bladder cancer: A development from bench to bedside prototype spectroscopic device

Urinary bladder cancer (UBC) is one of the most common cancers and has notoriously high risk of recurrence and mortality across the globe. Current clinical initial diagnostic approaches are either invasive or lacks sensitivity. In this study, an attempt has been made to invent a cost-effective, novel, portable diagnostic device based on the environmental sensitive fluorophores namely Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB). They act as sensing agents for detecting volatile organic compounds (VOC) exclusively present in the urine sample of UBC patients and differentiate the UBC samples from the healthy control group. Upon exposure with a particular group of VOCs, a significant amount of increment in fluorescence intensities of NR, EY and RB were detected and recorded in our indigenously developed “NABIL” device. To check the performance of NABIL, the data collected from the device was compared with the conventional techniques by arranging a clinical trial with 21 healthy controls and 52 UBC patients. With the assistance of our analysis technique based on LabVIEW platform, very high sensitivity and accuracy from healthy controls have been achieved. For UBC patients, it shows impressive diagnostic results. In addition, depending on the sample processing mechanism, NABIL device can also reveal the grade of UBC and prognosis under treatment. Overall, this study contributes a novel, non-invasive, easy-to-use, inexpensive, real-time, accurate method for selectively UBC diagnosis, which can be useful for personalized care/diagnosis and postoperative surveillance, resulting in saving more lives.

CyTOF analysis identifies unusual immune cells in urine of BCG-treated bladder cancer patients.

High grade non-muscle-invasive bladder tumours are treated with transurethral resection followed by recurrent intravesical instillations of Bacillus Calmette Guérin (BCG). Although most bladder cancer patients respond well to BCG, there is no clinical parameter predictive of treatment response, and when treatment fails, the prognosis is very poor. Further, a high percentage of NMIBC patients treated with BCG suffer unwanted effects that force them to stop treatment. Thus, early identification of patients in which BCG treatment will fail is really important. Here, to identify early stage non-invasive biomarkers of non-responder patients and patients at risk of abandoning the treatment, we longitudinally analysed the phenotype of cells released into the urine of bladder cancer patients 3-7 days after BCG instillations. Mass cytometry (CyTOF) analyses revealed a large proportion of granulocytes and monocytes, mostly expressing activation markers. A novel population of CD15+CD66b+CD14+CD16+ cells was highly abundant in several samples; expression of these markers was confirmed using flow cytometry and qPCR. A stronger inflammatory response was associated with increased cell numbers in the urine; this was not due to hematuria because the cell proportions were distinct from those in the blood. This pilot study represents the first CyTOF analysis of cells recruited to urine during BCG treatment, allowing identification of informative markers associated with treatment response for sub-selection of markers to confirm using conventional techniques. Further studies should jointly evaluate cells and soluble factors in urine in larger cohorts of patients to characterise the arms of the immune response activated in responders and to identify patients at risk of complications from BCG treatment.

Identification of hexosamine biosynthesis pathway as a novel prognostic signature and its correlation with immune infiltration in bladder cancer

Background: Urinary bladder cancer (UBC) is one of the common urological malignancies, lacking reliable biomarkers to predict clinical outcomes in UBC patients. Thus, it is needed to identify the novel diagnostic/prognostic biomarkers to stratify the high-risk UBC patients. As a shunt pathway of glycolysis, the hexosamine biosynthesis pathway (HBP) has been implicated in carcinogenesis. However, its prognostic value in UBC remains unclear. Methods: The RNA sequencing and mRNA microarray datasets were downloaded from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus databases. The expression levels of five HBP genes were analyzed in normal and UBC samples, and their associations with stage, grade and survival were plotted. The performance of HBP risk group was evaluated by receiver-operating characteristics (ROC) curve. The HBP signature was generated by Gene Set Variation Analysis (GSVA) and its association with clinicopathological parameters and survival were analyzed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were carried out to examine the potential biological functions of HBP using DAVID online tool. The infiltration estimation fraction of immune cells was performed using CIBERSORT-ABS algorithm. Gene set enrichment analysis (GSEA) was used to explore the potential function of HBP in tumor immunoregulation. Results: Four HBP genes were upregulated in UBCs compared to normal tissues in TCGA-BLCA dataset. The upregulation of all five HBP genes was significantly associated with tumor grade and stage of UBC in three independent UBC datasets. The expression of HBP genes predicted poor clinical outcomes in UBC patients in both TCGA-BLCA and GSE13507 datasets. The high-risk group based on HBP genes showed a poor prognosis. Furthermore, HBP signature was positively associated with tumor grade and stage in TCGA-BLCA dataset and with tumor grade, stage, distal metastasis and poor survival in GSE13507 dataset. Interestingly, high-HBP signature group exhibited a high infiltration of immune cells, particularly the macrophage population. Conclusion: We identified that HBP was a promising prognostic biomarker in UBC patients and strongly associated with immune infiltration.

Interferon gamma expression and mortality in unselected cohorts of urothelial bladder cancer patients.

BackgroundThe role of interferon gamma (IFN-γ) expression in long-term survival has not been studied in patients with urinary bladder cancer (UBC). IFN-γ expression was characterized among various UBC patient cohorts to assess if IFN-γ status is associated with overall survival (OS).MethodsA tumor-based IFN-γ gene signature was evaluated among adult UBC patients newly diagnosed between 2004 and 2017 from two hospital systems in New York. Patient cohorts included metastatic (stage IV or progressing to stage IV [MBC]), muscle-invasive (stages T2a to T4a [MIBC]), and non–muscle-invasive (carcinoma in situ or stages 0a, 0is, and I [NMIBC]) disease. Descriptive analyses were conducted comparing IFN-γ signature in the highest tertile to those in the lowest two tertiles.Results234 patients with bladder cancer were evaluated (56 MBC, 38 MIBC, and 140 NMIBC). Median OS was only reached in the MIBC cohort for those with an IFN-γ signature in the lowest two tertiles (15.03 months [95% CI, 8.50–50.60]). Those with an IFN-γ signature in the highest tertile had a decreased risk of mortality in all cohorts indicating better survival, but this was statistically significant in only the MIBC cohort (adjusted HR = 0.09 [95% CI, 0.01–0.73]).ConclusionIFN-γ signature status was associated with a decreased mortality risk in all cohorts, particularly MIBC, indicating that it may be a prognostic marker of survival in patients with UBC.

Characterization of novel small non-coding RNAs and their modifications in bladder cancer using an updated small RNA-seq workflow.

Background: Bladder cancer (BLCA) is one of the most common cancer types worldwide. The disease is responsible for about 200,000 deaths annually, thus improved diagnostics and therapy is needed. A large body of evidence reveal that small RNAs of less than 40 nucleotides may act as tumor suppressors, oncogenes, and disease biomarkers, with a major focus on microRNAs. However, the role of other families of small RNAs is not yet deciphered. Recent results suggest that small RNAs and their modification status, play a role in BLCA development and are promising biomarkers due to their high abundance in the exomes and body fluids (including urine). Moreover, free modified nucleosides have been detected at elevated levels from the urine of BLCA patients. A genome-wide view of small RNAs, and their modifications, will help pinpoint the molecules that could be used as biomarker or has important biology in BLCA development. Methods: BLCA tumor tissue specimens were obtained from 12 patients undergoing transurethral resection of non-muscle invasive papillary urothelial carcinomas. Genome-wide profiling of small RNAs less than 40 bases long was performed by a modified protocol with TGIRT (thermostable group II reverse transcriptase) to identify novel small RNAs and their modification status. Results: Comprehensive analysis identified not only microRNAs. Intriguingly, 57 ± 15% (mean ± S.D.) of sequencing reads mapped to non-microRNA-small RNAs including tRNA-derived fragments (tRFs), ribosomal RNA-derived fragments (rRFs) and YRNA-derived fragments (YRFs). Misincorporation (mismatch) sites identified potential base modification positions on the small RNAs, especially on tRFs, corresponding to m1A (N1-methyladenosine), m1G (N1-methylguanosine) and m2 2G (N2, N2-dimethylguanosine). We also detected mismatch sites on rRFs corresponding to known modifications on 28 and 18S rRNA. Conclusion: We found abundant non-microRNA-small RNAs in BLCA tumor samples. Small RNAs, especially tRFs and rRFs, contain modifications that can be captured as mismatch by TGIRT sequencing. Both the modifications and the non-microRNA-small RNAs should be explored as a biomarker for BLCA detection or follow-up.

Lamprey immunity protein enables early detection and recurrence monitoring for bladder cancer through recognizing Neu5Gc-modified uromodulin glycoprotein in urine

The clinical management of bladder cancer (BCa) is hindered by the lack of reliable biomarkers. We aimed to investigate the potential of lamprey immunity protein (LIP), a lectin that specifically binds to multi-antennary sialylated N-glycolylneuraminic acid (Neu5Gc) structures on UMOD glycoproteins in the urine of BCa patients. Primary BCa patients had higher levels of LIP-bound Neu5Gc in urine than healthy participants and patients receiving postoperative treatment did. In addition, lectin chip assay and mass spectrometry were used to analyze the glycan chain structure, which can recognize the UMOD glycoprotein decorated with multi-antennary sialylated Neu5Gc structures. Furthermore, compared with urine samples from healthy patients (N = 2821, T/C = 0.12 ± 0.09) or benign patients (N = 360, T/C = 0.11 ± 0.08), the range of the urine T/C ratio detected using LIP test paper was 1.97 ± 0.32 in patients with bladder cancer (N = 518) with significant difference (P < 0.0001). Our results indicate that LIP may be a tool for early BCa identification, diagnosis, and monitoring. Neu5Gc-modified UMOD glycoproteins in urine and Neu5Gc-modified N-glycochains and sialyltransferases may function as potential markers in clinical trials.