Evaluation of a New Survivin ELISA and UBC® Rapid for the Detection of Bladder Cancer in Urine.
ABSTRACT: Urine-based biomarkers for non-invasive diagnosis of bladder cancer are urgently needed. No single marker with sufficient sensitivity and specificity has been described so far. Thus, a combination of markers appears to be a promising approach. The aim of this case-control study was to evaluate the performance of an in-house developed enzyme-linked immunosorbent assay (ELISA) for survivin, the UBC®Rapid test, and the combination of both assays. A total of 290 patients were recruited. Due to prior bladder cancer, 46 patients were excluded. Urine samples were available from 111 patients with bladder cancer and 133 clinical controls without urologic diseases. Antibodies generated from recombinant survivin were utilized to develop a sandwich ELISA. The ELISA and the UBC®Rapid test were applied to all urine samples. Receiver operating characteristic (ROC) analysis was used to evaluate marker performance. The survivin ELISA exhibited a sensitivity of 35% with a specificity of 98%. The UBC®Rapid test showed a sensitivity of 56% and a specificity of 96%. Combination of both assays increased the sensitivity to 66% with a specificity of 95%. For high-grade tumors, the combination showed a sensitivity of 82% and a specificity of 95%. The new survivin ELISA and the UBC®Rapid test are both able to detect bladder cancer, especially high-grade tumors. However, the performance of each individual marker is moderate and efforts to improve the survivin assay should be pursued. A combination of both assays confirmed the benefit of using marker panels. The results need further testing in a prospective study and with a high-risk population.
Project description:Background: Non-muscle invasive bladder cancer (NMIBC) is associated with high rates of recurrence, resulting in frequent follow-up cystoscopies. We evaluated the use of two point-of-care tests - the nuclear matrix protein 22 (NMP22) and urinary bladder cancer antigen (UBC) Rapid - compared to routine follow-up in patients with a previous history of NMIBC. Methods: 31 patients with cystoscopy-verified active bladder cancer, and 44 follow-up patients without disease as confirmed by cystoscopy were prospectively enrolled. All urine samples were analyzed by voided urine and bladder washing cytology, NMP22 and UBC rapid test (qualitatively and quantitatively). The best cutoff (highest Youden index; ≥6.7 ng/ml) for the quantitative UBC was determined by receiver operating characteristic curves. Results: Voided urine and barbotage cytology resulted in a sensitivity of 25.8% and 32.3%, and a specificity of 100% and 100%, while the NMP22 showed a sensitivity and specificity of 12.9% and 100%, respectively. The qualitative and quantitative UBC Rapid revealed a sensitivity of 61.3% and 64.5%, with a specificity of 77.3% and 81.8%. Barbotage cytology and qualitative UBC test proved to be the best dual combination with the highest overall sensitivity (77.4%). In contrast to barbotage cytology alone, sensitivity increased from 21.4% to 50% for detecting low-grade tumors, and from 43.8% to 100% for high-grade cancers, but reducing specificity from 100% to 77.3%. Conclusion: Compared to urinary cytology, UBC tests alone as well as UBC tests in combination with bladder washing cytology revealed higher sensitivities in detecting low- and high-grade tumors, but at the expense of a lower specificity. Thus, currently cystoscopy cannot be replaced by any of the evaluated methods.
Project description:Recent studies showed the clinical utility of next-generation sequencing of urinary cell-free DNA (cfDNA) from patients with urothelial bladder cancer (UBC). In this study, we aimed to develop urinary cfDNA analysis by droplet digital PCR (ddPCR) as a high-throughput and rapid assay for UBC detection and prognosis. We analyzed urinary cfDNA of 202 samples from 2 cohorts. Test cohort was designed for investigating clinical utility of urinary cfDNA, and was composed of 74 samples from patients with UBC, and 52 samples of benign hematuria patients. Validation cohort was designed for validation and assessment of clinical utility comparing urinary cfDNA with UroVysion (Abbott, Illinois, USA), and was composed of 40 samples from patients with UBC, and 36 prospectively collected samples from patients under surveillance after surgery for urothelial carcinoma. We performed ddPCR analysis of hotspot gene mutations (TERT promoter and FGFR3). In the test cohort, the sensitivity of urinary cfDNA diagnosis was 68.9% (51/74) and the specificity was 100% in patients with UBC. The sensitivity increased to 85.9% when used in conjunction with urine cytology. In addition, patients with high TERT C228T allele frequency (?14%) had significantly worse prognosis in bladder tumor recurrence than patients with low TERT C228T allele frequency or negative TERT C228T (p = 0.0322). In the validation cohort, the sensitivity of urinary cfDNA was 57.5% (23/40) and the specificity was 100% in UBC patients. The sensitivity of the combination of urine cytology with our hotspot analysis (77.5%) was higher than that of urine cytology with UroVysion (68.9%). In the post-surgical surveillance group, patients positive for the TERT C228T mutation had significantly worse prognosis for bladder tumor recurrence than mutation negative patients (p < 0.001). In conclusion, ddPCR analysis of urinary cfDNA is a simple and promising assay for the clinical setting, surpassing UroVysion for detection and prognosis determination in UBC.
Project description:ELISA is the main approach for the sensitive quantification of protein biomarkers in body fluids and is currently employed in clinical laboratories for the measurement of clinical markers. As such, it also constitutes the main methodological approach for biomarker validation and further qualification. For the latter, specific assay performance requirements have to be met, as described in respective guidelines of regulatory agencies. Even though many clinical ELISA assays in serum are regularly used, ELISA clinical applications in urine are significantly less. The scope of our study was to evaluate ELISA assay analytical performance in urine for a series of potential biomarkers for bladder cancer, as a first step towards their large scale clinical validation. Seven biomarkers (Secreted protein acidic and rich in cysteine, Survivin, Slit homolog 2 protein, NRC-Interacting Factor 1, Histone 2B, Proteinase-3 and Profilin-1) previously described in the literature as having differential expression in bladder cancer were included in the study. A total of 11 commercially available ELISA tests for these markers were tested by standard curve analysis, assay reproducibility, linearity and spiking experiments. The results show disappointing performance with coefficients of variation>20% for the vast majority of the tests performed. Only 3 assays (for Secreted protein acidic and rich in cysteine, Survivin and Slit homolog 2 protein) passed the accuracy thresholds and were found suitable for further application in marker quantification. These results collectively reflect the difficulties in developing urine-based ELISA assays of sufficient analytical performance for clinical application, presumably attributed to the urine matrix itself and/or presence of markers in various isoforms.
Project description:The TERT promoter and FGFR3 gene mutations are two of the most common genetic events in urothelial bladder cancer (UBC), and these mutation assays in patient urine have been shown to be promising biomarkers for UBC diagnosis and surveillance. These results were obtained mainly from studies of patients with UBC in Western countries, and little is known about such information in Han Chinese patients with UBC. In the present study, we addressed this issue by analyzing tumors from 182 Han Chinese patients with UBC and urine samples from 102 patients for mutations in the TERT promoter and FGFR3 and TERT mRNA expression in tumors and/or urine. TERT promoter and FGFR3 mutations were identified in 87 of 182 (47.8%) and 7 of 102 (6.7%) UBC cases, respectively. In 46 urine samples from patients with TERT promoter mutation-carrying tumors, the mutant promoter was detected in 24 (52%) prior to operation and disappeared in most examined urine samples (80%) taken 1 week after operation. TERT mRNA was detected in urine derived from 46 of 49 patients (94%) that was analyzed before operation independently of the presence of TERT promoter mutations. Collectively, FGFR3 mutations occur at a very low rate in Han Chinese UBC and cannot serve as diagnostic markers for Chinese patients. Han Chinese patients with UBC have relatively low TERT promoter mutation frequency compared with patients in Western countries, and simultaneous detection of both mutant TERT promoter and TERT mRNA improves sensitivity and specificity of urine-based diagnosis.
Project description:Urinary biomarkers for bladder cancer detection are constrained by inadequate sensitivity or specificity. Here we evaluate the diagnostic accuracy of Mcm5, a novel cell cycle biomarker of aberrant growth, alone and in combination with NMP22.1677 consecutive patients under investigation for urinary tract malignancy were recruited to a prospective blinded observational study. All patients underwent ultrasound, intravenous urography, cystoscopy, urine culture and cytologic analysis. An immunofluorometric assay was used to measure Mcm5 levels in urine cell sediments. NMP22 urinary levels were determined with the FDA-approved NMP22® Test Kit.Genito-urinary tract cancers were identified in 210/1564 (13%) patients with an Mcm5 result and in 195/1396 (14%) patients with an NMP22 result. At the assay cut-point where sensitivity and specificity were equal, the Mcm5 test detected primary and recurrent bladder cancers with 69% sensitivity (95% confidence interval?=?62-75%) and 93% negative predictive value (95% CI?=?92-95%). The area under the receiver operating characteristic curve for Mcm5 was 0.75 (95% CI?=?0.71-0.79) and 0.72 (95% CI?=?0.67-0.77) for NMP22. Importantly, Mcm5 combined with NMP22 identified 95% (79/83; 95% CI?=?88-99%) of potentially life threatening diagnoses (i.e. grade 3 or carcinoma in situ or stage ?pT1) with high specificity (72%, 95% CI?=?69-74%).The Mcm5 immunoassay is a non-invasive test for identifying patients with urothelial cancers with similar accuracy to the FDA-approved NMP22 ELISA Test Kit. The combination of Mcm5 plus NMP22 improves the detection of UCC and identifies 95% of clinically significant disease. Trials of a commercially developed Mcm5 assay suitable for an end-user laboratory alongside NMP22 are required to assess their potential clinical utility in improving diagnostic and surveillance care pathways.
Project description:AIM: Survivin molecular beacons can be used to detect bladder cancer cells in urine samples non-invasively. The aim of this study is to improve the specificity of detection of bladder cancer cells using survivin dual fluorescence resonance energy transfer molecular beacons (FRET MBs) that have fluorophores forming one donor-acceptor pair. METHODS: Survivin-targeting dual fluorescence resonance energy transfer molecular beacons with unique target sequences were designed, which had no overlap with the other genes in the apoptosis inhibitor protein family. Human bladder cancer cell lines 5637, 253J and T24, as well as the exfoliated cells in the urine of healthy adults and patients with bladder cancer were examined. Images of cells were taken using a laser scanning confocal fluorescence microscope. For assays using dual FRET MBs, the excitation wavelength was 488 nm, and the emission detection wavelengths were 520±20 nm and 560±20 nm, respectively. RESULTS: The human bladder cancer cell lines and exfoliated cells in the urine of patients with bladder cancer incubated with the survivin dual FRET MBs exhibited strong fluorescence signals. In contrast, no fluorescence was detected in the survivin-negative human dermal fibroblasts-adult (HDF-a) cells or exfoliated cells in the urine of healthy adults incubated with the survivin dual FRET MBs. CONCLUSION: The results suggest that the survivin dual FRET MBs may be used as a specific and non-invasive method for early detection and follow-up of patients with bladder cancer.
Project description:BACKGROUND:Urothelial bladder cancer (UBC) is the 5th most common cancer in Western societies. The most common symptom of UBC is haematuria. Cystoscopy the gold standard for UBC detection, allows direct observation of the bladder, but is expensive, invasive, and uncomfortable. This study examines whether an alternative new urine-based diagnostic test, the DCRSHP, is cost-effective as a triage diagnostic tool compared to flexible cystoscopy in the diagnosis of UBC in haematuria patients. METHODS:A model-based cost-utility analysis using cost per quality adjusted life year and life year gained, parameterised with secondary data sources. RESULTS:If the DCRSHP is targeted at haematuria patients at lower risk of having bladder cancer e.g. younger patients, non-smokers, then it can be priced as high as £620, and be both effective and cost-effective. Sensitivity analysis found that DCRSHP is approximately 80% likely to be cost-effective across all willingness to pay values (for a QALY) and prevalence estimates. CONCLUSION:This analysis shows the potential for a non-invasive test to be added to the diagnostic pathway for haematuria patients suspected of having UBC. If the DCRSHP is applied targeting haematuria patients at low risk of UBC, then it has the potential to be both effective and cost-effective.
Project description:Bladder cancer is one of the most common urinary tract carcinomas in the world. Urine metabolomics is a promising approach for bladder cancer detection and marker discovery since urine is in direct contact with bladder epithelia cells; metabolites released from bladder cancer cells may be enriched in urine samples. In this study, we applied ultra-performance liquid chromatography time-of-flight mass spectrometry to profile metabolite profiles of 87 samples from bladder cancer patients and 65 samples from hernia patients. An OPLS-DA classification revealed that bladder cancer samples can be discriminated from hernia samples based on the profiles. A marker discovery pipeline selected six putative markers from the metabolomic profiles. An LLE clustering demonstrated the discriminative power of the chosen marker candidates. Two of the six markers were identified as imidazoleacetic acid whose relation to bladder cancer has certain degree of supporting evidence. A machine learning model, decision trees, was built based on the metabolomic profiles and the six marker candidates. The decision tree obtained an accuracy of 76.60%, a sensitivity of 71.88%, and a specificity of 86.67% from an independent test.
Project description:Because of the faltering sensitivity and/or specificity, urine-based assays currently have a limited role in the management of patients with bladder cancer. The aim of this study was to externally validate our previously reported protein biomarker panel from multiple sites in the United States and Europe.This multicenter external validation study included a total of 320 subjects (bladder cancer = 183). The 10 biomarkers (IL8, MMP9, MMP10, SERPINA1, VEGFA, ANG, CA9, APOE, SDC1, and SERPINE1) were measured using commercial ELISA assays in an external laboratory. The diagnostic performance of the biomarker panel was assessed using receiver operator curves (ROC) and descriptive statistical values.Utilizing the combination of all 10 biomarkers, the area under the ROC for the diagnostic panel was noted to be 0.847 (95% confidence interval, 0.796-0.899), outperforming any single biomarker. The multiplex assay at optimal cutoff value achieved an overall sensitivity of 0.79, specificity of 0.79, positive prediction value of 0.73, and negative prediction value of 0.84 for bladder cancer classification. Sensitivity values of the diagnostic panel for high-grade bladder cancer, low-grade bladder cancer, muscle invasive bladder cancer, and non-muscle invasive bladder cancer were 0.81, 0.90, 0.95, and 0.77, respectively.Urinary levels of the biomarker panel enabled discrimination of patients with bladder cancer and controls, and the levels of biomarker subsets were associated with advancing tumor grade and stage.If proven to be reliable, urinary diagnostic biomarker assays can detect bladder cancer in a timely manner such that the patient can expect improvements in overall survival and quality of life.
Project description:BACKGROUND:The muscle invasive form of urothelial bladder cancer (UBC) is a deadly disease. Currently, the therapeutic approach of UBC is mostly based on surgery and standard chemotherapy. Biomarkers to establish appropriate drugs usage are missing. Deficiency of the tumor suppressor CCDC6 determines PARP-inhibitor sensitivity. The CCDC6 levels are modulated by the deubiquitinase USP7. In this work we scored CCDC6 and USP7 expression levels in primary UBC and we evaluated the expression levels of CCDC6 in correlation with the effects of the PARP-inhibitors combined with the USP7 inhibitor, P5091, in vitro. Since PARP-inhibitors could be enhanced by conventional chemotherapy or DNA damage inducers, we tested the new agent RRx-001, able to induce DNA damage, to prove the benefit of combined treatments in bladder cancer cells. METHODS:The J82, T24, 5637 and KU-19-19 bladder cancer cells were exposed to USP7 inhibitor P5091 in presence of cycloheximide to analyse the CCDC6 stability. Upon the CCDC6 degradation induced by P5091, the cells sensitivity to PARP-inhibitor was evaluated by cell viability assays. The ability of the DNA damage inducer RRx-001 to modulate CCDC6 protein levels and H2AX phosphorylation was detected at immunoblot. The combination of USP7 inhibitor plus RRx-001 enhanced the PARP-inhibitor sensitivity, as evaluated by cell viability assays. The results of the scores and correlation of CCDC6 and USP7 expression levels obtained by UBC primary biopsies staining were used to cluster patients by a K-mean cluster analysis. RESULTS:P5091 determining CCDC6 degradation promoted bladder cancer cells sensitivity to PARP-inhibitor drugs. RRx-001, by inducing DNA damage, enhanced the effects of the combined treatment. The immunohistochemical staining of both CCDC6 and USP7 proteins allowed to cluster the high grade (G3) UBC patients, on the basis of CCDC6 expression levels. CONCLUSIONS:In high grade UBC the identification of two clusters of patients based on CCDC6 and USP7 expession can possibly indicate the use of PARP-inhibitor drugs, in combination with USP7 inhibitor in addition to the DNA damage inducer RRx-001, that also acts as an immunomodulatory agent, offering novel therapeutic strategy for personalized medicine in bladder cancer patients.