The early detection of bladder cancer (BCa) is pivotal for successful patient treatment and management. Through genomic and proteomic studies, we have identified a number of bladder cancer-associated biomarkers that have potential clinical utility. In a case-control study, we examined voided urines from 127 subjects: 64 tumor-bearing subjects and 63 controls. The urine concentrations of the following proteins were assessed by enzyme-linked immunosorbent assay (ELISA); C-C motif chemokine 18 (CCL18), Plasminogen Activator Inhibitor 1 (PAI-1) and CD44. Data were compared to a commercial ELISA-based BCa detection assay (BTA-Trak©) and voided urinary cytology. We used analysis of the area under the curve of receiver operating characteristic curves to compare the ability of CCL18, PAI-1, CD44, and BTA to detect BCa in voided urine samples. Urinary concentrations of CCL18, PAI-1, and BTA were significantly elevated in subjects with BCa. CCL18 was the most accurate biomarker (AUC; 0.919; 95% confidence interval [CI], 0.8704-0.9674). Multivariate regression analysis highlighted CCL18 (OR; 18.31; 95% CI, 4.95-67.70, p<0.0001) and BTA (OR; 6.43; 95% CI, 1.86-22.21, p = 0.0033) as independent predictors of BCa in voided urine samples. The combination of CCL18, PAI-1 and CD44 improved the area under the curve to0.938. Preliminary results indicate that CCL18 was a highly accurate biomarker for BCa detection in this cohort. Monitoring CCL18 in voided urine samples has the potential to improve non-invasive tests for BCa diagnosis. Furthermore using the combination of CCL18, PAI-1 and CD44 may make the model more robust to errors to detect BCa over the individual biomarkers or BTA.
Accurate urinary assays for bladder cancer (BCa) detection would benefit both patients and healthcare systems. Through genomic and proteomic profiling of urine components, we have previously identified a panel of biomarkers that can outperform current urine-based biomarkers for the non-invasive detection of BCa. Herein, we report the diagnostic utility of various multivariate combinations of these biomarkers. We performed a case-controlled validation study in which voided urines from 127 patients (64 tumor bearing subjects) were analyzed. The urinary concentrations of 14 biomarkers (IL-8, MMP-9, MMP-10, SDC1, CCL18, PAI-1, CD44, VEGF, ANG, CA9, A1AT, OPN, PTX3, and APOE) were assessed by enzyme-linked immunosorbent assay (ELISA). Diagnostic performance of each biomarker and multivariate models were compared using receiver operating characteristic curves and the chi-square test. An 8-biomarker model achieved the most accurate BCa diagnosis (sensitivity 92%, specificity 97%), but a combination of 3 of the 8 biomarkers (IL-8, VEGF, and APOE) was also highly accurate (sensitivity 90%, specificity 97%). For comparison, the commercial BTA-Trak ELISA test achieved a sensitivity of 79% and a specificity of 83%, and voided urine cytology detected only 33% of BCa cases in the same cohort. These datashow that a multivariate urine-based assay can markedly improve the accuracy of non-invasive BCa detection. Further validation studies are under way to investigate the clinical utility of this panel of biomarkers for BCa diagnosis and disease monitoring.
To investigate whether elevated urinary levels of vascular endothelial growth factor (VEGF), carbonic anhydrase 9 (CA9) and angiogenin are associated with BCa.
This is a case-control study in which voided urines from 127 patients: control subjects (n = 63) and tumor bearing subjects (n = 64) were analyzed. The urinary concentrations of VEGF, CA9, angiogenin and BTA were assessed by enzyme-linked immunosorbent assay (ELISA). We used the area under the curve (AUC) of receiver operating characteristic curves to determine the ability of VEGF, CA9, and angiogenin to detect BCa in voided urine samples. Data were also compared to a commercial ELISA-based BCa detection assay (BTA-Trak©). Sensitivity, specificity, positive and negative predictive values were calculated.
Urinary concentrations of VEGF, CA9, angiogenin and BTA were significantly elevated in BCa. VEGF was the most accurate urinary biomarker (AUC: 0.886; 95% confidence interval [CI]: 0.8301–0.9418). Furthermore, multivariate regression analysis highlighted VEGF (OR: 5.90; 95% CI: 2.60–13.40, p < 0.0001) as an independent variable. The sensitivities and specificities for VEGF (sensitivity, 83% and specificity, 87%) outperformed BTA (sensitivity, 80% and specificity, 84%).
VEGF may be a valuable addition to voided urine sample analysis for the detection of BCa. Larger, prospective studies are needed to determine the clinical utility of urinary VEGF and angiogenin as biomarkers in the non-invasive evaluation of BCa patients.
angiogenin; bladder cancer; biomarkers; diagnosis; VEGF
To assess the possibility of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) as a biological marker for detecting Bladder cancer (BCa), we examined the expression of HIP/PAP in both BCa specimens and BCa cell lines and measured HIP/PAP levels in urine from patients with BCa.
HIP/PAP expression in BCa samples was evaluated by western blot analysis, and urinary levels of HIP/PAP in patients with BCa were measured by enzyme-linked immunosorbent assay. Urine samples were collected from 10 healthy volunteers and 109 with benign urological disorders as controls, and from 101 patients who were diagnosed with BCa.
HIP/PAP was highly expressed in BCa samples as compared with control bladder. Urinary HIP/PAP concentrations were significantly higher in BCa patients than in controls (median value; 3.184 pg/mL vs. 55.200 pg/mL, P <0.0001, by Mann–Whitney U test). Urinary HIP/PAP levels in BCa patients correlated positively with pathological T stages and progression-risk groups among non-muscle invasive BCa (P = 0.0008, by Kruskal-Wallis test). Regarding the recurrence-risk classifications of non-muscle invasive BCa, the urinary levels of HIP/PAP were significantly higher in the intermediate than in the low risk group (P = 0.0002, by Mann–Whitney U test). Based on a cut-off of 8.5 pg/mL, the ability of urinary HIP/PAP levels to detect BCa had a sensitivity of 80.2%, specificity of 78.2%, positive predictive value (PPV) of 75.7%, and negative predictive value (NPV) of 82.3%.
HIP/PAP was abundantly expressed in BCa, and the urinary levels of HIP/PAP could be a novel and potent biomarker for detection of BCa, and also for predicting the risks of recurrence- and progression-risk of non-muscle invasive BCa. A large scale study will be needed to establish the usefulness of this biomarker.
Bladder cancer; Urinary marker; HIP/PAP; ELISA; ROC
The commercial NMP-22 urine assays for bladder cancer (BCa) detect nuclear mitotic apparatus protein 1 (NUMA1) using monoclonal antibodies. It remains unclear whether these assays are monitoring a tumor antigen or some other phenomenon associated with the disease state. In this study, we investigated the influence of urinary cellular and protein concentration, and hematuria on the performance of the NMP-22 tests in an experimental model.
Pooled urine from healthy subjects were spiked with varying concentrations of benign (UROtsa) cells, cancer cells (RT4, T24, KU-7 and UM-UC-14), whole blood or serum, prior to analysis with both NMP22® Bladder Cancer ELISA test and the NMP22® BladderChek® point-of-care test.
Urines from control subjects were negative for NMP-22. The addition of whole blood at 50ul/10 ml, but not serum, resulted in a false-positive result. Furthermore, the addition of a high concentration of benign urothelial cells (106) or the cell lysate from these cells (306 μg protein) resulted in a false-positive result. High concentrations of pooled-cancer cells (106) or cell lysate (30.6 μg and above) resulted in a positive NMP-22 assay. Concordance between the NMP-22 ELISA assay and the NMP-22 point of care assay was >90%.
Rather than detecting a specific tumor antigen, urinary NMP-22 assays may be measuring the cellularity or amount of cell turnover that may be introduced into the urine by a variety of conditions, including surface shedding from bladder tumors. The absence of significant urinary cellularity in some cases due to lesion characteristics or the timing of sampling may result in false-negative NMP-2 assays.
Bladder cancer; Urine; NMP-22
Bladder cancer is the fourth most common cancer in men and the ninth most common cancer in women in Canada. Early detection of tumours is essential for improved prognosis and long-term survival. The standard method for detection and surveillance is cystoscopy together with urine cytology. Cystoscopy is relatively sensitive but is expensive and invasive. Urinary cytology is a noninvasive method that has poor sensitivity but high specificity; it is relied on for the detection of carcinoma in situ. Currently, several urinary-based bladder tumour biomarkers with USFDA/Health Canada approval are available commercially, but none have been widely adopted by urologists despite their offering high sensitivity and/or specificity. We present here a review of recent studies evaluating 7 commercial biomarker assays for the detection and/or surveillance of bladder cancer.
Sensitivity and specificity ranges, respectively, for each marker were reported as follows: BTA Stat (Polymedco), 52.5%–78.0% and 69.0%–87.1%; BTA Trak (Polymedco), 51%–100% and 73%–92.5%; cytology, 12.1%–84.6% and 78.0%–100%; hematuria dipstick, 47.0%–92.6% and 51.0%–84.0%; NMP22 Bladder Cancer Test (Matritech), 34.6%–100% and 60.0%–95.0%; NMP22 BladderChek (Matritech), 49.5%–65.0% and 40.0%–89.8%; ImmunoCyt/uCyt+ (DiagnoCure), 63.3%–84.9% and 62.0%–78.1%; ImmunoCyt/uCyt+ and cytology, 81.0%–89.3% and 61.0%–77.7%; and UroVysion (Abbott Molecular)/florescence in situ hybridization, 68.6%–100% and 65.0%–96.0%.
We find that no currently available bladder cancer urinary marker is sensitive enough to eliminate the need for cystoscopy. In addition, cytology remains integral to the detection of occult cancer. However, owing to their relatively high sensitivities, these markers may be used to extend the period between cystoscopies in the surveillance of patients with transitional cell carcinoma. Further study is required to determine which markers, alone or in panel, would best accomplish this.
Current methods in the noninvasive detection and surveillance of bladder cancer via urine analysis include voided urine cytology (VUC) and some diagnostic urinary protein biomarkers; however, due to the poor sensitivity of VUC and high false-positive rates of currently available protein assays, detection of bladder cancer via urinalysis remains a challenge. In the study presented here, a rapid, high-sensitivity technique was developed to profile the N-linked glycoprotein component in naturally micturated human urine specimens. Concanavalin A (Con A) affinity chromatography coupled to nanoflow liquid chromatography was utilized to separate the complex peptide mixture prior to a linear ion trap MS analysis. Of 186 proteins identified with high confidence by multiple analyses, 40% were secreted proteins, 18% membrane proteins, and 14% extracellular proteins. In this study, the presence of several proteins appeared to be associated with the presence of bladder cancer, including α-1B-glycoprotein that was detected in all tumor-bearing patient samples but in none of the samples obtained from non-tumor-bearing individuals. The combination of Con A affinity chromatography and nano-LC/MS/MS provides an initial investigation of N-glycoproteins in complex biological samples and facilitates the identification of potential biomarkers of bladder cancer in noninvasively obtained human urine.
glycosylation; bladder cancer; lectin
Bladder Cancer (BCa) is the most common malignancy arising from the urinary tract. One of the mainstays of diagnosis, staging, and therapeutic decision-making for BCa is accurate and appropriate imaging. The ability to identify metastatic disease preoperatively is of utmost importance in determining treatment. Advances in standard cross sectional imaging techniques like Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) have improved imaging of bladder cancer. Over the last decade, 18F-fluorodeoxyglucose (FDG) Positron Emission Tomography (PET) in combination with CT (18F-FDG PET/CT) has become an important non-invasive imaging modality for the preoperative staging of various malignancies. 18F-FDG PET/CT is useful for detection of metastatic disease in BCa, but the ability to detect primary bladder wall lesions remains to be elucidated. To overcome the problem with urinary excretion of 18F-FDG, new PET tracers are being tested. MRI is an accurate technique for the local staging of BCa due to its superior spatial and contrast resolution. Anatomical MRI has a modest utility in NM-staging of BCa. However, incorporation of functional MR techniques, such as diffusion weighted MRI can improve the results for lesion detection and staging and multi-parametric MRI`s role is yet to be explored widely. The aim of this review is to present the recent advances in PET/CT and MRI in BCa, with particular focus on improvements in staging.
Positron Emission Tomography/Computed Tomography (PET/CT); Magnetic Resonance Imaging (MRI); Urothelial cancer; Bladder cancer
We have evaluated the feasibility of using nanoparticle (NP)-based assays for improving detection sensitivity of HIV-1 p24 antigen. The first assay is a gold NP-based biobarcode amplification (BCA) assay which could detect HIV-1 p24 antigen at levels as low as 0.1 pg/ml. Compared with BCA, the lower limit of detection (LOD) for enzyme-linked immunosorbent assay (ELISA) was 10 ~ 15 pg/ml. These results demonstrate that the HIV-1 p24 BCA assay offers 100 ~ 150-fold enhancement in the detection limit over the traditional colorimetric ELISA. Furthermore, the BCA assay detected HIV-1 infection 3 days earlier than ELISA in seroconversion samples. A second assay is the europium (Eu+) NP-based immunoassay (ENIA), which uses Eu+ NPs to replace gold NPs in the BCA assay to further simplify the detection method and decrease the incubation time. For detection of HIV-1 p24, the lower LOD for ENIA was 0.5 pg/ml. These results indicate that the universal labeling technology based on NPs and its application may provide a rapid and sensitive testing platform for clinical diagnosis and laboratory research.
Although, increased oxidative stress and hypomethylation of long interspersed nuclear element-1 (LINE-1) associate with bladder cancer (BCa) development, the relationship between these alterations is unknown. We evaluated the oxidative stress and hypomethylation of the LINE-1 in 61 BCa patients and 45 normal individuals. To measure the methylation levels and to differentiate the LINE-1 loci into hypermethylated, partially methylated and hypomethylated, peripheral blood cells, urinary exfoliated cells and cancerous tissues were evaluated by combined bisulfite restriction analysis PCR. The urinary total antioxidant status (TAS) and plasma protein carbonyl content were determined. The LINE-1 methylation levels and patterns, especially hypomethylated loci, in the blood and urine cells of the BCa patients were different from the levels and patterns in the healthy controls. The urinary TAS was decreased, whereas the plasma protein carbonyl content was increased in the BCa patients relative to the controls. A positive correlation between the methylation of LINE-1 in the blood-derived DNA and urinary TAS was found in both the BCa and control groups. The urinary hypomethylated LINE-1 loci and the plasma protein carbonyl content provided the best diagnostic potential for BCa prediction. Based on post-diagnostic samples, the combination test improved the diagnostic power to a sensitivity of 96% and a specificity of 96%. In conclusion, decreased LINE-1 methylation is associated with increased oxidative stress both in healthy and BCa subjects across the various tissue types, implying a dose-response association. Increases in the LINE-1 hypomethylation levels and the number of hypomethylated loci in both the blood- and urine-derived cells and increase in the oxidative stress were found in the BCa patients. The combination test of the urinary hypomethylated LINE-1 loci and the plasma protein carbonyl content may be useful for BCa screening and monitoring of treatment.
Syndecan binding protein (SDCBP), an adapter protein containing PDZ domains, contributes to the tumorigenicity and metastasis of many malignant tumors, such as malignant melanoma. Our study aimed in revealing the expression profile of SDCBP in breast cancer (BCa) and its role in tumor cell proliferation, and then exploring its value in the targeted treatment of BCa.
We first evaluated the SDCBP expression by immunohistochemistry in normal breast and BCa tissues. Then we explored the expression profile of SDCBP in different BCa cell lines. By constructing SDCBP-silenced BCa cell clones, we further assessed the effects of SDCBP suppression on tumor cells in vitro by cell culture and in vivo by tumorigenicity. SDCBP expression was detected in 80.6% (n = 160) of BCa tissues, in contrast to its expression in 13% (n = 23) of normal breast tissues (P<0.001). Among the tumors, the level of its expression was positively correlated with histological grade and tumor staging while negatively correlated with the estrogen receptor (ER) expression. Higher expression of SDCBP was also noted in ER-negative BCa cell lines. It was also identified that SDCBP expression was down-regulated in a dose-dependent mode by 17-β estradiol in estrogen-responsive MCF-7. Furthermore, SDCBP silence inhibited ER-negative tumor cell growth in vivo and in vitro. Cell cycle studies showed that SDCBP silence increased G1 cell population and resulted in related cell-cycle-regulator changes: up-regulation of p21 and p27 while down-regulation of cyclin E.
Our results suggested that SDCBP played an important role in tumor growth of ER-negative BCas. In these tumors where the estrogen signaling pathway is not available, SDCBP probably contribute to tumor growth through an alternative signaling pathway by promoting tumor cells passing the G1/S checkpoint into the cell cycle. Suppression of SDCBP expression may have its potential to become a targeted therapy for ER-negative BCas.
While alterations in xenobiotic metabolism are considered causal in the development of bladder cancer (BCa), the precise mechanisms involved are poorly understood. In this study, we used high-throughput mass spectrometry to measure over 2,000 compounds in 58 clinical specimens, identifying 35 metabolites which exhibited significant changes in BCa. This metabolic signature distinguished both normal and benign bladder from BCa. Exploratory analyses of this metabolomic signature in urine showed promise in distinguishing BCa from controls, and also non-muscle from muscle-invasive BCa. Subsequent enrichment-based bioprocess mapping revealed alterations in phase I/II metabolism and suggested a possible role for DNA methylation in perturbing xenobiotic metabolism in BCa. In particular, we validated tumor-associated hypermethylation in the CYP1A1 and CYP1B1 promoters of BCa tissues by bisulfite sequence analysis and methylation-specific PCR, and also by in vitro treatment of T-24 BCa cell line with the DNA demethylating agent 5-aza-2′-deoxycytidine. Further, we showed that expression of CYP1A1 and CYP1B1 was reduced significantly in an independent cohort of BCa specimens compared to matched benign adjacent tissues. In summary, our findings identified candidate diagnostic and prognostic markers and highlighted mechanisms associated with the silencing of xenobiotic metabolism. The metabolomic signature we describe offers potential as a urinary biomarker for early detection and staging of BCa, highlighting the utility of evaluating metabolomic profiles of cancer to gain insights into bioprocesses perturbed during tumor development and progression.
Bladder cancer has an incidence of 15 cases per 100,000 persons in the global population and is the most common tumor of the urinary tract. Imaging techniques, cytoscopy, and cytology are either invasive or not sufficiently accurate to detect early stage tumors, and the need for new diagnostic markers still remains. Among the markers most recently proposed to improve diagnostic accuracy and especially sensitivity, increasing attention has been focused on the role of the ribonucleoprotein, telomerase. Relevant papers on the etiology, diagnosis, and evaluation of bladder cancer using telomerase in urine were searched for and considered. The PubMed search was performed using the text terms “bladder cancer”, “diagnosis”, and “telomerase”. Previous studies have shown that the quantitative Telomerase Repeat Amplification Protocol (TRAP) assay performed in voided urine is an important non-invasive tool for the diagnosis of bladder tumors since it has very high sensitivity and specificity, even for early stage and low grade tumors. The main limitation of this test is the rate of false positive results due to the presence of inflammatory or non-tumor cells (i.e., epithelial cells from the lower genital tract), which express telomerase activity (TA). Consequently, an in situ analysis would seem to be important to identify the nature of telomerase-positive cells. Immunocytochemical detection of the hTERT subunit by a specific antibody seemed to open up the possibility to identify different cellular components of urine. However, the lack of a strict relationship between hTERT protein expression and telomerase activity has, to a certain extent, made this approach less relevant. In conclusion, telomerase activity in urine determined by TRAP seems to be marker of great potential, even more advantageous in cost/benefit terms when used in selected symptomatic patients or professionally high-risk subgroups.
Bladder cancer; early diagnosis; telomerase; telomerase repeat amplification protocol
Survivin as a tumor marker in the diagnosis of bladder cancer has not been completely confirmed yet and there are few reports about using Survivin enzyme-linked immunosorbent assay (ELISA) kit to detect the urine of bladder cancer patients. This study aimed to develop a Survivin ELISA and validate its value in the detection of bladder cancer.
Through square matrix titration, different combinations of coating antibody and detecting antibody, a Survivin ELISA was constructed. This assay was evaluated according to intra-assay precision, inter-assay precision and minimum detectable dose (MDD). Survivin levels were detected and analyzed in 102 bladder cancer patients and 102 healthy people by established ELISA. Then cutoff value was defined according to the analysis of receiver operating characteristic (ROC) curve. The sensitivity and specificity of detection were calculated on the basis of cutoff value to diagnose bladder cancer patients. Furthermore, the value of Survivin expression detected by ELISA among different clinicopathological characteristics of patients was also compared.
Through optimization of different conditions, intra-assay precision was 8.39%, inter-assay precision 8.57% and MDD 0.0625 ng/mL in this assay. When the optical density at 450 nm (OD450) was 0.09, it could get the optimized diagnostic cutoff value. According to this value, the sensitivity and specificity of diagnosis in bladder cancer patients were 70.6% and 89.2%, respectively. The associations between patients’ clinical variables and OD450 were not significant except tumor numbers in patients.
This experiment has preliminarily developed a Survivin ELISA and confirmed Survivin as a biomarker which owned a practical and significant value in the diagnosis of bladder cancer.
Survivin; bladder cancer; enzyme-linked immunosorbent assay (ELISA); tumor marker; diagnosis
For bladder cancer (BCa) patients undergoing bladder-sparing treatments, molecular markers may aid in accurately predicting progression to muscle invasion and recurrence. Hyaluronic acid (HA) is a glycosaminoglycan that promotes tumor metastasis. Hyaluronoglucosaminidase 1 (HYAL-1)–type hyaluronidase (HAase) promotes tumor growth, invasion, and angiogenesis. Urinary HA and HAase levels are diagnostic markers for BCa.
We evaluated whether HA and HYAL-1 can predict progression to muscle invasion and recurrence among patients with non–muscle-invasive BCa.
Design, setting, and participants
Based on tissue availability, tissue microarrays were prepared from a cohort of 178 BCa specimens (144 non–muscle invasive, 34 muscle invasive). Follow-up information was available on 111 patients with non–muscle-invasive BCa (mean follow-up: 69.5 mo); 58 patients recurred and 25 progressed to muscle invasion (mean time to progress: 22.3 mo).
HA and HYAL-1 expression was evaluated by immunohistochemistry and graded for intensity and area of staining. Association of HA and HYAL-1 staining with BCa recurrence and muscle invasion was evaluated by univariate and multivariate models.
Results and limitations
HA and HYAL-1 expression correlated with tumor grade, stage, and multifocality (p < 0.05). In non–muscle-invasive BCa specimens, HYAL-1 staining was higher (234.3 ± 52.2; 200.6 ± 61.4) if patients experienced progression to muscle invasion or recurrence when compared with no progression or recurrence (164.1 ± 48.2; 172.1 ± 57; p < 0.001). HA staining correlated with muscle invasion (p < 0.001). In univariate analysis, age (p = 0.014), multifocality (p = 0.023), and HYAL-1 staining (p < 0.001) correlated with muscle invasion, whereas only HYAL-1 correlated with recurrence (p = 0.013). In multivariate analysis, significantly associated with muscle invasion (p < 0.001; 76.8% accuracy) and recurrence (p = 0.01; 67.8% accuracy).
HYAL-1 is a potential prognostic marker for predicting progression to muscle invasion and recurrence.
Bladder cancer; Hyaluronic acid; Hyaluronidase; HYAL-1; non-muscle invasive bladder cancer; Prognostic markers; Tissue microarray
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.
The urinary proteome is a potential easily accessible source of biomarkers for inflammatory bladder diseases including interstitial cystitis. In the present study, we subjected rat urine to multiplex cytokine analysis in an attempt to identify an inflammatory signature of the temporal course of cyclophosphamide (CYP) - induced cystitis.
Rat urine was collected for 12h following CYP injection (150mg/kg) for multiplex analysis of 14 cytokines by a multiple antigen bead assay (Luminex™ 100 IS). Urine from each void was collected and voiding frequency was determined. Bladder tissue was analyzed for cytokines levels and histological evidence of inflammation.
Significant fold changes were noticed in urine levels of all cytokines with respect to baseline at 2, 4, 6 and 10h after CYP injection. Elevation was noticed at all times for most cytokines except for MCP-1 that showed a five fold decrease at 2h time point. Urine and tissue levels of IL-1β, IL-4 and GRO/KC were significantly correlated, with a positive spearman correlation also noticed for GM-CSF, MCP-1, IL-18 and IFN-γ. Tissue levels for most cytokines except IL-2 and urinary frequency were significantly elevated in CYP treated rats over control vehicle treated rats. The hints of severe inflammation in bladder indicated by urinary cytokines were confirmed by bladder histology and tissue cytokine levels on animal sacrifice.
The progression of CYP-induced cystitis is clearly reflected in the urine matrix by temporal and quantitative changes in cytokine levels. Further delineation of urine and bladder tissue cytokine expression may yield biomarkers for cystitis.
cystitis; multiplex analysis; rat; cyclophosphamide; cytokine; interstitial cystitis
Matrix Metalloproteinases (MMPs) are key molecules for tumor growth, invasion and metastasis. Over-expression of different MMPs in tumor tissues can disturb the homeostasis and increase the level of various body fluids. Many MMPs including high molecular weights (HMWs) were detected in the urine of prostate and bladder cancer patients. Our aim here is to assess the usefulness of HMW MMPs as non invasive biomarkers in bilharzial bladder cancer in Egyptian patients.
The activity of different MMPs including HMW species was determined using zymographic analysis technique in the urine samples procured from sixty six bladder cancer patients (bilharzial and non-bilharzial) as well as hundred healthy control subjects. Also, the correlation between these HMW MMPs activities and different clinico-pathological parameters was investigated.
High frequency of urine MMPs (uMMPs) activity was determined in 63.6% of examined tumor cases, however, none of the control cases showed any uMMPs activity. MMP-9 had the highest activity (62%) followed by MMP9/NGAL (60%), MMP-2 (54.5%), MMP-9 dimer (53%), ADAMTS (25.6%), and the lowest one was MMP-9/TIMP-1 (12%) only. There was no correlation between uMMPs and any of clinico-pathological parameters including age, gender, tumor size and type, bilharziasis, grade, lymph node involvement, and invasion to the prostate. A significant correlation was established only between MMP-9/TIMP-1 activities with the tumor size.
This study revealed that the detection of urinary MMPs including HMWs activity might be sensitive biomarkers for prediction of bladder cancer. It is also demonstrate that the detection of these urinary HMW gelatinases could not differentiate between bilharzial and non bilharzial bladder cancer subtypes.
Bladder cancer; High molecular weight matrix metalloproteinases; Early detection; Biomarkers
The early detection of urological cancers is pivotal for successful patient treatment and management. The development of molecular assays that can diagnose disease accurately, or that can augment current methods of evaluation, would be a significant advance. Ideally, such molecular assays would be applicable to non-invasively obtained body fluids, enabling not only diagnosis of at risk patients, but also asymptomatic screening, monitoring disease recurrence and response to treatment. The advent of advanced proteomics and genomics technologies and associated bioinformatics development is bringing these goals into focus. In this article we will discuss the promise of biomarkers in urinalysis for the detection and clinical evaluation of the major urological cancers, including bladder, kidney and prostate. The development of urine-based tests to detect urological cancers would be of tremendous benefit to both patients and the healthcare system.
Biomarkers; body fluids; diagnosis; non-invasive; urinary; urological cancer
Urine has emerged as an attractive biofluid for the noninvasive detection of prostate cancer (PCa). There is a strong imperative to discover candidate urinary markers for the clinical diagnosis and prognosis of PCa. The rising flood of various omics profiles presents immense opportunities for the identification of prospective biomarkers. Here we present a simple and efficient strategy to derive candidate urine markers for prostate tumor by mining cancer genomic profiles from public databases. Prostate, bladder and kidney are three major tissues from which cellular matters could be released into urine. To identify urinary markers specific for PCa, upregulated entities that might be shed in exosomes of bladder cancer and kidney cancer are first excluded. Through the ontology-based filtering and further assessment, a reduced list of 19 entities encoding urinary proteins was derived as putative PCa markers. Among them, we have found 10 entities closely associated with the process of tumor cell growth and development by pathway enrichment analysis. Further, using the 10 entities as seeds, we have constructed a protein-protein interaction (PPI) subnetwork and suggested a few urine markers as preferred prognostic markers to monitor the invasion and progression of PCa. Our approach is amenable to discover and prioritize potential markers present in a variety of body fluids for a spectrum of human diseases.
BCA2 is an E3 ligase linked with hormone responsive breast cancers. We have demonstrated previously that the RING E3 ligase BCA2 has autoubiquitination activity and is a very unstable protein. Previously, only Rab7, tetherin, ubiquitin and UBC9 were known to directly interact with BCA2.
Here, additional BCA2 binding proteins were found using yeast two-hybrid and bacterial-II-hybrid screening techniques with Human breast and HeLa cDNA libraries. Co-expression of these proteins was analyzed through IHC of TMAs. Investigation of the molecular interactions and effects were examined through a series of in vivo and in vitro assays.
Ten unique BCA2 interacting proteins were identified, two of which were hHR23a and 14-3-3sigma. Both hHR23a and 14-3-3sigma are co-expressed with BCA2 in breast cancer cell lines and patient breast tumors (n = 105). hHR23a and BCA2 expression was significantly correlated (P = < 0.0001 and P = 0.0113) in both nucleus and cytoplasm. BCA2 expression showed a statistically significant correlation with tumor grade. High cytoplasmic hHR23a trended towards negative nodal status. Binding to BCA2 by hHR23a and 14-3-3sigma was confirmed in vitro using tagged partner proteins and BCA2. hHR23a and 14-3-3sigma effect the autoubiquitination and auto-degradation activity of BCA2. Ubiquitination of hHR23a-bound BCA2 was found to be dramatically lower than that of free BCA2, suggesting that hHR23a promotes the stabilization of BCA2 by inactivating its autoubiquitination activity, without degradation of hHR23a. On the other hand, phosphorylated BCA2 protein is stabilized by interaction with 14-3-3sigma both with and without proteasome inhibitor MG-132 suggesting that BCA2 is regulated by multiple degradation pathways.
The interaction between BCA2 and hHR23a in breast cancer cells stabilizes BCA2. High expression of BCA2 is correlated with grade in breast cancer, suggesting regulation of this E3 ligase is important to cancer progression.
Bladder cancer is the fifth most commonly diagnosed malignancy in the United States and one of the most prevalent worldwide. It harbors a probability of recurrence of >50%, thus rigorous, long-term surveillance of patients is advocated. Flexible cystoscopy coupled with voided urine cytology (VUC) is the primary diagnostic approach, but cystoscopy is an uncomfortable, invasive procedure and the sensitivity of VUC is poor in all but high-grade tumors. Thus, improvements in non-invasive urinalysis assessment strategies would benefit patients. We applied gene expression microarray analysis to exfoliated urothelia recovered from bladder washes obtained prospectively from 46 patients with subsequently confirmed presence or absence of bladder cancer. Data from microarrays containing 56,000 targets was subjected to a panel of statistical analyses to identify bladder cancer-associated gene signatures. Hierarchical clustering and supervised learning algorithms were used to classify samples on the basis of tumor burden. A differentially expressed geneset of 319 gene probes was associated with the presence of bladder cancer (P<0.01), and visualization of protein interaction networks revealed VEGF and AGT as pivotal factors in tumor cells. Supervised machine learning and a cross-validation approach were used to build a 14-gene molecular classifier that was able to classify patients with and without bladder cancer with an overall accuracy of 76%. Our results show that it is possible to achieve the detection of bladder cancer using molecular signatures present in exfoliated tumor urothelia. Further investigation and validation of the cancer-associated profiles may reveal important biomarkers for the non-invasive detection and surveillance of bladder cancer.
Urinary bladder cancer; diagnostic signature; microarray profiling; urinalysis; VEGF
Results of the Uristat test (Shield Diagnostics Ltd.), a novel enzyme-linked immunosorbent assay (ELISA) for detection of urine antibodies to seven common bacterial pathogens, were compared with results of urine culture, urinalysis, and clinical history to determine the usefulness of Uristat in the diagnosis of urinary tract infections (UTIs). Midstream, catheterized, and indwelling catheter urine specimens sent to the laboratory for culture were included in the study. Quantitative cultures were performed on both 5% sheep blood agar and eosin-methylene blue agar. Uristat ELISAs were performed according to the manufacturer's instructions. By using a Bacillus subtilis bioassay technique, antibacterial activity was detected in the urine of 236 (22.2%) of 1,061 patients. Probable, possible, or asymptomatic UTIs were diagnosed for 258 (24.3%) of the 1,061 patients. Of those infections, 219 (84.9%) were caused by bacterial species whose antibodies were detectable by Uristat. Uristat's sensitivity and specificity were 76.7 and 56.0%, respectively. Uristat's predictive values of positive and negative results were 31.2 and 90.2%, respectively. Further development of the Uristat test is necessary before it can be of assistance in the diagnosis of UTIs.
The purpose of the study was to determine, in addition to well-known prognostic factors, histological grade, stage, tumour size and multiplicity, the correlation of BTA stat Test on disease free interval (DFI) on primary superficial bladder cancer. A total of 116 patients with newly diagnosed bladder cancer were evaluated in a prospective multicentre study. A voided urine sample was obtained prior to TURB and split for culture, cytology and BTA stat testing. Follow-up data for the patients were collected until the first recurrence or the last visit and the DFI was analysed by Kaplan–Meier method and Cox analysis. Ninety-seven of the 116 (83.6%) patients were eligible for analysis. The BTA stat Test was positive in 73 (75.3%) patients, whereas cytology detected 20 (20.6%) cases. The DFI was found to be shorter among patients with a positive BTA stat Test, and also among those with intermediate or high-grade tumours. The BTA stat Test result divided patients with grade 2 tumours into two prognostic groups, in that those testing positive had 68.6% risk of recurrence during the first year compared to 42.9% risk of those with a negative test result (P = 0.041). Although the effect of tumour size on DFI was notable, the difference did not reach statistical significance (P = 0.064). Number of tumours was not related to DFI, nor was the difference between different stage of tumour of significance. BTA stat Test is not only sensitive in detection of primary bladder cancer, but also might have some independent prognostic significance. © 2001 Cancer Research Campaign http://www.bjcancer.com
bladder neoplasms; tumour marker; prognosis
Bladder cancer has increased incidence during last decades. For those patients with nonmuscle involved tumors, noninvasive diagnosis test and surveillance methods must be designed to avoid current cystoscopies that nowadays are done regularly in a lot of patients. Novel urine biomarkers have been developed during last years. Telomerase is important in cancer biology, improving the division capacity of cancer cells. Even urinary telomerase could be a potentially useful urinary tumor marker; its use for diagnosis of asymptomatic and symptomatic patients or its impact during surveillance is still unknown. Moreover, there will need to be uniformity and standardization in the assays before it can become useful in clinical practice. It does not seem to exist a real difference between the most classical assays for the detection of urine telomerase (TRAP and hTERT). However, the new detection methods with modified TeloTAGGG telomerase or with gold nanoparticles must also be taken into consideration for the correct development of this diagnosis method. Maybe the target population would be the high-risk groups within screening programs. To date there is no enough evidence to use it alone and to eliminate cystoscopies from the diagnosis and surveillance of these patients. The combination with cytology or FISH is still preferred.