CCL18 in a multiplex urine-based assay for the detection of bladder cancer.

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 to 0.938. 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.

Systematic quantification of peptides/proteins in urine using selected reaction monitoring.

The evaluation of biomarkers in bodily fluids necessitates the development of robust methods to quantify proteins in a complex background, using large sets of samples. The ability to multiplex numerous analytes in a single assay expedites the process. Liquid chromatography-mass spectrometry (LC-MS) analyses performed in selected reaction monitoring (SRM) in conjunction with stable isotope dilution MS present an effective way to detect and quantify biomarker candidates in bodily fluids. The strategy presented involves an initial qualification of predefined sets of proteins in urine. The technique was applied to detect and quantify peptides in urine samples as surrogates for a few endogenous proteins. Multiplexed assays were developed to analyze proteins associated with bladder cancer; a few exogenous proteins were added as internal standards. The sample preparation and the analytical protocols were optimized to ensure reproducibility, analytical precision, and quantification limits in the low nanogram per milliliter range. Analyses were performed using known amounts of isotopically labeled peptides. Systematic replication of the measurements indicated intra-assay and inter-assay variability, with CVs in the range of 10%. The differences measured for two targeted proteins were correlated with their level of expression in the corresponding tumors using immunohistochemistry.

Comparison of CD44 and cytokeratin 20 mRNA in voided urine samples as diagnostic tools for bladder cancer.

OBJECTIVES: We evaluated the diagnostic efficacy of urinary CD44 and cytokeratin 20 (CK20) mRNA in comparison with voided urine cytology (VUC) for the detection of bladder cancer. DESIGN AND METHODS: A total of 136 Egyptian patients provided a single voided urine sample for CD44, CK20 mRNA and VUC before cystoscopy. Of the 136 cases, 111 were histologically diagnosed as bladder cancer whereas the remaining 25 had benign urological disorders. A group of 20 healthy volunteers was also included in this study. Voided urine was centrifuged and the urine sediment was used for cytology, estimation of CD44 by ELISA and RNA extraction. CK20 mRNA was detected by conventional RT-PCR and quantitative real-time RT-PCR. RESULTS: The best cutoff values for CD44 and relative CK20 mRNA detected by real-time RT-PCR were calculated by receiver operating characteristic curve. The positivity rates and the mean ranks for CD44 and CK20 mRNA showed significant difference among the three investigated groups (p=0.001). Quantitative real-time RT-PCR results were comparable to conventional RT-PCR for the detection of CK20 mRNA. The positivity rate of CD44 was significantly associated with schistosomiasis and urine cytology. The overall sensitivity and specificity were 52.3% and 88.9% for VUC, 63.1% and 88.9% for CD44, and 82.0% and 97.8% for CK20 mRNA. Combined sensitivity of VUC with CD44 and CK20 mRNA together (95.5%) was higher than either the combined sensitivity of VUC with CD44 (78.4%) or with CK20 mRNA (91.0%) or than that of the biomarker alone. CONCLUSION: Urinary CD44 and CK20 mRNA had higher sensitivities compared to VUC. However, when the diagnostic efficacy was considered, CK20 mRNA by either conventional RT-PCR or real-time RT-PCR had the highest sensitivity and specificity compared to CD44 and VUC.

Molecular markers in bladder cancer: a critical appraisal.

The diagnosis of both primary and recurrent bladder tumors currently relies upon the urine cytology and cystoscopy. Neither of these diagnostic tools is completely accurate. Prognostication of bladder cancer is largely based on pathologic tumor grade and stage. Over the past 2 decades, there is accumulating evidence that like many other cancers, bladder cancer, too, has a distinct molecular signature that separates it from other cancers and normal bladder tissue. Bladder tumors of different grades and stages even possess unique, and specific genotypic and phenotypic characteristics. Although recognition of several of these molecular alterations is possible by analyzing tumor tissue, urine, and serum samples, few if any of these "molecular markers" for bladder cancer are widely used in clinical practice. These markers include some that can be applied during the diagnostic work-up of symptoms (e.g., hematuria), those under surveillance for recurrence of superficial disease and forecasting long-term prognosis, or response to chemotherapy. In this review of molecular markers for bladder cancer, effectiveness of markers in each of these categories that are identifiable in the urine of patients with bladder cancer was examined. Many of the diagnostic markers appear to hold an advantage over urine cytology in terms of sensitivity, especially for the detection of low-grade superficial tumors. However, most markers tend to be less specific than cytology, yielding more false-positives. This result is more commonly observed in patients with concurrent bladder inflammation or other benign bladder conditions. Although there are several candidate markers for assessing prognosis or response to chemotherapy, studies of large patient populations are lacking. Further studies involving larger numbers of patients are required to determine their accuracy and widespread applicability in guiding treatment of bladder cancer.

[Clinical evaluation of BTAstat, NMP22, HA, survivin, CD44v6, vEGF and VUC in bladder cancer diagnosis].

OBJECTIVE: To evaluate the sensitivity and specificity of BTAstat, NMP22, HA, Survivin, CD44v6, VEGF, and VUC in detection of bladder cancer. METHODS: We detect VUC, BTAstat, NMP22, HA, Survivin, CD44v6, VEGF in the urine of 10 normal case (healthy volunteers), 11 benign urological diseases patients and 52 bladder cancer patients. The sensitivity, specificity the coefficient of variation, the examining time duration and the checking costs of each marker and combined markers were assessed to evaluate the clinical value. RESULTS: There is a significant difference between the cancer group and the two control groups. The overall sensitivity and specificity of urinary tumor markers were: 42.3% and 100% for VUC; 78.8% and 90.5% for BTAstat; 76.9% and 81.0% for NMP22; 86.5% and 90.5% for HA; 67.3% and 85.7% for Survivin; 50.5% and 85.7% for CD44 and 69.2% and 95.2% for VEGF. The highest sensitivity of combined markers was 96.2% for NMP22 + HA and HA + CD44v6, whereas the lowest sensitivity of combined markers was 67.3% for VUC + CD44v6. The highest specificity (95.2%) was the combined use of VUC + NMP22 and combined use of VUC + VEGF, whereas, method that achieved the lowest specificity (66.7%) was the combined use of HA + Survivin. The most convenient examining method was the detection for BTAstat; the lowest cost examining method was the detection for HA; methods which had the best repeatability were the detection for BTAstat and urine cytology examination. CONCLUSION: Each marker had achieved its obvious clinical value in diagnosis of bladder cancer. The sensitivity of all the markers was increased with the progression of tumor grades and clinical stages, except the CD44v6. The combined use of BATstat and HA is the best examining method concerning sensitivity, specificity, feasibility and cost in each different method.

Over expression of CD44V8-10 in urinary exfoliated cells as an independent prognostic predictor in patients with urothelial cancer.

PURPOSE: CD44 is a widely expressed cell surface adhesion molecule, of which various isoforms arise from alternative RNA splicing mechanisms. Over expression of specific CD44 splice variants, namely CD44v8-10, is evident in various malignant tumors and is considered to be associated with disease progression. In this study, we investigated whether the transcriptional level of CD44v8-10 relative to that of the standard CD44 isoform would predict the extent and prognosis of urothelial cancer. MATERIALS AND METHODS: The CD44v8-10- to -standard CD44 ratio was measured in the tissue (40 urothelial cancer specimens and corresponding normal urinary tissue) and spontaneously voided urine samples of 150 patients with urothelial cancer and 50 with benign urological disease by reverse transcriptase-polymerase chain reaction using the set of primers capable of amplifying all CD44 splice variant isoforms. RESULTS: Initially any CD44 variant isoforms were barely detectable in normal urinary tissues, whereas CD44v8-10 was predominantly expressed in most urothelial cancer specimens. Furthermore, the CD44v8-10- to -standard CD44 ratio in urothelial cancer was closely associated with tumor progression. We then compared the ratio in urothelial cancer tissue and urinary exfoliated cells, and noted a linear and significant correlation of these 2 values in the same patients. Therefore, we investigated whether the CD44v8-10- to -standard CD44 ratio in urinary exfoliated cells would predict the prognosis and disease progression. The mean ratio in the urinary exfoliated cells of patients with invasive urothelial cancer was significantly higher than in those with superficial urothelial cancer. Of the patients with superficial bladder cancer disease-free survival rate of those with an elevated versus a normal ratio was significantly lower. Moreover, of the patients with advanced urothelial carcinoma who underwent complete resection disease-free survival of those with an elevated CD44v8-10- to -standard CD44 ratio was significantly lower than that of patients with a normal ratio. CONCLUSIONS: These results indicate that CD44v8-10 is strongly expressed in tumor tissue and evident at high levels in urinary exfoliated cells of patients with invasive versus superficial urothelial cancer. An elevated CD44v8-10- to -standard CD44 ratio in urinary exfoliated cells may serve as a novel prognostic predictor and indicator of disease extent in patients with urothelial cancer.

Noninvasive diagnosis of bladder carcinoma by enzyme-linked immunosorbent assay detection of CD44 isoforms in exfoliated urothelia.

The expression of variant isoforms of the adhesion molecule CD44 is correlated with the onset of neoplasia in many carcinomas. We have previously shown that noninvasive detection of bladder carcinoma is possible by analysis of anomalous CD44 expression in exfoliated urothelia. Although the sensitivity and specificity values obtained for the detection of bladder tumors using RT-PCR and Western blotting methods were superior to those obtained using urine cytology, the application of such techniques is inconvenient for routine diagnostic use. We now report the design and development of a sandwich-ELISA system for the reliable detection of CD44 protein extracted from sedimented urothelial cells in voided urine. Naturally micturated urine samples were obtained from 53 patients with newly diagnosed bladder cancer and from 65 subjects with no evidence of disease; patients with gross hematuria were excluded because of interference with the assay. To demonstrate the diagnostic potential of the system, a "gate" was imposed at N (max), i.e., the highest absorbance value obtained from a sample known to be tumor free. All values above this value were assumed to be indicative of the presence of a tumor. Using this parameter, 42 of 53 (81.1%) patients with histologically confirmed bladder tumors were correctly diagnosed. Correspondingly, under these conditions, the assay is 100% specific for tumor detection, with a sensitivity of 81.1%, which equates to a positive predictive value of 100% and a negative predictive value of 81.1%. A further 54 patients who had previously received treatment for bladder cancer but were currently clinically disease-free were also investigated. Of these, 47 of 54 (87%) were correctly diagnosed to be tumor-free, which in this group equates to a positive predictive value of 87% and a negative predictive value of 100%. The data presented demonstrate that the rapid and accurate detection of elevated levels of CD44 protein isoforms in exfoliated urothelial cells is applicable to the identification and monitoring of primary and recurrent bladder cancer.

[Expression of CD44 variant isoform in urine samples of urothelial cancer patient].

CD44v8-10 variant isoform is frequently expressed in many kinds of cancers. We have already reported that 77% of bladder cancer specimens expressed CD44v8-10 and using CD44v8-10/CD44v10 competitive reverse transcription-polymerase chain reaction (CC-RT-PCR), we detected exfoliated urothelial cancer cells in urine samples of urothelial cancer patients (Int J Cancer 79: 560, 1998, J Urol 160: 2004). In this paper, we review the expressing of CD44 variant isoform in various kinds of cancers, and the principle of CC-RT-PCR which can be a novel screening method for urothelial cancer.

Expression of CD44V2 in transitional cell carcinoma of the urinary bladder and in urine.

CD44 is the principal cell surface receptor for hyaluronate. Variant forms of the receptor, produced by alternative splicing, have been found to be associated with tumor progression in a variety of cancers. Based on investigations at the RNA level, it has recently been proposed that expression of CD44 variant V2 was present in urothelial cancer but not in normal urothelium. Since a distinctive marker for urothelial cancer would be extremely useful, frozen sections of normal urothelium and urothelial cancer were examined for expression of standard CD44 and CD44V2. Frozen sections of specimens of 35 patients with transitional cell carcinoma of the bladder, 16 specimens of normal bladder and 5 ureters were examined. Immunohistochemical staining was performed using a polyclonal antibody to CD44V2 (PAB CD44V2), a monoclonal antibody to CD44V2 (MAB CD44V2) and a monoclonal antibody to CD44S (MAB CD44S). CD44V2 and CD44S were also measured in lysates of urine sediments from 21 patients by enzyme-linked immunoabsorbent assay (ELISA). All investigated transitional cell carcinomas expressed CD44V2. There was no differentiation between invasive and noninvasive carcinoma. CD44V2 was also expressed in normal urothelium. Standard CD44 was expressed by the transitional cell carcinoma, normal urothelium, musculature and interstitial tissue. The amount of CD44V2 and CD44S in lysates of urine sediments is not correlated to diagnosis. In contrast to investigations at the RNA level, CD44V2 on the protein level seems not to be a distinctive marker for urothelial cancer. Therefore, CD44V2 will not be a useful diagnostic marker for detection of transitional cell carcinoma.