MicroRNA-126 inhibits invasion in bladder cancer via regulation of ADAM9.

BACKGROUND: The miRNA deregulation is commonly observed in human malignancies, where they act as tumour suppressors or oncogenes. Despite the association of several miRNAs with bladder cancer, little is known about the miRNAs that contribute to bladder cancer progression from non-muscle invasive (NMI) to muscle-invasive (MI) disease. METHODS: We first profiled the expression of miRNAs and mRNAs in a cohort of urothelial carcinomas and further characterised the role of miR-126 in invasion, as it emerged as the most downregulated miRNA between MI and NMI tumours. RESULTS: We found that restoration of miR-126 levels attenuated the invasive potential of bladder cancer cells. Mechanistically, we identified the role of miR-126 in invasion through its ability to target ADAM9. Notably, a significant inverse correlation between miR-126 and ADAM9 expression was observed, where ADAM9 was upregulated in MI bladder cancer cells. While knockdown of ADAM9 attenuated the invasiveness of cells with low miR-126 levels, experimental upregulation of ADAM9 recapitulated the invasive phenotype. Furthermore, ADAM9 expression assessed by immunohistochemistry significantly correlated with poor prognosis in patients with urothelial carcinoma. CONCLUSIONS: In this study we describe the role of miR-126 in bladder cancer progression, identifying miR-126 and ADAM9 as potential clinical biomarkers of disease aggressiveness.

The neuroendocrine component in bladder tumors.

Neoplastic urothelium has the capacity to display enormous plasticity and divergent differentiation. Neuroendocrine tumors arise as a result of such capacity. Neuroendocrine tumors of the bladder represent a limited number of neoplasms characterized by neuroendocrine hormone secretion and a poor outcome. These tumors can be displayed as pure neuroendocrine neoplasms or more frequently as a neuroendocrine counterpart mixed with classical urothelial bladder cell carcinomas, adenocarcinoma, sarcomatoid carcinoma or mixtures of these components. Their heterogeneous character and clinical aggressiveness remain a challenge for clinical, pathological diagnosis and for therapy selection. Several types have been described, although small cell carcinoma represents the major subgroup of neuroendocrine tumors as compared to large cell carcinoma and carcinoid subtypes. In this review, epidemiology, presentation, macroscopic and microscopic features, and clinical prognostic and therapeutic implications of the major subgroups are described. Special focus is given to discuss how immunohistochemistry protein patterns and laboratory determinations may aid to characterize this type of tumors and to improve the clinical management of this highly aggressive type of bladder cancer patients.

Fluorescence in situ hybridization analysis of CCND3 gene as marker of progression in bladder carcinoma.

The aim of this study was to assess patterns of CCND3 gene amplification in bladder cancer and correlate gene status with recurrence-free and progression-free survival. A sequential cohort series of 102 primary bladder tumor samples in which there was enough tissue material to assess CCND3 gene status by fluorescent in situ hybridization (FISH) was the study group. CCND3 gene FISH amplification present in 31.4 percent of bladder carcinomas, was related to tumor progression (p=0.021) and lower time to progression (mean+-SD; 25.75+-15.25 months) as compared to 33.29+-11.0 months in the CCND3 not amplified group (p=0.05). By immunohistochemistry, Cyclin D3 labeling index was higher in the CCND3 amplified group (mean+-SD, 76.69+-27.51) than in not amplified (mean+-SD, 21.57+-7.02) (p less than 0.0001). The univariate survival analysis showed CCND3 gene amplification to be associated to a shorter progression-free survival (p=0.020) together with WHO histological grade (p=0.001) and pT stage category (p less than 0.0001). Cox’s regression analysis selected CCND3 amplification as an independent predictor of progression-free survival (p= 0.030, RR3.561, 95 percent CI 1.128-11.236) together with pT category (p less than 0.0001, RR5.834, 95 percent CI 2.364-14.395). Our FISH analysis suggests that CCND3 gene amplification is a marker of aggressiveness and might be a predictor of tumor progression in bladder urothelial carcinoma.

Putative tumour suppressor gene necdin is hypermethylated and mutated in human cancer.

BACKGROUND: Necdin (NDN) expression is downregulated in telomerase-immortalised normal human urothelial cells. Telomerase-immortalised normal human urothelial cells have no detected genetic alterations. Accordingly, many of the genes whose expression is altered following immortalisation are those for which epigenetic silencing is reported. METHODS: NDN expression was examined in normal tissues and tumour cell lines by quantitative real-time PCR and immunoblotting. Immunohistochemistry was performed on urothelial carcinoma (UC). Urothelial carcinoma and UC cell lines were subject to HumanMethylation27 BeadChip Array-based methylation analyses. Mutation screening was performed. The functional significance of NDN expression was investigated using retroviral-mediated downregulation or overexpression. RESULTS: NDN protein was widely expressed in normal tissues. Loss of expression was observed in 38 out of 44 (86%) of UC cell lines and 19 out of 25 (76%) of non-UC cell lines. Loss of NDN protein was found in the majority of primary UC. Oncomine analysis demonstrated downregulation of expression in multiple tumour types. In UC, tumour-specific hypermethylation of NDN and key CpG sites where hypermethylation correlated with reduced expression were identified. Six novel mutations, including some of predicted functional significance, were identified in colorectal and ovarian cancer cell lines. Functional studies showed that NDN could suppress colony formation at low cell density and affect anchorage-independent growth and anoikis in vitro. CONCLUSION: NDN is a novel tumour suppressor candidate that is downregulated and hypermethylated or mutated in cancer.

Discovery of myopodin methylation in bladder cancer.

Myopodin is an actin-binding protein that shuttles between the nucleus and the cytoplasm. After identifying an enriched CpG island encompassing the transcription site of myopodin, we aimed at evaluating the potential relevance of myopodin methylation in bladder cancer. The epigenetic silencing of myopodin by hypermethylation was tested in bladder cancer cells (n=12) before and after azacytidine treatment. Myopodin hypermethylation was associated with gene expression, being increased in vitro by this demethylating agent. The methylation status of myopodin promoter was then evaluated by methylation-specific polymerase chain reaction (MS-PCR) analyses. Myopodin was revealed to be frequently methylated in a large series of 466 bladder tumours (68.7%). Myopodin methylation was significantly associated with tumour stage (p<0.0005) and tumour grade (p=0.037). Myopodin expression patterns were analysed by immunohistochemistry on tissue arrays containing bladder tumours for which myopodin methylation was assessed (n=177). The presence of low nuclear myopodin expression alone (p = 0.031) or combined with myopodin methylation (p=0.008) was associated with poor survival. Moreover, myopodin methylation in 164 urinary specimens distinguished patients with bladder cancer from controls with a sensitivity of 65.0%, a specificity of 79.8%, and a global accuracy of 75.3%. Thus, myopodin was identified to be epigenetically modified in bladder cancer. The association of myopodin methylation and nuclear expression patterns with cancer progression and clinical outcome, together with its ability to detect bladder cancer patients using urinary specimens, suggests the utility of incorporating myopodin methylation assessment in the clinical management of patients affected by uroepithelial neoplasias.

Identification of DNA hypermethylation of SOX9 in association with bladder cancer progression using CpG microarrays.

CpG island arrays represent a high-throughput epigenomic discovery platform to identify global disease-specific promoter hypermethylation candidates along bladder cancer progression. DNA obtained from 10 pairs of invasive bladder tumours were profiled vs their respective normal urothelium using differential methylation hybridisation on custom-made CpG arrays (n=12 288 clones). Promoter hypermethylation of 84 clones was simultaneously shown in at least 70% of the tumours. SOX9 was selected for further validation by bisulphite genomic sequencing and methylation-specific polymerase chain reaction in bladder cancer cells (n=11) and primary bladder tumours (n=101). Hypermethylation was observed in bladder cancer cells and associated with lack of gene expression, being restored in vitro by a demethylating agent. In primary bladder tumours, SOX9 hypermethylation was present in 56.4% of the cases. Moreover, SOX9 hypermethylation was significantly associated with tumour grade and overall survival. Thus, this high-throughput epigenomic strategy has served to identify novel hypermethylated candidates in bladder cancer. In vitro analyses supported the role of methylation in silencing SOX9 gene. The association of SOX9 hypermethylation with tumour progression and clinical outcome suggests its relevant clinical implications at stratifying patients affected with bladder cancer.

Aplicación de arrays de anticuerpos en el estudio del cáncer vesical / Use of antibodies arrays in the study of bladder cancer

Los arrays de anticuerpos representan una tecnología de alto rendimiento muy versátil que permite la detección de múltiples proteínas simultáneamente. Al permitir cuantificar los niveles proteicos multiparamétricamente y analizar modificaciones post-translacionales, permiten mejorar la caracterización funcional de los procesos moleculares asociados con procesos neoplásicos. Más aun, la identificación de los patrones de expresión proteicos característicos de la progresión tumoral y de los distintos subtipos tumorales puede mejorar el manejo clínico del paciente con cáncer vesical y permitir en un futuro próximo el diseño de terapias adaptadas a la agresividad de cada tumor

Antibodies arrays represent a very versatile high-throughput technology that enables multiple protein detection simultaneously. It allows quantifying protein levels multiparametrically, and analyzing their post-translational modifications, thus improving the funtional characterization of molecular processes associated with neoplasic diseases. Moreover, the identification of protein expression patterns characteristic of tumor progression and of different tumor subtypes can improve the clinical management of patients with bladder cancer. In the near future, they may allow establishing tailored therapies according to the aggresiveness of each specific tumor

[Use of antibodies arrays in the study of bladder cancer]. / Aplicación de arrays de anticuerpos en el estudio del cáncer vesical.

Antibodies arrays represent a very versatile high-throughput technology that enables multiple protein detection simultaneously. It allows quantifying protein levels multiparametrically, and analyzing their post-translational modifications, thus improving the funtional characterization of molecular processes associated with neoplasic diseases. Moreover, the identification of protein expression patterns characteristic of tumor progression and of different tumor subtypes can improve the clinical management of patients with bladder cancer. In the near future, they may allow establishing tailored therapies according to the aggresiveness of each specific tumor.

Applications of array technology: identification of molecular targets in bladder cancer.

High-throughput microarrays are being used in expression profiling analyses with the objectives of gene and pathway discovery, functional characterization of genes, and tumor subclassification. This review summarizes bladder cancer studies dealing with both in vitro models and clinical specimens, using distinct microarray platforms for target discovery.

Marcadores en orina para el diagnóstico y la monitorización de los pacientes con cáncer vesical / Urinary tumor markers for the diagnosis and monitoring of patients with bladder cancer

La cistoscopia, el método de referencia para el diagnóstico del cáncer de vejiga, no posee una sensibilidad del 100 por ciento y lamentablemente resulta invasiva e incómoda para el paciente. La citología urinaria como método no invasivo de referencia presenta limitaciones en su sensibilidad y en su gran variabilidad interobservador. Por estos motivos siguen siendo necesariso métodos alternativos no invasivos y objetivos para el diagnóstico de la enfermedad. Los marcadores tumorales en orina representan una opción de gran interés para la detección precoz de la enfermedad y en el segumiento de los pacientes con cáncer vesical. La sensibilidad de la mayor parte de estos marcadores es superior a la citología urinaria, aunque su especificidad es inferior en los tumores de alto grado. El uso de criterios de exclusión puede aumentar la especificidad de los marcadores, tanto en el seguimiento como en el diagnóstico inicial de la enfermdad. Los marcadores tumorales podrían llegar a reemplazar la citología urinaria en la práctica clínica habitual. Sin embargo, no se ha demostrado que la sensibilidad a nivel global sea lo suficientemente alta como para reemplazar la cistoscopia como método de referencia para el diagnóstico de la enfermedad. Siguen siendo necesarios estudios prospectivos y multicéntricos que permitan consensualizar cómo mejorar la sensibilidad de los marcadores y determinar criterios de exclusión apropiados para aumentar su especifidad (AU)

Diagnostic performance of the urinary bladder carcinoma antigen ELISA test and multiparametric DNA/cytokeratin flow cytometry in urine voided samples from patients with bladder carcinoma.

BACKGROUND: The objective of the current study was to comparatively analyze the sensitivity and specificity of flow cytometric DNA/cytokeratin 8/18 measurements and the urinary bladder carcinoma antigen (UBC) enzyme linked immunoabsorbent assay (ELISA) test for the detection of bladder carcinoma in voided urine samples. METHODS: Eighty-one fresh urine voided samples, preserved frozen for a maximum period of 3 months, belonging to patients with an active bladder carcinoma (n = 37), patients who were free of disease as confirmed by cystoscopy (n = 19), patients receiving intravesical therapy (n = 17), and individuals with other benign and malignant conditions (n = 8), were collected. Flow cytometry measurements of thawed samples were based on the detection of cytokeratin (CK) 8+ and CK18+ cells using the 3F3 and 6D7 monoclonal antibodies alone or in combination with the measurement of cell DNA contents, after propidium iodide staining. Urinary bladder carcinoma antigen test was measured by ELISA. RESULTS: Patients were grouped according to the presence (n = 44) or absence (n = 29) of bladder carcinoma as confirmed by cystoscopy, and taking cutoffs of 9.7 microg/L for UBC-ELISA, 75% for the percentage of 3F3 (+) and 6D7 (+) cells, and 10.6% for the proportion of hyperdiplod cells that suggested a specificity of 83%, the individual sensitivity obtained for each parameter was 77%, 5%, 9%, and 77%, respectively. The presence of DNA aneuploid populations showed a relatively low sensitivity (36%) although it was the most specific parameter (93%). Combining UBC antigen test with the proportion of cells showing DNA content higher than 2n increased to 89% the sensitivity of the UBC antigen alone. However, false-positive results for both techniques were found in individuals with urologic diseases other than bladder carcinoma and in patients receiving intravesical therapy. CONCLUSIONS: The authors' results suggest that the combined use of the UBC antigen test and DNA/cytokeratin flow cytometry double stainings for the analysis of freshly obtained urine voided samples, cryopreserved to assure cellular integrity, is of great clinical utility for the detection of tumor recurrence in patients with bladder carcinoma.

Utility of serial urinary tumor markers to individualize intervals between cystoscopies in the monitoring of patients with bladder carcinoma.

BACKGROUND: Cross-section studies have shown the diagnostic characteristics of certain urinary tumor markers for the detection of bladder carcinoma. However, the role of serial urinary tumor markers in the monitoring of patients with bladder carcinoma in daily clinical surveillance has not been completely defined yet. METHODS: The study comprised 1185 urine samples belonging to 232 patients with a previous bladder carcinoma: 106 patients under follow-up (Group 1) and 126 bladder carcinoma patients receiving intravesic instillations (Group 2). Patients were monitored with urinary tumor markers during a one-year follow-up period. Urine samples were collected before cystoscopies and in the intercystoscopic periods for patients in Group 1 and before intravesic instillations for patients Group 2. Urinary bladder carcinoma antigen (UBC), CYFRA 21-1 and nuclear matrix proteins (NMP22) were measured by immunoassays. RESULTS: Monitoring of the disease with urinary tumor markers could detect recurrence sooner than scheduled cystoscopies in 27 patients (87%) for UBC, 27 patients (87%) for CYFRA 21-1, and 26 patients (84%) for NMP22 out of 31 Group 1 patients who recurred; and in 16 patients (67%) for UBC, 17 patients (71%) for cytokeratin fragments (CYFRA) 21-1, and 13 patients (54%) for NMP22 out of 24 Group 2 patients who recurred. The most relevant finding was that persistence of negative urinary markers during follow-up was largely indicative of disease free status in 65 of 75 (87%) patients of Group 1 and 31 of 102 (30%) cases of Group 2. Although false positive results were present, they were mainly associated with sporadic urinary tract infections in 10 of 75 (13%) cases of Group 1 and in 36 of 102 (35%) patients of Group 2; and with urine samples collected in the first two months at the beginning of intravesic therapy in 35 of 102 patients (34%) in Group 2. CONCLUSIONS: Monitoring of bladder carcinoma patients with serial urinary tumor markers could anticipate detection of recurrence. Persistent negative results might postpone and reduce the number of cystoscopies. Once the limitations leading to false positive results are controlled by urinalysis and by starting sample collection when basal levels are reached in patients with intravesic therapy, urinary tumor markers might eventually individualize the intervals between cystoscopies in the surveillance of patients with bladder carcinoma.

Serial urinary IL-2, IL-6, IL-8, TNFalpha, UBC, CYFRA 21-1 and NMP22 during follow-up of patients with bladder cancer receiving intravesical BCG.

BACKGROUND: We evaluated the potential role of serial preinstillation levels of several interleukins, TNFalpha and urinary tumor markers to monitor patients with bladder cancer receiving intravesical BCG. PATIENTS AND METHODS: 121 urine samples were collected from: patients with bladder cancer treated with BCG (group 1); patients with bladder cancer receiving other intravesical treatment (group 2) and patients with urinary tract infections (group 3). Cytokines [IL-2, IL6 and [L8] and TNFalpha and urinary tumor markers [UBC, CYFRA 21-1 and NMP22] were measured by immunoassays. RESULTS: In 3 out of 15 BCG non-responders that recurred over the period of the study, no cytokine peak for IL-2, IL-6 or TNFa were detected. Urinary tumor markers increased in 2 out of 3 of these patients earlier than scheduled cystoscopies. Cytokine measurement was heterogeneous among 12 out of 15 BCG-responding patients: there were low levels of IL-6 and TNFalpha and peaks of IL-2 and IL-8 in 10 out of 12 and 4 out of 12 patients, respectively. During responding patients' follow-up we observed false-positive results in 7 out of 65 urine samples for UBC, 8 out of 65 for CYFRA 21-1 and 20 out of 65 for NMP22. Urinary tract infections were the main factor associated with non-specific elevations of IL-6 and IL-8 and urinary tumor markers in all groups of patients. CONCLUSION: Although larger series are required to confirn our preliminary observations, our data argue for a potential predictive role for IL-2 of favourable response to BCG therapy. Monitoring BCG with urinary tumor markers could early detect recurrence in non-responding patients.

Comparative predictive values of urinary cytology, urinary bladder cancer antigen, CYFRA 21-1 and NMP22 for evaluating symptomatic patients at risk for bladder cancer.

PURPOSE: We study the potential diagnostic use of urinary bladder cancer antigen, CYFRA 21-1 and NMP22*; for evaluating symptomatic patients who present with microscopic hematuria and are at risk for bladder cancer. MATERIALS AND METHODS: Urinary tumor markers were determined in 187 samples from 112 patients symptomatic of bladder cancer (group 1), and 75 with benign and other urological conditions (group 2). Immunoassays were used to measure the 3 selected biomarkers. Sensitivity and specificity were established by previously defined cut points. Biomarker results were reported as corrected and uncorrected for urinary creatinine. Urinalysis was performed in all samples. RESULTS: Positive and negative predictive values were 85.5%, 80.5% and 81.1%, and 80.8%, 79.2% and 76.5% for urinary bladder cancer antigen, CYFRA 21-1 and NMP22, with the cutoffs 9.7 microg./l., 5.4 microg./l and 10.0 units per ml., respectively. These predictives values were 85.2% and 72.5%, respectively, for urinary cytology. The combination of biomarkers decreased the positive predictive values to 72.3% to 78.6% and increased negative predictive values to 84.2% to 86.1%. Urinary tract infection, inflammation and malignancy associated with other genitourinary organs were the primary cause for false-positive test results in the 3 assays evaluated. CONCLUSIONS: With a single biomarker, around 80% of the positive results would have correctly identified symptomatic patients for cystoscopy. Of the negative results 75% would have correctly reduced the number of cystoscopies. Sensitivity and negative predictive values could be improved with the combination of biomarkers but with a loss of specificity and positive predictive values. Urinary tract inflammation and other genitourinary malignancies might contribute to the reduction in specificity of these tests.

Evaluation of two new urinary tumor markers: bladder tumor fibronectin and cytokeratin 18 for the diagnosis of bladder cancer.

Our objectives were to evaluate the diagnostic value of two new urinary tumor markers, cytokeratin 18 (CK18) and bladder tumor fibronectin (BTF), for the detection and monitoring of bladder cancer. The study comprised 931 urine samples belonging to 402 subjects: 112 individuals under suspicion for a primary bladder tumor (group 1); 104 bladder cancer patients under scheduled follow-up (group 2); 109 bladder cancer patients receiving intravesical instillations (group 3); 45 patients with other urological diseases (group 4); and 32 healthy subjects (group 5). Voided urine samples were collected before cystoscopies, between them and before intravesical instillations. CK18 and BTF tests were measured by chemiluminescent immunoassays. Optimal receiver operating characteristic cutoffs of 7.4 microg/L for CK18 and 52.8 microg/liter for BTF rendered overall sensitivities of 66.2% for CK18 and 80.0% for BTF at specificities of 88.4 and 74.7%, respectively. Urinary cytology provided a sensitivity of 29.2% at a specificity of 99.1%. Sensitivities were 80.8, 74.2, and 82.3% for BTF and 71.1, 77.4, and 64.7% for CK18 for groups 1 to 3, respectively. False positive rates were higher for BTF in all groups of patients. Elevated urinary tumor markers during the monitoring of patients with bladder cancer could detect recurrence sooner than scheduled cystoscopies. Persistence of negative markers was greatly indicative of free of disease status in follow-up. CK18 and BTF in urine may eventually prove to be of benefit for specific patients with bladder carcinoma given its higher sensitivity compared with cytology. In selected patients, namely those with persistent negative urinary CK18 and BTF, the number of cystoscopies could be reduced.

[Cytokeratins (UBC and CYFRA 21-1) and nuclear matrix proteins (NMP22) as urine tumor markers in the diagnosis of bladder cancer]. / Citoqueratinas (UBC y CYFRA 21-1) y proteínas de la matriz nuclear (NMP22) como marcadores tumorales en la orina en el diagnóstico del cáncer vesical.

BACKGROUND: The development of urinary tumor markers such as UBC, CYFRA 21-1 and NMP22 appeared to be non invasive alternative methods for the detection of bladder cancer. We compared the individual and combined sensitivity of the urinary tumor markers in the detection of bladder cancer, contrasting them with the conventional diagnostic procedures. PATIENTS AND METHODS: 237 voided urines from subjects under risk for bladder cancer were collected immediately before the endoscopic examinations: 44 patients under suspicion of a primary bladder tumor and 193 patients under follow-up of a previous bladder cancer were included. UBC and NMP22 were measured by enzyme-immunoabsorbent-assays and CYFRA 21-1 by an electro-chemiluminescense-immunoassay. RESULTS: Taking the cutoffs of 9.7 micrograms/l for UBC, 5.4 ng/ml for CYFRA 21-1 and 10.0 U/ml for NMP22 sensitivities were 70%, 69% and 67% for UBC, CYFRA 21-1 and NMP22 at specificities of 95%, 94% y 80%, respectively. All tumor markers showed higher sensitivities than urinary cytology (7%), microhematuria (62%) and gross hematuria (10%) at specificities of 99%, 78% and 99%, respectively. The combinations of NMP22 plus CYFRA 21-1 reached the highest sensitivity (79%), slightly lower than simultaneously measuring the three tumor markers (80%). CONCLUSIONS: The sensitivities of the urinary markers UBC, CYFRA 21-1 and NMP22 appeared to be high enough so as to substitute urinary cytology. The diagnostic similarity between cytokeratins individually and in each type of patients might not recommend their simultaneous determination. The combined measurement of NMP22 and one cytokeratin marker (CYFRA 21-1 or UBC) appeared to be the most recommended.

Urinary tissue polypeptide-specific antigen for the diagnosis of bladder cancer.

OBJECTIVES: To evaluate the diagnostic characteristics of the urinary measurement of cytokeratin tissue polypeptide-specific antigen (TPS) for the detection of bladder cancer. METHODS: Three hundred thirty-five individuals in five groups were studied: group 1, subjects with microhematuria under suspicion for primary bladder cancer; group 2, patients being followed up with scheduled cystoscopic examinations; group 3, patients in follow-up receiving chemotherapy instillations; group 4, patients with other urologic diseases; and group 5, healthy subjects. Urine samples belonging to subjects from groups 1, 2, and 3 were collected immediately before cystoscopy. Additionally, patients from groups 2 and 3 were monitored with urinary TPS for a minimum period between two cystoscopies. TPS was measured by an enzyme immunosorbent assay. RESULTS: Receiver operating characteristic analysis gave a sensitivity of 64% and a specificity of 84% at a threshold value of 279 U/L. The positive and negative predictive value was 66% and 82%, respectively; accuracy was 77%. TPS could discriminate the presence of bladder tumor sooner than the scheduled cystoscopies in 9 of 19 follow-up patients with recurrence. False-positive results during follow-up were found in 112 urine samples, one third of which were associated with urinary tract infections. TPS did not appear to be specific for bladder cancer, with elevated results in 45% of patients from group 4, which might lead to clinical misinterpretation of urinary TPS results. CONCLUSIONS: Urinary TPS might provide additional information for the detection of bladder cancer as an adjunct to cystoscopy. Considering the false-positive rates, different urologic diseases should be ruled out before making clinical decisions on the basis of elevated urinary TPS results.

Comparative sensitivity of urinary CYFRA 21-1, urinary bladder cancer antigen, tissue polypeptide antigen, tissue polypeptide antigen and NMP22 to detect bladder cancer.

PURPOSE: We compare the individual and combined sensitivity of urinary CYFRA 21-1, urinary bladder cancer antigen, tissue polypeptide antigen and NMP22 to detect bladder cancer, evaluate the false-positive rates for different pathological conditions, and assess differential sensitivity regarding histological and clinical characteristics of disease. MATERIALS AND METHODS: A total of 267 subjects entered the study. Sensitivities of the tests were evaluated in 111 patients with active bladder cancer and 76 with no evidence of disease. False-positive rates were evaluated in 80 symptomatic and asymptomatic controls, including patients with benign urological conditions and nonbladder malignancies, and healthy subjects. CYFRA 21-1 was determined by electrochemoluminescent immunoassay in the Elecsys 2010, urinary bladder cancer antigen was quantified by enzyme linked immunosorbent assay (IDL Biotech), tissue polypeptide antigen was measured by the Prolifigen TPA-IRMA and NMP22 was assayed by enzyme linked immunosorbent assay (Matritech). Cutoffs were obtained by the 95% percentile in patients with no evidence of disease, which gave a 95% specificity for all biomarkers. Differences in sensitivity of urinary biomarkers regarding stage, grade, tumor size, pattern of growth, focality and recurrence were evaluated. RESULTS: At a specificity of 95% cutoffs were 5.4 ng./ml. for CYFRA 21-1, 15.5 microg./l. for urinary bladder cancer antigen, 760.8 U./l. for tissue polypeptide antigen and 14.6 U./ml. for NMP22. Using these cutoffs sensitivities were 75.7% for NMP22, 83.8% for CYFRA 21-1, 73.9% for urinary bladder cancer antigen quantitative and 80.2% for tissue polypeptide antigen. The additional determination of cytokeratins increased the sensitivity of NMP22. Cytokeratins did not appear to be specific for bladder cancer, and false-positives rates were between 20% for urinary bladder cancer antigen and 36% for tissue polypeptide antigen for benign urological conditions, and between 40% and 52%, respectively, for nonbladder malignancies. NMP22 showed lower false-positives rates, mainly for benign diseases. Urinary tumor markers appeared to be associated with some of the most relevant histological and clinical parameters of bladder cancer. CONCLUSIONS: Our preliminary evaluation showed the tests to be potential noninvasive adjuncts to help determine the need for cystoscopy. The combination of 2 tumor markers, NMP22 and 1 cytokeratin (CYFRA 21-1 or urinary bladder cancer antigen), seemed to be the most effective. Further comparative studies are needed to assess the promising diagnostic role of these markers.

New electrochemiluminescent immunoassay for the determination of CYFRA 21-1: analytical evaluation and clinical diagnostic performance in urine samples of patients with bladder cancer.

BACKGROUND: A new electrochemiluminescent immunoassay (ECLIA) has been developed for the determination of cytokeratin 19 (CYFRA 21-1) in the Elecsys 2010 immunoassay system. Urinary CYFRA 21-1 might have a role in the diagnosis of bladder cancer. METHODS: We performed an analytical evaluation of the CYFRA 21-1 ECLIA for serum and urine samples. The clinical value of urinary CYFRA 21-1 for the detection of bladder cancer was evaluated through its measurement in 226 urine samples from symptomatic and asymptomatic controls. RESULTS: At concentrations of 2-30 microg/L, within-assay imprecision (CV) was below 2.1% for sera and 3.3% for urines, with interassay CVs below 3.3% for sera and 4.9% for urines. The day-to-day CV was <20% at concentrations >0.2 microg/L (functional sensitivity). Measurement of diluted samples showed that the assay estimated CYFRA 21-1 between 98% and 103% for sera and 98% and 105% for urines. Recovery of added CYFRA 21-1 was 99-105% for sera and 96-115% for urines. We separately compared serum and urine CYFRA 21-1 ECLIA results with those obtained with an IRMA (CIS bio international). Regression analysis for sera was: CYFRA 21-1 (ECLIA) = 0.520 + 1.018 CYFRA 21-1 (IRMA); [95% confidence interval (CI) (y-intercept), -0.260 to 1.309]; 95% CI (slope), 0.978-1.060; n = 100; S(y|x) = 3.242; r(2) = 0.987. For urine samples it was: CYFRA 21-1 (ECLIA) = 0.716 + 0.966 CYFRA 21-1 (IRMA); 95% CI (y-intercept), 0.009-1.422; 95% CI (slope), 0.956-0.976; n = 100; S(y|x) = 4.136; r(2) = 0.986. In urine samples voided by patients with and without bladder cancer, the best ROC analysis discrimination provided 81.0% (95% CI, 72.7-87.7%) sensitivity and 97.2% (95% CI, 90.2-99.6%) specificity at a threshold value of 5.7 microg/L. CONCLUSIONS: Our initial evaluation showed reliable analytical performance for urinary CYFRA 21-1, which might assist urologists in the detection of bladder cancer as a noninvasive adjunct to cystoscopy.

Evaluation of nuclear matrix protein 22 as a tumour marker in the detection of transitional cell carcinoma of the bladder.

OBJECTIVES: To evaluate the sensitivity and specificity of urinary nuclear matrix protein-22 (NMP22) in detecting bladder cancer, to compare the diagnostic performance of NMP22 alone and when corrected by urinary creatinine level, and to correlate NMP22 level with the histological and clinical characteristics of bladder cancer. PATIENTS, SUBJECTS AND METHODS: The study included 267 patients classified into five groups: group 1 comprised 111 patients with active transitional cell carcinoma (TCC) of the bladder; group 2 included 76 patients who had had bladder TCC but were being followed and were free of disease, as confirmed by cystoscopy; group 3 comprised 25 patients with benign urological diseases; group 4 included 25 patients with other malignant pathological conditions; group 5 constituted a control group of 30 healthy subjects free of urological diseases. Urinary NMP22 was measured using an enzyme-linked immunosorbent assay. Receiver operating characteristic (ROC) curves were constructed to obtain the thresholds which gave optimal sensitivity and specificity for combinations of NMP22 alone and when corrected by urinary creatinine level. Stage, grade, tumour size, pattern of growth, focality and the presence of recurrence were recorded and their associations with NMP22 evaluated. RESULTS: The mean levels of NMP22 were 122.8, 5.1, 3.7, 2.3 and 0.3 U/mL for groups 1-5, respectively; overall, these values were significantly different (P<0.001). The mean (95% CI) optimal combination of 78.2% (69.3-85.5) sensitivity and 95.5% (87.3-99.0) specificity was obtained from the ROC analysis with a threshold value of 13.7 U/mL NMP22. When values were corrected by urinary creatinine levels, the threshold given by the best combination of sensitivity and specificity, at 73.2% (63.2-81.7) and 97.0% (89.6-99.5), respectively, was 3.0 U/mg creatinine. NMP22 level was statistically associated with stage, grade, tumour size and focality. CONCLUSIONS: Urinary NMP22 appeared to be a potential tumour marker for detecting TCC of the bladder; when corrected by urinary creatinine level, it might provide a better interpretation than when used alone. NMP22 correlated with the most relevant variables in bladder cancer. As a noninvasive adjunct, NMP22 might have a role in guiding urologists about the need for cystoscopy in such patients.