Detection of hyaluronidase RNA and activity in urine of schistosomal and non-schistosomal bladder cancer.

Diagnosis of bladder cancer is done by cystoscopy and cytology. In the last decade, many urine-based tests for bladder cancer have been developed and tested in different populations. Hence, it was relevant to assess the diagnostic significance of urinary hyaluronidase RNA and its enzyme activity in bladder cancer. Seventy patients with bladder cancer, 56 patients with benign bladder lesions, and 49 healthy controls were enrolled in this study. Voided urine samples from all subjects were used for estimation of urinary HAase RNA by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and determination of its enzymatic activity by zymography. There was a significant difference in the mean ranks and positivity rates of HAase RNA expression (P < 0.01) and its enzymatic activity among the three investigated groups: malignant, benign, and normal (P < 0.01). In detecting bladder cancer, the sensitivity of urine cytology (42.83 %) was improved to 100 % when combined with urinary Hyal RNA or Hyal enzyme activity. Detection of urinary Hyal RNA and its enzyme activity is promising noninvasive tests with high sensitivities and specificities for detection of bladder cancer.

Aberrant methylation of RARbeta2 and APC genes in voided urine as molecular markers for early detection of bilharzial and nonbilharzial bladder cancer.

BACKGROUND: Bladder cancer cells illustrate major disruptions in their DNA methylation patterns as compared with normal ones. Authors aimed to identify epigenetic molecular markers in urine for early detection of bladder cancer. MATERIALS AND METHODS: We retrospectively analyzed the methylation status of RARß(2) and APC genes in urine samples from 210 bladder cancer patients, 61 patients with benign urological diseases, and 49 healthy volunteers by using methylation-specific PCR. RESULTS: Methylated RARß(2) and APC were significantly higher in bladder cancer patients (62.8%, 59.5%) than benign (16.4%, 5%) but not detected in healthy volunteers (0%) at (P < 0.0001). Both methylated genes showed no significant difference among clinicopathologic factors; however, they were detected in all grades and stages. Among the 128 patients with bilharzial bladder cancer, 94 (73.4%) showed methylated RARß(2) and 86 (67.2%) showed methylated APC. Homoplasmic methylation pattern of both genes were only detected in bilharzial bladder cancer cases. Both sensitivities and specificities of the methylated genes for bladder cancer detection were superior to urine cytology and when altogether combined, the sensitivities improved to (91.8%), (93.5%), (91.9%), and (80.9%) in detection of: bladder cancer, non-muscle invasive bladder cancer, low-grade tumors, and bilharzial associated bladder cancer, respectively. CONCLUSION: Thus, methylated RARß(2) and APC genes might be valuable urinary molecular markers for early detection of bilharzial and nonbilharzial bladder cancer.

Expression of HYAL1 and survivin RNA as diagnostic molecular markers for bladder cancer.

PURPOSE: Urinary tumor markers that help in the early detection of bladder cancer promise a significant improvement in sensitivity, specificity and convenience over conventional, invasive diagnostic tests. We assessed the diagnostic efficacy of hyaluronidase (HYAL1) and survivin for early bladder cancer detection. MATERIALS AND METHODS: The study included 166 patients diagnosed with bladder carcinoma, 112 with benign bladder lesions and 100 healthy volunteers who served as controls. All underwent serological assessment of schistosomiasis antibody, urine cytology, and hyaluronidase (HYAL1) and survivin RNA estimation by qualitative and semiquantitative reverse transcriptase-polymerase chain reaction in urothelial cells from voided urine. RESULTS: Positivity rates of HYAL1 RNA and survivin RNA on qualitative reverse transcriptase-polymerase chain reaction were significantly different among the 3 groups. Mean rank using semiquantitative method was increased in the malignant vs the other groups. The best cutoff for HYAL1 and survivin RNA was 0.25 each. Using these cutoffs HYAL1 and survivin RNA sensitivity was 91% and 75%, respectively, with absolute specificity. HYAL1 RNA detected all patients with stages 0 and I bladder cancer (p <0.037). Urine cytology sensitivity improved when combined with hyaluronidase or survivin RNA on semiquantitative reverse transcriptase-polymerase chain reaction. CONCLUSIONS: The detection of urinary HYAL1 and survivin RNA is a promising noninvasive test for bladder cancer early detection. HYAL1 RNA was more sensitive and specific than urine cytology. Semiquantitative reverse transcriptase-polymerase chain reaction is favored for its high sensitivity and specificity.

A panel of angiogenic factors for early bladder cancer detection: enzyme immunoassay and Western blot.

PURPOSE: Angiogenesis is tightly regulated by a large number of pro-angiogenic factors, including vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor and angiogenin. We adapted and evaluated the measurement of these factors using enzyme-linked immunosorbent assay and compared the results with Western blot and voided urine cytology. MATERIALS AND METHODS: This study included 240 patients diagnosed with bladder carcinoma, 108 with benign bladder lesions and 110 healthy individuals who served as controls. All participants underwent serological schistosomiasis antibody assay in serum, urine cytology and estimation of angiogenic factors in voided urine. RESULTS: Intra-assay and interassay CVs of the investigated markers were 10.3 to 12.3 and 10 to 13.7, respectively. The recovery rate of the added angiogenic factor to the urine pool was 98% to 103%, 97% to 103%, 98% to 104% and 97% to 100% for vascular endothelial growth factor, basic fibroblast growth factor, angiogenin and hepatocyte growth factor, respectively. The concordance rate with Western blot was 97.5%. The levels and positive rates of urinary angiogenic markers and urine cytology were significantly higher in the malignant group than in the benign and healthy groups. Basic fibroblast growth factor increased significantly in bladder squamous cell carcinoma cases. Moreover, basic fibroblast growth factor and hepatocyte growth factor significantly correlated with tumor grade. Angiogenic markers showed significant association with clinical stage. CONCLUSIONS: Quantitative measurement of urinary angiogenic factors in voided urine samples by enzyme-linked immunosorbent assay was reliable. The sensitivity of basic fibroblast growth factor and hepatocyte growth factor was superior to that of the other investigated markers and of cytology in low grade and early stage cases, suggesting their convenience as sensitive, noninvasive diagnostic and screening tools for bladder cancer.

Detection of bladder carcinoma by combined testing of urine for hyaluronidase and cytokeratin 20 RNAs.

BACKGROUND: A new, sensitive, noninvasive method for the detection of urothelial carcinomas of the urinary bladder would open new possibilities in both the diagnosis and followup of patients. METHODS: This study included 228 patients diagnosed with bladder carcinoma, 68 patients with benign bladder lesions, and 44 healthy persons served as the control group. All were subjected to: serologic schistosomiasis antibody assay in serum, urine cytology, estimation of urine hyaluronic acid (HA) by enzyme-linked immunosorbent assay, and detection of CK-20 and hyaluronidase (HAase) by reverse transcription polymerase chain reaction (RT-PCR) in urothelial cells from voided urine. RESULTS: HA mean rank was higher in benign and malignant groups than in the healthy group (P < 0.0001) and was significantly related to tumor grade (P = 0.021). HA best-cutoff, determined using receiver operating characteristic curve to discriminate between malignant and nonmalignant groups, was 58.5 units/mg protein at 85.8% sensitivity and 60.7% specificity. HAase RNA showed superior sensitivity (90.8%) over cytology (68.9%) and CK-20 (78.1%) with specificity of 93.4%, 98.1% and 80.2%, respectively. The sensitivity reached 94.7% at a specificity of 91.5% when combined with CK-20. All 4 of the investigated markers were related to grade at P <0.05. Whereas only HAase and CK-20 were significantly related to stage (P < 0.05). As to schistosomiasis, only HAase RNA positivity was significantly associated (P = 0.038). CONCLUSIONS: HAase RNA is a promising noninvasive test with high sensitivity and specificity in bladder carcinoma detection.

HER2/neu expression in bladder cancer: relationship to cell cycle kinetics.

BACKGROUND AND OBJECTIVES: The incidence of overexpression of HER2/neu in bladder cancer is one of the highest among all human malignancies tested; such overexpression is thought to play a role in the aberrant proliferation of cancer cells. This study was conducted to evaluate the quantitative assessment of HER2/neu expression by enzyme immunoassay (EIA) and its prognostic significance in differentiating between high and low proliferating tumors. PATIENTS AND METHODS: Tissue samples were collected from 35 patients with benign bladder lesions, 28 with bilharzial bladder cancer, and 25 with nonbilharzial bladder cancer. Twenty normal samples were obtained from normal safety margin areas in nonbilharzial bladder cancer patients. Out of the malignant samples, 22 were found to be squamous cell carcinoma and 31 were transitional cell carcinoma. All samples were examined for HER2/neu expression by EIA and Western blot (WB). Flow cytometric (FCM) analysis was also performed in all the samples provided. RESULTS: HER2/neu was found to be significantly overexpressed in the malignant group compared to the benign and normal groups (P < 0.001) and no significant difference was found between the bilharzial and nonbilharzial cancer groups or between the transitional and squamous cell carcinoma groups. HER2/neu was significantly correlated to ploidy (P = 0.001), synthetic phase fraction, SPF (P = 0.012), and DNA index (P = 0.002). No significant correlation was found between HER2/neu and stage or grade while it was significantly associated with lymph node status of the tumour (P = 0.02). CONCLUSION: HER2/neu can be measured reliably by the EIA method as confirmed by WB. The quantitative assessment of HER2/neu expression in malignant tumors aided by other proliferation markers such as SPF, DI, and ploidy could be useful in selecting patients for more aggressive treatment or for predicting outcome.

Cell cycle regulators in bladder cancer: relationship to schistosomiasis.

Dysregulation of cell cycle control may lead to genomic instability, neoplastic transformation and tumor progression. In terms of the particular roles in regulation of the cell-cycle, p21(WAF1) causes growth arrest through inhibition of cyclin-dependant kinases required for G1/S transition. P16 (INK4A) and p15 (INK4B) are thought to act as tumor suppressors, since their inactivation and/or deletion are observable in various types of malignancies. Cyclin D1 is hypothesized to control cell cycle progression through the G1-S check point. The present study evaluated p21 expression, p16 and p15 gene deletion and cylin D1 expression in bladder carcinoma among Egyptian patients, in relation to different clinicopathological features of the tumors and presence or absence of bilharziasis. Tissue specimens were obtained from 132 patients with bladder carcinoma and 50 normal tissue samples from the same patients served as control. P21 was determined by Western blot (WB) and enzyme immunoassay (EIA), p16 and p15 gene deletions were examined by polymerase chain reaction (PCR) and Cyclin D1 was detected by WB. Levels of p21 were lower in malignant tumors than in normal tissues. Lower expression of p21 was evident in lymph node positive, well differentiated tumors and squamous cell carcinoma (SCC) than in lymph node negative, poorly differentiated tumors and transitional cell carcinoma (TCC). In all normal samples, p15 and p16 genes were detected while cyclin D1 was not detected. P16 and p15 genes were deleted in 38.7% (41/106) and 30.2% (32/106) of bladder tumors respectively. The deletion of both genes was associated with poor differentiation grade and presence of bilharziasis. P16 deletion was also correlated to advancing tumor stage. Cyclin D1 was expressed in 57.5% of bladder tumors (69/120), where its expression was correlated to early stage, well differentiation grade, schistomiasis, and low levels of p21. Cell cycle is dysregulated in bladder carcinoma. This was evident from the increased expression of cyclin D1, the decreased levels of p21 and the deletion of p15 and p16 genes. Moreover, p16 and p15 gene deletion was related to tumor progression and might have a role in bilharzial bladder carcinogenesis. Cyclin D1 over-expression appears to be an early event in bladder cancer and might explain bilharzial associated bladder carcinogenesis.

Comparison of cytokeratin 20 RNA and angiogenin in voided urine samples as diagnostic tools for bladder carcinoma.

BACKGROUND: We evaluated the diagnostic efficacy of urinary angiogenin (ANG) and cytokeratin 20 (CK-20) mRNA in comparison with voided urine cytology in the detection of bladder cancer patients. OBJECTIVES AND METHODS: A total of 97 Egyptian patients provided a single voided urine sample for ANG, CK-20 and cytology before cystoscopy. Of the 97 cases, 63 were histologically diagnosed as bladder cancer; 33 with transitional cell carcinoma (TCC) and 30 with squamous cell carcinoma (SCC), whereas the remaining 34 had benign urological disorders. A group of 46 healthy volunteers were also included in this study. Voided urine was centrifuged and the supernatant was used for estimation of ANG by EIA and confirmed by Western blotting (WB). The urine sediment was used for cytology and RNA extraction. CK-20 RNA was detected by RT-PCR. RESULTS: The best cutoff value for ANG was calculated by a ROC curve as 322.7 ng/mg protein. The median urinary ANG level in bladder carcinoma, benign urological disorders and healthy volunteer groups was: 802.7, 425 and 33 pg/mg protein, respectively. The positivity rate for urinary CK-20 mRNA of the control, benign and malignant groups was 0%, 2.9% and 82.3%, respectively (P = 0.000); while the rates for ANG were 11.6%, 54.8% and 75.4%, respectively (P = 0.000). There was no significant difference in positivity rates of CK-20 and ANG with respect to sex, smoking, schistosomiasis, urine cytology, tumor grade, tumor stage, hematuria or pus cells. The overall sensitivity and specificity were 71.4% and 90% for voided urine cytology, 75.4% and 70.3% for ANG, and 82.3% and 98.8% for CK-20. Combined sensitivity of voided urine cytology with ANG and CK-20 together (98.2%) was higher than either the combined sensitivity of voided urine cytology with ANG (96.5%) or with CK-20 (91.6%) or than that of the biomarker alone. We demonstrated significant positive correlation between CK-20 positivity with age (P = 0.043) and nodal involvement (P = 0.037); however, there was no significant correlation between CK-20 and ANG with the other clinicopathological parameters. CONCLUSIONS: Our data indicate that CK-20 and ANG in voided urine had higher sensitivities compared to voided urine cytology. However, when specificity was considered, CK-20 alone had superior sensitivity and specificity compared to ANG and voided urine cytology.

Detection of bladder tumours: role of cytology, morphology-based assays, biochemical and molecular markers.

PURPOSE OF REVIEW: Cystoscopy is currently considered the gold standard for the detection of bladder tumours. The role of urine cytology in the initial detection and follow-up of patients is under discussion. Many efforts have been made to increase the detection rates and to predict the outcome of bladder cancer. In this subject review, a series of morphology-based, biochemical and molecular markers were compared with urine cytology for the detection of bladder cancer. RECENT FINDINGS: Among the various markers reviewed, the average published sensitivity and specificity for the Bard tumour antigen test was 60 and 77%; for the nuclear matrix protein 22 test it was 67 and 72%; for the hyaluronic acid and hyaluronidase test it was 91 and 84%; for the ImmunoCyt it was 90 and 75%; for fluorescence in-situ hybridization it was 85 and 95%; for the telomerase assay it was 77 and 85%; and for the microsatellite assay it was 89 and 100%. DNA ploidy measurements, recent molecular markers and immunoassays designed to detect keratins, proteins, cell adhesion molecules, fibrinogen degradation products, and fibrinolysis markers were also included. SUMMARY: As is clear from the brief summary of available assays, the optimal method of application is not yet clear. The integration of an assay into clinical practice takes more than just the documentation of its sensitivity and specificity. However, several of the procedures have received considerable support from urologists as assisting them in the management of their patients.