Soluble syndecan-1 (SDC1) serum level as an independent pre-operative predictor of cancer-specific survival in prostate cancer.

BACKGROUND: PSA-screening detects many cases of clinically non-aggressive prostate cancer (PC) leading to significant overtreatment. Therefore, pre-operatively available prognostic biomarkers are needed to help therapy decisions. Syndecan-1 (SDC1) is a promising prognostic tissue marker in several cancers including PC but serum levels of shedded SDC1-ectodomain (sSDC1) have not been assessed in PC. METHODS: A total of 150 patients with PC were included in this study (n = 99 serum samples, n = 103 paraffin-embedded samples (FFPE), n = 52 overlap). SDC1 protein expression and cellular localization was evaluated by immunohistochemistry (IHC), while sSDC1 serum concentrations were measured by ELISA. Serum sSDC1 levels were compared to those of MMP7, which is known to be a protease involved in SDC1 ectodomain-shedding. Clinico-pathological and follow-up data were collected and correlated with SDC1 tissue and serum levels. Disease (PC)-specific (DSS) and overall-survival (OS) were primary endpoints. RESULTS: Median follow-up was 167 months in the serum- and 146 months in the FFPE-group. SDC1-reactivity was higher in non-neoplastic prostate glands compared to PC. In addition, cytoplasmatic, but not membranous SDC1 expression was enhanced in PC patients with higher Gleason-score >6 PC (P = 0.016). Soluble SDC1-levels were higher in patients with Gleason-score >6 (P = 0.043) and metastatic disease (P = 0.022) as well as in patients with progressed disease treated with palliative transurethral resection (P = 0.002). In addition, sSDC1 levels were associated with higher MMP7 serum concentration (P = 0.005). In univariable analyses, only sSDC1-levels exhibited a trend to unfavorable DSS (P = 0.077). In a multivariable pre-operative model, high pre-operative sSDC1-level (>123 ng/ml) proved to be an independent marker of adverse OS (P = 0.048) and DSS (P = 0.020). CONCLUSIONS: The present study does not confirm the prognostic relevance of SDC1-IHC. The significant higher sSDC1 serum levels in advanced cases of PC, suggest that SDC1 shedding might be involved in PC progression. Additionally, high sSDC1-level proved to be an independent factor of adverse OS and DSS in a multivariable pre-operative model, making evaluation of sSDC1-levels a promising tool for pre-operative risk-stratification and/or therapy monitoring. Prostate 76:977-985, 2016. © 2016 Wiley Periodicals, Inc.

Circulating and Tissue Expression Levels of YKL-40 in Renal Cell Cancer.

PURPOSE: Blood levels of YKL-40 are elevated in various malignancies and other inflammatory diseases. Higher YKL-40 levels have consequently been shown to correlate with poor prognosis in several cancers. We investigated the prognostic value of circulating and tissue levels of YKL-40 in renal cell cancer. MATERIALS AND METHODS: Preoperative YKL-40 serum/plasma levels were determined in 222 surgically treated patients with renal cell cancer and in 35 controls. Postoperative serum samples were analyzed in 19 of the 222 renal cell cancer cases. Gene expression levels were assessed in 101 renal cell cancer frozen tissue samples using quantitative real-time reverse transcriptase-polymerase chain reaction. Finally immunohistochemical analysis was done in 37 renal cell cancer cases to assess tissue localization of YKL-40. Results were correlated with clinicopathological and followup data. RESULTS: YKL-40 serum but not tissue gene expression levels were higher in patients with renal cell cancer compared to controls (p = 0.050). Serum YKL-40 levels significantly increased following nephrectomy (p <0.001). High circulating YKL-40 concentrations were independently associated with shorter survival in the serum and plasma cohorts. YKL-40 gene expression did not correlate with patient prognosis. CONCLUSIONS: Preoperatively elevated circulating levels of YKL-40 predict survival in patients treated with nephrectomy for renal cell cancer independently of levels determined in serum or plasma. Tumor cells do not seem to be the main source of increased serum/plasma YKL-40 levels in patients with renal cell cancer.

Urachal Carcinoma of the Bladder: Impact of Clinical and Immunohistochemical Parameters on Prognosis.

PURPOSE: Urachal carcinoma of the bladder is a rare malignancy. Its histological phenotype is similar to that of primary bladder and colorectal adenocarcinoma. The aim of this study was to explore the expression and prognostic relevance of 6 select protein markers of urachal carcinoma of the bladder, including p53, Ki67, RHAMM, BGN, IMP3 and MMP-7, which were formerly shown to be prognostic in urothelial carcinoma and colorectal adenocarcinoma. MATERIALS AND METHODS: Clinical and followup data were obtained on a total of 26 patients with urachal carcinoma of the bladder treated at 2 university hospitals. Immunohistochemical analysis of p53, Ki67, RHAMM, BGN, IMP3 and MMP-7 expression was performed in samples from 15 patients. Clinicopathological parameters and immunohistochemical results were tested for prognostic value on univariable and multivariable analyses. RESULTS: Followup was 50 months. Five-year overall and progression-free survival was 46% and 32%, respectively. On multivariable analysis a positive resection margin was an independent predictor of poor overall survival (p = 0.025). RHAMM (p = 0.0431), IMP3 (p = 0.0052), Ki67 (p = 0.0006) and p53 (p = 0.0024) expression rates were significantly increased in urachal carcinoma of the bladder cells compared to normal urothelium. IMP3 was elevated in Sheldon tumor stage IIIA compared to IIIB or greater (p = 0.0048). None of the analyzed protein markers was associated with survival. CONCLUSIONS: The independent prognostic value of a positive resection margin underlines the importance of complete surgical removal of urachal carcinoma of the bladder combined with en bloc resection of the median umbilical ligament and umbilicus. Our results in a limited number of samples show that Ki67, p53, RHAMM and IMP3 expression is enhanced but has no prognostic significance in urachal carcinoma of the bladder.

Mistletoe Plant Extract in Patients with Nonmuscle Invasive Bladder Cancer: Results of a Phase Ib/IIa Single Group Dose Escalation Study.

PURPOSE: We determined the maximum tolerated dose, safety and effectiveness of intravesical instillation of mistletoe extract after transurethral resection of nonmuscle invasive bladder cancer. MATERIALS AND METHODS: In this single group dose escalation study patients with nonmuscle invasive bladder cancer were treated with weekly instillations of mistletoe extract for 6 weeks. Four weeks before instillation therapy all patients underwent transurethral resection of bladder tumors. During this procedure a marker tumor was left. At 12 weeks after the start of instillation therapy transurethral resection of the marker tumor or biopsy of the former marker tumor location was done so that patients were tumor free when entering followup until week 48. During the followup clinical assessment laboratory tests for safety and cystoscopy were done every 12 weeks. RESULTS: A total of 36 patients were treated with increasing doses of mistletoe extract. We found no dose limiting toxicity up to a dose of 675 mg of plant extract. Besides local reactions we saw hints that pyrexia may develop. All adverse events were well manageable. At 12 weeks a marker tumor remission rate of 55.6% (95% CI 38.1 to 72.1) was achieved. At 1 year a recurrence rate of 26.3% (95% CI 9.1 to 51.2) was observed. CONCLUSIONS: In this study intravesical instillation of mistletoe extract as treatment in patients with nonmuscle invasive bladder cancer was shown to be safe and well tolerated. Promising data on efficacy were observed and will be further investigated in a phase III study.

Second-Line Treatment of Advanced Urothelial Cancer with Paclitaxel and Everolimus in a German Phase II Trial (AUO Trial AB 35/09).

OBJECTIVE: The efficacy of second-line treatment after failure of platinum-based chemotherapy in patients with advanced urothelial cancer is limited. Based on encouraging preclinical and clinical phase I data, we evaluated the safety and efficacy of the combination of paclitaxel and everolimus in these patients. METHODS: In this trial, patients having failed to respond to prior platinum-based combination treatment of urothelial cancer were treated with paclitaxel (175 mg/m(2) i.v., 3-weekly) and the mTOR-inhibitor everolimus (10 mg p.o., once daily). The patients were treated until tumor progression or until a maximum of 6 cycles was completed. A one-stage design was used to evaluate the objective response rate (ORR) as the primary endpoint. RESULTS: A total of 27 patients (67% male; median age 63 years) were enrolled. The most frequent grade III/IV toxicities were anemia (28%), peripheral neuropathy (28%), and fatigue (24%). No treatment-related deaths were reported. Complete and partial remissions were observed in 0/24 and 3/24 patients eligible for efficacy analysis, respectively (ORR 13%). Progression-free survival was 2.9 months [95% confidence interval (95% CI) 1.9-4.2], and the median overall survival was 5.6 months (95% CI 4.8-10.2). CONCLUSION: The combination of paclitaxel and everolimus has not achieved the expected efficacy in second-line treatment of urothelial cancer and should not be further explored.

Renal cell carcinoma and tumour thrombus in the inferior vena cava: clinical outcome of 98 consecutive patients and the prognostic value of preoperative parameters.

OBJECTIVES: To evaluate the outcome of patients after nephrectomy and removal of tumour thrombus and to assess the prognostic value of preoperative parameters. PATIENTS AND METHODS: Ninety-eight patients who were surgically treated between 2002 and 2011 were included. Patients' charts were reviewed, and patients with renal cell carcinoma (RCC) and concomitant tumour thrombus in the renal vein (RV) were compared with those with extended inferior vena cava (IVC) thrombus. Wilcoxon rank-sum test, Kaplan-Meier analysis and uni- and multivariate Cox regression analysis were used for statistical evaluation. RESULTS: Follow-up was 36 months (20-122 months), and 5-year disease-specific survival (DSS) and overall survival were 68.4 and 54.1 %, respectively. Patients with extended thrombus (levels 2-4) had higher intraoperative transfusion rates of concentrated red cells (CRC) and fresh-frozen plasma (FFP) compared with patients with thrombus confined to the RV (CRC: 5.8 vs. 1.5, p < 0.0001; FFP: 2.3 vs. 0.4, p = 0.0032). Surgery time (190 vs. 107 min, p < 0.0001), duration of hospitalisation (16 vs. 11 days, p = 0.0269), serum phosphate (3.64 vs. 3.29 mmol/l, p = 0.0369) and CRP levels (6.7 vs. 4.4 mg/dl, p = 0.0194) as well as aPTT were increased (33.7 vs. 29.6 s, p = 0.0059) in extended thrombus disease. In multivariate analysis, the presence of distant metastasis (p = 0.03) and lymphovascular invasion (p = 0.001), high platelet counts (p = 0.001) and high serum potassium levels (p = 0.032) proved to be independent prognostic factors. CONCLUSION: The surgical treatment of RCC with tumour thrombus in the RV or IVC has favourable results. Extended thrombus disease requires multidisciplinary approach. High serum potassium levels and platelet counts are associated with reduced DSS.

Prognostic value of tissue and circulating levels of IMP3 in prostate cancer.

Tissue levels of the oncofetal protein insulin-like growth factor 2 (IGF2) messenger RNA-binding protein 3 (IMP3) have been associated with poor prognosis in multiple human malignancies. However, its circulating levels have not yet been analyzed. Therefore, the aim of this study was to assess the prognostic value of both serum and tissue levels of IMP3 in prostate cancer (PC). IMP3 protein expression was analyzed in 124 PC and 13 benign prostate hyperplasia (BPH) patients using immunohistochemistry. Gene expression levels of IMP3 and its molecular target IGF2 were analyzed in 29 frozen and 26 paraffin-embedded PC tissues using real-time polymerase chain reaction and immunohistochemistry. Serum IMP3 levels were assessed in 94 PC and 20 BPH patients as well as in 20 controls using enzyme-linked immunosorbent assay. IMP3 immunostaining was present in 0% (0/13) of BPHs, 15% (15/101) of clinically localized PCs and 65% (15/23) of palliatively treated metastatic PCs (p < 0.001). Accordingly, serum IMP3 concentrations were significantly higher in PC compared to BPH patients which were higher than those in controls (p < 0.001 each). The highest concentrations were detected in metastatic PC patients (p = 0.036). In patients who underwent radical prostatectomy high IMP3 serum levels were independently associated with poor cancer-specific survival. IMP3 gene and protein expressions were not correlated with those of IGF2. In conclusion, we found enhanced IMP3 levels in tissue and serum samples of PC patients compared to non-PC men. Moreover, IMP3 was associated with metastasis and PC-specific survival. The tumor promoting effect of IMP3 appears to be independent from its regulatory role on IGF2 in PC.

Prospective randomized double-blind multicentre phase II study comparing gemcitabine and cisplatin plus sorafenib chemotherapy with gemcitabine and cisplatin plus placebo in locally advanced and/or metastasized urothelial cancer: SUSE (AUO-AB 31/05).

OBJECTIVE: To evaluate the efficacy and safety of gemcitabine and cisplatin in combination with sorafenib, a tyrosine-kinase inhibitor, compared with chemotherapy alone as first-line treatment in advanced urothelial cancer. PATIENTS AND METHODS: The study was a randomized phase II trial. Its primary aim was to show an improvement in progression-free survival (PFS) of 4.5 months by adding sorafenib to conventional chemotherapy. Secondary objectives were objective response rate (ORR), overall survival (OS) and toxicity. The patients included in the trial had histologically confirmed locally advanced and/or metastatic urothelial cancer of the bladder or upper urinary tract. Chemotherapy with gemcitabine (1250 mg/qm on days 1 and 8) and cisplatin (70 mg/qm on day 1) repeated every 21 days, was administered to all patients in a double-blind randomization of additional sorafenib (400 mg twice daily) vs placebo (two tablets twice daily) on days 3-21. Treatment continued until progression or unacceptable toxicity, the maximum number of cycles was limited to eight. The response assessment was repeated after every two cycles. RESULTS: Between October 2006 and October 2010, 98 of 132 planned patients were recruited. Nine patients were ineligible. The final analysis included 40 patients in the sorafenib and 49 patients in the placebo arm. There were no significant differences between the two arms concerning ORR (sorafenib: complete response [CR] 12.5%, partial response [PR] 40%; placebo: CR 12%, PR 35%), median PFS (sorafenib: 6.3 months, placebo: 6.1 months) or OS (sorafenib: 11.3 months, placebo: 10.6 months). Toxicity was moderately higher in the sorafenib arm. Diarrrhoea occurred significantly more often in the sorafenib arm and hand-foot syndrome occurred only in the sorafenib arm. The study was closed prematurely because of slow recruitment. CONCLUSION: Although the addition of sorafenib to standard chemotherapy showed acceptable toxicity, the trial failed to show a 4.5 months improvement in PFS.

Validation of a 3D-printed robot-assisted partial nephrectomy training model.

Objectives: Most renal tumours can be treated with a partial nephrectomy, with robot-assisted partial nephrectomy becoming the new gold standard. This procedure is challenging to learn in a live setting, especially the enucleation and renorraphy phases. In this study, we attempted to evaluate face, content, and preliminary construct validity of a 3D-printed silicone renal tumour model in robotic training for robot-assisted partial nephrectomy. Materials and Methods: We compared the operative results of three groups of surgeons with different experience levels (>20 partial nephrectomies, 1-20 partial nephrectomies and no experience at all) performing a robotic tumour excision of a newly developed silicone model with four embedded 3D-printed renal tumours. We evaluated the participants' performance using surgical margins, excision time, total preserved parenchyma, tumour injury and GEARS score (as assessed by two blinded experts) for construct validity. Postoperatively, the participants were asked to complete a survey to evaluate the usefulness, realism and difficulty of the model as a training and/or evaluation model. NASA-TLX scores were used to evaluate the operative workload. Results: Thirty-six participants were recruited, each group consisting of 10-14 participants. The operative performance was significantly better in the expert group as compared to the beginner group. NASA-TLX scores proved the model to be of an acceptable difficulty level.Expert group survey results showed an average score of 6.3/10 on realism of the model, 8.2/10 on the usefulness as training model and 6.9/10 score on the usefulness as an evaluation tool. GEARS scores showed a non-significant tendency to improve between trials, emphasizing its potential as a training model. Conclusion: Face and content validity of our 3D renal tumour model were demonstrated. The vast majority of participants found the model realistic and useful for training and for evaluation. To evaluate construct and predictive validity, we require further research, aiming to compare the results of 3D-model trained surgeons with those of untrained surgeons in real-life surgery.

Serum endostatin levels correlate with enhanced extracellular matrix degradation and poor patients' prognosis in bladder cancer.

Endostatin, the proteolytic fragment of collagen XVIII, is an inhibitor of angiogenesis and tumor growth. Interestingly, elevated circulating endostatin levels have been found to correlate with poor patients' prognosis in several cancers. The aim of this study was to assess the prognostic value of endostatin in bladder cancer (BC) and to gain insight into the mechanisms involved in its production. This retrospective study included a total of 337 patients with BC and 103 controls. Collagen XVIII gene expression was analyzed using real-time PCR (n = 82). Endostatin tissue localization was assessed by immunohistochemistry (n = 27). Endostatin serum (n = 87) and urine (n = 153) levels were determined by ELISA. In 12 cases, both serum and paraffinized tissue samples from the same patients were available. We found decreased collagen XVIII tissue expression and increased endostatin urine and serum concentration in samples of patients with BC compared to controls. High serum endostatin levels correlated with the presence of lymph node metastases and MMP-7 concentrations and were independently associated with poor metastasis-free and disease-specific survival. Immunohistochemical analysis revealed a strong endostatin staining in the wall of tumor associated blood vessels in superficial but not in muscle-invasive BCs. Based on these, we concluded that elevated endostatin levels in patients with BC are the consequence of enhanced extracellular matrix degradation and are independent from collagen XVIII expression. Furthermore, serum endostatin levels may provide prognostic information independent from histopathological parameters and may therefore help to optimize therapy decisions. Loss of endostatin expression in tumor associated blood vessels might represent an important step supporting tumor-induced angiogenesis.

High insulin-like growth factor mRNA-binding protein 3 (IMP3) protein expression is associated with poor survival in muscle-invasive bladder cancer.

UNLABELLED: What's known on the subject? and What does the study add? Insulin-like growth factor mRNA-binding protein 3 (IMP3) is an oncofetal protein found to be re-expressed in a series of human cancers including bladder cancer. In vitro analyses showed an invasion and proliferation promoting effect for IMP3. Further in vitro studies suggested that IMP3 is able to bind to the mRNAs of CD44 and insulin-like growth factor 2 (IGF2), enhancing their stability and expression. However, this molecular interaction has not yet been analysed in tumour samples. In the present study, we identified for the first time high IMP3 tissue protein expression as an independent predictor of poor patients' survival in muscle-invasive bladder cancer. Furthermore, there was no correlation between IMP3 and its molecular targets in bladder carcinoma specimens and concluded that the tumour-promoting effect of IMP3 is not related to its regulatory action on IGF2 and CD44. OBJECTIVE: To assess the prognostic value and molecular actions of the oncofetal protein insulin-like growth factor mRNA-binding protein 3 (IMP3) in muscle-invasive bladder cancer (BC). PATIENTS AND METHODS: IMP3 expression was analysed by immunohistochemistry, real-time polymerase chain reaction and Western blot analysis in 224 patients with BC. The molecular targets of IMP3; CD44, insulin-like growth factor 2 (IGF2) and its receptor the IGF1 receptor (IGF1-R) were also investigated. Expression levels were correlated with clinical follow-up data by using both univariate and multivariate Cox regression analyses. RESULTS: IMP3 mRNA and protein levels were significantly elevated in high-stage and high-grade muscle-invasive BC. In muscle-invasive BC IMP3 protein but not gene expression proved to be an independent predictor of disease-specific (hazard ratio [HR] 2.58, 95% confidence interval [CI] 1.28-4.56, P = 0.004) and overall survival (HR 2.07, 95% CI 1.12-3.82, P = 0.020). The expression levels of IGF2 and CD44 showed no correlation with that of IMP3. CONCLUSIONS: High IMP3 protein levels may identify patients with BC at high risk of disease progression and may therefore select patients for a more intensive therapy or for a strict follow-up. Its high expression in high-grade bladder carcinoma cells makes IMP3 for an attractive target for therapy. The tumour promoting effect of IMP3 is independent from its regulatory action on IGF2 and CD44 expression.

Chronic Kidney Disease and Pregnancy. Guideline of the DGGG, OEGGG, DGfN (S2k Level, AWMF Registry No. 015 - 090).

Aim As diagnostic and therapeutic options have improved in recent years, women with limited renal function of varying etiologies are now able to become pregnant. Depending on the extent of disease and the patients' comorbidities, the care of these women can be especially challenging. This guideline aims to improve the interdisciplinary care offered to pregnant women with kidney disease. Methods A selective literature search was carried out. This S2k guideline was then compiled using a structured consensus-based process which included representatives from different medical specialties and professional societies. Recommendations Recommendations for the care of pregnant women with renal disease were developed to cover preconception counseling, the recording of risks, special aspects of prenatal care and prenatal screening, as well as the specific treatment options for the underlying disease in women wanting to have children and pregnant women.

Elevated serum matrix metalloproteinase 7 levels predict poor prognosis after radical prostatectomy.

Elevated matrix metalloproteinase-7 (MMP-7) tissue expression and serum concentration have been shown to be associated with cancer progression and metastasis. The aim of our study was to assess the prognostic value of preoperative circulating MMP-7 levels in serum samples of patients with clinically localized prostate cancer. Furthermore, we compared the serum MMP-7 levels between patients with organ confined and metastatic prostate cancer. MMP-7 levels were measured in 93 patients with localized prostate cancer, 13 patients with distant bone metastasis and in sera of 19 controls using enzyme-linked immunosorbent assay. The results were compared to the clinical follow-up data. We did not find any significant difference in MMP-7 serum levels between patients and controls (p = 0.268). Circulating MMP-7 serum concentration was significantly elevated in patients with distant metastasis (p < 0.001). For the detection of distant prostate cancer metastasis, using a cut-off value of 3.7 ng/ml, a specificity of 69% and a sensitivity of 92% were observed. Multivariate analysis identified high MMP-7 serum concentration as an independent risk factor for prostate cancer-related death both in a preoperative and a postoperative model (p = 0.003 and 0.018, respectively). Furthermore, the evaluation of predictive models revealed that addition of serum MMP-7 levels to the preoperatively available predictors improves prognostic accuracy (the concordance index increased from 0.631 to 0.734 when MMP-7 was included). Based on these, we concluded that MMP-7 is a potential marker to identify patients with metastatic prostate cancer. In clinically localized prostate cancer, MMP-7 may provide independent prognostic information, thereby helping to optimize therapy decisions.

Urinary matrix metalloproteinase-7 level is associated with the presence of metastasis in bladder cancer.

OBJECTIVE: • To assess the presence of matrix metalloproteinase (MMP)-7 in urine samples of patients with bladder cancer and to investigate the correlation between MMP-7 urine concentration and clinicopathological variables. PATIENTS AND METHODS: • The presence of MMP-7 in the urine of patients with bladder cancer was tested in 32 representative cases using immunoprecipitation followed by western blot analysis. • Urinary MMP-7 concentration levels were analyzed in 132 patients with bladder cancer and 96 controls using an enzyme-linked immunosorbent assay. RESULTS: • MMP-7 levels did not differ significantly between patients with localized bladder cancer and controls (P= 0.174). On the other hand, we detected a fourfold, significantly elevated MMP-7 concentration in urine samples of patients with bladder cancer with regional or distant metastasis (P= 0.003). • Using a threshold value of 6.88 ng/ml, determined by receiver-operating characteristic curve analysis, a specificity of 82% and a sensitivity of 78% were observed. • Western blot analysis revealed that the 55-kDa tissue inhibitor of metalloproteinase 1 complexed MMP-7 is the dominant form of urinary matrilysin. CONCLUSIONS: • MMP-7 is present in detectable amounts in the urine of patients with bladder cancer. Its concentrations are significantly elevated in patients with metastatic disease. • Determination of urinary matrilysin level could help to detect bladder cancer metastasis, and may therefore provide a more reliable prognosis and influence therapy decisions.

Correlation of pathological and cytological-cytometric grading of transitional cell carcinoma of the urinary tract.

INTRODUCTION: Cystoscopy and cytology are standard procedures for diagnosis and follow-up of patients with bladder cancer. Urinary cytodiagnosis is a descriptive method for tumor characterization. We correlated histopathologic diagnosis of noninvasive urothelial carcinomas with cytological evaluation and, furthermore, we validated cytology by cytometric analysis. PATIENTS AND METHODS: 94 patients with a history of bladder cancer were included in the study. 25 patients were negative for tumors, 22 showed pTa G1 carcinomas, 25 had pTaG2 and 22 patients had G3 carcinomas. All patients underwent cytological and cytometric evaluation. Nuclear diameter and circumference were measured for 15 representative nuclei per specimen. Statistical evaluation was performed using Graph Pad Software. RESULTS: Cytology showed excellent tumor detection for G2 and G3 carcinomas, with a sensitivity of 100% combined with a specificity of 100%. Using cytometry, we can significantly distinguish between unsuspicious patients and G1 carcinomas on the one hand and high-grade carcinomas on the other. Furthermore, in 6 of 25 patients (24%) with noninvasive G2 carcinomas, but G3 cytological evaluation, tumor progression occurred. CONCLUSIONS: Urinary cytology is an excellent instrument for detection of clinically relevant high-grade bladder cancer. Descriptive alterations of the cytopathology can be validated by objective data using cytometric analysis.

Inhibition of Rho-kinase abrogates migration of human transitional cell carcinoma cells: results of an in vitro study.

INTRODUCTION: Migration of cells involves a complex signaling network. The aim of the present study was to elucidate the impact of Rho-kinase (ROK) on G protein-coupled receptor-induced migration of human transitional cell carcinoma cells in an in vitro experimental setting. MATERIALS AND METHODS: Intracellular calcium concentration ([Ca(2+)](i)) was measured with the indicator dye Fura-2 in response to lysophosphatidic acid, thrombin and sphingosine-1-phosphate. Phospholipase C activity was determined in myo-[(3)H]inositol- (0.5 µCi/ml) labeled cells. Migration was performed using a Boyden chamber. Transient transfection of a dominant-negative mutant of ROK was done with calcium phosphate. For staining of actin filaments, tetramethylrhodamine isothiocyanate-conjugated phalloidin was used. RESULTS: Lysophosphatidic acid, thrombin and sphingosine-1-phosphate cause increases in [Ca(2+)](i), cellular responses being accompanied by an enhancement of phospholipase C activity and sensitive to the G(i) inhibitor pertussis toxin. Agonists potently stimulated migration of T24 and J82 cells. Inhibition of Rho proteins by Clostridium difficile toxin B abrogated cell migration. Inhibition of ROK using HA1077 and Y-27632 mimicked the properties of toxin B. Expression of a ROK mutant drastically reduced migration. CONCLUSIONS: G protein-coupled receptors potently stimulated cell migration in T24 and J82 cells. Rho proteins and ROK play a pivotal role in this signaling cascade. Rho and ROK may be putative targets for new therapy options in bladder cancer.

Matrix metalloproteinase-7 as a marker of metastasis and predictor of poor survival in bladder cancer.

Matrix metalloproteinases (MMPs) play an important role in tumor progression and metastasis. Here, we investigated the prognostic relevance of MMP-7 in urinary bladder cancer. MMP-7 gene expression was measured in tissue samples of 101 patients using quantitative real-time PCR. Circulating MMP-7 serum levels of 98 individuals (79 patients and 19 controls) were analyzed by enzyme-linked immunosorbent assay. The results were compared with the clinical follow-up data, performing Kaplan-Meier log-rank test as well as univariate and multivariate Cox analysis. In representative cases, immunohistochemical analysis for MMP-7 was performed. We detected significantly elevated MMP-7 levels both in tissue and serum samples of patients with metastatic disease (P = 0.001 and P = 0.002). Multivariate analysis revealed that high MMP-7 tissue expression and serum concentration are stage- and grade-independent predictors of both metastasis-free (hazard ratio [HR] = 3.80, 95% confidence interval [CI], 1.29-11.23, P = 0.016, and HR = 2.53, 95% CI, 1.01-6.37, P = 0.048) and disease-specific survival (HR = 1.89, 95% CI, 1.00-3.55, P = 0.050 and HR = 1.95, 95% CI, 1.03-3.71, P = 0.041). Based on these findings, we conclude that MMP-7 is a promising marker to detect present and to predict future metastasis. Serum MMP-7 analysis provides information about the risk of metastasis before surgery which could help to optimize therapeutic procedures. Furthermore, high MMP-7 tissue and/or serum levels could identify patients most likely to benefit from early adjuvant chemotherapy.

Angiogenic switch of angiopietins-Tie2 system and its prognostic value in bladder cancer.

PURPOSE: Vascular endothelial growth factor (VEGF), angiopoietins (Ang-1 and Ang-2), and their receptor Tie2 are critically involved in both normal and pathologic angiogenesis. The aim of this study was to explore the role of Ang-1, Ang-2, VEGF, and Tie2 in the development and progression of bladder cancer as well as to examine their prognostic value in this tumor type. EXPERIMENTAL DESIGN: Tumor samples of 113 bladder cancer patients, normal bladder epithelium of 5 noncancer patients, and two low-grade (UMUC3 and RT4) and two high-grade (J82 and T24) bladder cancer cell lines were analyzed by quantitative real-time PCR. The expression data were analyzed performing Wilcoxon rank-sum and Kaplan-Meier log-rank tests as well as univariate Cox analyses and Cox proportional hazards regression model. RESULTS: In tissues of noninvasive bladder tumors, Ang-1 expression was significantly lower (P < 0.001), whereas VEGF expression was significantly higher (P = 0.031) than in normal bladder tissue. These findings were also confirmed at the protein level by immunohistochemistry. In contrast, Tie2 and Ang-2 abundance in tumor did not differ significantly from that in normal bladder tissue. Multivariate analysis identified Ang-2 as a strong and independent predictor of tumor recurrence [hazard ratio (HR), 10.18; 95% confidence interval (95% CI), 2.69-38.49; P < 0.001] and Tie2 expression as an independent favorable prognostic factor for both metastasis (HR, 0.31; 95% CI, 0.11-0.89; P = 0.029) and disease-specific survival (HR, 0.25; 95% CI, 0.10-0.62; P = 0.003). CONCLUSIONS: These data show the strongest change in expression of VEGF and Ang-1 in superficial bladder cancer in comparison with normal bladder epithelium and the invasive tumor stages. The prognostic significance of Ang-2 and Tie2 underlines the essential role of angiopoietins-Tie2 system in progression of bladder cancer.

Rap2B-dependent stimulation of phospholipase C-epsilon by epidermal growth factor receptor mediated by c-Src phosphorylation of RasGRP3.

Receptor tyrosine kinase regulation of phospholipase C-epsilon (PLC-epsilon), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors. PLC and Ca(2+) signaling by the EGF receptor, which activated both PLC-gamma1 and PLC-epsilon, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B. EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-epsilon, and Rap2B-dependent translocation of PLC-epsilon to the plasma membrane. GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca(2+) and expression of lipase-inactive PLC-gamma1 but not of PLC-epsilon. Expression of RasGRP3, a Ca(2+)/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca(2+) signaling by the EGF receptor. EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation. Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-gamma1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-epsilon.

Validation of the diagnostic and prognostic relevance of serum MMP-7 levels in renal cell cancer by using a novel automated fluorescent immunoassay method.

PURPOSE: Despite encouraging results in other cancers, in renal cell cancer, no consensus exists regarding the diagnostic and prognostic relevance of MMP-7. The aim of this study was to assess the diagnostic and prognostic potential of serum MMP-7 levels in renal cell cancer. Furthermore, parallel to the widely used ELISA method, we tested a new, fluid-phase, fluorescent immunoassay (B.R.A.H.M.S KRYPTOR®) for the quantitation of MMP-7. METHODS: We analyzed the serum samples of 174 individuals (77 patients and 97 age-matched healthy controls) by a commercially available sandwich ELISA and by a novel, automated, fluid-phase immunofluorescent assay (B.R.A.H.M.S KRYPTOR®). Results were correlated with the clinicopathological and follow-up data. RESULTS: MMP-7 concentrations showed a high concordance level (R (2) = 0.979) between the two methods (p < 0.001). Serum MMP-7 concentrations were significantly higher in patients compared to controls. At a cutoff value of 3.15 ng/ml, a specificity and a sensitivity of 70 and 82 % for the detection of RCC was found. Patients with metastasis had significantly higher MMP-7 levels as those without metastasis (p = 0.038 by KRYPTOR, p = 0.011 by ELISA). High MMP-7 levels proved to be independently associated with shorter overall, disease-specific and metastasis-free survival, regardless of the analytical method. CONCLUSIONS: Based on these results, serum MMP-7 levels have both diagnostic and prognostic potential. The KRYPTOR method provided comparable results to the standard ELISA analysis, with a high concordance level and can therefore be considered as a surrogate method. Its flexibility and automated operation make the KRYPTOR MMP-7 assay suitable for routine laboratory use in the daily practice.