Tibor Szarvas

Matrix metalloproteinases and their clinical relevance in urinary bladder cancer.

Matrix metalloproteinases (MMPs) have important roles in several cancer-supporting cellular processes, such as extracellular matrix (ECM) remodeling, angiogenesis, apoptosis, epithelial-to-mesenchymal transition and cell proliferation. This broad range of activity has led to considerable interest in the use of MMPs in the clinical setting as diagnostic or prognostic biomarkers and as therapeutic targets. Levels of the different MMPs can be measured in several sample types, including paraffin-embedded or fresh frozen tissue, serum, plasma and urine, and by various analytical methodologies, such as immunohistochemistry, real-time PCR, western and northern blot analyses, enzyme-linked immunosorbent assay and zymography. Several MMPs have been identified as having potential diagnostic or prognostic utility, whether alone or in combination with currently available diagnostic tests or imaging modalities. Although the early broad-spectrum anti-MMP agents showed a lack of efficacy, our continually improving understanding of the complex physiologic and pathologic roles of MMPs might enable the development of new MMP-specific and tumor-specific therapies. Accordingly, MMPs will continue to be the subjects of intensive research 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.

Detection of Bladder Cancer from the Urine using Fluorescence in situ Hybridization Technique

The authors report on their first experiences with the UroVysion fluorescencein situ hybridization (FISH) kit developed for the detection of bladder cancer. This new non-invasive diagnostic application of the FISH technique in the field of urology was elaborated to replace cystoscopy. The special urine examination method detects genetic alterations of the urothelial cells found in the urine, using fluorescent directlabeled DNA probes binding to the peri-centromeric regions of chromosomes 3, 7 and 17 as well as on the 9p21 locus. We aimed to evaluate the utility of UroVysion test in the light of the histological diagnosis. Urine samples from 43 bladder cancer patients and 12 patients with no or benign alterations were studied using a new application of FISH technique: the UroVysion reagent kit. The obtained FISH results were compared with the histological findings of the transurethral surgical resection specimens. The study rated the specificity and sensitivity of the technique 100% and 87%, respectively. Therefore, the technique could well fit into the diagnostic process of bladder carcinomas. Statistical analyses showed significant correlation between tumor progression and the severity of the genetic alterations detected by this FISH technique. Furthermore, positive correlation was found between tumor grade and the proportion of tumor cells showing genetic abnormality. The noninvasiveness, the robustness of evaluation and the high specificity/sensitivity are all in favor of this technique. The disadvantages are the higher costs of the technical background and the required future clinical studies to determine whether this technique can replace cystoscopy.

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.

Serum Levels of Angiogenic Factors and their Prognostic Relevance in Bladder Cancer

Angiogenesis plays a critical role in tumor growth. VEGF, angiopoietins (Ang-1, Ang-2) and their tyrosine kinase receptor Tie2 are major regulators of angiogenesis. The aim of this study was to evaluate the prognostic value of the serum levels of these factors in bladder cancer. We analyzed the serum samples of 117 bladder cancer patients and 64 healthy volunteers by enzyme linked immunosorbent assay (ELISA) for Ang-1, Ang-2, VEGF and the extracellular domain of Tie2. The statistical evaluation of the obtained data was performed via Kaplan–Meier log-rank test, univariate Cox analyses as well as Cox proportional hazards regression model. Serum Ang-1 levels of bladder cancer patients were significantly higher (p < 0.001), while soluble Ang-2 and Tie2 levels were significantly lower (p = 0.016 and p = 0.001 respectively) in patients than those in controls. Cox univariate analysis revealed high sTie2 serum level as a risk factor for metastasis and as a borderline significant risk factor for disease related death (p = 0.022 and p = 0.081 respectively). These correlations were independent from tumor stage and grade in a Cox multivariate model (p = 0.016 and p = 0.069). These data indicate that the serum levels of analyzed angiogenic factors do change characteristically in bladder cancer. The soluble extracellular serum level of Tie2 may provide a stage and grade independent diagnostic tool to select a high risk group of bladder cancer patients.

The long noncoding RNA HOTAIR has tissue and cell type-dependent effects on HOX gene expression and phenotype of urothelial cancer cells

BackgroundUrothelial carcinoma (UC) is the fifth most common cancer in the developed world. Delineation of differentiation subtypes in UC highlighted the importance of aberrant differentiation. Understanding underlying mechanisms may facilitate diagnosis and development of efficient therapy strategies. It is well accepted that epigenetic mechanisms are involved. Long noncoding RNAs (lncRNAs), a new class of epigenetic factors, are thought to mediate molecular differences between cell types to control cellular identity. The present study focuses on the lncRNA HOTAIR, originating from the HOXC locus. Its overexpression induces an aggressive phenotype in many cancers and aberrant expression of homeotic HOX transcription factors, especially HOXD10, that regulate differentiation and tissue homeostasis. The aim of the present study was to determine the functional role of HOTAIR in UC with regard to aggressive phenotype, regulation of aberrant differentiation and altered HOX gene expression.MethodsWe determined RNA expression levels of HOTAIR and HOX genes in UC tissues and cell lines. Knockdown of HOTAIR and ectopic overexpression was performed to determine the effect on reported target genes in UC. Cell lines were stably transfected with HOTAIR to investigate changes in phenotype and HOX gene expression.ResultsHOTAIR was overexpressed in approximately half of UC tissues and cell lines. Effects of HOTAIR overexpression differed between cell lines. Whereas VM-CUB1 cells acquired the expected phenotype with increased proliferation, clonogenicity, anchorage independent growth, migratory activity and epithelial-to-mesenchymal transition, 5637 cells grew more slowly displaying induction of senescence and related immune response genes. Other UC lines showed intermediate effects. Expression profiling revealed divergent effects on HOX genes, cell cycle regulators and differentiation according with the phenotypic differences between HOTAIR-overexpressing VM-CUB1 and 5637 cells.ConclusionsOur data indicate that HOTAIR overexpression may affect differentiation state and aggressiveness of UC cells, but in a cell-type dependent manner. Our functional studies and the comparison of our expression data sets with those from other cancer cell types, which revealed minimal overlaps, indicate that effects of HOTAIR are strongly tissue-dependent and can even differ within one cancer type. Thus, HOTAIR functions and target genes cannot simply be transferred from one cancer type to the other.

Validation of Circulating MMP-7 Level as an Independent Prognostic Marker of Poor Survival in Urinary Bladder Cancer

Molecular marker analyses aiming a more accurate disease characterization and risk stratification of cancer patients provided several promising marker candidates in the last few years. However, recent reviews underlined the paramount importance of validation, since many of the initially promising results could not be confirmed in independent patient cohorts. If serum or plasma is a more appropriate sample to test for prognostic markers is a matter of debate. We recently found serum MMP-7 levels to correlate with poor patients’ prognosis in urinary bladder cancer. In this study, we examined associations of the MMP-7 plasma levels with clinical follow-up data in an independent cohort of bladder cancer patients to validate our former results and to assess if plasma is also suitable for MMP-7 analysis. Plasma levels of 97 patients and 22 controls were analyzed, using enzyme-linked immunosorbent assay. Associations between MMP-7 plasma concentrations and clinical data were assessed applying both univariate and multivariate analysis. Plasma MMP-7 levels were significantly higher in patients than in controls. Similarly to our former findings in sera, high MMP-7 plasma levels proved to be significant and independent predictors of both overall and disease-specific survival. In addition, we observed a metastasis-specific difference in MMP-7 levels between serum and plasma. In summary, we confirmed the prognostic relevance of circulating MMP-7 levels in an independent cohort of patients and concluded that circulating MMP-7 levels may help to identify bladder cancer patients at high-risk of disease progression who could benefit from an adjuvant chemotherapy or from an extended lymph node dissection.

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

Whats known on the subject? and What does the study add?

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

Study Type – Therapy (case series)
Level of Evidence 4