Juergen Serth

Correlation of bFGF expression in renal cell cancer with clinical and histopathological features by tissue microarray analysis and measurement of serum levels

The prognostic value of bFGF for surgically treated renal cell cancer (RCC) patients was evaluated by immunohistochemistry (IHC) and the tissue microarray technique (TMA). Additionally, preoperative serum bFGF levels were correlated to tumour stage and the presence of metastases at initial diagnosis. Serum levels of bFGF were measured by ELISA in 39 healthy volunteers, in 37 patients with benign urologic diseases and in 74 RCC patients, 26 of whom revealed lymph node or distant metastases. bFGF expression as detected by IHC was investigated in 777 tissue cores from 259 different RCC patients [median follow-up: 138 (36–240) months]. Eighty eight patients died from tumour progression. For each patient, the TMA slides contained a tissue core from the primary tumour, its invasion front and the normal renal parenchyma. bFGF serum levels were higher in RCC patients vs healthy volunteers (P<0.01) and vs patients with benign urologic diseases (P<0.01). Metastasized patients revealed higher bFGF serum levels than organ-confined specimens (P<0.01). As detected by IHC only increased bFGF expression in the invasion front tissue correlated with the patients’ long-term survival (log rank test) (P=0.03). In multivariate analysis regional LN metastases (P<0.01), the histological grading (P<0.01), and an increased bFGF expression in the invasion front (P=0.04) independently predicted the patients’ clinical prognosis. Not the expression of bFGF in the primary tumour but in its invasion front reflects the aggressiveness of RCC, hereby indicating a different biological potential within both areas. The value of bFGF serum levels as indicators of systemic tumour dissemination remains to be determined.

Galectin-1 and Galectin-3 mRNA expression in renal cell carcinoma

BackgroundGalectins are known to regulate cell differentiation and growth as well as cell adhesion and apoptosis. Galectins have been discussed as possible prognosticators for survival in renal cell cancer (RCC) and other urological tumors. They might also play an emerging role as possible new marker-proteins for RCC. In this study, we analyzed the expression of galectin-1 and galectin-3 mRNA in order to further investigate their clinical significance in RCC.MethodsTissue samples were obtained from 106 patients undergoing surgery for RCC. The expression of galectin-1 and galectin-3 mRNA in normal kidney and corresponding cancer tissue was analyzed using quantitative real time PCR. Differences in expression levels of paired tissue samples were assessed using paired two-sample tests. Associations of relative mRNA expression levels in tumor tissues with clinical findings were analyzed using univariate logistic regression.ResultsThe expression of galectin-1 (p < 0.001) and -3 (p < 0.001) mRNA were significantly higher in RCC when compared to the adjacent normal kidney tissue. For clear cell RCC, an association of male gender with higher galectin-1 and galectin-3 mRNA expression (p = 0.054, p = 0.034) was detected. For all RCCs, galectin-1 mRNA expression failed to show a significant association with advanced disease as well as a higher rate of lymph node metastases (p = 0.058, p = 0.059).ConclusionThe mRNA expression of galectin-1 and galectin-3 is significantly increased in RCC cancer tissue. The higher mRNA expression in tumor tissue of male patients raises the question of a functional connection between galectins and the higher prevalence of RCC in men. Associations with advanced disease might lead to new ways of identifying patients at higher risk of recurrent disease and might even facilitate early metastasectomy with curative intent.

GATA5 CpG island hypermethylation is an independent predictor for poor clinical outcome in renal cell carcinoma

Transcriptional inactivation and CpG island (CGI) methylation of GATA transcription factor family members GATA3 and GATA5 have been reported for a few types of human cancer. Whether high-density CGI methylation of GATA3 or GATA5 is associated with the clinical course of patients with renal cell cancer (RCC) has not been clarified. Quantitative methylation-specific PCR assays were carried out to analyze 25 tumor cell lines including 6 RCC lines and 119 RCC and 87 adjacent normal tissues for the presence of densely methylated sequences. Methylation values were statistically compared with clinicopathological and recurrence-free survival (RFS) data for patients. Comparison of GATA3 and GATA5 methylation in different tumor cell lines revealed a marker-specific methylation characteristic with high and frequent signals for both methylation marks in RCC lines. GATA3 and GATA5 CGI relative methylation levels were found to be strongly associated with the state of metastasis (P=0.003 and P<0.001, respectively) and advanced disease (P=0.024 and P<0.001, respectively). Moreover, an independent decrease in RFS in Cox proportional hazard analysis was found for tumors exhibiting high GATA5 methylation (P<0.001, hazard ratio, 19.3; 95% confidence interval, 4.58–81.6). Epigenetic alterations in GATA family members may be associated with aggressive tumor phenotypes in RCC, and in the case of GATA5, may serve as a new independent molecular marker for aggressiveness and disease progression.

Heterogeneous p27Kip1 Expression within Primary Renal Cell Cancers, Their Invasive Margins and Peritumoral Renal Parenchyma Correlation with Pathological and Prognostic Features

Introduction: The expression of the negative cell cycle regulator p27Kip1 is frequently found to be deregulated in various human cancer types. Whether expression of p27Kip1 can be used as prognostically relevant biological variables for renal cell cancer patients still remains to be clarified. Therefore, in the present investigation the expression within different tissue areas obtained from renal cell carcinomas was determined. Patients and Methods: For analysis of p27Kip1 in 420 tumor nephrectomy specimens obtained from 420 consecutively included patients, tissue microarrays were used comprising of 1,260 tissue samples each obtained from the tumor itself, the invasive front as well as non-malignant surrounding parenchyma. A sufficient follow-up after surgical therapy was available in 251 cases. Results: In univariate survival analysis, decreased expression of p27Kip1 within tissue cores obtained from the invasion front was significantly correlated with the patients’ disease-specific long-term survival (p = 0.02, log-rank test). In contrast, expression of p27Kip1 protein within the primary tumors was not identified to reveal any prognostically important information. In Cox regression analysis, histological stage and grade (p < 0.01), the presence of regional lymph node (p < 0.01) or distant metastases at the time of surgery (p < 0.01) as well as decreased expression of p27Kip1 (p = 0.04) within the invasion front tissue samples independently predicted the disease-specific long-term survival following surgery. Conclusion: Our analysis demonstrated that p27Kip1 is heterogeneously expressed in renal cell carcinomas. Moreover, the result of the present study supports the prognostic value of p27Kip1 protein expression for patients diagnosed with renal cell carcinoma.

Rho GDP dissociation inhibitor beta ARHGDIB in renal cell cancer.

474 Background: Rho GDP dissociation inhibitor 2 (ARHGDIB) is an important mediator of cellular signaling. The expression of ARHGDIB correlates with tumor growth and metastasis in a variety of non genitourinary cancers, however the role of ARHGDIB in renal cell cancer (RCC) has not yet been evaluated. Methods: Tissue samples from 106 patients undergoing surgery for RCC were obtained. The expression of ARHGDIB mRNA in normal kidney tissue and in corresponding cancer tissue was analyzed by means of quantitative real time PCR. Differences in mRNA expression levels were assessed using paired two-sample tests. Associations of relative mRNA expression levels and clinicopathological parameters were statistically analyzed using an univariate logistic regression model. Relative mRNA expression levels in healthy renal tissue compared to cancerous tissue from the same kidney was assessed using a paired t-test. Results: When comparing 74 tissues from kidney tumors with adjacent histologically normal appearing paired ...

Association of reduced GATA1 mRNA expression with clinicopathology and shortened recurrence-free survival in clear cell renal cell carcinoma.

521 Background: GATA1, a zinc-finger transcription factor and member of the GATA family proteins 1-6, is known to be involved in cell growth and apoptosis, especially in the erythroid lineage. Recent studies demonstrated that GATA1 interacts with p53 and its overexpression leads to an inhibition of the p53 gene function. Increased p53 expression levels have been shown to be associated with prognosis and tumor progression in renal cell carcinoma. Methods: Quantitative real-time reverse-transcribed polymerase chain reaction was applied to measure relative GATA1 mRNA expression levels in 135 kidney tissue samples, including 77 clear cell RCC (ccRCC) tissues and 58 paired adjacent normal renal tissue samples. Relative GATA1 expression levels were determined using the ΔΔCt method. Results: The mean GATA1 expression levels were significantly decreased in tumor tissues compared to adjacent normal tissues (p < 0.001, paired t-test). In univariate logistic regression analysis decreased GATA1 mRNA expression was as...

DNA hypermethylation biomarkers to predict therapy response of antiangiogenic therapy in metastatic renal cell carcinoma patients.

459 Background: Antiangiogenic therapy has demonstrated to increase progression-free (PFS) as well as overall survival (OS) in RCC patients. Clinical scoring systems like the Memorial Sloan Kettering Cancer Center (MSKCC) help to stratify patients for treatment options. However, identification of molecular phenotypes of RCC patients would improve stratification of specific targeted therapies. We investigated whether DNA hypermethylation based markers can predict the PFS following first-line therapy. Methods: We examined primary tumors from formalin-fixed paraffin embedded tissue specimens obtained from 18 patients receiving anti-VEGF based targeted therapy. Quantitative methylation-specific PCRs were carried out following bisulphite conversion of DNA for CpG island methylation analysis of the five candidate genes, CST6, GATA5, LAD1, hsa-miR124-3 and hsa-miR9.1 identified in advance as potential prognosticators for RCC. For Kaplan Meier survival analysis relative methylation values were uniformly dichotomi...

CpG island hypermethylation of corticotropin-releasing hormone-binding protein in kidney cancer and association with clinicopathological parameters.

414 Background: Significance of Urocortin (Ucn or UcnI), Ucn2, Ucn3, and their receptors, Corticotropin Releasing Factor Receptor 1 and 2 (CRFR1 and CRFR2), and the binding protein, Corticotropin-Releasing Hormone-Binding Protein (CRHBP) in oncology is growing rapidly. Recently we found that the UCN system may also be a relevant structure in renal cell cancer. Here we investigate whether CRHBP CGI methylation occurs in cc-RCC and whether its methylation is associated with clinicopathological parameters of patients. METHODS Tumoral tissues of 109 patients with renal cell cancer and their corresponding normal tissues have been used. Combined bisulfite restriction analysis (COBRA) for detection of relative degree of CpG island (CGI) methylation and pyrosequencing have been performed. RESULTS We found an approximately five-fold increase in mean methylation of the CRHBP CGI for tumor tissues (p < 0.00005). Higher DNA methylation of the CRHBP CGI showed a positive correlation with advanced disease as well (p = 0.024). CONCLUSIONS To our knowledge we report for the first time the CGI methylation of CRHBP in clear cell renal cell carcinoma indicating a contribution of epigenetic alteration of CRHBP to RCC tumorigenesis. Moreover, our results suggest CRHBP as a potential molecular marker for assessment of progression and aggressiveness of tumors.

CpG-island methylation of GATA-family members GATA3 and GATA5 in renal cell carcinoma and association with clinicopathological parameters and progression-free survival.

370 Background: Transcriptional inactivation and CGI methylation of GATA3 and -5 has been reported to be involved in mammary carcinoma, pancreatic cancer, colorectal and gastric carcinogenesis. A recent study demonstrated that a loss of GATA-3 expression due to partially methylation silencing in several renal cell carcinoma (RCC) patients. We quantitatively investigated GATA3 and -5 CGI methylation in RCC and analyzed its association with clinical characteristics as well as progression free survival of patients. METHODS Methylation data were obtained from a quantitative methylation-specific polymerase chain reaction assay (QMSP) for both genes. We investigated 108 RCC and 77 paired tissue samples as well as six RCC cell lines. Statistical analyses were carried out using the paired t-test for matched tumor (TU) and adjacent normal (adN) samples, logistic regression for comparisons of independent samples and cox regression for survival analysis. RESULTS In paired samples we found a significant higher methylation in TU compared to adN for GATA3 (P=0.007) and for GATA5 (P=3.6*10-9) for all RCCs. GATA5 showed also strong correlations between methylation and status of metastasis (P=0.05) and advanced (pT≥3 and/or N+, M+) tumor samples compared to localized (pT≤2, N0, M0) tumors (P=4.7*10-9). A decreased progression free survival in cox proportional hazard model analysis could be demonstrated for patients with a high GATA5 methylation (P=0.0006, HR=6.5) and a trend could also be seen for GATA3 methylated patients (P=0.06). CONCLUSIONS GATA3 and -5 were identified to demonstrate tumor-specific CGI hypermethylation in renal cell cancer patients. The association of GATA5 CGI methylation with metastasis, advanced disease and progression free survival of patients indicates that epigenetic alterations of both genes are involved in renal cell carcinogenesis. GATA5 methylation could serve as a biomarker for tumor progression. Further prospective and functional investigations are necessary to clarify whether CGI methylation of GATA family members can provide independent information for future clinical management of patients with RCC.

Molecular characterization of temsirolimus-induced response in human renal and bladder cancer cell lines.

295 Background: Targeted therapies like mTOR inhibition is a clinically esthablished treatment modality for advanced renal cell cancer (RCC). We hypothesize that common elements of molecular signalling exists in RCC and transitional cell carcinoma of the bladder (TCC) that could provide a rational of the usage of this novel compound in human TCC. Therefore the goal of this investigation was to measure the in vivo and in vitro effect of temsirolimus/CCI-779 on human RCC and TCC cell lines on the molecular level. METHODS For in vivo experiments 3 RCC (786-O, A498, ACHN) cell lines and 7 TCC (T24, 5637, RT112, EJ-28, CLS-439, HB-CLS-1, HB-CLS-2) cell lines were compared. Effect of temsirolimus/CCI-779 was measured by real time impedance analysis (XCelligence, Roche). Following mRNA isolation microarray based mRNA expression analysis with 45.015 oligoprobes (G4112F, Agilent Technologies) was performed for molecular comparison of RCC and TCC cell lines. Expression patterns of 15 pathways were analyzed using the statistical software R (2.12.0) and the LIMMA package. RESULTS RCC and TCC cell lines demonstrated dose dependent inhibition of cellular growth with IC50 values of 10-20nM of temsirolimus/CCI-779 as measured by quantitative real time impedance analysis. Furthermore six out of 15 pathways including the mTOR and VEGF signalling were found with similar expression patterns following treatment with CCI-779 in both tumor entities. CONCLUSIONS In vivo and in vitro analysis of temsirolimus mTOR inhitibtion on human bladder cancer cell lines support the hypothesis that a common molecular architectur exists in both tumor entities suggesting inhibition of mTOR in TCC as a possible target for further experimental therapeutic studies.