Background: Fibronectin 1 (FN1) is a glycoprotein involved in cellular adhesion and migration processes. The aim of this study was to elucidate the role of FN1 in development of renal cell cancer (RCC) and to determine a prognostic relevance for optimal clinical management. Methods: 212 renal tissue samples (109 RCC, 86 corresponding tissues from adjacent normal renal tissue and 17 oncocytomas) were collected from patients undergoing surgery for renal tumors and subjected to total RNA extraction. Detection of FN1 mRNA expression was performed using quantitative real time PCR, three endogenous controls, renal proximal tubular epithelial cells (RPTEC) as biological control and the ΔΔCt method for calculation of relative quantities. Results: Mean tissue specific FN1 mRNA expression was found to be increased approximately seven fold comparing RCC and corresponding kidney control tissues (p < 0.001; ANOVA). Furthermore, tissue specific mean FN1 expression was increased approx. 11 fold in clear cell compared to papillary RCC (p = 9×10^-5; Wilcoxon rank sum test). Patients with advanced disease had higher FN1 expression when compared to organ-confined disease (p < 0.001; Wilcoxon rank sum test). Applying subgroup analysis we found a significantly higher FN1 mRNA expression between organ-confined and advanced disease in the papillary and not in the clear cell RCC group (p = 0.02 vs. p = 0.2; Wilcoxon rank sum test). There was an increased expression in RCC compared to oncocytoma (p = 0.016; ANOVA). Conclusions: To our knowledge, this is the first study to show that FN1 mRNA expression is higher in RCC compared to normal renal tissue. FN1 mRNA expression might serve as a marker for RCC aggressiveness, indicating early systemic progression particularly for patients with papillary RCC.

Whether methylation of the microRNA (mir)-124-3 CpG island is of relevance for the clinical course of a solid cancer and whether it shows association with clinicopathology or survival of patients with renal cell cancer (RCC) is not known as yet. In a cross-sectional study, relative methylation of mir-124-3 was measured in 111 RCC samples and 77 paired normal appearing tissues using quantitative methyl-specific PCR. Results were statistically compared with tumour histology, clinicopathological parameters and disease recurrence. We found tumour-specific hypermethylation of mir-124-3 in samples of RCCs with clear cell histology (ccRCC) compared with paired normal appearing tissues (P<0.0001). Methylation was significantly increased in tumours with state of advanced disease (P<0.0001). Higher relative methylation was associated with worse recurrence-free survival in both univariate (hazard ratio=9.37; P=0.0005) as well as bivariate Cox regression analyses considering age, sex, diameter of tumours and state of advanced disease, metastasis and lymph node metastases as covariates (hazard ratios=5.9–18.2; P-values of 0.0003–0.008). We identified mir-124-3 CpG islands (CGI) methylation as a relevant epigenetic mark for ccRCC thus underlining the need for functional studies of potentially affected signalling pathways in kidney tumour models. Methylation of mir-124-3 is suggested as an independent prognosticator for ccRCC.

Background: Epigenetic silencing of the RAS association domain family 1A (RASSF1A) tumor suppressor gene promoter has been demonstrated in renal cell carcinoma (RCC) as a result of promoter hypermethylation. Contradictory results have been reported forRASSF1Amethylation in normal kidney, thus it is not clear whether a significant difference betweenRASSF1Amethylation in normal and tumor cells of the kidney exists. Moreover, RASSF1A expression has not been characterized in tumors or normal tissue as yet.Results: Using combined bisulfite restriction analysis (COBRA) we compared RASSF1A methylation in 90 paired tissue samples obtained from primary kidney tumors and corresponding normal tissue. Bisulfite sequence analysis was carried out using both pooled amplicons from the tumor and normal tissue groups and subclones obtained from a single tissue pair. Expression of RASSF1A was analyzed by the use of tissue arrays and immunohistochemistry. We found significantly increased methylation in tumor samples (mean methylation, 20%) compared to corresponding normal tissues (mean methylation, 11%;P< 0.001). Densely methylated sequences were found both in pooled and individual sequences of normal tissue. Immunohistochemical analysis revealed a significant reduced expression of RASSF1A in most of the tumor samples. Heterogeneous expression patterns of RASSF1A were detected in a subgroup of histologically normal tubular epithelia.Conclusion: Our methylation and expression data support the hypothesis thatRASSF1Ais involved in early tumorigenesis of renal cell carcinoma.

VEGF-targeted therapy increases both the progression-free (PFS) and overall survival (OS) of patients with metastasized renal cell cancer (mRCC). Identification of molecular phenotypes of RCC could improve risk-stratification and the prediction of the clinical disease course. We investigated whether gene-specific DNA hypermethylation can predict PFS and OS among patients undergoing anti-VEGF-based therapy. Primary tumor tissues from 18 patients receiving targeted therapy were examined retrospectively using quantitative methylation-specific PCR analysis of CST6, LAD1, hsa-miR-124-3, and hsa-miR-9-1 CpG islands. PFS and OS were analyzed for first-line and sequential antiangiogenic therapies using the log rank statistics. Sensitivity and specificity were determined for predicting first-line therapy failure. Hypermethylation of CST6 and LAD1 was associated with both a shortened PFS (log rank p = 0.009 and p = 0.004) and OS (p = 0.011 and p = 0.043). The median PFS observed for the high and low methylation groups of CST6 and LAD1 was 2.0 vs.11.4 months. LAD1 methylation had a specificity of 1.0 (95% CI 0.65–1.0) and a sensitivity of 0.73 (95% CI 0.43–0.90) for the prediction of first-line therapy. CST6 and LAD1 methylation are candidate epigenetic biomarkers showing unprecedented association with PFS and OS as well as specificity for the prediction of the response to therapy. DNA methylation markers should be considered for the prospective evaluation of larger patient cohorts in future studies.

Caveolae play a significant role in disease phenotypes such as cancer, diabetes, bladder dysfunction, and muscular dystrophy. The aim of this study was to elucidate the caveolin-1 ( CAV1) protein expression in renal cell cancer (RCC) and to determine its potential prognostic relevance. 289 clear cell RCC tissue specimens were collected from patients undergoing surgery for renal tumors. Both cytoplasmic and membranous CAV1 expression were determined by immunohistochemistry and correlated with clinical variables. Survival analysis was carried out for 169 evaluable patients with a median follow up of 80.5 months (interquartile range (IQR), 24.5 - 131.7 months). A high CAV1 expression in the tumor cell cytoplasm was significantly associated with male sex (p = 0.04), a positive nodal status (p = 0.04), and poor tumor differentiation (p = 0.04). In contrast, a higher than average (i.e. > median) CAV1 expression in tumor cell membranes was only linked to male sex (p = 0.03). Kaplan-Meier analysis disclosed significant differences in 5-year overall (51.4 vs. 75.2%, p = 0.001) and tumor specific survival (55.3 vs. 80.1%, p = 0.001) for patients with higher and lower than average cytoplasmic CAV1 expression levels, respectively. Applying multivariable Cox regression analysis a high CAV1 protein expression level in the tumor cell cytoplasm could be identified as an independent poor prognostic marker of both overall (p = 0.02) and tumor specific survival (p = 0.03) in clear cell RCC patients. Over expression of caveolin-1 in the tumour cell cytoplasm predicts a poor prognosis of patients with clear cell RCC. CAV1 is likely to be a useful prognostic marker and may play an important role in tumour progression. Therefore, our data encourage further investigations to enlighten the role of CAV1 and its function as diagnostic and prognostic marker in serum and/or urine of RCC patients.