In renal cell carcinoma (RCC), single members of the Wnt/β-catenin signaling cascade were recently identified to contribute to cancer progression. However, the role of Wnt1, one of the key ligands in β-catenin regulation, is currently unknown in RCC. Therefore, alterations of the Wnt1/β-catenin axis in clear cell RCC (ccRCC) were examined with regard to clinicopathology, overall survival (OS) and cancer specific survival (CSS). Corresponding ccRCCs and benign renal tissue were analyzed in 278 patients for Wnt1 and β-catenin expression by immunohistochemistry in tissue microarrays. Expression scores, including intensity and percentage of stained cells, were compared between normal kidney and ccRCCs. Data was categorized according to mean expression scores and correlated to tumor and patients’ characteristics. Survival was analyzed according to the Kaplan-Meier and log-rank test. Univariable and multivariable Cox proportional hazard regression models were used to explore the independent prognostic value of Wnt1 and β-catenin. In ccRCCs, high Wnt1 was associated with increased tumor diameter, stage and vascular invasion (p ≤ 0.02). High membranous β-catenin was associated with advanced stage, vascular invasion and tumor necrosis (p ≤ 0.01). Higher diameter, stage, node involvement, grade, vascular invasion and sarcomatoid differentiation (p ≤ 0.01) were found in patients with high cytoplasmic β-catenin. Patients with a high cytoplasmic β-catenin had a significantly reduced OS (hazard ratio (HR) 1.75) and CSS (HR 2.26), which was not independently associated with OS and CSS after adjustment in the multivariable model. Increased ccRCC aggressiveness was reflected by an altered Wnt1/β-catenin signaling. Cytoplasmic β-catenin was identified as the most promising candidate associated with unfavorable clinicopathology and impaired survival. Nevertheless, the shift of membranous β-catenin to the cytoplasm with a subsequently increased nuclear expression, as shown for other malignancies, could not be demonstrated to be present in ccRCC.

What's known on the subject? and What does the study add? The loss of p27Kip1 correlates with poor prognosis in various human cancers, and was postulated as a biomarker in RCC. Up to now p27Kip1 analysis in RCC was focused on its nuclear localization. We confirmed higher p27Kip1 expression in the nucleus and cytoplasm of RCC and correlated high cytoplasmic p27Kip1 with an unfavourable clinic and a reduced survival. OBJECTIVES To analyse the cytoplasmic and nuclear differences of p27Kip1 expression and localization in benign and clear cell renal cell carcinoma (ccRCC) with regard to overall survival (OS) and cancer‐specific survival (CSS). p27Kip1 is considered to contribute to the progression of ccRCC and is targeted by next generation dual‐therapies. PATIENTS AND METHODS In 140 patients, ccRCC and corresponding benign kidney tissue were analyzed for nuclear and cytoplasmic staining of p27Kip1 by immunohistochemistry using a tissue microarray technique. Staining intensity and percentage of positive stained cells are given as expression scores. p27Kip1 expression was categorized as high if ccRCC tissue stained stronger than the respective level of the corresponding benign tissue and categorized as low if ccRCC tissue stained less than or equal to the corresponding benign tissue. Differences in OS and CSS were analyzed by log‐rank analysis and expression levels were correlated with tumour and patient characteristics using Fisher's exact test. RESULTS Cytoplasmatic (mean [sd]: 13.8% [1.2%] vs 10.7% [1.7%]; P= 0.04) and nuclear (mean [sd]: 75.6% [2.7%] vs 13.6% [2.1%]; P < 0.001) staining of p27Kip1 were significantly stronger in ccRCC tissues compared to benign tissue. High cytoplasmic p27Kip1 expression was significantly associated with a higher T and N stage, Fuhrman grade and the presence of metastatic disease (P < 0.001). The median follow‐up time was 38.2 months. There was no difference in OS between the low and high expression groups, although CSS was significantly lower in patients with high cytoplasmic p27Kip1 (P < 0.001) and CSS heavily tended to be lower in the nuclear low expression group (P= 0.069). CONCLUSIONS High cytoplasmic p27Kip1 levels in renal cancer tissues are associated with advanced disease and reduced cancer specific survival, whereas low nuclear expression levels appear to be beneficial. The present study corroborates the consideration of cytoplasmic p27Kip1 for future diagnostic and targeted therapeutic approaches in RCC establishing a potential protective shift of p27Kip1 from the cytoplasm to the nucleus.

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INTRODUCTION AND OBJECTIVES: Although it is well known that deregulation of VHL-HIF1 pathway is responsible for the carcinogenesis of clear cell renal cell carcinoma (ccRCC), molecular mechanisms responsible for the development of metastasis are still poorly investigated. The first aim of this study is to identify copy number alteration (CNA) that is specifically detected in metastasis but not in primary lesion. The second aim is to identify a gene whose deregulation is responsible for the development of metastasis.METHODS: For the array comparative genomic hybridization (CGH) analysis, genomic DNAs were extracted from the formalin-fixed, paraffin-embedded tissues. Twenty cases of primary ccRCCs and their corresponding metastases were analyzed using 4x44K oligonucleotide CGH array (Agilent Inc.). For the gene expression analysis, total RNA was extracted from frozen samples of 30 primary ccRCCs. Expression levels of the 58 genes were analyzed by qRT-PCR. To investigate the biologic function of candidate genes, RCC cell lines, 786-O and 769-P were used. Cell proliferation, invasiveness and apoptosis were analyzed before and after the infection of lenti-virus encoding candidate genes to the RCC cell lines.RESULTS: By comparing CNAs of primary ccRCCs with their corresponding metastases, we found that gene copy number loss at 9p24.1-p13.3 was more frequently found in metastases than in primary lesions. Out of 58 genes located at 9p24.1-p13.3, we identified 2 genes, NDUFB6 and LRRC19, both of which were down-regulated due to copy number loss. Next, using lenti-virus encoding each gene, we introduced the two genes into 786-O and 769-P cells in which both the 2 genes were down-regulated. We found that, when NDUFB6 was overexpressed in both cell lines, cell proliferation was significantly suppressed (figure shown below), although apoptotic activity and invasion activity were unchanged.CONCLUSIONS: Down-regulation of NDUFB6 caused by gene copy number loss at 9p24.1-p13.3 may bring about growth advantage to metastatic ccRCC cells. We propose that NDUFB6 is a new candidate for a tumor suppressor and that its down-regulation may play an important role in the development of metastasis of ccRCC.

465 Background: Members of the Wnt/ß-catenin signaling pathway are abnormally expressed in renal cell carcinoma (RCC). This study analyzed the role of Wnt1/ß-catenin alterations in clear cell RCC (ccRCC) with regard to clinicopathology, overall survival (OS) and cancer specific survival (CSS) to prove the options of a Wnt targeted therapy. Methods: Corresponding ccRCCs and benign renal tissue were analyzed in 278 patients for Wnt1 and ß-catenin by immunohistochemistry in tissue microarrays. Expression scores including intensity and percentage of stained cells were compared between normal kidney and ccRCCs. Data was categorized according to the mean expressions core and correlated to tumor and patients’ characteristics and analyzed for OS and CSS according to the Kaplan-Meier and log-rank test. To identify prognostics value of wnt1 and ß-catenin, univariable and multivariable Cox proportional hazard regression models were used. Results: High Wnt1 tumor expression was associated with increased tumor diameter (p=0.01), stage (p=0.004) and vascular invasion (p=0.02). Positive membranous ß-catenin was associated with advanced stage (p=0.003), vascular invasion (p=0.01) and tumor necrosis (p=0.004). Increased tumor diameter (p=0.01), stage (p=0.003), node involvement (0.04), grade (p=0.001), vascular invasion (p=0.002), and sarcomatoid differentiation (p=0.01) were found in patients with high cytoplasmic ß-catenin. Furthermore, this patient subgroup showed reduced OS (p=0.03) and CSS (p=0.009). Conclusions: Significant clinicopathological associations underline the oncogenic potential of Wnt1/ß-catenin pathway parameters. Whereas, cytoplasmic ß-catenin was identified as the most valuable parameter with regard to clinico-prognostic implications for future targeted therapies.