Expression In Renal Cell Carcinoma Research Articles

CD40 expression in renal cell carcinoma is associated with tumor apoptosis, CD8+ T cell frequency and patient survival

The co-stimulatory molecule, CD40, is expressed in renal cell carcinoma (RCC) and a variety of inflammatory diseases in the kidney. We investigated the relationship between tumor-associated CD40 expression, immune milieu of the tumor microenvironment, tumor stage and survival of patients with RCC. The expression of CD40, TUNEL and CD8 in human renal cell carcinomas was analyzed by immunohistochemistry performed on tissue samples obtained at the time of surgery. Computer-assisted quantitation of protein expression was used to analyze results in connection with patient survival and tumor stage. We show for the first time that tumor-associated CD40 expression is associated with prolonged survival in RCC patients. Tumor apoptosis (TUNEL) and CD8 immunostaining were also associated with patient survival. No relation was observed between CD40 expression and tumor stage. Our results suggest CD40 may be a prognostic biomarker indicative of prolonged RCC patient survival. Strategies that up-regulate CD40 expression in some RCC patients may thus improve survival, supporting further studies of agonistic CD40 antibodies in RCC.

P0150 Survivin expression in renal cell carcinoma and its correlation with clinicopathological parameters

Background The aim of the current study is to cast further light on the issues related to prognostication of renal cell carcinoma (RCC), assessing the expression of survivin in a subset of primary RCC and determining its relation to different clinicopathological features and disease-free survival. Methods The present series consisted of tissue samples obtained from 37 Libyan patients with stage I, II, III, or IV RCC. Survivin expression in these tumours was assessed by immunohistochemistry using an automated staining system. Different grading systems were tested for expression of survivin. Findings Expression of survivin was significantly associated with venous invasion (tumour thrombus; p = 0.042), larger tumour size ( p = 0.051), higher primary T classification ( p = 0.013), advanced tumour stage ( p = 0.033), and borderline associated ( p = 0.068) with tumour location. In univariate Kaplan–Meier survival analysis, surviving expression showed a borderline association ( p = 0.089) with disease-free survival. However, there was no significant correlation between survivin expression and gender, age, histological grade, distance metastasis, lymph node involvement, perinephric fat and capsular invasion, status at end point and recurrence. Interpretation Survivin expression in RCC may identify patients at risk of a more aggressive disease and a worse prognosis. Further investigations, on a larger and more heterogeneous population, should be carried out to validate and extend our results.

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.

Down-Regulation of UNC5D in Bladder Cancer: UNC5D as a Possible Mediator of Cisplatin Induced Apoptosis in Bladder Cancer Cells

Identifying potential targets would improve therapeutic planning anddisease management. Therefore, we investigated whether the novel identified dependence receptor UNC5D acts as a tumor suppressor in bladder malignancies. We assessed the UNC5D level in a panel of 15 primary bladder carcinomas and 6 cell lines using real-time reverse transcriptase-polymerase chain reaction and Western blot. MTT assay, TUNEL staining, colony formation assay and Western blot were done in cells untransfected and transfected with UNC5D vector, siUNC5D or siDAPK. UNC5D was dramatically down-regulated in bladder cancer tissue samples and malignant cell lines. Restoration of UNC5D expression in bladder cancer cells lacking endogenous UNC5D expression suppressed cell proliferation and survival. Cisplatin treatment significantly induced UNC5D expression and DAPK dephosphorylation while UNC5D knockdown decreased bladder cancer cell sensitivity to cisplatin. DAPK silencing significantly inhibited the effect of UNC5D on apoptosis induced by cisplatin. Our study suggests that UNC5D may have important roles as a novel suppressor in bladder cancer via the UNC5D/DAPK pathway.

Discrimination between Von Hippel-Lindau gene and hypoxia-regulated alterations in the metabolism and protein expression in renal cell carcinoma using ome-based strategies.

447 Background: The objective of this project is to dissect the von Hippel-Lindau- and hypoxia-regulated alterations in human renal cell carcinoma to define both novel prognostic markers as well as therapeutic targets. Methods: For this, the VHL-negative RCC cell line 786-O next to recently established gain of function VHL transfectants thereof were used as a model system and subsequently cultured under normoxic or hypoxic conditions. Whereas direct effects on the altered energy metabolism were determined by lactate, pyruvate and pH measurements. the resulting effects at the gene/protein expression profiles were determined by comparative cDNA micro array analysis/ 2DE-based proteome analyses, respectively. Some of the differentially expressed genes/proteins were verified by qPCR and/or Western blot analysis in a panel of RCC cell lines. Results: By employing the VHL-/VHL+RCC model system VHL-dependent, HIF-dependent as well as VHL/HIF-independent alterations in the gene and protein expression pattern were found. Some of the differentially expressed genes/proteins were either associated with the loss of VHL function, the hypoxic conditions or both. The genes/proteins defined as differentially expressed under these conditions are mainly involved in the cellular metabolism and predominantly localized in the cytoplasm or mitochondrion. The verification of the differentially expressed genes/proteins by qPCR and Western blot analysis revealed a heterogeneous expression in RCC cells and lesions. One such gene/protein is represented by the glutamine gamma glutamyl transferase (TGM2). Its expression was associated with the loss of VHL and induction of hypoxia. Furthermore, TGM2 might be an important marker for both VHL-independent and/or hypoxia-induced responses. Conclusions: The data demonstrate that VHL functionality and/or hypoxia induce distinct effects on the metabolic switch of RCC.

Programmed cell death protein 4 expression in renal cell carcinoma, penile carcinoma and testicular germ cell cancer

Programmed cell death protein 4 expression in renal cell carcinoma, penile carcinoma and testicular germ cell cancer

Impact of Gender in Renal Cell Carcinoma: The Relationship of FABP7 and BRN2 Expression with Overall Survival

OBJECTIVETo investigate the relationship between gender differences in fatty acid-binding protein7 (FABP7) and BRN2 (POU class 3 homeobox 2) expression in renal cell carcinoma (RCC) and the prognosis of patients with RCC.MATERIALS AND METHODSimmunohistochemical (IHC) staining as well as reverse transcription-polymerase chain reaction (RT-PCR) was performed in renal tissues from 103 patients (83 men, mean age = 63.6 years old; 20 women, mean age = 63.1 years old) underwent radical nephrectomy from January 1, 2001 through December 31, 2010. The probability of overall patient survival was estimated using the Kaplan-Meier method.RESULTSFABP7 mRNA expression was more frequent in men (P = 0.07) while BRN2 protein expression was significantly more frequent in women (P = 0.029). In particular, FABP7 was expressed in 100% of G1 renal cell carcinoma both in mRNA and protein levels. In women, FABP7 (−) and BRN2 (+) groups had a worse prognosis both in mRNA level (P = 0.038) and protein level (P = 0.058). BRN2 was expressed 100% of papillary RCC both in mRNA and protein levels.CONCLUSIONSOur results demonstrated that gender was a key factor in FABP7 and BRN2 expression in RCC, and the combination with FABP7 and BRN2 stratified by gender could be a new potential prognostic factor in patients with RCC.

Proliferation inhibition and the underlying molecular mechanisms of microRNA-30d in renal carcinoma cells

To investigate the inhibitory effects of microRNA-30d (miR-30d) on renal carcinoma cell proliferation and the underlying molecular mechanisms, miR-30d expression in renal cell carcinoma (RCC) specimens was analyzed by quantitative polymerase chain reaction (qPCR). The inhibition of the proliferation of miR-30d on renal carcinoma cells (ACHN cell line) was analyzed by MTT and colony formation assays. The effects of miR-30d on cyclin E2 expression were detected by the luciferase activity of the reporter gene. In addition, the effects of miR-30d on endogenous cyclin E2 expression at the RNA and protein levels were investigated by qPCR and western blot analysis, respectively. Cell cycles were analyzed by flow cytometry. The results showed the following: i) Expression of miR-30d was significantly downregulated in renal carcinoma tissues compared with paraneoplastic tissues; ii) overexpression of miR-30d inhibited renal carcinoma cell proliferation and colony formation; iii) miR-30d inhibited cyclin E2 3′ untranslated region-mediated reporter gene expression; and iv) overexpression of miR-30d downregulated endogenous cyclin E2 expression and blocked the cell cycle at the G1 phase. In conclusion, miR-30d functions as a tumor suppressor gene in RCC and inhibits renal carcinoma cell proliferation. Cell cycle regulatory factor cyclin E2 is a target gene of miR-30d. miR-30d inhibits renal carcinoma cell proliferation via the regulation of cyclin E2 expression at the post-transcriptional level.

Prognostic significance of claudin-1 expression in renal cell carcinoma

Purpose Renal cell carcinoma (RCC) is the most common renal cancer in adults comprising ∼3% of all human malignancies. Claudin-1 is a tight junction protein widely expressed in liver and kidney tissue. The role of claudins in cancer has not been clearly defined. We aimed to assess the prognostic significance of claudin-1 expression in RCC. Patients and methods Immunohistochemical staining for 51 RCCs was performed, and the results were correlated with clinical, histopathologic, and prognostic parameters. Results Claudin-1 was expressed in 52.9% of RCC cases. Only 46.7% of conventional RCCs were positive for claudin-1, whereas 68.75% of papillary RCCs showed positive claudin-1 expression. Claudin-1 expression was associated with markers of unfavorable tumor biology in conventional and papillary RCC. Claudin-1 positivity was a prognosticator of shortened patient survival in conventional and papillary RCC (P=0.06 and 0.007, respectively). Conclusion Upregulation of claudin-1 was associated with dedifferentiation, aggressiveness, and shortened patient survival in RCC.

Differential Expression of SDF-1 Isoforms in Bladder Cancer

SDF-1 is a ligand of the chemokine receptors CXCR4 and 7. The 6 known SDF-1 isoforms are generated by alternative mRNA splicing. While SDF-1 expression has been detected in various malignancies, only few groups have reported differential expression of SDF-1 isoforms and its clinical significance. We evaluated the expression of 3 SDF-1 isoforms (α, β and γ) in bladder cancer. Using quantitative polymerase chain reaction we measured SDF-1α, β and γ mRNA levels in 25 normal and 44 bladder cancer tissues, and in 210 urine specimens (28 normal, 74 benign, 57 bladder cancer, 35 bladder cancer history, 8 other cancer history and 8 other cancer) from consecutive patients. Levels were correlated with clinical outcome. Of the SDF-1 isoforms only SDF-1β mRNA was significantly over expressed 2.5-fold to sixfold in bladder cancer compared to normal bladder tissues. SDF-1α was expressed in bladder tissues but SDF-1γ was undetectable. On multivariate analysis SDF-1β was an independent predictor of metastasis and disease specific mortality (p=0.017 and 0.043, respectively). In exfoliated urothelial cells only SDF-1β mRNA levels were differentially expressed with 91.2% sensitivity and 73.8% specificity for detecting bladder cancer. In patients with a bladder cancer history increased SDF-1β levels indicated a 4.3-fold increased risk of recurrence within 6 months (p=0.0001). SDF-1 isoforms are differentially expressed in bladder tissues and exfoliated urothelial cells. SDF-1β mRNA levels in bladder cancer tissues predict a poor prognosis. Furthermore, SDF-1β mRNA levels in exfoliated cells detect bladder cancer with high sensitivity and they are a potential predictor of future recurrence.

CD40 expression in renal cell carcinoma correlates with tumor apoptosis, CD8 T cell frequency and patient survival

Meeting abstracts To investigate the relationship between tumor-associated CD40 expression, RCC patient survival and tumor stage. The expression of CD40, TUNEL and CD8 in human renal cell carcinomas was analyzed by immunohistochemistry. Computer-assisted quantitation of protein expression was used

Myoglobin expression in renal cell carcinoma is regulated by hypoxia

Myoglobin is a member of the hemoprotein superfamily, which additionally includes hemoglobin, neuroglobin and cytoglobin. Cytoplasmic localized myoglobin functions as a radical scavenger and prevents hypoxia. Besides muscle tissue MB expression could also be observed in other tissues as well as in different types of cancer.For the correlation between the expression of myoglobin, hypoxia-inducible-factor-1α, and capillary density tissue of 86 different renal cell carcinomas were immunohistochemically stained with myoglobin-specific and hypoxia-inducible-factor-1α-specific antibodies as well as with CD31 antibody. Four different renal carcinoma cell lines were cultivated under hypoxic conditions and the expression of myoglobin and hypoxia-inducible-factor-1α was evaluated by real-time PCR and Western blot.Renal cell carcinoma including clear cell, papillary, and chromophobe subtypes expressed myoglobin with an inverse relationship to capillary density being highly significant for clear cell renal cell carcinoma. For hypoxia-inducible-factor-1α a significant correlation with capillary density could also be observed in clear cell RCC. In renal cell carcinoma cell lines hypoxia induced a significant increase of myoglobin expression up to 62 fold, whereas hypoxia-inducible-factor-1α only increased up to 5 fold. The PCR results of myoglobin expression could be confirmed by Western blot.Myoglobin seems to be a sensitive marker for hypovascularized tumor entities especially during the early phase of hypoxia. Such neoplasias may benefit from an antiangiogenic therapy.

Differential microRNA expression in renal cell carcinoma

The present study aimed to detect microRNA expression levels in the tissues and sera of patients with clear cell renal cell carcinoma (ccRCC). The association of microRNA expression with ccRCC clinical pathology was analyzed, and the potential of the microRNAs as ccRCC serum markers and the significance of their expression in the clinical diagnosis, staging, prognosis and selection of new therapeutic targets for ccRCC were discussed. Specific microRNAs were selected according to the associated literature. TaqMan quantitative polymerase chain reaction (qPCR) technology was used to determine the expression levels of selected microRNAs. miR-34a, miR-224 and miR-21 were upregulated, whereas miR-141, miR-149 and miR-429 were downregulated in the ccRCC tissues (P<0.01). The expression of miR-221 and miR-211 was not significant in the ccRCC tissues (P>0.05). miR-34a, miR-21 and miR-224 were upregulated and miR-141 was downregulated in the sera of patients with ccRCC (P<0.01), while the expression of miR-149 and miR-429 was not significant (P>0.05). The serum miR-21 expression levels were significantly correlated with the clinical staging of the patients with ccRCC (P<0.05). miR-34a, miR-21 and miR-224 are upregulated in the tissues and sera of patients with ccRCC, whereas miR-141 is downregulated. miR-21 and miR-141 are associated with ccRCC and are, thus, potential ccRCC serum markers.

The Tumor-Suppressive Function ofUNC5Dand Its Repressed Expression in Renal Cell Carcinoma

As a newly added member of the UNC5H receptors, the function of UNC5D/H4 in tumorigenesis remains poorly defined. The aim of this study was to examine the expression of UNC5D in primary renal cell carcinomas (RCC), analyze the mechanisms responsible for its downregulation in RCC, and assess its functional relevance to tumor growth and migration. Forty-four paired primary RCCs and corresponding adjacent noncancerous tissues were collected. The mRNA and protein expression level of UNC5D was assessed by reverse transcriptase-PCR, real-time PCR, or immunohistochemistry. Epigenetic alterations in UNC5D promoter and LOH in the UNC5D locus were also analyzed. Ectopic expression of UNC5D in renal cancer cells with silenced expression of UNC5D was used for analysis of the biologic functions of UNC5D. UNC5D expression was attenuated in multiple carcinoma cell lines including renal cancer cells. Similar reduction was also observed in primary RCC tissues as compared with paired adjacent noncancerous tissues. Methylation-specific PCR showed hypermethylation in UNC5D promoter in a significant proportion (18 of 44) of tumor tissue (40.9%). LOH of UNC5D was observed in 13 of 44 patients with RCCs (29.5%). Restoration of UNC5D expression in renal cancer cells significantly inhibited cell proliferation, anchorage-dependent and -independent growth, as well as migration and invasion, whereas knockdown of UNC5D promoted cell growth. Furthermore, ectopic expression of UNC5D induced G2-M cell-cycle arrest. UNC5D is a functional tumor suppressor that is frequently downregulated in RCCs due to promoter hypermethylation and LOH.

Epigenetic regulation of microRNA expression in renal cell carcinoma

The underlying mechanisms of microRNA deregulation in cancer cells include epigenetic modifications, which play a crucial role in carcinogenesis. We demonstrate that numerous microRNAs are induced in renal cell carcinoma cell lines after treatment with inhibitors of the DNA-methyltransferase (5-aza-2′-deoxycytidine) and the histone-deacetylase (suberoylanilide hydroxamic acid). We provide evidence that enrichment of H3 and H3K18 acetylation at the miR-9 promoter is causative for re-expression, while DNA hypermethylation remains unchanged. Our experiments show that the treatment with the epigenetic drugs causes re-expression of silenced microRNAs with putative tumor suppressive function in ccRCC cell lines.

Feline sarcoma‐related protein expression correlates with malignant aggressiveness and poor prognosis in renal cell carcinoma

Feline sarcoma-related protein (Fer) is a ubiquitously expressed non-receptor protein tyrosine kinase associated with proliferation in various cancer cells. However, no reports have described the pathological roles and prognostic value of Fer expression in renal cell carcinoma (RCC). We investigated Fer expression in three RCC cell lines (ACHN, Caki-1, and Caki-2) and in normal tubule cells (HK-2) by immunoblotting. Fer expression was highest in ACHN cells, with Caki-1 showing intermediate levels and Caki-2 showing low levels, and was undetectable in HK-2. RNA interference was therefore used to assess the effects of Fer knockdown in ACHN. Knockdown of Fer expression was found to inhibit RCC cell proliferation and colony formation. Immunohistochemical analysis of 131 human RCC tissues (110 conventional, 11 chromophobe, and 10 papillary) investigated relationships between Fer expression and clinicopathological features, including cancer cell proliferation, apoptosis, and prognostic value for survival. In human tissues, Fer expression was significantly higher in cancer cells than in normal tubules. In addition, expression levels correlated with cancer cell proliferation, but not with apoptosis. Multivariate analysis indicated associations of Fer expression with pT stage, tumor grade, and metastasis (P < 0.001). Fer expression was also prognostic for cause-specific survival according to multivariate analysis (hazard ratio, 3.89; 95% confidence interval, 1.02-14.84, P = 0.047). Fer expression correlates with RCC cell proliferation both in vitro and in vivo, and with tumor progression and survival. This represents useful information for discussing the pathological and clinical significance of Fer in RCC.

Abstract 5080: Inhibiting the ALK1/BMP9 signaling pathway with dalantercept as an antiangiogenic therapy.

Abstract Activin receptor-like kinase 1 (ALK1) is an endothelial cell-specific receptor for bone morphogenetic proteins (BMP) 9 and 10, and is a key regulator of angiogenesis and vascular morphogenesis. Preclinical data suggest that the BMP9/ALK1 pathway is operating in the resolution and maturation stage of angiogenesis, which is distinct from the VEGF/VEGFR pathway which drives the initiation stage of angiogenesis. Dalantercept is an ALK1 extracellular domain-Fc fusion protein that inhibits the BMP9/ALK1 pathway. Thus, dalantercept may prove useful either as a monotherapy to inhibit tumor angiogenesis or in combination with VEGFR inhibitors to target different phases of vascular network formation and potentially to overcome acquired resistance to anti-VEGF therapy. To test this hypothesis, the A498 renal cell carcinoma xenograft model was used to test dalantercept efficacy either alone or in combination with sunitinib. In this model, tumor progression was defined as a 2 mm increase in tumor length. In the placebo group, mean (± SD) tumor progression was 5.8 ±0.6 days. Treatment with dalantercept delayed tumor progression to 10.8 ± 1.7 days (p&amp;lt;0.01 vs placebo) and sunitinib delayed tumor progression to 13.8 ± 2.5 days (p&amp;lt;0.01 vs placebo). The combination treatment of dalantercept plus sunitinib had a greater beneficial effect with a delay in tumor progression to 35 ± 12.6 days (p=0.01 vs sunitinib). We used immunohistochemistry (IHC) analysis to evaluate effects of dalantercept on tumor vasculature and to look for potential biomarkers of response to treatment in A498 tumors. Id1 is a transcription factor expressed in endothelial cells and is known to be regulated via the BMP9/ALK1/SMAD pathway. We observed a decrease in Id1 protein expression in endothelial cells of A498 xenografts treated with dalantercept, consistent with our previous experiments. Because BMP9 is one of the main target ligands of dalantercept, IHC analysis of archived human tumor samples was performed to determine BMP9 expression levels in various tumor types. In squamous cell carcinoma of the head and neck it was shown that out of 28 tumor samples 79% had high or medium levels of BMP9 staining, with little to no staining in normal tissues. Data from BMP9 expression in renal cell carcinoma and hepatocellular carcinoma will also be presented. In a Phase 1 study in patients with advanced, refractory solid tumors, dalantercept was generally well tolerated and exhibited signs of clinical activity including patients with objective response and prolonged stable disease. Dalantercept is currently being tested in several Phase 2 oncology studies, including a study of dalantercept in combination with the anti-VEGFR TKI axitinib in second-line advanced renal cell carcinoma. Citation Format: Marat Alimzhanov, Nicolas Solban, Michael Lee, Phaethon Philbrook, Xiaoen Wang, Lin Wei, Jiaxi Song, Sabina Signoretti, R. Scott Pearsall, Matthew L. Sherman, Ravindra Kumar, Rupal S. Bhatt. Inhibiting the ALK1/BMP9 signaling pathway with dalantercept as an antiangiogenic therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5080. doi:10.1158/1538-7445.AM2013-5080

Fibulin-1 is Down-Regulated Through Promoter Hypermethylation and Suppresses Renal Cell Carcinoma Progression

Renal cell carcinoma is one of the most common cancers worldwide but the molecular mechanisms that underlie it are not fully understood. Fibulin-1, a multi-functional extracellular matrix protein, is involved in many types of cancer but its function in renal cell carcinoma is unclear. We investigated fibulin-1 expression and function in renal cell carcinoma. We performed real-time polymerase chain reaction, Western blot analysis and immunohistochemistry to determine fibulin-1 expression in renal cell carcinoma cells and patient tissues. Methylation specific polymerase chain reaction and quantitative sequencing were done to examine the methylation status of the FBLN1 gene promoter. Eukaryotic expression plasmid and a lentiviral vector were used to over express fibulin-1 in ACHN and 786-O renal cell carcinoma cells to investigate its function in vitro and in vivo. Fibulin-1 was significantly down-regulated in renal cell carcinoma cell lines and patient tissues. Dysregulation was associated with renal cell carcinoma progression. The FBLN1 gene promoter region was hypermethylated and its methylation status correlated with fibulin-1 expression. Fibulin-1 over expression led dramatically to decreased cell growth, enhanced tumor cell apoptosis, decreased cell motility and angiogenesis in cultured renal cell carcinoma cells and in xenograft tumors in nude mice. Fibulin-1 is down-regulated in renal cell carcinoma through promoter hypermethylation. It functions as a tumor suppressor and angiogenesis inhibitor in renal cell carcinoma.

Impact of Ischemia and Procurement Conditions on Gene Expression in Renal Cell Carcinoma

Previous studies have shown that ischemia alters gene expression in normal and malignant tissues. There are no studies that evaluated effects of ischemia in renal tumors. This study examines the impact of ischemia and tissue procurement conditions on RNA integrity and gene expression in renal cell carcinoma. Ten renal tumors were resected without renal hilar clamping from 10 patients with renal clear cell carcinoma. Immediately after tumor resection, a piece of tumor was snap frozen. Remaining tumor samples were stored at 4°C, 22°C, and 37°C and frozen at 5, 30, 60, 120, and 240 minutes. Histopathologic evaluation was conducted on all tissue samples, and only those with greater than 80% tumor were selected for further analysis. RNA integrity was confirmed by electropherograms and quantitated using RNA integrity number index. Altered gene expression was assessed by paired, two-sample t test between the zero time point and aliquots from various conditions obtained from the same tumor. One hundred and forty microarrays were conducted. Some RNA degradation was observed 240 minutes after resection at 37°C. The expression of more than 4,000 genes was significantly altered by ischemia times or storage conditions. The greatest gene expression changes were observed with longer ischemia time and warmer tissue procurement conditions. RNA from kidney cancer remains intact for up to 4 hours post surgical resection regardless of storage conditions. Despite excellent RNA preservation, time after resection and procurement conditions significantly influence gene expression profiles. Meticulous attention to preacquisition variables is of paramount importance for accurate tumor profiling.

Expression of EZH2 in renal cell carcinoma as a novel prognostic marker

Enhancer of zeste homolog 2 (EZH2) is a member of the Polycomb group proteins and a part of Polycomb repressive complex 2. EZH2 is important for transcriptional regulation through nucleosome modification and interaction with other transcription factors. Particularly, aberration of EZH2 has been implicated in oncogenesis and progression of various neoplasms. The objective of this study was to evaluate EZH2 expression in renal cell carcinoma (RCC), especially clear cell RCC (CRCC) and correlate the expression with prognostic factors. EZH2 expression was determined by immunohistochemical staining with additional Western blotting. High expression of EZH2 was significantly correlated with higher pT stage or more frequent distant metastases (P= 0.001 and 0.024, respectively). Survival analyses displayed that patients with high EZH2 expression had a significantly shorter disease-free survival than those with low expression (P= 0.019). High expression of EZH2 tended to reduce the overall survival, however, differences did not reach statistical significance (P= 0.066). From our results, we propose that EZH2 is a useful prognostic marker for aggressive behavior of CRCC and may be applicable as a therapeutic target molecule.