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Purpose: Our purpose was to investigate the association between family history of renal cell carcinoma (RCC) and RCC risk., Materials and Methods: RCC cases diagnosed in Sweden between 2005 and 2014 and 10 matched controls were identified using the Renal Cell Cancer Database Sweden, with linkage to the Multigeneration Register and the Swedish Cancer Registry. The association between a family history of RCC and RCC was investigated, overall and by sex and age groups., Results: Among 9416 RCC cases, 294 (3.1%) had 1 or more parent or sibling (first-degree relative [FDR]) with RCC. Median age at diagnosis for cases with an affected FDR was 65 years (IQR 59-71) and 68 years (IQR 60-75) for all cases. The proportion of women was significantly higher among familial RCC compared to sporadic RCC (44.6% vs 38.5% , P = .035). RCC was twice as likely with 1 or more FDR with RCC (OR 1.9; CI 1.65-2.16). Stratified analysis showed an OR of 2.4 for women (CI 1.93-2.92) and 1.6 for men (CI 1.35-1.93). Two or more FDRs was associated with a sixfold increased risk (95% CI 2.37-15.5). Familial RCC was strongly associated with bilateral and multifocal tumors (OR 5.5; CI 2.36-13.0, OR 3.5; CI 1.89-6.49)., Conclusions: In this Swedish data set, 3.1% of RCC patients have 1 or more FDR diagnosed with RCC. There was no statistical difference in median age between sporadic RCC and familial RCC. Having 1 or more FDR with RCC approximately doubles the risk of RCC with a higher risk increase for women than for men. People with 2 FDRs with RCC constitute a small high-risk group that may benefit from screening.

Purpose: We quantified the familial risk of renal cell cancer (RCC) among first-degree relatives (FDRs) on a population level, and examined interactions between family history and body mass index or blood glucose., Materials and Methods: Using the National Health Insurance database, which covers the entire Korean population, and the National Health Screening Program, we constructed a cohort of 5,524,403 individuals with blood-related FDRs and their lifestyle factors from 2002 to 2018. We calculated familial risk using incidence risk ratios (IRRs) with 95% confidence intervals, which compares the risk of individuals with and without FDR. The combined effect and interaction of a given risk factor and family history of RCC were measured by the relative excess risk due to interaction., Results: Individuals with affected FDRs showed a 2.29-fold (95% CI 1.68-3.13) increased risk of disease. Familial risk adjusted for lifestyle factors showed minimal attenuation (IRR 2.25; 95% CI: 1.65-3.08), suggesting that genetic predisposition is the main contributor in the familial aggregation of RCC. Individuals with both a positive family history and overweight/obesity (IRR 3.71, 95% CI 2.50-4.92) or hyperglycemia (IRR 4.52, 95% CI 2.59-6.45) had a significantly higher risk that exceeded the sum of their individual risks, suggesting an interaction that was statistically significant (relative excess risk due to interaction 95% CI: 0.91, -0.21-2.12; 2.21, 0.28-4.14)., Conclusions: Our findings suggest an interaction between genetic and environmental factors, namely obesity and hyperglycemia. Individuals with both factors should be considered a high-risk group and advised to undergo genetic counseling.

Purpose: In various malignancies RNA fragments are dysregulated. Our study was designed to determine the expression of 4, 5'-tRNA halves in the tissue and serum of patients with clear cell renal cell carcinoma., Materials and Methods: Tissue and serum samples of patients with clear cell renal cell carcinoma and nonmalignant disease were collected prospectively in our biobank. We isolated total RNA from 95 clear cell renal cell carcinomas and 50 normal renal tissues as well as serum RNA from 27 patients with clear cell renal cell carcinoma and 13 with nonmalignant urological disease. To specifically determine the expression of 5'-tRNA halves we dephosphorylated and ligated an adaptor nucleotide to the 3' end of the tRNA halves. The expression levels of 4, 5'-tRNA halves (5'-tRNA-Arg-CCT, 5'-tRNA-Glu-CTC, 5'-tRNA-Leu-CAG and 5'-tRNA-Lys-TTT) were then measured by TaqMan® based quantitative reverse transcription-polymerase chain reaction., Results: All studied 5'-tRNA halves were down-regulated in clear cell renal cell carcinoma tissues, indicating a potential role as a tumor suppressor. Furthermore, we noted decreased expression of 5'-tRNA halves in patients with adverse clinicopathological parameters. All 5'-tRNA halves were expressed at lower levels in nonorgan confined clear cell renal cell carcinoma. The 5'-tRNA-Lys-TTT halves inversely correlated with ISUP (International Society of Urological Pathology) grade. In patients with clear cell renal cell carcinoma 5'-tRNA-Arg-CCT, 5'-tRNA-Glu-CTC and 5'-tRNA-Lys-TTT halves circulated at lower levels than in control subjects, indicating relevance as noninvasive biomarkers., Conclusions: In patients with clear cell renal cell carcinoma 5'-tRNA halves have potential as diagnostic and prognostic biomarkers. The 5'-tRNA halves may act in a tumor suppressive manner, which requires further research to confirm., (Copyright © 2018 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Mutations of SETD2 occur in 3% to 16% of clear cell renal cell carcinoma cases. Previous studies identified an association between SETD2 mutation and prognosis of patients with nonmetastatic clear cell renal cell carcinoma. In this study we explored the prognostic and predictive value of SETD2 expression in patients with metastatic renal cell carcinoma treated with targeted therapy., Materials and Methods: We retrospectively enrolled 138 patients with metastatic renal cell carcinoma treated with sunitinib or sorafenib at a single institution from 2007 to 2014. SETD2 expression was assessed by immunohistochemistry on tissue microarrays., Results: After excluding those patients with loss of followup or unavailable tissue samples, 111 were included in the study. Low SETD2 expression was associated with reduced overall survival (p <0.001) and progression-free survival (p=0.001). After adjustment for histological type, Heng risk group and drugs used for targeted therapy, SETD2 was defined as an independent prognostic marker for overall survival (HR 2.535 [95% CI 1.429-4.497], p=0.001) and progression-free survival (HR 1.755 [95% CI 1.031-2.988], p=0.038). Its prognostic value for overall survival was more predominant in patients with clear cell renal cell carcinoma (p <0.001) or patients in the intermediate risk group of Heng risk criteria (p <0.001), while its predictive value for progression-free survival was more predominant in patients treated with sorafenib (p <0.001). SETD2 could also be combined with the Heng risk model for better overall survival prediction., Conclusions: SETD2 is a potential prognostic biomarker for overall survival and progression-free survival prediction in patients with metastatic renal cell carcinoma receiving targeted therapy. However, it remains to be seen whether this is generalizable to other ethnicities and prospective external validation is required., (Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Renal cell carcinoma is the most common highly metastatic kidney malignancy. Adhesion has a crucial role in the metastatic process. TGF (transforming growth factor)-β1 is a pleiotropic cytokine that influences cancerous transformation. We hypothesized that 1) changes in the expression of adhesion related genes may influence survival rate of patients with renal cell carcinoma and 2) TGF-β1 may contribute to changed expression of adhesion related genes., Materials and Methods: Two-step quantitative real-time polymerase chain reaction arrays were used to analyze the expression of adhesion related genes in 77 tumors and matched pair controls. The prognostic significance of genes was evaluated in TCGA (The Cancer Genome Atlas) data on 468 patients with renal cell carcinoma. Quantitative real-time polymerase chain reaction and Western blot were applied for TGF-β1 analysis. TGF-β1 mediated regulation of gene expression was analyzed by TGF-β1 supplementation of Caki-2 cells and quantitative real-time polymerase chain reaction., Results: The expression of 19 genes related to adhesion and extracellular matrix remodeling was statistically significantly disturbed in renal cell carcinoma compared with controls. The 10-gene expression signature (COL1A1, COL5A1, COL11A1, FN1, ICAM1, ITGAL, ITGAM, ITGB2, THBS2 and TIMP1) correlated with poor survival (HR 2.85, p = 5.7e-10). TGF-β1 expression was 22 times higher in renal cell carcinoma than in controls (p <0.0001). TGF-β1 induced expression of TGFBI, COL1A1, COL5A1, COL8A1, FN1, ITGA5, ITGAM and TIMP1 in a renal cell carcinoma derived cell line., Conclusions: Disturbed expression of genes involved in adhesion and extracellular matrix remodeling develops early during renal cell carcinoma carcinogenesis and correlates with poor survival. TGF-β1 contributes to changed expression of extracellular matrix and adhesion related genes. Bioinformatic analysis performed on a broad panel of cancers of nonkidney origin suggests that disturbed expression of genes related to extracellular matrix and adhesion may be a universal feature of cancerous progression., (Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Sarcomatoid changes in renal cell carcinoma are associated with a poor prognosis. The identification of genetic alterations that drive this aggressive phenotype could aid in the development of more effective targeted therapies. In this study we aimed to pinpoint unique copy number alterations in sarcomatoid renal cell carcinoma compared to classical renal cell carcinoma subtypes., Materials and Methods: Genomic copy number analysis was performed using single nucleotide polymorphism based microarrays on tissue extracted from the tumors of 81 patients who underwent renal mass excision, including 17 with sarcomatoid renal cell carcinoma., Results: Sarcomatoid renal cell carcinoma showed a significantly higher number of copy number alterations than clear cell, papillary and chromophobe renal cell carcinoma (mean 18.0 vs 5.8, 6.5 and 7.2, respectively, p <0.0001). Copy number losses of chromosome arms 9q, 15q, 18p/q and 22q, and gains of 1q and 8q occurred in a significantly higher proportion of sarcomatoid renal cell carcinomas than in the other 3 histologies. Patients with sarcomatoid renal cell carcinoma demonstrated significantly worse overall survival compared to those without that condition on Kaplan-Meier analysis (p = 0.0001). Patients with 9 or more copy number alterations also demonstrated significantly worse overall survival than those with fewer than 9 copy number alterations (p = 0.004)., Conclusions: Sarcomatoid changes in renal cell carcinoma are associated with a high rate of chromosomal imbalances with losses of 9q, 15q, 18p/q and 22q, and gains of 1q and 8q occurring at significantly higher frequencies in comparison to nonsarcomatoid renal cell carcinoma. Identifying candidate driver genes or tumor suppressor loci in these chromosomal regions may help identify targets for future therapies., (Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: In clear cell renal cell carcinoma BAP1 and PBRM1 are 2 of the most commonly mutated genes (10% to 15% and 40% to 50%, respectively). We sought to determine the prognostic significance of PBRM1 and BAP1 expression in clear cell renal cell carcinoma., Materials and Methods: We used immunohistochemistry to assess PBRM1 protein expression in 1,479 primary clear cell renal cell carcinoma tumors that were previously stained for BAP1. A centralized pathologist reviewed all cases and categorized tumors as positive or deficient for PBRM1 and BAP1. Kaplan-Meier and Cox regression models were used to evaluate association of PBRM1 and BAP1 expression with the risk of death from renal cell carcinoma and the risk of metastasis after adjustment for age and the Mayo Clinic SSIGN (stage, size, grade and necrosis) score., Results: PBRM1 and BAP1 expression was PBRM1+ BAP1+ in 40.1% of tumors, PBRM1- BAP1+ in 48.6%, PBRM1+ BAP1- in 8.7% and PBRM1- BAP1- in 1.8%. The incidence of PBRM1 and BAP1 loss in the same tumor was significantly lower than expected (actual 1.8% vs expected 5.3%, p <0.0001). Compared to patients with PBRM1+ BAP1+ tumors those with PBRM1- BAP1+ lesions were more likely to die of renal cell carcinoma (HR 1.39, p = 0.035), followed by those with PBRM1+ BAP1- and PBRM1- BAP1- tumors (HR 3.25 and 5.2, respectively, each p <0.001). PBRM1 and BAP1 expression did not add independent prognostic information to the SSIGN score., Conclusions: PBRM1 and BAP1 expression identified 4 clinical subgroups of patients with clear cell renal cell carcinoma who had divergent clinical outcomes. The clinical value of these biomarkers will be fully realized when therapies targeting pathways downstream of PBRM1 and BAP1 are developed., Competing Interests: §Financial interest and/or other relationship with Bethyl Laboratories., (Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: The role of androgen receptor in renal cell carcinoma is not well understood. In this study the correlation between androgen receptor mRNA expression and clinicopathological features in patients with localized renal cell carcinoma was investigated. Additionally, human renal cell carcinoma cell lines were examined for the presence and effect of androgen receptor., Materials and Methods: Androgen receptor mRNA expression was evaluated by quantitative real-time polymerase chain reaction in 115 tumor samples from patients with primary pathological stage T1 or T2 (pT1/pT2) renal cell carcinoma and 57 specimens of corresponding normal kidney tissue. Reverse transcriptase-polymerase chain reaction and Western blot were done to examine the expression of androgen receptor in human renal cell carcinoma cell lines. Effects on cellular proliferation were investigated after activating and blocking androgen signaling in tissue culture., Results: Androgen receptor mRNA expression levels were significantly higher in patients with pT2 tumors than in those with pT1 tumors (p = 0.011). Kaplan-Meier estimates revealed significant differences in time to progression and cancer specific survival between low and high androgen receptor mRNA expression groups regardless of gender. Multivariate Cox regression analysis demonstrated that the level of androgen receptor expression was an independent predictor of cancer specific survival (HR 15.546, 95% CI 1.320-183.131, p = 0.029). In tissue culture treatment with dihydrotestosterone caused proliferation in androgen receptor positive cell lines while enzalutamide resulted in reduced cell viability in a dose dependent manner., Conclusions: In patients with localized renal cell carcinoma the androgen receptor mRNA expression level is associated with prognosis. In addition, cell culture data suggest that enzalutamide may have an effect in limiting the growth of androgen receptor positive renal cell carcinoma., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Bone metastasis develops in 30% of all patients with renal cell carcinoma. We elucidated the mechanisms that lead to and predict bone metastasis of renal cell carcinoma., Materials and Methods: Nine renal cell carcinoma primary cell lines and 30 renal cell carcinoma tissue specimens (normal and tumor tissue) were collected from 3 patients with no metastasis and 10 with lung or bone metastasis within 5 years after nephrectomy. Cell migration was analyzed in a Boyden chamber and proliferation was assessed by bromodeoxyuridine incorporation. Adhesion to fibronectin, and collagen I and IV was determined after cell staining. The expression and/or activity of cellular signaling molecules was quantified by Western blot., Results: Compared to renal cell carcinoma cells from patients without metastasis, the migration of cells from patients with bone metastasis was enhanced 13.5-fold (p = 0.034), and adhesion to fibronectin and collagen I was enhanced 5.8-fold and 6.1-fold (p = 0.002 and 0.014, respectively). In general proliferation was decreased in metastasizing cells. In accordance with these results we detected higher activity of AKT (p = 0.011) and FAK (p = 0.054), higher integrin α5 expression (p = 0.052) and lower PTEN expression in primary cells from patients with bone metastasis compared to nonmetastasizing cells. An almost similarly altered expression pattern was also observed in the renal cell carcinoma tissue specimens and the normal renal tissue of patients with bone metastasis., Conclusions: We describe evidence that molecular predispositions determine the potential for bone metastasis to develop in renal cell carcinoma, which may serve as prognostic markers after initial tumor detection., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Accurate discrimination of benign oncocytoma and malignant renal cell carcinoma is useful for planning appropriate treatment strategies for patients with renal masses. Classification of renal neoplasms solely based on histopathology can be challenging, especially the distinction between chromophobe renal cell carcinoma and oncocytoma. In this study we develop and validate an algorithm based on genomic alterations for the classification of common renal neoplasms., Materials and Methods: Using TCGA renal cell carcinoma copy number profiles and the published literature, a classification algorithm was developed and scoring criteria were established for the presence of each genomic marker. As validation, 191 surgically resected formalin fixed paraffin embedded renal neoplasms were blindly submitted to targeted array comparative genomic hybridization and classified according to the algorithm. CCND1 rearrangement was assessed by fluorescence in situ hybridization., Results: The optimal classification algorithm comprised 15 genomic markers, and involved loss of VHL, 3p21 and 8p, and chromosomes 1, 2, 6, 10 and 17, and gain of 5qter, 16p, 17q and 20q, and chromosomes 3, 7 and 12. On histological rereview (leading to the exclusion of 3 specimens) and using histology as the gold standard, 58 of 62 (93%) clear cell, 51 of 56 (91%) papillary and 33 of 34 (97%) chromophobe renal cell carcinomas were classified correctly. Of the 36 oncocytoma specimens 33 were classified as oncocytoma (17 by array comparative genomic hybridization and 10 by array comparative genomic hybridization plus fluorescence in situ hybridization) or benign (6). Overall 93% diagnostic sensitivity and 97% specificity were achieved., Conclusions: In a clinical diagnostic setting the implementation of genome based molecular classification could serve as an ancillary assay to assist in the histological classification of common renal neoplasms., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Aberrant promoter methylation turns off gene expression and is involved in human malignancy. Studies show that first exon methylation has a tighter association with gene silencing compared to promoter methylation or gene mutation. However, to our knowledge the clinical importance of exonic methylation in renal cell carcinoma is unknown. We analyzed renal cell carcinoma for VHL gene exonic methylation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry., Materials and Methods: In 48 institutionally banked renal cell carcinoma patient tissue samples VHL exon sequencing was done as well as methylation analysis of promoter and exon 1 by mass spectrometry or conventional bisulfite analysis. Methylated human lymphocytic DNA (0% and 100%), nontemplate distilled H2O, and the UOK121 and UOK171 human renal cell carcinoma cell lines served as assay controls. Samples were considered hypermethylated if a CpG site showed greater than 50% methylation., Results: Nine of the 43 patient samples read by our exon 1 assay had methylated VHL exon 1 sites, including 3 showing hypermethylation. The exon 1 methylation assay was robust and reproducible. Samples with exon 1 hypermethylation showed no exonic mutations. All samples assayed at VHL exon 2 were hypermethylated., Conclusions: To assay renal cell carcinoma tumors for VHL methylation matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is robust and reproducible, and capable of quantifying the methylation status of individual DNA bases. Exon 1 methylation may be an alternate mechanism of VHL gene silencing in renal cell carcinoma in addition to mutation and promoter methylation. Applying this assay in patient populations may allow enhanced diagnosis or tumor typing in the future., (Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: We investigated the potential functions of miR-34a in CD44 transcriptional complexes in renal cell carcinoma., Materials and Methods: We detected miR-34a expression by quantitative real-time polymerase chain reaction. Oligonucleotides were used to over express miR-34a. Cell proliferation and xenograft assays, colony formation and flow cytometry were done to examine effects on cancer cell proliferation in vitro and in vivo. Luciferase assay was performed to verify the precise target of miR-34a., Results: Promoter methylation contributed to miR-34a loss in the ACHN, 786-O and SN12PM6 renal carcinoma cell lines. Ectopic over expression of miR-34a restrained cell growth, tube formation and migration/invasion, and significantly suppressed the growth of renal carcinoma xenografts and metastasis in nude mice. Dual luciferase assay revealed that CD44 was a direct target of miR-34a in renal cancer cells and CD44 knockdown by RNAi in renal cancer cells suppressed tumor progression. In contrast, CD44 ectopic expression partially reversed the antitumor effects of miR-34a in renal cancer cells., Conclusions: Our findings indicate that miR-34a targets CD44 in renal cancer cells and suppresses renal cancer cell growth, tube formation and metastasis in vitro and in vivo. Thus, miR-34a may be a potential molecular target for novel therapeutic strategies for clear cell renal carcinoma., (Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: We investigated the clinical implications of biallelic loss of PTEN in clear cell renal cell carcinoma and whether PTEN biallelic loss would induce p53 dependent cellular senescence., Materials and Methods: Data were obtained using the CGDS-R package from the TCGA data set kirc&#95;tcga&#95;pub. PTEN allelic status was classified into 3 groups, including biallelic PTEN loss (homozygous deletion or combined heterozygous deletion and mutation), monoallelic PTEN loss (heterozygous deletion or mutation) and absent allelic loss. Univariate and multivariate overall survival analysis was performed. TP53 allelic loss and mean expression of genes related to p53 dependent cellular senescence were compared., Results: Of 416 patients with clear cell renal cell carcinoma 11 (2.6%) had biallelic PTEN loss and 69 (16.6%) had monoallelic loss. PTEN allelic loss was associated with late tumor stage and high histological grade. Patients with biallelic loss showed worse overall survival after adjusting for age and AJCC tumor stage (HR 3.1, 95% CI 1.4-6.8, p = 0.005). About half of the patients with PTEN biallelic loss had accompanying TP53 allelic loss. Biallelic loss of PTEN did not increase the expression of genes related to p53 dependent cellular senescence., Conclusions: PTEN biallelic loss may be a prognostic marker for clear cell renal cell carcinoma. It does not seem to induce p53 dependent cellular senescence, partly due to allelic loss of TP53., (Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Several recently reported recurrent genomic alterations in clear cell renal cell carcinoma are linked to pathological and clinical outcomes. We determined whether any recurrent cancer gene mutations or copy number alterations identified in the TCGA (The Cancer Genome Atlas) clear cell renal cell carcinoma data set could add to the predictive accuracy of current prognostic models., Materials and Methods: In 413 patients who underwent nephrectomy/partial nephrectomy we investigated whole exome, copy number array analyses and clinical variables. We identified 65 recurrent genomic alterations based on prevalence and combined them into 35 alterations, including 12 cancer gene mutations. Genomic markers were modeled using the elastic net algorithm with preoperative variables (tumor size plus patient age) and in the postoperative setting using the externally validated Mayo Clinic SSIGN (stage, size, grade and necrosis) prognostic scoring system. These models were subjected to internal validation using bootstrap., Results: Median followup in survivors was 45 months. Several markers correlated with adverse cancer specific survival and time to recurrence on univariate analysis. However, most of them lost significance when controlling for tumor size with or without age in the preoperative models or for SSIGN score in the postoperative setting. Adding multiple genomic markers selected by the elastic net algorithm failed to substantially add to the predictive accuracy of any preoperative or postoperative model for cancer specific survival or time to recurrence., Conclusions: While recurrent copy number alterations and cancer gene mutations are biologically significant, they do not appear to improve the predictive accuracy of existing models of clinical cancer specific survival or time to recurrence for clear cell renal cell carcinoma., (Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Mutations in the FLCN gene are responsible for fibrofolliculoma, pulmonary and renal cysts, and renal cell carcinoma in patients with Birt-Hogg-Dubé syndrome. To explore therapeutic approaches to renal cell carcinoma in patients with Birt-Hogg-Dubé syndrome we investigated the anticancer effects of irradiation on folliculin deficient renal cancer cells., Materials and Methods: Folliculin deficient (UOK257 and ACHN-5968) and folliculin expressing (UOK257-2 and ACHN-sc) cell lines were used in this study. Clonogenic assays were used to determine the radiosensitivity of folliculin deficient and expressing renal cell carcinoma cells. Apoptosis was detected in these cells by DAPI and TUNEL assays after irradiation. Monodansylcadaverine analysis, GFP-LC3 assay and Western blot were performed to monitor the autophagic process., Results: Folliculin deficient cells were more sensitive to irradiation than their folliculin expressing counterparts. The enhanced effects of irradiation on folliculin deficient cells were mediated by increased autophagy but not by apoptosis. An increased Beclin 1 protein level and an activated mitogen-activated protein kinase pathway were identified as the key regulators of increased autophagy in these folliculin deficient cells. Inhibiting autophagy with 3-methyladenine or beclin 1 siRNA obviously increased radioresistance in folliculin deficient cells. Moreover, irradiation combined with autophagic inducer rapamycin significantly increased autophagy and radiosensitivity in folliculin deficient renal cell carcinoma cells., Conclusions: Findings suggest that folliculin deficient renal cell carcinoma cells are highly sensitive to irradiation due to increased autophagic cell death, unlike other types of renal cell carcinoma. Irradiation plus autophagy inducers, eg rapamycin, might be a potentially more effective therapeutic approach to folliculin deficient renal cell carcinoma., (Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Mutations in the tumor suppressor gene BAP1 occur in approximately 15% of clear cell renal cell carcinoma cases. Sequencing efforts demonstrated worse outcomes in patients with BAP1 mutated clear cell renal cell carcinoma. We investigated the clinicopathological significance and oncologic outcomes of BAP1 loss using a previously validated immunohistochemical assay., Materials and Methods: Immunohistochemistry for BAP1 was performed on tissue microarray sections from 559 nonmetastatic clear cell renal cell carcinoma cases treated with nephrectomy at multiple institutions. The association of BAP1 expression with clinicopathological parameters was analyzed using the Wilcoxon rank sum and Cochran-Mantel-Haenszel tests. Survival was assessed by Cox regression analysis, which also identified independent predictors of time dependent outcomes., Results: At a median followup of 50 months (range 0 to 183) 86 of 483 patients (17.8%) experienced recurrence and 121 of 559 (21.6%) had died. BAP1 was negative in 82 of 559 tumors (14.7%). BAP1 loss was associated with adverse clinicopathological variables, including high Fuhrman grade (p <0.0001), advanced pT stage (p = 0.0021), sarcomatoid dedifferentiation (p = 0.0001) and necrosis (p <0.0001). Cox regression revealed that patients with BAP1 negative tumors had significantly worse disease-free survival (HR 2.9, 95% CI 1.8-4.7, p <0.0001) and overall survival (HR 2.0, 95% CI 1.3-3.1, p = 0.0010) than patients with BAP1 positive tumors., Conclusions: Immunohistochemistry for BAP1 serves as a powerful marker to predict poor oncologic outcomes and adverse clinicopathological features in patients with nonmetastatic clear cell renal cell carcinoma. BAP1 assessment using immunohistochemistry on needle biopsy may benefit preoperative risk stratification and guide treatment planning in the future., (Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Epithelial-mesenchymal transition enhances tumor cell motility and has a critical role in invasion and metastasis in a number of carcinomas. A set of transcription factors acts as a master regulator of the epithelial-mesenchymal transition process. To our knowledge it is unknown whether epithelial-mesenchymal transition is important for clear cell renal cell carcinoma progression. Therefore, we comprehensively assessed mRNA levels of epithelial-mesenchymal transition associated genes in renal cell carcinoma as well as their prognostic relevance., Materials and Methods: We determined the expression of a set of 46 epithelial-mesenchymal transition related genes by oligonucleotide microarray and gene set enrichment analyses using RNA from 14 samples each of normal kidneys, and G1 and G3 primary renal cell carcinomas. Expression of select epithelial-mesenchymal transition genes was validated by real-time polymerase chain reaction in normal kidneys, primary renal cell carcinomas and metastases in an independent cohort of 112 patients. Results were combined with followup data for survival analysis., Results: The epithelial-mesenchymal transition gene set was preferentially expressed in primary renal cell carcinoma compared to normal tissue (false discovery rate 0.01). No difference was found between G1 and G3 tumors. Quantitative reverse transcriptase-polymerase chain reaction revealed down-regulation of critical epithelial-mesenchymal transition genes such as CDH2 and ZEB1 in metastases, suggesting epithelial-mesenchymal transition reversal during metastasis. Kaplan-Meier analysis demonstrated a better outcome in patients with low CXCR4, vimentin, fibronectin and TWIST1 mRNA levels. Multivariate analyses revealed that CXCR4 and VIM up-regulation represents an independent prognostic marker for poor cancer specific survival in patients with renal cell carcinoma., Conclusions: Taken together, our data provide strong evidence that epithelial-mesenchymal transition occurs in renal cell carcinoma. Thus, interference with epithelial-mesenchymal transition in renal cell carcinoma might represent a future therapeutic option., (Copyright © 2014. Published by Elsevier Inc.)

Purpose: Few risk factors have been identified for renal cell carcinoma. We performed a validation study in a population with a European background to identify the most significant variants previously identified in association with renal cell carcinoma risk., Materials and Methods: We performed a case-control validation study after recruiting 463 controls and 463 patients with a histologically confirmed diagnosis of clear cell renal cell carcinoma. For each patient and matched control we genotyped 8 single nucleotide polymorphisms selected from previous studies to evaluate the association between candidate single nucleotide polymorphisms and renal cell carcinoma susceptibility., Results: After adjusting for patient age, gender, smoking status and body mass index the AG + AA genotypes from rs7105934 (11q13) were associated with a decreased risk of renal cell carcinoma (OR 0.50, 95% CI 0.33-0.75, p = 0.001) and the AC + CC genotypes from rs1049380 (ITPR2) were associated with an increased risk (OR 1.66, 95% CI 1.28-2.16, p <0.001). Kidney cancer developed at an older age in patients carrying the dominant risk allele A for rs7105934 (mean age at diagnosis 73.1 vs 68.9 years, p = 0.002) and at a younger age in those carrying the dominant allele C for rs1049380 (mean 68.1 vs 70.8 years, p = 0.005)., Conclusions: In what is to our knowledge the first validation study of the main 8 single nucleotide polymorphism variants associated with renal cell carcinoma susceptibility we confirmed the association of 2 single nucleotide polymorphisms with the risk of renal cell carcinoma. Each variant influenced patient age at disease diagnosis., (Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Cowden syndrome is a hereditary cancer syndrome associated with a germline mutation in PTEN. Patients are predisposed to multiple malignancies including renal cell carcinoma., Materials and Methods: Patients with Cowden syndrome were evaluated as part of a clinical protocol. Those with a history of renal cell carcinoma underwent review of clinical features, tumor characteristics and family history. Renal tumors were evaluated for loss of heterozygosity., Results: Among 24 patients with Cowden syndrome 4 were identified with renal cell carcinoma (16.7%). Three patients had solitary tumors, 2 with papillary type I histology and 1 with clear cell histology. The fourth patient had bilateral, synchronous chromophobe tumors. No patients had a prior family history of renal cell carcinoma. All patients with renal cell carcinoma had dermatologic manifestations of Cowden syndrome and had macrocephaly. Loss of heterozygosity at the PTEN mutation was identified in 4 tumors (80%). No genotype-phenotype association was found, as the same mutation was identified in different renal cell carcinoma histologies., Conclusions: Renal cell carcinoma is an underappreciated feature of Cowden syndrome. As most patients lack a prior family history or a distinctive renal cell carcinoma histology, recognition of the associated nonrenal features should target referral for genetic counseling. PTEN loss of heterozygosity is common in Cowden syndrome renal tumors. Because loss of PTEN can activate mTOR and mTOR inhibitors are Food and Drug Administration approved to treat renal cell carcinoma, these agents have clinical potential in renal cell carcinoma associated with Cowden syndrome., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: PAR-1 mediates angiogenesis and impacts the process of tumor growth and disease progression. We evaluated the associations of the gene variations PAR-1 IVSn -14 A>T (rs168753), -506 Ins/Del (rs11267092) and -1426 C>T (rs32934) with renal cell carcinoma pathology and cancer specific survival., Materials and Methods: DNA was extracted from the peripheral blood leukocytes of 236 consecutive patients with renal cell carcinoma. Genotyping was done using restriction fragment length polymorphism analysis of polymerase chain reaction amplicons and sequencing., Results: The IVSn -14 AA genotype was associated with a 3.13-fold increased risk of distant metastases (p = 0.015). In addition, cancer specific survival of patients with IVSn -14 AA was significantly worse than in those with AT/TT (HR 2.98, p = 0.019). The 1 and 4-year cancer specific survival rate for AA vs AT/TT was 89% vs 99% and 82% vs 92%, respectively. After adjusting for the stage, size, grade and necrosis (SSIGN) score on multivariable analysis, IVSn -14 AA was identified as an independent adverse prognostic factor (HR 2.72, p = 0.044). The variations -506 Ins/Del and -1426 C>T were not significantly associated with pathological factors or cancer specific survival., Conclusions: Results suggest that the AA genotype of the PAR-1 variation IVSn -14 A>T is associated with an increased risk of metastasis and poorer prognosis of renal cell carcinoma. Therefore, assessing the individual risk based on genotypes may be a helpful adjunct to identify subgroups at high risk for a poor clinical outcome., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Our microRNA expression signature of renal cell carcinoma revealed that miR-218 expression was significantly decreased in cancer tissues, suggesting that miR-218 is a candidate tumor suppressor. We investigated the functional significance of miR-218 in cancer cells and identified what are to our knowledge novel miR-218 mediated cancer pathways in renal cell carcinoma., Materials and Methods: Gain of function studies using mature miR-218 were performed to investigate cell proliferation, migration and invasion in the A498 and 786-O renal cell carcinoma cell lines. To identify miR-218 mediated molecular pathways and responsible genes in renal cell carcinoma, we used gene expression and in silico database analyses. Loss of function assays were performed to investigate the functional significance of miR-218 target genes., Results: Restoration of mature miR-218 significantly inhibited RCC cell proliferation, migration and invasion. Gene expression studies and luciferase reporter assays showed that CAV2 involved in the focal adhesion pathway was directly regulated by miR-218. A silencing study of CAV2 revealed significant inhibition of cell proliferation, migration and invasion. CAV2 mRNA and protein expression was significantly up-regulated in renal cell carcinoma clinical specimens., Conclusions: Loss of tumor suppressive miR-218 enhances cancer cell migration and invasion through dysregulation of the focal adhesion pathway, especially CAV2 as an oncogenic function in renal cell carcinoma. Tumor suppressive microRNA mediated cancer pathways and responsible genes provide new insights into the potential mechanisms of renal cell carcinoma oncogenesis and metastasis., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Caspase-8 is a key regulator of apoptosis. Its cancer cell antigen induced cell death activity is strongly impacted by the insertion/deletion promoter polymorphism CASP8 -652 6N ins/del (rs3834129). We studied the association of this polymorphism with renal cell carcinoma risk and pathology., Materials and Methods: In this hospital based case-control study 500 Austrian patients were genotyped, including 250 with renal cell carcinoma, and 250 age and gender matched healthy controls. Polymerase chain reaction amplified genomic DNA was evaluated by restriction fragment length polymorphism analysis and automatic sequencing. We assessed associations with renal cell carcinoma risk and pathological factors, and performed a meta-analysis of the literature., Results: The CASP8 -652 6N ins/del polymorphism was significantly linked to renal cell carcinoma (chi-square for trend = 9.50, p = 0.002). Compared with ins/ins, del/del was associated with a 57% decreased risk of the disease (OR 0.43, 95% CI 0.26-0.73, p = 0.002). Furthermore, del/del was associated with a lower risk of distant metastases (p <0.05) but not with T stage, N stage or grade. On meta-analysis the CASP8 -652 6N ins/del polymorphism was associated with renal cell carcinoma risk (p <0.001)., Conclusions: The del/del genotype of the CASP8 -652 6N ins/del promoter polymorphism decreases the overall risk of renal cell carcinoma. It may be associated with a decreased risk of metastasis. Larger studies are warranted to validate our findings., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: 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., Materials and Methods: 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., Results: 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., Conclusions: 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., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Renal cell carcinoma often presents asymptomatically and patients are commonly diagnosed at the metastatic stage, when treatment options are limited and survival is poor. Since progression-free survival using current therapy for metastatic renal cell carcinoma is only 1 to 2 years and existing drugs are associated with a high resistance rate, new pharmacological targets are needed. We identified and evaluated the nuclear exporter protein CRM1 as a novel potential therapy for renal cell carcinoma., Materials and Methods: We tested the efficacy of the CRM1 inhibitors KPT-185 and 251 in several renal cell carcinoma cell lines and in a renal cell carcinoma xenograft model. Apoptosis and cell cycle arrest were quantified and localization of p53 family proteins was assessed using standard techniques., Results: KPT-185 attenuated CRM1 and showed increased cytotoxicity in renal cell carcinoma cells in vitro with evidence of increased apoptosis as well as cell cycle arrest. KPT-185 caused p53 and p21 to remain primarily in the nucleus in all renal cell carcinoma cell lines, suggesting that the mechanism of action of these compounds depends on tumor suppressor protein localization. Furthermore, when administered orally in a high grade renal cell carcinoma xenograft model, the bioavailable CRM1 inhibitor KPT-251 significantly inhibited tumor growth in vivo with the expected on target effects and no obvious toxicity., Conclusions: The CRM1 inhibitor protein family is a novel therapeutic target for renal cell carcinoma that deserves further intensive investigation for this and other urological malignancies., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: We identified microRNA driven mechanisms in clear cell renal cell carcinoma associated with the tumor response to the multitargeted receptor tyrosine kinase inhibitor sunitinib., Materials and Methods: We performed screening genome-wide microRNA real-time quantitative polymerase chain reaction on 20 freshly frozen clear cell renal cell carcinoma tissue samples of patients who received sunitinib as first line targeted therapy. Nine patients with progressive disease within 6 months after initiating therapy were considered poor responders and 11 with at least 1-year progression-free survival were considered good responders. We studied microRNA-141 function in vitro by stable up-regulation of microRNA-141, quantification of target gene expression and cell viability in normoxic and hypoxic conditions. Relative expression in clinical and cell line samples was determined by real-time quantitative polymerase chain reaction. Localization of microRNA-141 and its targets was assessed by microRNA in situ hybridization and immunohistochemistry. Hypoxia induced cytotoxicity was assessed by a luminescence adenosine triphosphate detection assay., Results: Compared to good responders, microRNA-141 was significantly down-regulated in tumors of poor responders to sunitinib. This seemed spatially linked to epithelial-to-mesenchymal transition in vivo. Reintroduction of microRNA-141 in vitro reversed epithelial-to-mesenchymal transition and decreased cell viability in hypoxic conditions., Conclusions: In our study microRNA-141 down-regulation driven epithelial-to-mesenchymal transition in clear cell renal cell carcinoma was linked to an unfavorable response to sunitinib therapy. Reintroduction of microRNA-141 in vitro led to epithelial-to-mesenchymal transition reversal and increased sensibility to a hypoxic environment. Future experiments should be done in vivo to see whether microRNA-141 driven reversal of epithelial-to-mesenchymal transition could affect the efficacy of sunitinib treatment., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: A key barrier to identifying tissue biomarkers of clear cell renal cell carcinoma is the heterogeneity of protein expression in tissue. However, by providing spectra for every 0.05 mm(2) area of tissue, imaging mass spectrometry reveals the spatial distribution of peptides. We determined whether this approach could be used to identify and map protein signatures of clear cell renal cell carcinoma., Materials and Methods: We constructed 2 tissue microarrays with 2 cores each of matched tumor and normal tissue from the nephrectomy specimens of 70 patients with clear cell renal cell carcinoma. Samples were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In each tissue microarray peptide signatures were identified that differentiated cancer from normal tissue. The signatures were then cross validated. Mass spectrometry/mass spectrometry sequencing was performed to determine the identity of select, differentially expressed peptides. Immunohistochemistry was used for validation., Results: In each tissue microarray peptide signatures were identified that had 94.7% to 98.5% classification accuracy for each 0.05 mm(2) spot (spectrum) and 96.9% to 100% accuracy for each tissue core. Cross validation across tissue microarrays revealed a classification accuracy of 82.6% to 84.7% for each spot and 88.9% to 92.4% for each core. We identified vimentin, histone 2A.X and α-enolase as proteins with greater expression in cancer tissue. This was validated by immunohistochemistry., Conclusions: Imaging mass spectrometry identified and mapped specific peptides that accurately distinguished malignant from normal renal tissue. This demonstrates its potential as a novel, high throughput approach to clear cell renal cell carcinoma biomarker discovery. Given the multiple pathways and known heterogeneity involved in tumors such as clear cell renal cell carcinoma, multiple peptide signatures that maintain their spatial relationships may outperform traditional protein biomarkers., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Recently, a new renal cell cancer syndrome has been linked to germline mutation of multiple subunits (SDHB/C/D) of the Krebs cycle enzyme, succinate dehydrogenase. We report our experience with the diagnosis, evaluation and treatment of this novel form of hereditary kidney cancer., Materials and Methods: Patients with suspected hereditary kidney cancer were enrolled on a National Cancer Institute institutional review board approved protocol to study inherited forms of kidney cancer. Individuals from families with germline SDHB, SDHC and SDHD mutations, and kidney cancer underwent comprehensive clinical and genetic evaluation., Results: A total of 14 patients from 12 SDHB mutation families were evaluated. Patients presented with renal cell cancer at an early age (33 years, range 15 to 62), metastatic kidney cancer developed in 4 and some families had no manifestation other than kidney tumors. An additional family with 6 individuals found to have clear cell renal cell cancer that presented at a young average age (47 years, range 40 to 53) was identified with a germline SDHC mutation (R133X) Metastatic disease developed in 2 of these family members. A patient with a history of carotid body paragangliomas and an aggressive form of kidney cancer was evaluated from a family with a germline SDHD mutation., Conclusions: SDH mutation associated renal cell carcinoma can be an aggressive type of kidney cancer, especially in younger individuals. Although detection and management of early tumors is most often associated with a good outcome, based on our initial experience with these patients and our long-term experience with hereditary leiomyomatosis and renal cell carcinoma, we recommend careful surveillance of patients at risk for SDH mutation associated renal cell carcinoma and wide surgical excision of renal tumors., (Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Although molecular targeted therapy has improved the clinical outcome of metastatic renal cell carcinoma, a complete response is rare and there are various side effects. Identifying novel target molecules is necessary to improve the clinical outcome of metastatic renal cell carcinoma. HMGA1 is over expressed in many types of cancer and it is associated with metastatic potential. It is expressed at low levels or not expressed in normal tissue. We examined HMGA1 expression and function in human renal cell carcinoma., Materials and Methods: HMGA1 expression in surgical specimen from patients with renal cell carcinoma was examined by immunoblot. HMGA1 expression in 6 human renal cell carcinoma cell lines was examined by immunoblot and immunofluorescence. The molecular effects of siRNA mediated knockdown of HMGA1 were examined in ACHN and Caki-1 cells., Results: Immunoblot using surgical specimen showed that HMGA1 was not expressed in normal kidney tissue but it was expressed in tumor tissue in 1 of 30 nonmetastatic (3%) and 6 of 18 metastatic (33%) cases (p=0.008). Immunoblot and immunofluorescence revealed significant nuclear expression of HMGA1 in ACHN and Caki-1 cells derived from metastatic sites. HMGA1 knockdown remarkably suppressed colony formation and induced significant apoptosis in ACHN and Caki-1 cells. HMGA1 knockdown significantly inhibited invasion and migration in vitro, and induced anoikis associated with P-Akt down-regulation in ACHN cells., Conclusions: HMGA1 is a potential target for novel therapeutic modalities for metastatic renal cell carcinoma., (Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: We identified regions of DNA copy number changes that are significantly associated with metastasis and clinical outcome in patients with clear cell renal cell carcinoma., Materials and Methods: We analyzed 53 primary clear cell renal cell carcinomas, including 31 metastasized and 22 nonmetastasized tumors, by array comparative genomic hybridization with a median resolution of 1 to 1.5 Mbp. To validate copy number aberrations with potential prognostic value we performed fluorescence in situ hybridization analysis using commercially available fluorescent probes., Results: We identified 5 recurrent chromosomal aberrations that were significantly associated with metastasis, including gains of 1q21.3, 12q13.12, 12q13.3q14.1 and 20q11.21q13.2, and loss of 9p21.3p24.1. The most prominent of them with the highest OR for metastatic risk were loss of 9p21.3p24.1, and gains of 1q21.3 and 20q11.21q13.32. Eight alterations involving chromosomes 7, 9, 12, 16 and 20 significantly correlated with shortened cancer specific survival. The lowest p values on Kaplan-Meier analysis showed losses of 9p21.3p24.1 and 9q32q33.1, and gains of 7q36.3 and 20q11.21q13.32. Fluorescence in situ hybridization done in the same cohort for the 4 select regions 1q21.3, 7q36.3, 9p21.3p24.1 and 20q11.21q13.32 clearly confirmed the results of array comparative genomic hybridization., Conclusions: Data suggest that specific chromosomal alterations in clear cell renal cell carcinoma can be used to predict metastasis and cancer specific survival in patients with clear cell renal cell carcinoma. It seems possible to design a combined fluorescence in situ hybridization assay based on these genetic targets for outcome prediction, which can be used for routine diagnostics., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Renal cell carcinoma is the eighth most common cancer in the United States and clear cell renal carcinoma is the most common type. Many signaling pathways are implicated in clear cell renal carcinoma development, including the inflammation pathway. However, less is known about how gene expression variation in this pathway influences clear cell renal carcinoma development and clinical outcomes., Materials and Methods: Gene expression in tumor and adjacent normal tissues from 93 patients was detected using a genome-wide expression array. A panel of 661 inflammation related genes was then analyzed. Differential expression patterns between tumor and normal tissues were identified. Association with recurrence or survival was evaluated with genes showing significant association tested further in a validation set of 258 tumors using an independent platform (quantitative real-time polymerase chain reaction)., Results: We identified 151 genes with at least a two-fold change in gene expression between adjacent normal tissue and tumor, of which most were up-regulated in tumors. A total of 20 genes significantly associated with recurrence and/or overall survival were selected for further validation. In the replication data set high expression of GADD45G was significantly associated with a 2.09-fold (95% CI 1.08-6.14, p = 0.034) increased risk of recurrence while high CARD9, NCF2 and CIITA expression was significantly associated with a 2.52-fold (95% CI 1.24-5.12, p = 0.010), 2.26-fold (95% CI 1.12-4.58, p = 0.023) and 2.11-fold (95% CI 1.05-4.27, p = 0.037) increased risk of death, respectively., Conclusions: Results suggest that inflammation gene expression may be significant prognostic biomarkers for the risk of recurrence (GADD45G) and death (CARD9, CIITA and NCF2) in patients with clear cell renal carcinoma., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Renal cell carcinoma is the most common neoplasm of the adult kidney. Currently to our knowledge there are no biomarkers for diagnostic, prognostic or predictive applications for renal cell carcinoma. miRNAs are nonprotein coding RNAs that negatively regulate gene expression and are potential biomarkers for cancer., Materials and Methods: We analyzed 70 matched pairs of clear cell renal cell carcinoma and normal kidney tissues from the same patients by microarray analysis and validated our results by quantitative real-time polymerase chain reaction. We also performed extensive bioinformatic analysis to explore the role and regulation of miRNAs in clear cell renal cell carcinoma., Results: We identified 166 miRNAs that were significantly dysregulated in clear cell renal cell carcinoma, including miR-122, miR-155 and miR-210, which had the highest over expression, and miR-200c, miR-335 and miR-218, which were most down-regulated. Analysis of previously reported miRNAs dysregulated in RCC showed overall agreement in the direction of dysregulation. Extensive target prediction analysis revealed that many miRNAs were predicted to target genes involved in renal cell carcinoma pathogenesis. In renal cell carcinoma miRNA dysregulation can be attributed in part to chromosomal aberrations, co-regulation of miRNA clusters and co-expression with host genes. We also performed a preliminary analysis showing that miR-155 expression correlated with clear cell renal cell carcinoma size. This finding must be validated in a larger independent cohort., Conclusions: Analysis showed that miRNAs are dysregulated in clear cell renal cell carcinoma and may contribute to kidney cancer pathogenesis by targeting more than 1 key molecule. We identified mechanisms that may contribute to miRNA dysregulation in clear cell renal cell carcinoma. Dysregulated miRNAs represent potential biomarkers for kidney cancer., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: The discovery of metastasis markers in clear cell renal cell carcinoma is of critical importance to define individual metastatic risk and select patients for new targeted therapies. We identified potential biomarkers for metastatic clear cell renal cell carcinoma by gene expression analysis., Materials and Methods: We performed transcriptional profiling of 16 primary metastatic and 18 nonmetastatic clear cell renal cell carcinomas with PIQOR™ microarrays. Differentially expressed genes were validated by quantitative real-time polymerase chain reaction., Results: Genes discriminating between metastatic and nonmetastatic tumors were identified at q <0.001 by significance analysis of microarrays. The metastatic signature contained 127 transcripts. In metastatic samples a greater than 4-fold decrease in expression was detected for the genes CD151 and IKBA (t/F statistic p <0.0001) while the genes MMP16, B7-H1, BCL2L2 and FRA2 showed greater than 4-fold increase of expression in metastatic primary tumors (p <0.0001). Quantitative real-time polymerase chain reaction revealed significant differences in expression among all metastatic tumors, including synchronously and metachronously metastasized tumors, and nonmetastatic tumors for FRA2 (p = 0.032) and CD151 (p = 0.005). In addition, the genes B7-H1 (p = 0.040), FRA2 (p = 0.035), CD151 (p = 0.004) and BCL2L2 (p = 0.035) showed significantly higher expression in early metastasized than in nonmetastatic tumor samples. Different B7-H1 (p = 0.002) and BCL2L2 (p = 0.007) expression levels were found in samples with late metastasis compared to those in synchronously metastasized tumors., Conclusions: We determined a metastatic signature of clear cell renal cell carcinoma by microarray analysis. Our data provide the possibility of defining the metastatic potential of primary clear cell renal cell carcinoma based on a select number of genes even in a localized situation., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Managing oncocytoma in the setting of bilateral renal masses is a challenging scenario. Nevertheless, to our knowledge the pathological concordance of an oncocytic neoplasm in 1 kidney with tumors in the contralateral kidney is not known. We evaluated the influence of germline Birt-Hogg-Dubé mutation on concordance rates to assist in managing these cases., Materials and Methods: We reviewed the records of patients at the National Institutes of Health between 1983 and 2009 who had bilateral renal masses, known pathology bilaterally and oncocytoma or an oncocytic neoplasm in at least 1 kidney. Oncocytoma or an oncocytic neoplasm in 2 renal units was considered concordant. Demographic, pathological and clinical data were collected., Results: The population consisted of 40 patients, including 23 with and 17 without a diagnosis of Birt-Hogg-Dubé syndrome. Patients with the syndrome were younger (p <0.01) but there were no other differences between the 2 groups. However, patients with the syndrome had statistically lower histological concordance between bilateral masses than patients without the diagnosis (Fisher's exact test p <0.01). Also, the 8 patients without Birt-Hogg-Dubé syndrome who had multifocal renal masses showed 100% oncocytoma concordance between renal units., Conclusions: Of patients with bilateral renal masses those with Birt-Hogg-Dubé syndrome have significantly lower histological concordance than those without the syndrome. Patients with Birt-Hogg-Dubé syndrome should be monitored and treated differently than those without detected genetic mutations, especially patients with multifocal oncocytomas. Genetic testing for Birt-Hogg-Dubé should be considered in the treatment algorithm of patients with bilateral renal masses and known oncocytoma., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Translocation renal cell carcinomas represent a distinct clinicopathological entity. Studying the natural history, biological behavior and potential prognostic factors are crucially warranted., Materials and Methods: We selected 54 patients with renal cell carcinoma with positive nuclear transcription factor E3 and transcription factor EB expression from the Juvenile RCC Network. Recurrence-free survival and overall survival were assessed., Results: Median patient age was 24 years (range 1 to 64) and the male-to-female ratio was 1:1.4. At diagnosis 35 patients (65%) had local disease while 19 (35%) presented with distant metastases. The latter patients were older (median age 36 years) and predominantly male (male-to-female ratio 2) whereas the former group had a median age of 16 years and a male-to-female ratio of 1:2.5. Overall 36 patients underwent complete tumor resection and of these 8 had recurring cancer. On univariate analysis only lymph node involvement and American Joint Committee on Cancer stage were associated with poor recurrence-free survival. When stratified according to lymph node status age 25 years or older was found to predict relapse (p = 0.03). With a median followup of 19.2 months (range 1 to 58) 3-year overall survival was 14.3% in patients with distant metastasis and 70.6% in those without distant metastasis. Distant metastasis developed in the 2 patients with ASPSCR1-TFE3 fusion vs 1 of 11 with other fusion genes., Conclusions: Transcription factor E3 and transcription factor EB renal cell carcinoma display different clinical behavior according to gender and age. Lymph node involvement represents the only factor that predicts recurrence. ASPSCR1-TFE3 might be the most aggressive among the transcription factor E3 fusion genes., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Papillary renal cell carcinoma is characterized histologically by tumors with cells arranged in a papillary pattern. Typically the cells have a chromophilic appearance but areas may show cells with clear cytoplasm, similar to those in clear cell renal cell carcinoma., Materials and Methods: We re-reviewed the histological slides of 148 patients with papillary renal cell carcinoma who underwent nephrectomy for the presence of clear cells. Results were correlated with other pathological features, immunohistochemical expression of 22 protein markers, cytogenetic analysis and overall survival., Results: Papillary renal cell carcinoma with clear cells was identified in 57 patients (39%). Clear cells were associated with higher T classification and grade, vascular invasion and type 2 papillary renal cell carcinoma. On immunohistochemistry these tumors revealed higher expression of epithelial vascular endothelial growth factor receptor-2 than papillary renal cell carcinoma without clear cells. All papillary renal cell carcinomas with clear cells expressed α-methylacyl-coenzyme A racemase and 76% expressed cytokeratin 7. Six of 8 tumors (75%) with chromosome 3p loss had clear cell features. The presence of clear cells was retained as an independent prognostic factor on multivariate analysis. In cases of papillary renal cell carcinoma with clear cells the loss of 3p material and absent cytokeratin 7 expression were associated with a worse outcome., Conclusions: Papillary renal cell carcinoma with clear cells is a novel entity with a unique clinical, immunohistochemical and cytogenetic phenotype. The presence of clear cells is associated with aggressive pathological characteristics and poorer prognosis., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Vascular endothelial cell growth factor is studied in different malignant tumors as a key endothelial cell mitogen. Many single nucleotide polymorphisms in the VEGF gene have been described. We compared VEGF gene polymorphisms between a control group and a renal cancer group., Materials and Methods: This study was performed in 202 control, white, healthy blood donors (control group) and in 51 consecutive patients with renal cell carcinoma. We studied VEGF genotype polymorphisms at positions -2549, -460, -1154, +405 and +936 using polymerase chain restriction fragment length polymorphism, and looked for correlations with clinical data., Results: No association was found between VEGF gene polymorphism and renal cell carcinoma prognostic parameters. However, in contrast as observed for controls and other polymorphisms the patient group displayed a heterozygote excess (p = 0.0179, 35.9% more than that expected) at the -460 polymorphism. Comparing the control group and the renal cell carcinoma group we detected a significantly increased risk of renal cell carcinoma in subjects with the C-460T polymorphism. T carrier genotypes and the T allele increased the risk of renal cell carcinoma with an OR of 14.15 (95% CI 1.900-105.41, p = 0.0017) and 2.14 (95% CI 1.34-3.419, p = 0.0018), respectively. The genotype at the -2549 polymorphism exhibited a nonsignificant trend for increased risk but the D allele was significantly associated with increased risk (p = 0.0305)., Conclusions: Our results suggest that the -460 polymorphism is a risk factor for renal cancer. An individual screening test could be proposed for high risk populations., (Copyright © 2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Identifying tumor suppressor genes silenced by promoter CpG methylation uncovers mechanisms of tumorigenesis and identifies new epigenetic biomarkers for early cancer detection. DLEC1 is located at 3p22.3, a critical tumor suppressor gene locus for renal cell carcinoma. We explored its epigenetic alteration in renal cell carcinoma and possible clinicopathological association., Materials and Methods: We examined DLEC1 expression and methylation by semiquantitative reverse transcriptase and methylation specific polymerase chain reaction in 9 renal cell carcinoma cell lines and 81 primary tumors. We also analyzed the relationship between DLEC1 methylation and clinicopathological features in patients with renal cell carcinoma. We assessed DLEC1 inhibition of renal cell carcinoma cell growth by colony formation assay., Results: DLEC1 methylation and down-regulation were detected in all renal cell carcinoma cell lines. Treatment with 5-aza-2'-deoxycytidine (Sigma) and/or trichostatin A (Cayman Chemical, Ann Arbor, Michigan) reversed methylation and restored DLEC1 expression, indicating that methylation directly mediates its silencing. Aberrant methylation was further detected in 25 of 81 primary tumors (31%) but only 1 of 53 nonmalignant renal tissues (2%) showed methylation. DLEC1 methylation status was significantly associated with TNM classification and grade in patients with renal cell carcinoma (chi-square test p = 0.01 and 0.04, respectively). DLEC1 ectopic expression in silenced renal cell carcinoma cells resulted in substantial tumor cell clonogenicity inhibition., Conclusions: To our knowledge we report for the first time that DLEC1 is often down-regulated by CpG methylation and shows tumor inhibitory function in renal cell carcinoma cells, indicating its role as a tumor suppressor. DLEC1 tumor specific methylation may serve as a biomarker for early detection and prognosis prediction of this tumor., (Copyright (c) 2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Despite aggressive screening patients with hereditary renal cancers can present with large multifocal tumors. We present oncological outcomes in patients with hereditary renal cell carcinoma treated with partial nephrectomy for multifocal solid tumors with the largest lesion greater than 4 cm., Materials and Methods: Between 1995 and 2008 we identified 58 patients with hereditary renal cell carcinoma treated at our institution with partial nephrectomy for solid tumors greater than 4 cm. Data collected included demographic parameters, tumor size, pathological findings and laterality. Overall and metastasis-free survival was calculated based on information from the most recent followup evaluation and imaging., Results: The cohort included 41 patients (71%) with von Hippel-Lindau disease, 10 (17%) with Birt-Hogg-Dubé syndrome and 7 (11%) with hereditary papillary renal carcinoma. Mean age was 43.7 years (range 18 to 63) and mean largest tumor size was 5.3 cm (range 4 to 13). A mean of 6.4 kidney tumors (range 1 to 44) was resected. There was a predominance of nuclear grade 2 tumors (51 cases or 85%) and clear cell histology (44 or 73%), followed by papillary type I histology (7 or 11.7%). Overall and metastasis-free survival rates were 93% and 96.5%, respectively, at a median followup of 45 months (range 2 to 163)., Conclusions: Metastasis-free and overall survival of our patients is similar to that in the literature of those who undergo partial nephrectomy for T1B tumors in the sporadic population. Multifocality does not affect oncological outcomes at intermediate followup. Partial nephrectomy can be offered to patients with hereditary disease who present with multifocal tumors greater than 4 cm., (Copyright (c) 2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: miRNAs are small, nonprotein coding RNAs that are differentially expressed in many malignancies. We previously identified 80 miRNAs that are dysregulated in clear cell renal cell carcinoma. In this study we validated over expression of the miR-17-92 cluster in clear cell renal cell carcinoma and tested the effect of 2 members of this cluster (miR-17-5p and miR-20a) on tumor proliferation. We also elucidated the role of miRNA in clear cell renal cell carcinoma pathogenesis with bioinformatics., Materials and Methods: miRNA expression was validated by quantitative reverse transcriptase-polymerase chain reaction. The cell proliferation effect of miR-17-5p and miR-20a was tested in a renal adenocarcinoma cell line model. Multiple in silico analyses were done of dysregulated miRNAs., Results: We validated miR-71-92 cluster over expression in clear cell renal cell carcinoma by quantitative reverse transcriptase-polymerase chain reaction. Transfection of miR-20a inhibitor significantly decreased cell proliferation in a dose dependent manner. Transfection of miR-17-5p, which is not endogenously expressed in the ACHN cell line, led to increased cell proliferation compared to control values. This effect was suppressed by miR-17-5p inhibitor. Bioinformatics analysis identified 10 clusters of miRNAs dysregulated in clear cell renal cell carcinoma that followed the same expression patterns. We also identified matching patterns between reported chromosomal aberration in clear cell renal cell carcinoma and miRNA dysregulation for 37.5% of the miRNAs. Target prediction analysis was done using multiple algorithms. Many key molecules in clear cell renal cell carcinoma pathogenesis, including HIFs, mTOR, VEGF and VHL, were potential targets for dysregulated miRNAs., Conclusions: A significant number of dysregulated proteins in clear cell renal cell carcinoma are potential miRNA targets. Also, many clear cell renal cell carcinoma dysregulated miRNAs are phylogenetically conserved., (Copyright 2010 American Urological Association. Published by Elsevier Inc. All rights reserved.)

Purpose: We assessed CA9 single nucleotide polymorphisms and mutations, and their association with CAIX protein expression, overall survival and response to interleukin-2 in white patients with metastatic clear cell renal cell carcinoma., Materials Methods: Genomic DNA was extracted from frozen tumor samples of 54 metastatic clear cell renal cell carcinomas. The CA9 gene exons and flanking regions were amplified by polymerase chain reaction and sequenced. The monoclonal antibody M75 was used to evaluate CAIX protein expression in the primary tumor by immunohistochemistry., Results: CA9 reference single nucleotide polymorphisms rs2071676, rs12553173, rs3829078 and rs1048638 were found in 59%, 15%, 11% and 33% of patients, respectively. The deletion c.376del393 was observed in 2 patients. CAIX expression was greater than 85% in 65% of patients. No single nucleotide polymorphisms were significantly associated with CAIX expression. Patients with the C allele variant of rs12553173 had improved median survival (27.3 vs 13.6 months, p = 0.0431) and a greater likelihood of an interleukin-2 response (57% vs 22%, p = 0.081) Likewise high CAIX expression was associated with longer median survival (25.5 vs 8.5 months, p <0.0001) and a greater interleukin-2 response rate (37% vs 8%, p = 0.070). In a multivariate Cox model the C allele variant of CA9 single nucleotide polymorphism rs12553173 and CAIX expression were retained as independent prognostic factors., Conclusions: CA9 single nucleotide polymorphisms are common in patients with metastatic clear cell renal cell carcinoma. The synonymous C allele variant of rs12553173 may be associated with improved overall survival and a greater likelihood of a response to interleukin-2. CA9 rs12553173 and CAIX are independent prognostic factors of overall survival and complementary for predicting the prognosis of metastatic clear cell renal cell carcinoma.