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METHODS: 48 institutionally-banked patient RCC tissue sam-ples underwent genomic DNA extraction, bisulfite DNA conversion,PCR, and T7 polymerase RNase A site-specific cleavage in preparationfor MALDI-TOF MS analysis. DNA samples also underwent Sangersequencing and methylation-specific PCR analysis at all three exons ofVHL and the VHL promoter site respectively. 0% and 100% methylatedhuman lymphocytic DNA, non-template dH2O, and 2 human RCC celllines (UOK121, UOK171) were used as assay controls. Samples werecalled hypermethylated if at least 1 CpG site exhibited greater than 50%methylation.

RESULTS: 43 patient samples were read by our exon 1 assay;9 had methylated VHL exon 1 sites, with 3 showing hypermethylation atexon 1. Only 1 sample was hypermethylated at both exon 1 and at theVHL promoter site. Specificity and sensitivity of the exon 1 methylationassay were both 100% when tested on RCC cell line controls. Sampleswith exon 1 hypermethylation did not exhibit exonic mutations. Allsamples assayed at VHL exon 2 were hypermethylated without regardto tumor stage, grade, or metastasis.

CONCLUSIONS: Using MALDI-TOF MS to assay RCC tumorsfor VHL methylation has increased specificity and sensitivitycompared to other methylation analysis techniques. Furthermore, wehave developed a successful MALDI-TOF MS-based VHL methyl-ation assay to detect VHL methylation in tumor tissue. From our re-sults, it appears that VHL exon 1 methylation is an alternative toexonic mutations and promoter methylation in VHL silencing. A high-throughput assay in large clinical studies is essential to understand-ing its role in the surveillance and possible detection of renalcell carcinoma.

Source of Funding: Emory University School of MedicineDepartment of Urology Departmental Funding, Evan CountyCares Foundation

MP23-16TCEB1-MUTATED RENAL CELL CARCINOMA: A DISTINCTGENOMIC AND MORPHOLOGIC SUBTYPE

A Ari Hakimi*, New York, NY; Yusuke Sato, Teppei Morikawa, Tokyo,Japan; Haruki Kume, Tokyo, Japan; Masashi Fukayama,Yukio Homma, Tokyo, Japan; Ying Bei Chen, Roy Mano, Paul Russo,New York, NY; Seishi Ogawa, Kyoto, Japan; James Hsieh,Victor Reuter, Satish Tickoo, New York, NY

INTRODUCTION AND OBJECTIVES: Recent integratedsequencing analysis identified a group of renal cell carcinoma (RCC)tumors characterized by hotspot mutations in TCEB1 (a gene thatcontributes to the VHL complex to ubiquitinate Hypoxia Inducible Factor(HIF)). We analyzed these tumors along with an expanded cohort toassess whether these tumors should be considered a distinct entityfrom clear cell renal cell carcinoma (ccRCC).

METHODS: A total of 12 tumors from three distinct cohorts(Sato et al, TCGA and MSKCC) were available for analysis. All tumorswere characterized by recurrent hotspot mutations in TCEB1 Y79C/S/F/N or A100P. Morphologic characteristics of the tumors H&E wereassessed by an expert genitourinary pathologist. Clinical and pathologicvariables, copy number alterations and mutations were assessed usingpublically available data.

RESULTS: TCEB1-mutated tumors all shared unique anddistinct morphology as illustrated; thick fibromuscular bands transectingthe tumor (star), pure clear cell cytology frequently with cells showingvoluminous cytoplasm, ccRCC-like acinar areas associated withinfolding tubular and focally papillary architecture (Figure1). All tumorswere VHL and PBRM1 wild type and contained distinct copy numberprofiles including loss of heterozygosity of chromosome 8 (whereTCEB1 resides). All tumors also lacked the ccRCC signature 3p loss.The majority of the tumors were low grade (11/12) and no metastaseswere detected at last followup.

CONCLUSIONS: TCEB1-mutated RCC is a distinct entity withrecurrent hotspot mutations, specific copy number alterations andunqiue morphologic characteristics. Further and expanded clinical

followup is needed to determine whether these tumors are more indo-lent compared to conventional ccRCC.

Source of Funding: This work has been supported by thePaula Moss Trust for the research into the cure and treatmentof kidney cancer (Hsieh), the Sidney Kimmel Center forProstate and Urologic Cancers, by funds provided by DavidH. Koch through the Prostate Cancer Foundation, theNational Cancer Institute T32 CA082088-12 training grant(Hakimi), and the Stephen P Hanson Family Fund Fellowshipin Kidney Cancer (Hakimi).

MP23-17BELLINI DUCT CARCINOMAS HAVE A DIFFERENT GENETICSIGNATURE COMPARED TO UROTHELIAL CARCINOMAS OF THEUPPER URINARY TRACT

Volker Jung*, Frank Becker, Martin Parr, Homburg/Saar, Germany;Arndt Hartmann, Erlangen, Germany; Susanne F€ussel, Dresden, Ger-many; Rainer Grobholz, Aarau, Switzerland; Marieta Toma, Dresden,Germany; Bernd Wullich, Erlangen, Germany; Arne Strauss,Carl Ludwig Behnes, G€ottingen, Germany; Wolfgang Otto, Regensburg,Germany; Michael St€ockle, Kerstin Junker, Homburg/Saar, Germany

INTRODUCTION AND OBJECTIVES: Bellini duct carcinomaor collecting duct carcinoma (CDC) is a rare renal neoplasm that isassociated with poor prognosis given its highly aggressive courseand limited response to immuno- or chemotherapy. Histologically,CDC is defined as a subtype of renal cell carcinomas, but in somecases, it is difficult to differentiate from urothelial carcinomas (UC).Therefore the aim of this study was to determine genetic alterations ofCDC in comparison to that of urothelial carcinomas of the upperurinary tract (UUT-UC) to clarify the histological origin of this raretumor entity.

METHODS: Twenty-nine CDC samples were obtained fromseven different German centers and compared with twenty-six urothelialcarcinomas of the upper urinary tract. Comparative genomic hybridi-zation (CGH) was used to investigate the genetic composition of pa-tients’ tumors and allowed the detection of losses and gains of DNAcopy numbers throughout the entire genome. The clinical data werecorrelated with CGH results.

RESULTS: CGH analysis of CDC revealed DNA aberrations inmany chromosomes. DNA losses were more frequently observed thangains, while high-level amplifications were not detected. The meanfrequency of CDC chromosomal aberrations (4.9/case) was slightlylower than that in UUT-UC (5.4/case). Recurrent CDC DNA lossesoccurred at 8p (n¼9/29), 16p (9/29), 1p (n¼7/29) and 9p (n¼7/29), andgains occurred in 13q (n¼9/29). In contrast to CDC, the most frequentlydetected UUT-UC DNA aberration was a loss at 9q (n¼13/26). DNAlosses at 9q, 13q and 8q as well as gains at 8p showed significantvariations in UUT-UC compared to CDC. There was no correlation

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between the patients’ clinical course and the presence or absence ofthese recurrent genetic alterations.

CONCLUSIONS: For the first time we have shown that DBCsare characterized by a different genetic pattern compared to UUT uro-thelial carcinomas. Regarding also the published data on renal cellcarcinoma, we conclude that CDC seems to be an unique entity ofkidney carcinomas.

Source of Funding: none

MP23-18FUNCTIONAL ROLE OF BONE MORPHOGENETIC PROTEIN 2 ASTUMOR SUPPRESSOR IS INACTIVATED BY PROMOTER CPGMETHYLATION IN HUMAN RENAL CELL CARCINOMA

Yozo Mitsui*, Miho Hiraki, Kohei Ogawa, Taichi Nagami, Haruki Anjiki,Chiaki Koike, Naoko Arichi, Shigenobu Nakamura, Takeo Hiraoka,Masahiro Sumura, Hiroaki Yasumoto, Hiroaki Shiina, Izumo, Japan

INTRODUCTION AND OBJECTIVES: Bone morphogeneticprotein (BMP)-2, a member of the transforming growth factor b super-family, has been shown to act as a tumor suppressor in several cancersby activating signaling cascades that cause cell cycle arrest. In addition,the expression level of BMP-2 is downregulated because of promoterCpG hypermethylation. We hypothesized that impaired regulation ofBMP-2 through epigenetic pathways is associated with the pathogen-esis of renal cell carcinoma.

METHODS: To test this hypothesis, CpG methylation of theBMP-2 gene was analyzed in 3 renal tumor cell lines and 96 renal tumorand matched normal renal tissue. Promoter CpG methylation wasanalyzed by methylation specific PCR using bisulfite-modified DNA astemplate. BMP-2 expression was measured by quantitative RT-PCR.

RESULTS: BMP-2 mRNA expression was significantlyenhanced after 5-aza-2€uf-deoxycytidine treatment in renal tumor celllines. The prevalence of BMP-2 promoter methylation was significantlyhigher and BMP-2 mRNA expression was significantly lower in renaltumor samples than in normal kidney samples (p<0.0001, p¼0.014,respectively). Furthermore, a significant correlation was found betweenBMP-2 promoter methylation and mRNA transcription in the tumors(p<0.001). A significant association of BMP-2 methylation was foundwith pathologic findings showing pT category and infiltrative growthpattern (p¼0.034). When the expression level of the BMP-2 mRNAtranscript was divided into 2 groups based on the mean value, a lowerexpression was significantly associated with overall survival (OS) after aradical nephrectomy (p¼0.004). Although both promoter CpG methyl-ation and BMP-2 mRNA transcript level were significantly associatedwith OS in univariate analysis, BMP-2 mRNA expression was shown tobe a more significantly independent predictor for OS than methylationstatus in multivariate analysis.

CONCLUSIONS: This is the first clinical study of inactivation ofthe BMP-2 gene via epigenetic pathways in renal cell carcinoma.Aberrant BMP-2 methylation and the resultant loss of BMP-2 expres-sion might become useful as a molecular marker for prediction of OSafter a radical nephrectomy.

Source of Funding: none

MP23-19EZH2 CONTRIBUTES TO PROLIFERATION AND METASTASIS INRENAL CELL CARCINOMA CELLS

Dong Zhang*, Xiaojie Yang, Hongliang Li, Tie Chong, Xi’an, China,People’s Republic of

INTRODUCTION AND OBJECTIVES: The enhancer of zesthomolog 2 (EZH2) gene has been recognized as a proto-oncogene andlinked to human malignancies. Here, we determined potential role ofEZH2 in prosurvival and metastasis of renal cell carcinoma (RCC) andits possible mechanisms.

METHODS: EZH2-overexpressing vector and siRNA for EZH2were transfected into 786-O and ACHN cells respectively. Growth ratewas determined by MTT. Cell cycle was detected by flow cytometryfollowing EZH2 alteration. Wound healing and Transwell assay wereused to determine migration and invasive potential in response to EZH2change. Real-time PCR and Western blot was used to determinealteration of MMPs and TIMPs.

RESULTS: EZH2 overexpressing promoted cell growth in 786-O cells, whereas EZH2 knockdown inhibit cell growth in ACHN cells.EZH2 stimulated 786-O cells to enter S phase, however, ACHN cellswere arrested in G1/S phase following EZH2 knockdown. In addition,EZH2 elevated migration and invasion potential in 786-O cells, andsuppress of EZH2 resulted in inhibition of migratory and invasivecapability in ACHN cells. Mechanically, EZH2 could repress TIMP-1activity rather than TIMP-2 or -3, furthermore, inhibition of EZH2markedly reduced the proteolytic activity of MMP-9, whereas MMP-2activity was found not to be changed significantly.

CONCLUSIONS: EZH2 plays a critical role in proliferation inRCC cells. In addition, EZH2 can enhance both migratory and invasivepotential of RCC cells in vitro. EZH2 plays an active role in the upre-gulation of MMP activity, downregulaion of TIMP, and subsequent in-vasion of RCC cells. Facilitating of invasion makes EZH2 a promisingtarget for management of RCC metastasis.

Source of Funding: The National Natural Science Foundationof China (NSFC No.81101937)

MP23-20INTEGRIN SIGNALING POTENTIATES TRANSFORMING GROWTHFACTOR-BETA 1 (TGF-B1) DEPENDENT DOWN-REGULATION OFE-CADHERIN EXPRESSION e IMPORTANT IMPLICATIONS FOREPITHELIAL TO MESENCHYMAL TRANSITION (EMT) IN RENALCELL CARCINOMA

Yury Rapoport, Boris Feldkoren, Wen-Xiu Zhang, Vitaly Margulis*,Dallas, TX

INTRODUCTION AND OBJECTIVES: Signal transductionthrough transforming growth factor-beta 1 (TGF-b1) pathway affectsepithelial to mesenchymal transition (EMT), partly by modulation of E-Cadherin expression. Concurrent impact of extracellular matrix drivenactivation of integrin signaling on evolution of EMT has not been wellcharacterized. We assess cumulative effect and molecular mechanismsof TGF-b1 and integrin signal transduction on E-Cadherin expression inrenal cell cancer (RCC).

METHODS: TGF-b1 driven alteration of EMT specific markerswas confirmed in an established in-vitro model of RCC. Globalchanges in expression of cell surface integrins were evaluated bymicroarray and confirmed by RT-PCR. Arginylglycylaspartic acid(RGD), in addition to TGF-b1, was utilized to mimic integrin signalingand evaluate cumulative effects on markers of EMT. Silencing of po-tential mediators of cumulative action of RGD and TGF-b1 was carriedout by small interfering RNA and confirmed by Western blotting orRT-PCR.

RESULTS: After stimulation of RCC cells with TGF-b1, a three-fold increased expression of integrin aVb3 was identified by microarrayand confirmed by RT-PCR. Pre-treatment of cells with RGD in additionto TGF-b1 demonstrated a significantly higher effect on expression ofmarkers of EMT (E-cadherin and Snail-1) than either ligand alone. Si-RNA mediated silencing of FAK and PINCH, independently andconclusively abrogated the cumulative effect of RGD and TGF-b1 onmarkers of EMT.

CONCLUSIONS: We have identified a novel mechanismthrough which extracellular matrix event transduction by integrinsfurther augments TGF-b1 related effects on the well establishedmarkers of EMT. Molecular machinery involved in integrin/TGF-b1interplay serves as an attractive therapeutic target in RCC.

Source of Funding: none