Eur Urol Suppl 2009;8(4):298 712 vHL Gene aLTeRaTIOnS pROMOTe canceR pROGReSSIOn In eaRLY-STaGe cLeaR ceLL Rcc THROUGH nUcLeaR localizzation oF hypoxia-inducible Factor-1฀ Minervini A. 1 , Di Cristofano C. 2 , Lessi F. 3 , Menicagli M. 4 , Lapini A. 5 , Masieri L. 5 , Lanzi F. 5 , Tuccio A. 5 , Siena G. 6 , Bevilacqua G. 7 , Serni G. 6 , Minervini R. 8 , Carini M. 6 1 University of Florence, Dept. of Urology, Florence, Italy, 2 I.C.O.T, Dept. of Experimental Medicine, Latina, Italy, 3 Division of Surgical, Molecular and Ultrastructural Pathology, Dept. of Oncology, Pisa, Italy, 4 Mgm, Dept. of Molecular Genetics and Medicine, Pisa, Italy, 5 Clinic of Urology, Dept. of Urology, Florence, Italy, 6 Clinic of Urology, Dept. of Urology, Florence, Italy, 7 Division of Surgical, Molecular and Ultrastructural Pathology, Dept. of Oncology, Pisa, Italy, 8 University of Pisa and University Hospital of Pisa, Dept. of Surgery, Division of Urology, Pisa, Italy Introduction & Objectives: Von Hipple-Lindau gene (VHL) inactivation represents the most frequent abnormality in clear cell RCC. VHL encodes two diferent proteins: VHL30 and VHL19 arising as a result of an alternative translation initiation. Hypoxia-inducible factor-1 (HIF-1) is a heterodimer composed of a α and ß subunit. HIF-1ß is constitutively expressed, while HIF-1α expression is regulated by O2 level. In normal O2 conditions VHL binds HIF- 1α, after hydroxylation in ODD (oxygen-dependent degradation domain) and it allows HIF-1α proteasomal degradation. In this domain a SNP at codon 582 is located. In hypoxia, VHL/ HIF-1α interaction is abolished and HIF-1α activates target genes in the nucleus. The study analyzes the VHL and codon 582 of HIF-1α gene (HIF-1α) status in clear cell RCC, correlating genetic alterations, protein expression and subcellular localization with prognosis. Material & Methods: Tissues microarray from 136 intracapsular clear cell RCC cases were constructed and two anti-pVHL Ab (Ig32 and Ig33) and a polyclonal anti-HIF-1α Ab were used for IHC analysis. Mean follow up was 117 months. It was performed mutational analysis for the whole VHL and for HIF-1α SNP (C1772T), promoter methylation of VHL and LOH (3p25). The results were correlated with survival (TSS). Results: VHL gene alteration was found in 57% of cases. VHL mutations, methylation and LOH were detected in 51%, 11% and 17% cases. Patients with biallelic VHL alteration had a shorter TSS (p=0.01). We found a statistical association between biallelic VHL alteration and no pVHL expression (p=0.004) and nuclear localization of HIF-1α (p=0,04). The SNP was associated only with a cytoplasmic localization of HIF-1α (p=0.007). pVHL negativity and HIF-1α nuclear positivity were correlated with a short TSS. conclusions: VHL and HIF-1α alterations infuence their protein expression and localization. Biallelic VHL gene alterations with loss of pVHL expression are associated to nuclear localization of HIF1-a and poor prognosis in early-stage clear cell RCC. Alteration of pVHL/HIF-1α pathway is an early event in clear cell RCC carcinogenesis and it is involved in cancer progression through nuclear localizzation of HIF-1α. 711 THe pROGnOSIc IMpORTance OF anGIOGenIc pHenOTYpe In RenaL ceLL caRcInOMa Bigot P. 1 , Edeline J. 2 , Vigneau C. 3 , Martin B. 4 , Jouan F. 4 , Denis M. 4 , Patard J.J. 1 , Rioux-Leclercq N. 1 1 Rennes University Hospital, Dept. of Urology, Rennes, France, 2 Rennes University Hospital, Dept. of Pathology, Rennes, France, 3 Rennes University Hospital, Dept. of Nephrology, Rennes, France, 4 Cnrs Umr6061 Genetics and Development, Ifr 140, University of Rennes 1, Rennes, France Introduction & Objectives: To assess the prognostic value of neoangiogenesis architecture along with its relationship with the VHL/VEGF pathway in a prospective series of patients with renal cell carcinoma (RCC). Material & Methods: 75 RCC patients who underwent a radical nephrectomy at our institution were prospectively included in the trial. Neoangiogenesis was analysed through its histological architecture (type 1 with mature aspect versus type 2 with immature aspect) and to microscopic vascular density (MVD). Intratumoral expressions of VEGF-A, CAIX and pVHL protein were assessed by immunohistochimical analysis. Abnormalities of the VHL gene (mutations, deletions, promoter hypermethylation) were analysed on frozen samples by PCR/sequencing techniques and MLPA. Results: Immature tumoral angiogenesis (type 2) was signifcantly associated to higher Fuhrman grades (p=0.0001), more advanced T stages (p=0.001), nodal invasion (p=0.03), presence of distant metastases (p=0.002), low CAIX expression (p=0,002), higher pVHL (p=0.001) and VEGF-A expressions (p=0.0001). Type 2 neoangiogenesis phenotype was also associated with low MVD (37.4 +/- 41.04 in type 2 vs. 10+/-27.5 in type 1, p=0.0001). Progression free survival time was signifcantly reduced in the immature RCC group (type 2) (p=0.003). Tumours with defective VHL gene were more likely to be associated with type 1 neoangiogenesis phenotype thus exhibiting good prognosis (p=0.01). conclusions: Two neoangiogenesis tumoral phenotypes seem to exist in RCC: a mature one carrying out a good prognosis, and an immature one driving a poor outcome. The neoangiogenesis phenotype is dependant on the VHL/VEGF pathway. It may be useful to assess neoangigenesis tumoral architecture as a predictive factor for antiangiogenic treatments efcacy. 709 THe pROGnOSTIc vaLUe OF TISSUe BIOMaRKeRS ReLaTed TO HYpOxIa-IndUced paTHWaY In RenaL ceLL caRcInOMa Ortiz F. 1 , Hardisson D. 2 , Blasco A. 1 , Hontoria J. 1 , Fernández C. 3 , Bajo A.M. 4 , Prieto J.C. 4 , Sánchez-Chapado M. 1 1 Alcalá University and Principe De Asturias Hospital, Dept. of Surgery and Urology, Alcala De Henares, Spain, 2 Autonoma University and La Paz Hospital, Dept. of Pathology, Madrid, Spain, 3 Complutense University, Dept. of Biostadistic, Madrid, Spain, 4 Alcalá University, Dept. of Biochemistry and Molecular Biology, Alcala De Henares, Spain Introduction & Objectives: Several inmunohistochemical markers have been suggested to help to improve the value of the classic prognostic factors. The aim of this study is to know the prognostic value of some of these markers related to hypoxia-induced pathway in renal cell carcinoma: vascular endothelial growth factor (VEGF), carbonic anhydrase IX (CAIX), P-AKT and phosphatase and tensin homologue delated on chromosome 10 (PTEN). Material & Methods: We analyzed 148 samples of renal cell carcinomas (109 clear cell carcinomas, 22 papillary carcinomas, 13 cromophobe carcinomas and 6 multilocular cystic renal cell carcinoma) in patients that underwent radical or nephron-sparing nephrectomy with curative intention. The median follow-up time was 65,3 months. We determined the percentage of cells and the intensity of expression of P-AKT and PTEN, and the expression of cytoplasmic PTEN, CAIX and VEGF with inmunohistochemical techniques. We have studied the survival relating it with the presence or absence of markers expression and their intensity. Results: In our study, the survival of the tumors that express VEGF is worse with regard to the ones that do not express it (Breslow p=0.012); with HR=2.89 (p=0.035) for tumors with weak expression, and HR=6.00 (p=0.040) and HR=1.10 for tumors with moderate and intense expression respectively. We do not have diferences in the evolution in tumors that express or not CAIX (Breslow p= 0,5). We have not found diferences as far as the evolution of the tumors stratifying by the percentage of cells (<25%, 25-50%, 51-75%, >75%) that express PTEN (Breslow p=0.963). We have not either found diferences in relation to the intensity of expression (weak, moderate or intense) (Breslow p=095). Diferences do not exist according to the expression of citoplasmic PTEN with respect to which they do not express it (Breslow p=0.131). We did not fnd diferences in the evolution either if we observed the number of cells that they express P-AKT (<25%, 25-50%, 51-75%, >75%) in relation to that they did not express it (Breslow p=0.085). On the contrary, the tumors that express P-AKT intensely have worse prognosis than those that do it weakly or they don´t do it (HR=3.48, p=0.024 versus HR= 2,07, p=0.074). conclusions: In our study, only the inmunohistochemical expression of VEGF and the intense expression of P-AKT can be related to a worse prognosis in renal cell carcinoma. More studies are required to determine the fnal value of these biomarkers. 710 BIOMaRKeRS OF SUnITnIB cLInIcaL ReSpOnSe In MeTaSTaTIc RenaL cLeaR ceLL caRcInOMa paTIenTS Bastien L. 1 , Paule B. 1 , Maille P. 1 , Azoulay S. 2 , Allory Y. 2 , Patard J.J. 3 , Rioux-Leclercq N. 4 , Crépel M. 3 , Wallerand H. 5 , Bernhard J.C. 5 , Game X. 6 , Mazerolles C. 7 , Guillotreau J. 6 , Rufon A. 8 , Decaussin-Petrucci M. 9 , Zini L. 10 , Leroy X. 11 , Berthon N. 10 , Culine S. 12 , Salomon L. 1 , Hoznek A. 1 , Vordos D. 1 , Abbou C.C. 1 , Mourah S. 1 , De La Taille A. 1 1 Hôpital Henri Mondor, Dept. of Urology, Creteil, France, 2 Hôpital Henri Mondor, Dept. of Anatomopathology, Creteil, France, 3 Centre Hospitalier Universitaire, Dept. of Urology, Rennes, France, 4 Centre Hospitalier Universitaire, Dept. of Anatomopathology, Rennes, France, 5 Centre Hospitalier Universitaire, Dept. of Urology, Bordeaux, France, 6 Hôpital De Rangueil, Dept. of Urology, Toulouse, France, 7 Hôpital De Rangueil, Dept. of Anatomopathology, Toulouse, France, 8 Centre Hospitalier Lyon Sud, Dept. of Urology, Lyons, France, 9 Centre Hospitalier Lyon Sud, Dept. of Anatomopathology, Lyons, France, 10 Centre Hospitalier Régional Universitaire, Dept. of Urology, Lille, France, 11 Centre Hospitalier Régional Universitaire, Dept. of Anatomopathology, Lille, France, 12 Hôpital Henri Mondor, Dept. of Oncology, Creteil, France Introduction & Objectives: Sunitinib inhibits VEGF pathway by targeting principally VEGF-R and PDGF-R. According to previously published studies, Sunitinib objective response rate is 47% but there is a lack of clinical or biological markers of clinical response. The goal of this study is to evaluate VEGF pathway according to the clinical response. Material & Methods: Primitive tumors of patients with metastatic renal cell carcinoma treated by sunitinib were included retrospectively from 6 diferent centers between 2005 and 2007. According to the RECIST criteria, objective response had been evaluated after 3 cycles of treatment. Immunohistochemistry with specifc antibodies against VEGF pathway markers (VEGF, VEGF-R, HIF-1a, PDGF-Rb, pKDR and CA IX) on Tissue Micro Array was performed in order to compare protein expression with tumor response. Staining intensity, percentage of positive cells and endothelial staining were evaluated. For VEGF, staining between the center and the front of the tumor was also analyzed. Results: 64 patients presenting metastatic renal cell carcinoma treated by sunitinib were studied. Objective response rate was 39% and was not related to MSKCC prognosis group, nor TNM classifcation or Führman grade. VEGF expression (intensity staining or percentage of positive cells) and other markers were not correlated with clinical response. However, surexpression of VEGF in the front compared to the center was related to a better response (p=0.015) with an OR= 9.3 [1.75-49.59]. Using this staining, 77% of the response could be predicted: if VEGF diference was observed, 80% of patients had clinical response. But if VEGF surexpression was absent 70% of the patients had no clinical response. conclusions: This study suggested that surexpression of VEGF in the front of metastatic renal cell carcinoma compared to the center using a simple method of immunohistochemistry could be a predictive factor of response to sunitinib. Further studies with a high number of patients are mandatory to confrm our results.