Vol. 189, No. 4S, Supplement, Sunday, May 5, 2013 THE JOURNAL OF UROLOGY姞 e249 pathologically proven tumor other than RCC and 50 patients with metastasis, 1,091 patients with a mean age of 54 years were finally included in the analysis. Clear cell, papillary, chromophobe, and other types of RCC occurred in 962 (88.8%), 80 (7.4%), 31 (2.9%), and 10 (0.8%), respectively, of the 1,091 study patients (Table). PDGFR-␤ expression was the highest in clear cell RCC, however VEGF and PDGF-␤ expression was the highest in papillary RCC. VEGF staining showed higher expression with the increasing T or N stage. After adjusting the T stage and Furhman nuclear grade using multivariate logistic regression analysis, papillary RCC showed significantly stronger expression of VEGF (OR⫽3.57, 95% CI 1.74 –7.32, p⬍0.001), VEGFR2 (OR⫽1.82, 95% CI 1.11–2.99, p⫽0.017), and PDGF-␤ (OR⫽2.46, 95% CI 1.49 – 4.06, p⫽0.019) compared to clear cell RCC. CONCLUSIONS: In this large series of immunohistochemical assay, we demonstrated different cytoplasmic expression of VEGF, VEGFR2, PDGF-␤, and PDGFR-␤ in tumor cell according to the pathologic stage and the cell type of RCC. It is noteworthy that VEGF and PDGF-␤ expression was the highest in papillary RCC, not in clear cell RCC. P value Strong PDGFR␤ Weak PDGFR␤ P value Strong PDGF Weak PDGF P value Strong VEGFR2 Weak VEGFR2 Weak VEGF P value Strong VEGF Table VEGF, VEGFR2, PDGF-␤, PDGFR-␤ expression according to cell types Cell type, n (%) 0.166 ⬍0.001 0.002 ⬍0.001 340 622 532 430 747 215 646 316 (35.3) (34.7) (55.3) (44.7) (77.7) (22.3) (67.2) (32.8) 9 71 34 46 44 36 58 22 Papillary (11.3) (88.8) (42.5) (54.5) (55.0) (45.0) (72.5) (27.5) 14 17 16 15 22 9 28 3 Chromophobe (45.2) (54.8) (51.6) (48.4) (71.0) (29.0) (90.3) (9.7) 1 9 5 5 8 2 10 0 Others (10.0) (90.0) (50.0) (50.0) (80.0) (20.0) (100.0) (0.0) Clear cell VEGF⫽vascular endothelial cell growth factor; VEGFR2⫽vascular endothelial cell growth factor receptor 2; PDGF-␤;⫽platelet-derived growth factor-␤; PDGFR␤⫽platelet-derived growth factor-␤ receptor Source of Funding: none Source of Funding: none 609 VEGF/VEGFR2 OR PDGF-␤/PDGFR-␤ EXPRESSION IN NON-METASTATIC, RENAL CELL CARCINOMA: A PROSPECTIVE STUDY WITH 1,091 CONSECUTIVE CASES Sang Hoon Song*, In Gab Jeong, Dalsan You, Jun Hyuk Hong, Bum Sik Hong, Cheryn Song, Seoul, Korea, Republic of; Jae Young Joung, New Brunswick, NJ; Kyung Hyun Moon, Ulsan, Korea, Republic of; Young Mi Cho, Hanjong Ahn, Choung-Soo Kim, Seoul, Korea, Republic of INTRODUCTION AND OBJECTIVES: We investigated the correlation between the expression of the vascular endothelial cell growth factor (VEGF), the platelet-derived growth factor-␤ (PDGF-␤), their receptors (VEGFR2, PDGFR-␤), and the pathologic stage or cell type in non-metastatic, renal cell carcinoma (RCC). METHODS: A prospective, observational study was conducted to assess the VEGF, VEGFR2, PDGF-␤, and PDGFR-␤ protein expression by immunohistochemical assay in tumor tissue samples obtained from surgical specimens after radical or partial nephrectomy performed at our center between January 2008 and March 2012. The intensity of these proteins were quantified by a single pathologist and rated on a scale of 0 to 3. We dichotomized the expression level as weak (scale 0-1) or strong (scale 2-3). The correlation between the expression of these proteins and the oncologic parameters, especially the pathologic classification according to the cell type of RCC, was analyzed. RESULTS: A total of 1,423 patients diagnosed with RCC underwent surgery during the study period. Excluding 292 patients with 610 DEVELOPMENT OF A TEST FOR RCC BY DETECTION OF VHL MUTATIONS IN TISSUE AND FLUIDS OF PATIENTS WITH RENAL CELL CARCINOMA Shilpa Sreedharan*, Rebecca S Arnold, Kimberly Kerley, Kristina B Mercer, Kerry Ressler, Viraj A Master, Kenneth Ogan, David L Roberts, John G Pattaras, John A Petros, Atlanta, GA INTRODUCTION AND OBJECTIVES: Surgery is the only highly effective therapy for renal cell carcinoma (RCC) and there is no effective screening or blood test for RCC. Somatic changes in the von Hippel-Lindau gene (VHL) are found in approximately 80% of clear cell RCC. Cancer cells and/or DNA may be recovered from body fluids of patients with RCC in nearly every case. The objective of this research is to develop a blood and/or urine test for RCC based upon the detection of small amounts of circulating tumor DNA characterized by the somatic mutations in VHL. METHODS: DNA from archived patient tissue and fluids was used to compile a panel of acquired VHL polymorphisms for use in a diagnostic urine or serum test identifying circulating RCC cells. The sensitivities of methods capable of such identification were contrasted to more easily detect mutations present in early stages of RCC at a time when they can be surgically cured. DNA from 50 archived renal cancer patient tissue, serum, and urine was purified, amplified, and sequenced using an ABI3100 DNA Sequencer. The mutations detected were confirmed with restriction fragment length polymorphism (RFLP), when possible. The sensitivity of RFLP was contrasted with Sequenom’s© ability to detect minute amounts of mutated DNA in urine and serum via a single base extension reaction and mass spectrometry. RESULTS: Sequencing of VHL revealed the presence of mutations which subsequently created alterations in restriction enzyme e250 THE JOURNAL OF UROLOGY姞 digestion. After analysis of the commonly mutated exon regions of VHL for 50 patients, 78% were found to have mutations. 17% of those mutations occurred in multiple patients. DNA from serum and/or urine from these patients was then examined for the identified mutations. 27% of patients had mutations detected in the DNA purified from their serum and/or urine. Contrasting the sensitivity of mutation detection via RFLP of body fluids versus detection via Sequenom indicated that Sequenom was able to identify mutations present in DNA as dilute as 0.05ng, compared to 12.5ng DNA required for RFLP detection. CONCLUSIONS: A panel of the most highly observed VHL polymorphism DNA biomarkers is a potential diagnostic tool for the detection of early stage RCC. High-throughput identification methods of such mutations, such as mass spectrometry, have an increased sensitivity and specificity compared to PCR/RFLP analysis and would therefore better translate into clinical application. VHL mutations identified in DNA purified from patient urine or serum via RFLP and mass spectrometry support the feasibility of developing such a test. Source of Funding: Institutional 611 MICRORNA AS NOVEL BLOOD-BASED BIOMARKERS IN CLEAR CELL RENAL CELL CARCINOMA A Ari Hakimi*, Anders Jacobsen, Nina Mikkilineni, Brandon Fiegoli, Sara Blass, Yevgeniy Grigoryev, Agnes Viale, Nicholas Socci, Martin H Voss, Robert Motzer, Victor E Reuter, Jonathan Coleman, Paul Russo, James J Hsieh, New York, NY INTRODUCTION AND OBJECTIVES: MicroRNAs (miRNA) are short, non-coding RNAs involved in post-transcriptional gene regulation. Several reports have assessed their role as blood based biomarkers given their tissue and cancer-specific expression. Using an integrative approach we sequenced the miRNA transcriptome of the plasma of several clear cell renal cell carcinoma (ccRCC) patients both before and after surgery as well as several controls. METHODS: We performed next generation miRNA sequencing (miRNAseq) on eight pairs (pre and postoperative plasma samples) and four non-cancer controls to identify potential biomarker candidates. We further integrated our data with the miRNAseq tumor data from the Cancer Genome Atlas (TCGA) study to determine whether plasma miRNA levels are representative of tumor miRNA expression in ccRCC. RESULTS: Overall, 930 unique miRNAs were detected, including 272 at greater than 10 read counts. There was a global shift of miRNA expression toward the non-cancer controls in the postoperative samples compared to preoperative. We further identified several stably expressed miRNAs across all samples and controls including miR-16, miR-191, and miR-103. We also identified several potential biomarker candidates by looking at differential expression both in terms of preoperative and postoperative status, as well as tumor vs control including miR-378 and miR-660. Intriguingly, the plasma miRNA expression patterns showed no relationship to the tumor expression patterns using the TCGA samples. CONCLUSIONS: Plasma miRNA expression patterns are consistently altered in ccRCC and, following surgery, globally revert to the non-cancerous levels of the controls. Several biomarker candidates have been identified and a panel is undergoing validation in a larger cohort. Plasma miRNA levels do not appear to reflect tumor levels in ccRCC. Source of Funding: Funding Sources: This work has been supported by the Paula Moss Trust for the research into the cure and treatment of kidney cancer (Hsieh), the Sidney Kimmel Center for Prostate and Urologic Cancers, by funds provided by David H. Koch through the Prostate Cancer Foundation, the National Cancer Institute T32 CA082088-12 training grant (Hakimi), and the Stephen P Hanson Family Fund Fellowship in Kidney Cancer (Hakimi). Vol. 189, No. 4S, Supplement, Sunday, May 5, 2013 612 PD-0332991, AN INHIBITOR OF CYCLIN-DEPENDENT KINASE 4/6, DEMONSTRATES INHIBITION OF PROLIFERATION IN RENAL CELL CARCINOMA AT NANOMOLAR CONCENTRATIONS AND MOLECULAR MARKERS PREDICT FOR SENSITIVITY Joshua Logan*, Nikayeh Mostofizadeh, Amrita Desai, Erika von Euw, Dylan Conklin, Veerauo Konkankit, Habib Hamidi, Mark Eckardt, Lee Anderson, Hsiao-Wang Chen, Charles Ginther, Eileen Taschereau, Los Angeles, CA; James Christensen, La Jolla, CA; Arie Belldegrun, Dennis Slamon, Fairooz Kabbinaar, Los Angeles, CA INTRODUCTION AND OBJECTIVES: Cell cycle dysregulation is a fundamental trait in cancer biology as evidenced by its prevalence in multiple malignancies, including renal cell carcinoma (RCC). PD-0332991 is an orally active, potent, and selective inhibitor of cyclin-dependent kinases (CDK) 4 and 6, blocking retinoblastoma (Rb) phosphorylation in nanomolar concentrations. We evaluated PD-0332991 in multiple renal cell lines to determine its effects on proliferation, phosphorylation of Rb, cell cycle and apoptosis. Lastly, we evaluated the response to drug for associations with copy number alterations and variances in transcript expression to identify potential molecular markers of response. METHODS: A panel of 28 RCC and immortalized kidney cell lines were used to examine the effects of PD-0332991 on proliferation to determine the half maximal inhibitory concentration (IC50) values. The effects of PD-0332991 on cell-cycle, apoptosis, and Rb phosphorylation were also assessed with flow cytometry and western blot analysis for five of the cell lines: RCC-HB and SW 156 (sensitive and malignant), R444 and Hs 891.T (resistant and malignant) and CCD 1103 (resistant and immortalized non-malignant). Molecular markers for response prediction, including p16, p15, CCND1, CCNE1, E2F1, Rb, CDK4 and CDK6, were studied using array CGH and gene expression profiling. RESULTS: A concentration-dependent inhibition of proliferation was identified; IC50 values ranged from 25.0nM up to 700nM, and five cell lines were completely resistant at 1000nM. CDK4/6 inhibition with PD-0332991 demonstrated G0/G1 cell cycle arrest, as well as induction of late apoptosis in SW 156, and Rb phosphorylation was blocked in a time-dependent fashion in both sensitive cell lines. Genotype and expression data of CDKN2A and CDKN2B were combined and a consensus was made regarding the status of p16 and p15, a significant association between loss and sensitivity to PD-0332991 was identified for p16 (p⫽0.027). No other copy number alterations were identified by array CGH in the other genes. Cell lines were then classified as having “high” or “low” expression for each of these markers; E2F1 was the only gene identified with expression levels significantly associated with response to PD-0332991 (p⫽0.041). CONCLUSIONS: PD-0332991 shows promising anti-proliferative activity in RCC through blockade of cell cycle progression. The decreased expression of select molecular markers p16 and E2F1 predict for sensitivity to PD-0332991 in RCC. Source of Funding: None 613 VALIDATION AND CLINICAL ASSOCIATIONS OF RECENTLY REPORTED RCC SUSCEPTIBILITY LOCI: A Ari Hakimi*, Irina Ostrovnaya, Kelly Stratton, James J Hsieh, Jonathan Coleman, Paul Russo, Robert J Klein, New York, NY INTRODUCTION AND OBJECTIVES: There have been several recent reports of RCC susceptibility loci from large genome wide association studies. To date there has been no analysis of the effect of these loci on clinical characteristics of RCC patients. METHODS: We identified six SNPs previously associated with kidney cancer using the NHGRI GWAS database; of these, five are either on the Affymetrix SNP 6.0 genotyping platform or are well tagged