Results SNP-distribution in patients with ccRCC

1. Role of VEGF C(-460)T single nucleotide polymorphism in the development of renal cell carcinoma S. Füssel1, S. Schneider1, A. Lohse-Fischer1, S. Tomasetti1, S. Fuessel1, T. Köhler2, A. Rost2, A. Meye1, M.P. Wirth1 1Department of Urology, Medical Faculty, Technical University of Dresden & 2AJ Roboscreen Leipzig granted by technology support with financial sources of the European Regional Development Fund and the State of Saxony heterozygous homozygous mutant homozygous wild-type Q Q Q Q Q Q R1 R1 R1 5` 5` 5` 3` 5` 3` R2 R2 R2 5` 5` 5` 5` 5` 5` 5` 5` 3` 3` 3` 3` R1 = FAM R2 = ROX Q = BHQ1 / BHQ2 Fig. 3 Genotyping of VEGF–C460T at position 3 Fig. 2 C(-460)T polymorphism of the VEGF promoter Fig. 1 TRIPLEHYB probe format: basic principle Fig. 4 Optimization of the SNP-detection assay TT Du-145 CC patient CT LNCaP TT Du-145 CC patient CT LNCaP FAM (for T-variant)ROX (for C-variant) • Objectives • Application of a new detection format • establishment of a novel detection format for real-time PCR supporting quantitative applications • suitability for genetic polymorphism detection • improved or same sensitivity, specificity, flexibility, and robustness compared to available formats • no need for external licenses • development of the TRIPLEHYB probe format • Results • SNP-distribution in patients with ccRCC • analysis of DNA from 99 pts with clear cell renal cell carcinoma • median age: 65 yrs (37-83 yrs), 62 male & 37 female • SNP-distribution in ccRCC: 22.2 % CC / 55.6 % CT / 22.2 % TT • first comparison with data from 116 healthy controls from the HapMap-study (CEU - people from Utah with ancestry from northern & western Europe): • 19.0 % CC / 46.6 % CT / 34.5 % TT • comparison of CT vs. TT: OR = 1.85  prevalence for RCC • next steps: • analysis of more ccRCC patients • analysis of matched healthy controls with this assay • dependence on tumor stage / grade / outcome • haplotype analysis of further VEGF-promoter SNPs D1 – D4: positions for the attachment of different dyes D1  fluorescent dye D2  quencher Materials & Methods Susanne Fuessel1, Susanne Schneider1, Andrea Lohse-Fischer1, Silke Tomasetti1, Thomas Koehler2, Anne-Katrin Rost2, Axel Meye1, Manfred P. Wirth1 • plasmids and cell lines as standards and controls for all variants • same results with DNA from tissue / whole blood / leukocytes • concordance with sequencing results (for 30 patient samples) • SNP in pos. 3 of up-probe  no binding to template  no signal • model system: SNP CT at position -460 of the VEGF promoter • association to different diseases/predisposition to tumorigenesis 3-step PCR (45 cycles; LC480): 95°C/15s; 45°C/1s; 59°C/40s; primers: 0.5µM each; probes: 0.3µM ROX-up/0.4µM FAM-up + 1.2µM do; Universal Master Mix + add. 5mM MgCl2 (ABI); template: 10 / 20 / 50 / 100ng DNA • Conclusions • biomolecular PCa detection on a given prostate specimen is conceivable as