1. Results 1 comparison for 5 systems containing the SNP at postion 1 to 5 of the up-stream probe  systems 2-5 work well, system 3 offers most stable & reliable discrimination (Fig.4) concordance between sequencing results & QPCR measurements based on system 3 same results with patient DNA from tissue, whole blood & leukocytes Objectives single nucleotide polymorphisms (SNPs) are frequently associated with the onset or progression of diseases including cancers SNPs in the promoter region of the vascular endothelial growth factor (VEGF) are reported to influence VEGF expression which is increased in several tumor types including renal cell carcinoma (RCC) analysis of SNP distribution supports risk assessment and prediction of cancer development determination of SNP variants by sequencing, array analysis or quantitative PCR (QPCR) QPCR approaches include melting curve analyses or the use of SNP-specific probes aim of the study:  establishment of a novel QPCR detection format for real-time PCR suitable for SNP detection  improved or same sensitivity, specificity, flexibility, and robustness compared to available formats  no need for external licenses (interest of the cooperating company AJ Roboscreen) development of the TRIPLEHYB probe format & its validation in a clinico-experimental application  development of the TRIPLEHYB probe format & its validation in a clinico-experimental application Role of VEGF C(-460)T single nucleotide polymorphism in the development of renal cell carcinoma S. Füssel 1, S. Schneider 1, A. Lohse-Fischer 1, S. Tomasetti 1, S. Fuessel 1, T. Köhler 2, A. Rost 2, A. Meye 1, M.P. Wirth 1 1 Department of Urology, Medical Faculty, Technical University of Dresden & 2 AJ Roboscreen Leipzig granted by technology support with financial sources of the European Regional Development Fund and the State of Saxony Fig. 1TRIPLEHYB probe format: basic principle D 1 – D 4 : positions for the attachment of different dyes D1  fluorescent dye D2  quencher Fig. 3Genotyping of VEGF–C460T at position 3 of the up-stream probe An fluorescence signal is only expected in case of perfect match:  homozygous mut & heterozygous wt/mut positive in FAM channel  homozygous wt & heterozygous wt/mut positive in ROX channel homozygous wild-type 3` 5` Q R1 3` 5` Q R1 3` 5` Q R1 3` 5` Q R2 heterozygous homozygous mutant 3` 5` Q R2 3` 5` Q R2 R1 = FAMR2 = ROXQ = BHQ1 / BHQ2 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 = mut) ROX (for C-variant = wt) plasmids and cell lines as standards and controls for all variants concordance with sequencing results (for 30 patient samples) 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 MgCl 2 (ABI); template: 10 / 20 / 50 / 100ng DNA Material & Methods selection of the SNP C/T at postion -460 of the VEGF promoter as model system since it is supposed to alter VEGF expression  analysis of this SNP in patients with clear-cell RCC C corresponds to wildtype (wt) & T to mutant (mut) design of a probe system based on the TRIPLEHYB format (Fig.1) consisting of:  the same forward & reverse primers used for both SNP variants  two up-stream probes each specific for one SNP variant  one down-stream probe matching to both up-stream probes  each one half of the up- & down-stream probes is complementary to the template and one half to the other forming a stem structure (Fig. 1)  labeling with fluorescent dyes (D1) and quencers (D2)  specific combination for each SNP variant: ROX + BHQ2 for wt and FAM + BHQ1 for mut (Fig.2)  fluorescence signal only expected in case of perfect match between up-stream probe and template  parallel detection of amplification signals in both channels  homozygous wt & heterozygous wt/mut positive in ROX channel, homozygous mut & heterozygous wt/mut positive in FAM channel (Fig.3) design and testing of different probe systems containing the nucleotides substitution at postions 1-5 of the up-stream probe  reliable discrimination of both SNP variants possible? testing on cloned model plasmids for each SNP variant using the LightCylcer 480 (Roche) and the Universal Master Mix (Applied Biosystems) supplemented with 5mM MgCl 2 use of DNA from cell lines or patients with known SNP-status as positive controls for each variant validation of the system 3 (with SNP at pos. 3 of the up-stream probe) on DNA samples from 30 patients with known SNP status (sequenced at AJ Innuscreen, Berlin) parallel determination of SNP status in patient DNA samples originating from tumor tissue, whole blood and leukocytes (isolated by standard protocols) SNP analysis on DNA (50-100ng per reaction) from leukocytes of 99 patients with clear-cell RCC comparison with SNP data from 116 healthy controls calculation of Odds ratios for different SNP variant as indicator of an increased chance to develop RCC Fig. 2C(-460)T polymorphism of the VEGF promoter SNP in pos. 3 of up-stream probe  no binding to template  no signal model system: SNP C  T at position -460 of the VEGF promoter labeling with fluorescent dyes (FAM, ROX) and quenchers (BHQ1+2) Q = BHQ1 or BHQ2 R2 = ROX Results 2 analysis of DNA from 99 pts with primary clear cell RCC median age: 65 yrs (37-83 yrs), 62 male & 37 female SNP-distribution in clear cell RCC: 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 http://urologie.uniklinikum-dresden.de / susanne.fuessel@uniklinikum-dresden.de