Materials & Methods prospective study: February 2006 – December 2008 inclusion criteria:

1. Materials & Methods • prospective study: February 2006 – December 2008 • inclusion criteria: • - patients undergoing transurethral resection (TUR-B) for newly diagnosed BCa, recurrent BCa & • cystoscopically suspicious bladder lesions • exclusion criteria: • - patients with prostate cancer and non-urothelial tumors • controls: • - BPH patients, cystitis patients, healthy volunteers • collection of urine and tissue samples: • pre-operative urine sample for every TUR-B • - intra-operative tumor tissue & “normal appearing” bladder mucosa • - same procedure for recurrences & cystectomies (Fig.1) • - controls: 1 urine sample • processing and measurement of urine and tissue samples: • - collection of up to 250 ml urine, processed immediately after transport to the laboratory • - preparation of cellular components from urine by centrifugation (10 min, 4°C, 830 x g) • - two washing steps in ice cold PBS • - resuspension of the cellular pellet in 700 µl RNA-stabilizing buffer (Invitek, Berlin), storage at -80°C • - collection of malignant and apparently non-malignant bladder tissue specimens during TUR-B • - immediate snap-freezing in liquid nitrogen • - preparation of cryosections (60-80 slices à 4 µM) and addition of 600 µl RNA-stabilizing buffer (Invitek, Berlin) • - isolation of total RNA by standard protocols (Spin Tissue/Cell RNA Mini Kit, Invitek, Berlin) • - cDNA-synthesis using up to 500 µg RNA, random hexamer primers (Amersham) and Superscript II Rnase H Reverse Transcriptase (Invitrogen) • - quantitative PCR for transcript levels of survivin, Ki67, XIAP & CK20 and the reference gene TBP in urine and tissue samples using gene-specific primers and probes (LightCycler, Roche), detection limit of 10 transcript molecules • urine cytology was performed by a single experienced examiner • correlation of the relative expression levels (internal normalization to TBP) of survivin, Ki67, XIAP & CK20 with clinico-pathological data using SPSS 12.0 • Patients and controls: Non-invasive detection of bladder cancer by measurement of tumor-related transcript markers in urine Catharina Rippel1, Juliane Schmidt1, Woei-Yun Siow2, Susanne Fuessel1, Anka Baldauf-Twelker1, Axel Meye1, Andrea Lohse1, Marc-Oliver Grimm1, Oliver W. Hakenberg1, Manfred P. Wirth1 1 Department of Urology, Technical University of Dresden, Germany, 2 Dept. of Urology, National University Hospital, Singapore • Introduction • Bladder cancer (BCa): 4th most common cancer in men & 9th leading cause of death worldwide • cystoscopy & urine cytology: current gold standards for diagnosis & surveillance of BCa • no ideal tumor marker for non-invasive diagnostic & surveillance at the moment • therefore new diagnostic methods and markers are needed, e.g. BCa-related transcript markers: • survivin (SVV) & XIAP: inhibitor of apoptosis proteins (IAP) • Ki67: proliferation marker, essential for cell cycle progression • cytokeratin 20 (CK20) • selectively over-expressed in most human malignancies incl. BCa • association between over-expression and higher stage & grade and with unfavorable prognosis • possible markers (tissue and urine specimens ) and therapeutic targets for BCa • Objectives • to establish methods for quantitative transcript measurements (qPCR) in urine and tissue specimens • to determine suitability of transcript levels of different BCa-related genes (survivin, Ki67, XIAP and CK20) as diagnostic markers of BCa in urine samples • to validate this bio-molecular technique as additional, non-invasive diagnostic tool in BCa assessment • Results • Tissuespecimens: • RNA quality satisfactory for reliable qPCR measurement, only few samples had negative reference gene expression • up-regulation (1.8-fold) of CK20 in tumor specimens compared to apparently tumorfree tissue samples (Fig.2) • Urine specimens: • negative correlation between reference gene expression & urinary contamination by RBCs, WBCs & bacteria up to 20% of the samples with negative reference gene expression (e.g. patients with infection or macro-hematuria) • Expression of all markers significantly higher in urines from BCa patients compared to control group (p< 0,001) (Fig.3) • Dependency on EAU- risk groups observed for transcript levels of SVV, Ki67, XIAP&CK20 (Fig.4) • Sensitivity for single marker expression ranged from 65% (XIAP) to 86% (CK20) (Fig.) • Specificity for single marker expression ranged from 64% (XIAP) to 84% (CK20) (Fig.) • Cytology showed a sensitivity of 80,7% And a specificity of 92,3% (Fig. ) • Combination of CK20 and cytology was the most promising for discrimination of BCa-patients from non-BCa subjects (sensitivity 96,8% and specificity 100%) (Fig.) Fig.1 Sensitivity and Specificity for Ki67, XIAP, SVV&CK20 and their combination with cytology Fig.2 Transcript markers in malignant and non-malignant bladder tissue specimens Distribution of the relative transcript levels of SVV, Ki67, XIAP&CK20 (normalized to TBP) in unpaired malignant and non-malignant bladder specimens collected during TUR-B. The solid lines within the boxplots represent the medians, numbers of samples per group are indicated. Ratios of the medians in the Tu and Tf groups are shown in the table, an up-regulation in these tumor tissues is observed only for CK20 and SVV. Fig.4 Transcript markers in urine correlated with tumor risk groups Distribution of the relative transcript levels of SVV, Ki67, XIAP&CK20 (normalized to TBP) in urine specimens collected before TUR-B correlated to tumor risk groups (EAU guidelines 2002). The solid lines within the boxplots represent the median transcript levels, numbers of samples per group are indicated. XIAP/TBP CK20/TBP SVV/TBP Ki67/TBP SVV/TBP Ki67/TBP tumor tumor-free tumor tumor-free tumor tumor-free tumor tumor-free Median values are presented. XIAP/TBP Fig.3 Transcript markers in urine specimens of BCa patients & controls The relative transcript levels of SVV, Ki67, XIAP&CK20 (normalized to TBP) in urine samples of BCa patients undergoing TUR-B are shown in comparison to control groups (patients with BPH or cystitis and healthy volunteers). The “no tumor”-group comprises patients undergoing TUR-B due to the suspicion of having a BCa which could no be proven histologically. The solid lines within the boxplots represent the median transcript levels, numbers of samples per group are indicated. BCa BPH cystitis healthy BT NT n 105 (85,37%) 18 (14,63%) 62 25 46 age* 71 y (22-93) 67,5 y (62-87) 32,5 y (18-88) 29 y (18-59) m:f 72:50 (59:41%) 1:24 (4:96%) 16:30 (34,8:65,2%) PSA* 1,56 (0-33,91) ng/ml 2,4 (0,05-16.85) ng/ml For healthy controls the absent values were substituted by zero. Median values are presented. • Conclusion • All markers are correlated with rising risk group • CK20 shows the highest sensitivity and specificity of all analysed markers • Combination of CK 20 with cytology very promising, but greater numbers needed Outlook • Correlation with follow-up data possible prediction of recurrence or progression? • Definition of risk groups (EAU guidelines 2002): • Low risk: (solitary, < 3 cm, low-grade/G1, Ta, without CIS): n=29 • Intermediate risk: (Ta-1, G1-2, multifocal > 3 cm): n=26 • High risk (T1, high-grade/G3, multifocal oder recurrent ± CIS): n=50 SVV/TBP Ki67/TBP XIAP/TBP CK20/TBP BCa controls BCa controls BCa controls BCa controls