Materials & Methods prospective study: February 2006 – December 2008

1. Materials & Methods • prospective study: February 2006 – December 2008 • patients with newly diagnosed BCa • controls: BPH patients, cystitis patients, healthy volunteers, patients with cystoscopically suspicious lesions (histologically proven benign) • intraoperative collection of tissue samples: • tumor tissue (=tumor) • macroscopically non-malignant tissue (=tumor free) • Collection of urine samples: • pre-operative urine sample of patients undergoing TUR-B or cystectomy • controls: 1 urine sample • processing and measurement of urine and tissue samples: • centrifugation of urine (10 min, 4°C, 830 x g) & PBS washings • respectively snap freezing of tissue samples • - isolation of total RNA from tissue and urine samples by standard protocols (Spin Tissue/Cell RNA Mini Kit, Invitek, Berlin) • cDNA-synthesis using up to 500 µg RNA • quantitative PCR for transcript levels of survivin, Ki67, XIAP & CK20 and the reference gene TBP in urine and tissue samples (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: SVV/TBP Ki67/TBP XIAP/TBP CK20/TBP 31 31 31 31 31 31 27 27 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 • no ideal tumor marker for non-invasive diagnostic & surveillance of bladder cancer (BCa) available • new diagnostic methods and markers are needed, e.g.: • survivin (SVV) & XIAP: inhibitor of apoptosis proteins (IAP) • Ki67: proliferation marker, essential for cell cycle progression • cytokeratin 20 (CK20): specific urothelial marker • 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 detection to improve diagnostic performance of cytology • Results • Tissuespecimens: • RNA quality satisfactory for reliable qPCR measurement, only in few samples reference gene expression was undetectable • up-regulation (1.8-fold) of CK20 in tumor specimens compared to macroscopically tumorfree tissue samples (Fig.1) • Urine specimens: • negative correlation between reference gene expression & urinary contamination by RBCs, WBCs & bacteria up to 20% of the samples with undetectable reference gene expression (e.g. patients with infection or macrohematuria) • Expression of all markers significantly higher in urines from BCa patients compared to control group (p< 0,001) (Fig.2) • Sensitivity and specificity of single markers and marker combination with cytology (see Fig. 3) • Sensitivity for single marker expression ranged from 65% (XIAP) to 86% (CK20) • Specificity for single marker expression ranged from 64% (XIAP) to 84% (CK20) • Cytology showed a sensitivity of 80,7% and a specificity of 92,3% • All markers are correlated with rising risk group, but only significant for Ki67 (p<0,004) (Fig. 4) Fig.1 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 paired malignant and non-malignant bladder specimens collected during TUR-B. The solid lines within the boxplots represents the median expression; numbers of samples per group are indicated. Ratios of the median expression in tumor and tumor-free tissue are shown in the table, an up-regulation in tumor tissue is observed only for CK20 (not significant). Fig.4 Transcript markers correlated with tumor risk groups in urine 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. Median values are presented. Fig.2 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 (individually and totally). The “no tumor”-group comprises patients undergoing TUR-B due to the suspicion of 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. Ratios of median expression of markers in BCa patients vs. controls are indicated. An upregulation in BCa patients is observed for all markers (p<0,001). SVV/TBP Ki67/TBP }of the same patient=paired tissue samples CK20/TBP XIAP/TBP For healthy controls the absent values were substituted by zero. Median values are presented. Ki67/TBP SVV/TBP CK20/TBP XIAP/TBP BCa controls BCa controls BCa controls BCa controls BCa BPH cystitis healthy BT NT n 105 (85,37%) 18 (14,63%) 62 25 46 • Conclusion • qPCR is a suitable method for transcript measurement in tissues • urine measurement is problematic due to a failure rate of ~20% • combination with biomarkers improves sensitivity and specificity of cytology • Single marker: • Sensitivity from 65% to 86% • Specificity from 64% to 84% • CK20 appears to be the most promising single marker • CK20 improves sensitivity and specificity of cytology considerably (96,8 and 100% respectively) Fig.3 Sensitivity and Specificity for Ki67, XIAP, SVV&CK20 and their combination with cytology in urine samples 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%) • 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