Woei-Yun Siow & Axel Meye & Oliver W. Hakenberg

1. Comparative quantitative evaluation of the XIAP, survivin & Ki67 transcript levels in urine & tissue samples of bladder cancer patients Woei-Yun Siow & Axel Meye & Oliver W. Hakenberg Juliane Schmidt & Susanne Füssel & Catharina Rippel

2. 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

3. Objectives • to establish methods for quantitative transcript measurements in urine and tissue specimens (TUR-BT) • to determine suitability of transcript levels of different BCa-related genes (survivin, Ki67 and XIAP) in urine samples as diagnostic, surveillance and prognostic markers of BCa • to analyze marker expression in corresponding BCa tissue specimens in comparison to urine samples

4. BCa-related genes • survivin & XIAP: inhibitor of apoptosis proteins (IAP) • Ki67: proliferation marker, essential for cell cycle progression • selectively over-expressed in most human malignancies incl. BCa • association between over-expression and higher stage & grade and with unfavorable prognosis • suitable markers (tissue and urine specimens ) and therapeutic targets for BCa

5. Materials & Methods 1 • prospective study: February 2006 - January 2007 • inclusion criteria: • patients undergoing transurethral resection (TUR-BT) for newly diagnosed BCa, recurrent BCa & cystoscopically suspicious bladder lesions • exclusion criteria: • patients with PCa and non-urothelial tumors • controls • BPH patients • cystitis patients • healthy volunteers • BCa patients before cystectomy

6. Materials & Methods 2 • BCa patients • pre-operative urine sample • intra-operative tumor tissue & “normal appearing” bladder mucosa • post-operative urine sample (1 POD) • for every TUR-BT (prim./sec./ tert., 4-6 weeks) • same procedure for recurrences • controls • 1 urine sample

7. recurrence primary TUR-BT 4-6 Wochen recurrence secondary TUR-BT cystectomy 4-6 weeks tertiaryTUR-BT cystectomy Course of treatment for BCa patients

8. Materials & Methods 3 • preparation of cellular components from urine • isolation of total RNA and cDNA-synthesis • quantitative PCR for transcript levels of survivin, XIAP & Ki67 and the reference gene TBP in urine and tissue samples • correlation of the relative expression levels (internal normalization to TBP) of survivin, XIAP & Ki67 with clinico-pathological data

9. BCa patients (n=) • age (median) = 70 yrs. (34 – 93) • M:F = 63 : 43 (59,4% : 40,6%) • newly diagnosed : recurrence = 92 : 14 (86,8% : 13,2%) • PSA (median; 58 pts.) = 1,195 (0,16 – 33,91) • tumor stage: NT= kein Tumor nachweisbar pTa pT1 >pT1 • cis: 92 :14 (86,6% : 13,2%) Cis • pos. : neg. = 5 : 54 (8.5% : 91.5%) • All pts with cis harbour high grade (G2/ G3) disease as well. • tumor grade: NT LMP (low malginancy potential) • low grade • high grade

10. BCa patients 3  59 primary TUR  42 second op (33 sec TUR, 9 cystec) • 8 third op (6 tertiary TUR, 2 cystec) Controls

11. Results 1 • 2 reference genes tested: TBP better than HPRT • urine specimens: negative correlation between reference gene expression & urinary contamination by RBCs, WBCs & bacteria  many samples with negative reference gene results (e.g. pts with infection or hematuria or post-TUR urines) • tissue specimens: less samples with negative reference gene results • target validation in tissue specimens, comparison Tu  Tf • target evaluation in urine specimens with regard to BCa diagnosis

12. Tumor markers in unpaired tissue specimens Median values presented.

13. Ki67 / TBPin unpaired tissue specimens

14. XIAP / TBP in unpaired tissue specimens

15. SVV / TBP for unpaired tissue specimens

16. Tumor markers in paired tissue specimens Median values presented.

17. Tumor markers in urine specimens of BCa patients & controls For healthy controls the absent values were substituted by zero Median values presented.

18. Ki67 / TBPin urine of BCa patients & controls

19. XIAP / TBP in urine of BCa patients & controls

20. SVV / TBP in urine of BCa patients & controls

21. Tumor markers in urine vs BCa stage Median values presented.

22. Ki67 in urine vs BCa stage

23. XIAP in urine vs BCa stage

24. Survivin in urine vs BCa stage

25. Tumor markers in urine vs BCa grade Median values presented.

26. Ki67 in urine vs BCa grade

27. XIAP in urine vs BCa grade

28. Survivin in urine vs BCa grade

29. Offene Fragen/Überlegungen 1 • Abfall der Tumormarker nach der primären TUR-BT? Korrelation mit histologischem Befund? • Ausschlusskriterium HWI: Bakterienzahl oder positive Urinkultur? • Möglicherweise Verfälschung der Werte durch hohe Leukozytenzahl • Kontrolle der vermeintlich Gesunden

30. Offene Fragen/Überlegungen 2 • Ausweitung der gesunden Kontrollgruppe: z.B. Zystitis-Patienten nach Abschluss der Therapie? • Statistische Signifikanz: möglich durch Kombination der Tumormarker ? • Ausweitung der Tumorgene sinnvoll (z.B. hTERT )? • Berechnung Sensitivität/Spezifität erst bei größeren Fallzahlen sinnvoll, dann Vergleich mit Urinzytologie

31. Offene Fragen/Überlegungen 3 • Geschlechtsspezifischer Test? ( Probleme durch Einfluss von BPH ) • Korrelation Tumormarker im Urin und korrespondierenden Gewebe • Postoperativer Urin oft nicht auswertbar (Blut, Kreisspülung usw.)  Stopp der Sammlung ? • Stopp der Gewebesammlung oder Fortführung für andere Projekte ?

32. Offene Fragen/Überlegungen 4 • Urinsammlung vor Cystektomie ? • Erhebung der klinischen Daten vollständig? • Gezielte Nachbeobachtung von Patienten ohne histologischen Tumornachweis?