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): 4 th most common cancer in men & 9 th leading cause of death worldwideBladder cancer (BCa): 4 th most common cancer in men & 9 th leading cause of death worldwide cystoscopy & urine cytology: current gold standards for diagnosis & surveillance of BCacystoscopy & urine cytology: current gold standards for diagnosis & surveillance of BCa no ideal tumor marker for non-invasive diagnostic & surveillance at the momentno 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)survivin & XIAP: inhibitor of apoptosis proteins (IAP) Ki67: proliferation marker, essential for cell cycle progressionKi67: proliferation marker, essential for cell cycle progression selectively over-expressed in most human malignancies incl. BCaselectively over-expressed in most human malignancies incl. BCa association between over-expression and higher stage & grade and with unfavorable prognosisassociation between over-expression and higher stage & grade and with unfavorable prognosis suitable markers (tissue and urine specimens ) and therapeutic targets for BCasuitable markers (tissue and urine specimens ) and therapeutic targets for BCa

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

6. Materials & Methods 2 BCa patientsBCa 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 controlscontrols –1 urine sample

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

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

9. BCa patients (n=74 o. 77??) age (median) = ?? yrs. (?? – ??)age (median) = ?? yrs. (?? – ??) M:F = 63 : 43 (59,4% : 40,6%)M:F = 63 : 43 (59,4% : 40,6%) newly diagnosed : recurrence = 92 : 14 (86,8% : 13,2%)newly diagnosed : recurrence = 92 : 14 (86,8% : 13,2%) PSA (median; 58 pts.) = 1,195 (0,16 – 33,91)PSA (median; 58 pts.) = 1,195 (0,16 – 33,91) tumor stage:16 o. 14 o.15??? NT= no tumor detectable 46?pTa 11?pT1 17? >pT1tumor stage:16 o. 14 o.15??? NT= no tumor detectable 46?pTa 11?pT1 17? >pT1 cis:92 :14 (86,6% : 13,2%) cis:92 :14 (86,6% : 13,2%) All pts with cis harbour high grade (G2/ G3) disease as well. tumor grade: 16 o. 14 o.15??? NTtumor grade: 16 o. 14 o.15??? NT no/excluded LMP (low malginancy potential) no/excluded LMP (low malginancy potential) »13 low grade »59 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 BPHcystitishealthy 531327 68 yrs (52-85) 64 yrs (19-85) 31 yrs (18-59) PSA median 2.9 (0.3 – 33.9) M : F 3 (23.1%) :10 (76.9%) M : F 7 (25.9 %) :20 (74.1 %)

11. Results 1 2 reference genes tested: TBP better than HPRT2 reference genes tested: TBP better than HPRT urine specimens: negative correlation between reference gene expression & urinary contamination by RBCs, WBCs & bacteriaurine 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) (e.g. pts with infection or hematuria or post-TUR urines) tissue specimens: less samples with negative reference gene resultstissue specimens: less samples with negative reference gene results target validation in tissue specimens, comparison Tu  Tftarget validation in tissue specimens, comparison Tu  Tf target evaluation in urine specimens with regard to BCa diagnosistarget evaluation in urine specimens with regard to BCa diagnosis

12. Tumor markers in unpaired tissue specimens unpaired tissue samples Ki67 / TBP SVV / TBP XIAP / TBP tumor tissue (36) tumor free (65) median Tu / median Tf Median values are presented.

13. Ki67 / TBP in unpaired tissue specimens Ki67 / TBP: Tu (n=36): medianmean Tf (n=65): medianmean median Tu/median Tf  clear difference

14. SVV / TBP for unpaired tissue specimens SVV / TBP: Tu (n=36): medianmean Tf (n=36): medianmean median Tu/median Tf  clear difference

15. XIAP / TBP in unpaired tissue specimens XIAP / TBP: Tu (n=36): medianmean Tf (n=65): medianmean median Tu/median Tf  no difference!

16. Tumor markers in paired tissue specimens Das drinne lassen??? Wie sehen die Daten aus??? paired tissue samples (??) Ki67 / TBP SVV / TBP XIAP / TBP tumor tissue tumor free median Tu / median Tf pairwise ratio Tu / Tf >1 <1 Median values are presented.

17. Tumor markers in urine specimens of BCa patients & controls clinical diagnosis Ki67 / TBPSVV / TBPXIAP / TBP BCa (69-77) (positive histo) no tumor (16) (negative histo) BPH (18-28) cystitis (5) healthy (34-40)   For healthy controls the absent values were substituted by zero. Median values are presented.

18. Ki67 / TBP in urine of BCa patients & controls Ki67 possibly suitable for discrimination

19. SVV / TBP in urine of BCa patients & controls SVV possibly suitable for discrimination

20. XIAP / TBP in urine of BCa patients & controls XIAP not rather suitable for discrimination

21. Tumor markers in urine vs BCa stage BT stage @ pri TUR Ki67 / TBP SVV / TBP XIAP / TBP healthy (32-40) no tumor (14-15) pTa (42-46) Pt1(9-11) > pT1(15-17) Median values are presented.

22. Ki67 in urine vs BCa stage continuous in Ki67 levels from superficial (pTa & pT1) to invasive BCa

23. Survivin in urine vs BCa stage difference in SVV levels between superficial and invasive BCa

24. XIAP in urine vs BCa stage clear difference in XIAP levels between superficial and invasive BCa

25. Tumor markers in urine vs BCa grade BT grade @ pri TUR Ki67 / TBP SVV / TBP XIAP / TBP healthy (32-40) no tumor (14-15) low grade (11-13) high grade (55-59) Median values are presented.

26. Ki67 in urine vs BCa grade increase of Ki67 levels with increasing grade

27. Survivin in urine vs BCa grade slight increase of SVV levels with increasing grade

28. XIAP in urine vs BCa grade increase of XIAP levels with increasing grade

29. Conclusions & outlook relative transcript levels of Ki67 and SVV possibly useful as BCa markers in urine samplesrelative transcript levels of Ki67 and SVV possibly useful as BCa markers in urine samples dependence on tumor stage and grade for both markersdependence on tumor stage and grade for both markers XIAP not suitable for discriminationXIAP not suitable for discrimination continuation of sample collection for better statistical calculationscontinuation of sample collection for better statistical calculations then definition of cut-off values for calculation of test performance in comparison to cytologythen definition of cut-off values for calculation of test performance in comparison to cytology correlation with follow-up data  possibly prediction of recurrence (SVV as well-known predictor)correlation with follow-up data  possibly prediction of recurrence (SVV as well-known predictor)