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

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

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 • BCa patients before cystectomy • exclusion criteria: • patients with PCa and non-urothelial tumors • controls • BPH patients • cystitis patients • healthy volunteers

6. Materials & Methods 2 • BCa patients • pre-operative urine sample • intra-operative tumor tissue & “normal appearing” bladder mucosa • post-operative urine sample (now stopped) • for every TUR-BT (prim./sec./ tert., 6-8 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 tertiary TUR-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=114) • age (median) = 70 yrs. (34 – 93yrs.) • M:F = 68 : 46 (59,6% : 40,4%) • newly diagnosed : recurrence = 96 : 18 (84,2% : 15,8%) • PSA (median; 63 pts.) = 1.124 (0.16 – 33.91) • tumor stage: 18 pts. NT= no tumor detectable 57 pts. pTa 16 pts. pT1 19 pts. >pT1 (3 pts. pTis only; for 1pt. tumor stage n.a.) • tumor grade: 18 pts.NT 7 pts. LMP (low malignancy potential) 19 pts.low grade 70 pts.high grade

10. BCa patients 3  114 primary TUR  69 second op (56 sec. TUR, 13 cystec) • 11 third op (6 tert. TUR, 5 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) • tissues: less samples with negative reference gene results • marker validation in tissue specimens, comparison Tu  Tf • marker evaluation in urine specimens with regard to BCa diagnosis

12. unpaired tissue samples Ki67 / TBP SVV / TBP XIAP / TBP tumor tissue (36) tumor free (65) median Tu / median Tf Tumor markers in unpaired tissue specimens 3.59 1.13 14.66 1.87 0.72 14.97 1.92 1.57 0.98 Median values are presented.

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

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

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

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

17. Ki67 / TBPin urine of BCa patients & controls Ki67 possibly suitable for discrimination

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

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

20. BT stage @ pri TUR Ki67 / TBP SVV / TBP XIAP / TBP healthy (40) no tumor (18) pTa (57) pT1(16) > pT1(19) Tumor markers in urine vs BCa stage 0 0 0 0.62 1.7 42.87 1.56 2.41 38.31 2.07 2.55 86.7 4.3 6.93 74.09 Median values are presented.

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

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

23. XIAP in urine vs BCa stage difference in XIAP levels between pTa and pT1 / >pT1

24. BT grade @ pri TUR Ki67 / TBP SVV / TBP XIAP / TBP healthy (n=40) no tumor (n=18) low grade (n=19) high grade (n=70) Tumor markers in urine vs BCa grade 0 0 0 0.62 1.7 42.87 2.7 0.87 32.09 2.45 3.9 57.93 Median values are presented.

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

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

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

28. Conclusions & outlook • relative transcript levels of Ki67 and SVV possibly useful as BCa markers in urine samples • dependence on tumor stage and grade for both markers • XIAP not suitable for discrimination • continuation of sample collection for better statistical calculations • then 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)