“Regina Elena” National Cancer Institute Michele Gallucci Anna Cianciulli

1. Highlights in the management of Urogenital Cancer Rome, May 9-10 2008 Biomarkers associated with bladder cancer progression: a potential tool for individualized therapy? “Regina Elena” National Cancer Institute Michele Gallucci Anna Cianciulli

2. Multivariables prognostic model • Pathological variables Michele Gallucci • Biological markers Anna Cianciulli

3. Pathological prognostic factors • 70 % of Ta bladder tumours • 7% of these are G3 tumours (WHO 1973) • CIS 5-10% R. J. SILVESTER TSW UROLOGY (2006) 1 (S2), 15-23

4. Pathological prognostic factors 5 years follow up 906 TURB Ta-T1 47% of the patients recurred 9 % progressed to muscle invasive disease 1/3 of patients with CIS died R. J. SILVESTER TSW UROLOGY (2006) 1 (S2), 15-23

5. CLINICAL PROGRESSION

8. HYSTOLOGY • UROTHELIUM • LAMINA PROPRIA MEAN DEEP 1.4 mm * • MUSCULARIS MUCOSAE/ VESSELS • SUBMUCOSA • MUSCULARIS PROPRIA * Cheng L. et al., Cancer 1999; 85: 2638 - 47

9. INVASIVE UROTHELIAL CARCINOMA SUBSTAGING OF T1 DISEASE T1a: tumors above or into muscularis mucosae (or large vessels). T1b: tumors below muscularis mucosae (or large vessels).

10. T1SUBSTAGING T1a MUSCULARIS MUCOSAE muscularis propria -------------------------------------------------------------------- T1b MUSCULARIS MUCOSAE muscularis propria

11. INVASIVE UROTHELIAL CARCINOMA T1a

12. INVASIVE UROTHELIAL CARCINOMA T1b

13. INVASIVE UROTHELIAL CARCINOMA T2

14. INVASIVE UROTHELIAL CARCINOMA TUMORS INVADING BEYOND THE MUSCULARIS MUCOSAE TEND TO BEHAVE LIKE MUSCULARIS PROPRIA INVASIVE (T2) TUMORS. Epstein JI et al. in Bladdere biopsy interpretation. Ed. Lippincott Williams & Wilkins, 2004, pag. 95.

15. T1a and T1b as variables predictive of disease progression staging number of cases progression rate • 1994 Hasui et al. T1a 60 6.7 T1b 28 53.5 • 1997 Holmang et al. T1a 26 36 T1b 38 58 • 1999 Cheng et al. T1a 23 11 T1b 21 32 • 2000 Kondylis et al. T1a 32 22 T1b 17 29 • 2003 Trias et al. T1a 11 9 T1b 13 30.7 • 2005 Orsola et al. T1a 25 8 T1b 34 34

16. CARCINOMA IN SITU CARCINOMA OF THE BLADDER INVADING THE MUSCULARIS MUCOSAE (T1b) ASSOCIATED WITH CIS HAVE AN INCREASED RISK OF PROGRESSION Bernardini S et al., J Urol 2001; 165: 42 - 46

17. T1 SUBSTAGING SUBSTAGING OF T1 BLADDER CARCINOMA ACCORDING TO THE DEPTH INVASION (MEASURED BY MICROMETER) PROVIDES SIGNIFICANT PROGNOSTIC INFORMATION, AND WE RECOMMEND THAT IT BE INCORPORATED INTO A FUTURE TNM SYSTEM Cheng L et al.: Substaging of T1 bladder carcinoma based on the depth of invasion as measured by micrometer. Cancer 1999, vol. 15: 1036-1042

18. IF THE BIOPSY IS SUPERFICIAL AND LACKS MUSCULARIS PROPRIA, A SECOND BIOPSY SHOULD BE CONSIDERED Epstein JI et al. in Bladder biopsy interpretation. Ed. Lippincott Williams & Wilkins, 2004, pag. 118

19. UROTHELIAL CARCINOMA HISTOLOGICAL SUBTYPES Urothelium Nested Tubular variant

20. MICROPAPILLARY CARCINOMA Ca in situ Micropapillary carcinoma

21. ‘SIGNET RING CELL TYPE’ UROTHELIAL CARCINOMA Signet ring cell

22. SMALL CELL UROTHELIAL CARCINOMA

24. Early vs. deferred cystectomy

25. Presence of CIS Early cystectomy Deferred cystectomy

26. Biological markers

27. BIOLOGICAL MARKERS To provide additional important informations in patients with muscle invasive carcinomas concerning their metastatic potential and response to adjuvant regimens To identify and monitor those patients presenting with superficial tumors who are likely to develop recurrent or progressive disease

28. PHENOTYPIC MARKERS • Blood Group Antigens ( ABH antigens ) • Tumor Associated Antigens ( BTA, Nuclear Matrix Proteins) • Proliferating Antigens ( Ki-67, Proliferating Cell Antigen) • Cellular Adhesion Molecules (e-cadherin, integrin)

29. GENOTYPIC MARKERS • CYTOGENETIC ALTERATIONS • MOLECULAR ALTERATIONS OF ONCOGENES • MOLECULAR ALTERATIONS OF TUMOR SUPPRESSOR GENES

31. MOLECULAR ALTERATIONS OF ONCOGENES • H-ras • c-myc • mdm2 • c-erB-2

33. MOLECULAR ALTERATIONS OF TUMOR SUPPRESSOR GENES Studies on loss of heterozygosity have identified specific allelic deletions in many bladder cancer. The Rb gene(13q) and the p53 gene (17p) are the best studied tumor suppressor genes.

34. RETINOBLASTOMA TUMOR SUPPRESSOR GENE “ Inactivation of the Rb gene is thought to be an important step in bladder cancer progression “ ( Jung I et al, Cancer Control 2000 ) “ Patients with muscle invasive bladder tumors who had lost Rb expression had a statistically significant shorter 5-year survival ( p=0.001) than those with normal Rb protein expression “ ( Cordon-Cardo et al., Scand J Urol Nephrol 2000)

35. p53 TUMOR SUPPRESSOR GENE “ Increased p53 immunoreactivity has been noted in higher grade and stage bladder cancers and is associated with disease progression, and decreased overall and disease specific survival “ ( Chatterjee SJ et al, J. J Clin Oncol 2004 )

36. Richard J. Cote *, Ram H. Datar, 2006

37. Prospective evaluation of p53 as prognostic marker in T1 transitional cell carcinoma of the bladder 2007, BJU INT

38. p53 MUTATIONS: CHEMORESISTENCE VERSUS CHEMOSENSITIVITY Gemcitabine is effective in TCC cell lines independent of p53 status. Urology 2003

40. Why are the results contradictory? • One difference is methodological • p53 affects a remarkable number of cellular processes. Perhaps defects in damage-induced checkpoints enhance chemosensitivity, whereas defects in apoptosis promote drug resistence • Examination of p53 status alone cannot determine whether the p53 pathway is intact

41. p21 TUMOR SUPPRESSOR GENE “ Loss of p21 expression was strongly associated with an increased probability of recurrence and decreased probability of survival in patients with lymphonode negative organ confined and lymphonode negative extrabladder disease “ ( Shariat SF, 2004)

42. Richard J. Cote *, Ram H. Datar, 2006

43. Normal Urothelium p53,EGF-R 9p-,9q- Rb-,p16, EGF-R, 17p,7 Low-grade TCC Ta High-grade TCC Superficial Carcinoma in situ 9p-,9q- p53? Stage T1 8p-,11p,erbb2 Rb-,8p-,3p High-grade TCC Ta p53? Stage T2-4 N+,M+ Metastasis

44. CHROMOSOME 9 The genes,p16 and p15, are found in tandem at p21 region.These genes encode members of a new family of negative cell cycle regulators, which product function as cyclin-dependent kinase inibitory molecules

45. CHROMOSOME 7 Pycha et al reported that in patients with recurrence ,61% has trisomy 7. Other investigators have also shown that increased copy number of chromosome 7 is associated with progression

46. CHROMOSOME 17 Cordon-Cardo et al, revealed that deletions of 17p occur only in invasive tumors and are involved in the progression of bladder cancer

47. Our experience Interphase cytogenetics of bladder cancer progression ( Cianciulli et al., 2000) Genetic instability in superficial bladder cancer and adjacent mucosa ( Cianciulli et al., 2003) Genetic instability in advanced bladder cancer and adjacent mucosa ( Gallucci et al, 2005) Adverse genetic prognostic profile identification ( Gallucci et al, 2007)

48. Interphase cytogenetics of bladder cancer progression: relationship between aneusomy, DNA ploidy pattern, histopathology and clinical outcome Cianciulli et al. Int J Clin Lab Res2000

49. The aims of this study were: To gain more insight into genetic changes at the chromosomal level during different histopathological stages To compare the sensitivity of FISH and FCM for detection of disturbed DNA content To combine pathological variables with genetic markers in order to identify the phenotypes likely to progress

50. p value; Mann-Whitney test 70 patients