1. Prognostic value of occult breast cancer cells in blood and bone marrow Gro Wiedswang Surgical unit, Ullevål University Hospital & The Micrometastasis lab, Radiumhospital-Rikshospital Oslo Norway Rome 16.11.06

2. - why occult tumour cells - what is occult tumour cells - occult tumour cells in bone marrow - timing of bone marrow examination - occult tumour cells in blood - clinical trials - futher perspectives

3. Challenge of breast cancer Better diagnostics Screening Improved surgical treatment Improved adjuvant treatment 25-30% of N0 patients die whitin 5 years 40% of N1 patients survive > 10 år Overgaard NEJM 1997

4. Challenge of breast cancer Therapeutic desicions –size of tumour –lymph node involvement –histological grade –hormone receptor –HER-2 status Improved prognostication! Feature of the patient??

5. Why micrometastasis? Cancer incidence: > 2500 women/year in Norway > 1mill women/ year wordwide Still 20% 5 year mortality in Norway Current treatment strategies does not catch patients at risk / overtreat low-risk patients Micrometastasis / occult tumour cells: - prognostic tool - identify high/low risk patients - monitoring therapy - identify therapeutic targets

6. Why micrometastasis ? Patients do relapse after removing the whole breast and negative lymph nodes Early, subclinical dissemination of tumour cells TNM staging does not catch all patients at risk Isolated tumour cells for prognostication ?

7. Mikrometastasis Groups of tumour cells 0.2-2mm in another tissue than primary tumour (AJCC 2002, UICC 2002) bone marrow / lymph nodes / blood signs of invastion and organisation (Diel & Cote, Ca Treat Reviews 2000)

8. Occult tumour cells disseminated tumour cells circulating tumour cells minimal residual disease micrometastasis early cancer spread

9. Pilot studies Ref. Salvadori 1990 Mansi 1991 Diel 1994 Harbeck 1994 BM+ 17% 25% 45% 38% Follow- up (mnd) 48 76 77 39 Relapse BM- 24% 25% 23% 15% Relapse BM+ 30% 48% 77% 39% p- value ns <0.005

10. Collaboration between several hospitals in the Oslo region aspiration of 40 ml bone marrow from crista iliaca ant and post peroperatively 920 patients included (1995-98) Oslo Micrometastasis project

11. Isolation of mononuclear cells

12. Immunocytochemical detection Single cell suspension 2 x 10 6 cells/test Spinns to slides fixation - acetone Anti-epitelial antibody AE1/AE3 Enzymatic visualisation of antibody - APAAP Y Y Y

14. Tumour cell

15. Standardization of the immunocytochemical detection of cancer cells in BM and blood: I. Establishment of objective criteria for the evaluation of immunostained cells The European ISHAGE Working Group for Standardization of Tumor cell detection E. Borgen et al. 1999. Cytotherapy 1:377-388

16. Histopatological data n=920 Nodal status Tumor status N0 N+ T1 T2 T3-4 63% 33% 58% 29% 6% frequency Naume et al Clin Can Res 2001;7:4122-9

17. Detection of tumour cells T1 11.2% T2 15.0% T3 22.6% N0 9.9% N+ 20.6% Tumour status Nodal status BM + Naume et al Clin Can Res 2001;7:4122-9

18. Correlation of bone marrow positvity to other known prognostic characteristica ER/PgR p53 Catepsin D c-erbB2 Vascular infiltration +/-,-/+,+/+ -/- Pos Neg Pos Neg Pos Neg Yes No 12.0 % 17.6 % 16.2 % 11.9 % 13.1 % 14.6 % 23.9 % 12.3 % 17.4 % 12.6 % 0.05 5 0.12 8 0.57 5 0.02 4 0.04 5 BM+ p-value Naume et al Clin Can Res 2001;7:4122-9

19. Clinical follow-up No of patients with infiltrating cancer, M0 and evaluable immunocytology Obs.time median (range) No of relapses No of systemic relapses No of locoregional relapses No died of breast cancer 817 49 (½-85) mnd 174 (21%) 127 (16%) 47 (6%) 94 (11%) Wiedswang JCO, 18; 3469, 2003

20. Localisation of systemic relapse Wiedswang JCO, 18; 3469, 2003 Frequency % Skeleton Liver Lung CNS 10.2 5.8 5.2 1.9

21. Survival according to bone marrow status p<0.001 DDFS p<0.001 BCSS BM- BM+

22. Survival lymph node-pos (N+) p=0.008 p=0.001 DDFS BCSS BM+ BM-

23. Survival lymph node-negative (N0) BCSS DDFS

24. Survival T1N0 without adjuvant treatment p=0.014 P<0.001

25. Uni -& multivariat analysis Cox-regression: –BM+ versus BM- –histological grade –T1 versus T2-4 –N0 versus N1 –hormone receptor (ER+ or/and PgR+ versus both-) –p53 –c-erbB2 –catepsinD –vascular infiltration

26. Multivariat analysis (Cox regression) p -values breast cancer specific survival T-status <0.001 <0.001 ns N-status <0.001 BM 0.018 0.015 ns hormonreceptor <0.001 <0.001 ns histologic grade 0.002 0.017 0.021 All N+ N0

27. Multifactorial survial analyses with BM status P<0.001p=0.003 BM+ BM-

28. Mansi et al Patient inclusion: 1981-86 Mansi et al. 1987/91/99

29. Occult tumour cells in bone marrow and clinical outcome Braun et al, 2000, NEJM

34. Persisting tumor cells in BM after chemotherapy - the impact on survival Braun et al. 2000

35. Second bone marrow aspiration 3 years after surgery

36. Pasient characteristics (n=356) –70.2% T1 –71.9% N0 –80.1% hormon receptor positiv 14.9% BM+ –correlates to nodal status and adjuvant treatment

37. Second bone marrow aspiration 3 years after surgery Clinical data: –26 mnd after BM2 –66 mnd after primary surgery 32 relapses –12 local –20 systemic 10 pasients died of breast cancer

38. Distant disease free survival Breast cancer spesific survival p<0.001 Wiedswang et al Clin Ca Res 2004 Survival according to second bone marrow status

39. Breast cancer specific survival N+ (n=93) N0 (n=256) p=0.003p=ns Wiedswang et al Clin Ca Res 2004

40. BM1 & BM2 n= 356 patients 4 groups: ++ 17 patients +- 54 ” -+ 33 ” -- 231 ”

41. BM1 & BM2 Distant disease free survival Breast cancer specific survival p<0.001 ++ Wiedswang et al Clin Ca Res 2004

42. ” Magasinet”, Dagbladet april 03

43. Occult tumour cells in blood and bone marrow, correlated to clinical outcome (n=341) BM PB Months after operation Cumulative survival p<0.001 p=0.001 BM+ BM- BM+ BM- PB+ PB- PB+ PB- p=0.002 Events/total no of pts: BM-: 17/293 BM+: 10/48 Events/total no of pts: PB-: 20/307 PB+: 7/34 Events/total no of pts: PB-: 8/307 PB+: 6/34 Events/total no of pts: BM-: 8/293 BM+: 6/48 p<0.001 BM+ BM- PB+ PB- p<0.001 p=0.001 Events/total no of pts: PB-: 28/307 PB+: 9/34 Events/total no of pts: BM-: 23/293 BM+: 14/48 DDFSBCSSDFS Wiedswang et al Int J Ca 2006

47. Xenidis JCO 2006,24:3756-62

48. Benoy et al Br J Ca 2006 RT-PCR BM and blood 147 M0/M+ pts Bone marrow superior to blood in predicting outcome