Prognostic value of occult breast cancer cells

in blood and bone marrow

Gro WiedswangSurgical unit,

Ullevål University Hospital

&The Micrometastasis lab,

Radiumhospital-RikshospitalOslo

Norway

Rome 16.11.06

- 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

Challenge of breast cancer

• Better diagnostics• Screening• Improved surgical

treatment• Improved adjuvant

treatment

25-30% of N0 patients die whitin 5 years40% of N1 patients survive > 10 år

Overgaard NEJM 1997

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??

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

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 ?

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)

Occult tumour cells

• disseminated tumour cells• circulating tumour cells• minimal residual disease• micrometastasis• early cancer spread

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

<0.005

<0.005

•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

Isolation of mononuclear cells

Immunocytochemical detection

• Single cell suspension2 x 106 cells/test

• Spinns to slides• fixation - acetone

• Anti-epitelial antibodyAE1/AE3

• Enzymatic visualisation of antibody - APAAP

YY

Y

Tumour cell

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

Histopatological datan=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

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

Correlation of bone marrow positvity to other known prognostic characteristica

ER/PgR

p53

Catepsin D

c-erbB2

Vascular infiltration

+/-,-/+,+/+-/-

PosNeg

PosNeg

PosNeg

YesNo

12.0 %17.6 %

16.2 %11.9 %

13.1 %14.6 %

23.9 %12.3 %

17.4 %12.6 %

0.055

0.128

0.575

0.024

0.045

BM+ p-value

Naume et al Clin Can Res 2001;7:4122-9

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

Localisation of systemic relapse

Wiedswang JCO, 18; 3469, 2003

Frequency %

Skeleton Liver Lung CNS 10.2 5.8 5.2 1.9

Survival according to bone marrow status

p<0.001

DDFS

p<0.001

BCSS

BM- BM-

BM+

BM+

Survival lymph node-pos (N+)

p=0.008p=0.001

DDFS BCSS

BM+

BM+

BM-BM-

Survival lymph node-negative (N0)

BCSSDDFS

Survival T1N0 without adjuvant treatment

BCSS T1N0-adj

TTDØD

100806040200

Cu

m S

urv

iva

l

1,0

,8

,6

,4

,2

0,0

DIICPNEN

2,00

2,00-censored

1,00

1,00-censored

BCSS T1N0-adj

TTDØD

100806040200

Cu

m S

urv

iva

l

1,0

,8

,6

,4

,2

0,0

GR1_2VS3

2,00

2,00-censored

1,00

1,00-censored

p=0.014 P<0.001

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

Multivariat analysis(Cox regression)

p -values breast cancer specific survival

T-status <0.001 <0.001 nsN-status <0.001BM 0.018 0.015 nshormonreceptor <0.001 <0.001 nshistologic grade 0.002 0.017 0.021

All N+ N0

Multifactorial survial analyses with BM

statusN0 pasienter

Tid til død (mnd)

100806040200

Ove

rle

vels

e

1,0

,8

,6

,4

,2

0,0

multifaktoriell +/-

2,00

2,00-censored

1,00

1,00-censored

N1 pasienter

Tid til død (mnd)

100806040200

Ove

rle

vels

e

1,0

,8

,6

,4

,2

0,0

multifaktoriell +/-

2,00

2,00-censored

1,00

1,00-censored

P<0.001 p=0.003

BM+

BM+

BM-BM-

Mansi et al Patient inclusion: 1981-86

Mansi et al. 1987/91/99

Occult tumour cells in bone marrow and clinical outcome

Braun et al, 2000, NEJM

Persisting tumor cells in BM after chemotherapy - the

impact on survival

Braun et al. 2000

Second bone marrow aspiration 3 years after surgery

Second bone marrow aspiration 3 years after

surgery

• 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

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

Distant disease free survival

Breast cancer spesific survival

p<0.001 p<0.001Wiedswang et al Clin Ca Res 2004

Survival according to second bone marrow status

Breast cancer specific survival N+

(n=93)

N0(n=256)

p=0.003 p=ns

Wiedswang et al Clin Ca Res 2004

BM1 & BM2

• n= 356 patients• 4 groups:

++ 17 patients+- 54 ” -+ 33 ”-- 231 ”

BM1 & BM2

Distant disease free survival

Breast cancer specific survival

p<0.001 p<0.001

++++

Wiedswang et al Clin Ca Res 2004

”Magasinet”, Dagbladet april 03

Occult tumour cells in blood and bone marrow, correlated to clinical

outcome (n=341)

BM

PB

Months after operation

Cum

ulat

ive

surv

ival

Cum

ulat

ive

surv

ival

p<0.001 p=0.001

BM+

BM-

BM+

BM-

PB+

PB-

PB+

PB-

p=0.002

Events/total no of pts:BM-: 17/293BM+: 10/48

Events/total no of pts:PB-: 20/307PB+: 7/34

Events/total no of pts:PB-: 8/307PB+: 6/34

Events/total no of pts:BM-: 8/293BM+: 6/48

p<0.001

BM+

BM-

PB+

PB-

p<0.001

p=0.001

Events/total no of pts:PB-: 28/307PB+: 9/34

Events/total no of pts:BM-: 23/293BM+: 14/48

DDFS BCSSDFS

Wiedswang et al Int J Ca 2006

Xenidis JCO 2006,24:3756-62

Benoy et alBr J Ca 2006

RT-PCR

BM and blood

147 M0/M+ pts

Bone marrow superior to blood in predicting outcome