Staining of Circulating Tumor Cells - as easy as a blood picture. Staining is moch more sensitive than isolation of CTCs.

1. Maintrac®
A tool for monitoring therapy success in solid tumors:
circulating epithelial tumor cells
Based on the presentation of Prof. Dr. med Katharina Pachmann MD
Transfusion Medicine Center Bayreuth TZB, Germany

2. Circulating Tumor Cells
from solid tumors
S Carcinomas are from
epithelial origin
S Carcinomas dissiminate
epithelial cells
⇒ CETCs
(circulating epithelial
tumor cells)
Y.
Shiozawa,
A
M
Havens,
K
J
Pienta
and
R
S
Taichman,
Leukemia
(2008)
22,
941–950

3. Testing
Microscope based
semi-automated image
evaluation
Recording of
S any solid tumor
S not for lymphoma
or leukemia

4. Method
Maintrac liquid biopsy
cell staining allows
quantitative detection
of vital circulating
tumor cells.
NO fixation.
NO isolation.
NO enrichment.
Fluorochrome
labeled antibody
FITC
Circulating
tumor cell
Surface
antigen
EpCAM
=NO cell loss

5. Cell gallery
of a patient
red stained nucleus
= dead cell

6. Monitoring therapy success
using Circulating Tumor Cells
monitor response to treatment with a particular therapeutic
product for the purpose of adjusting treatment to achieve
improved safety or effectiveness.
(‚Companoin Diagnostics‘: FDA)

7. Monitoring
Monitoring the Response of Circulating Epithelial Tumor
Cells to Adjuvant Chemotherapy in Breast Cancer Allows
Detection of Patients at Risk of Early Relapse
Katharina Pachmann, Oumar Camara, Andreas Kavallaris, Sabine Krauspe, Nele Malarski,
Mieczyslaw Gajda, Torsten Kroll, Cornelia Jo¨rke, Ulrike Hammer, Annelore Altendorf-Hofmann,
Carola Rabenstein, Ulrich Pachmann, Ingo Runnebaum, and Klaus Ho¨ffken
From the Clinic for Internal Medicine II,
Institution for Pathology, and Women’s
Hospital, Friedrich Schiller University;
Tumorzentrum, Jena; and Transfu-
sionsmedizinisches Zentrum, Bayreuth,
Germany.
Submitted July 25, 2007; accepted
November 2, 2007.
Authors’ disclosures of potential con-
flicts of interest and author contribu-
tions are found at the end of this
article.
Corresponding author: Katharina Pach-
mann, MD, PhD, Department of Experi-
mental Hematology and Oncology,
Clinic for Internal Medicine II, Friedrich
Schiller Universita¨t Jena, Erlanger Allee
101, D-07747, Jena, Germany; e-mail:
katharina.pachmann@med.uni-jena.de.
© 2008 by American Society of Clinical
Oncology
0732-183X/08/2608-1208/$20.00
DOI: 10.1200/JCO.2007.13.6523
A B S T R A C T
Purpose
To demonstrate that it is possible to monitor the response to adjuvant therapy by repeated analysis
of circulating epithelial tumor cells (CETCs) and to detect patients early who are at risk of relapse.
Patients and Methods
In 91 nonmetastatic primary breast cancer patients, CETCs were quantified using laser scanning
cytometry of anti–epithelial cell adhesion molecule–stained epithelial cells from whole unsepa-
rated blood before and during adjuvant chemotherapy.
Results
Numbers of CETCs were analyzed before therapy, before each new cycle, and at the end of
chemotherapy. The following three typical patterns of response were observed: (1) decrease in
cell numbers (Ͼ 10-fold); (2) marginal changes in cell numbers (Ͻ 10-fold); and (3) an (sometimes
saw-toothed) increase or an initial decrease with subsequent reincrease (Ͼ 10-fold) in numbers of
CETCs. Twenty relapses (22%) were observed within the accrual time of 40 months, including one
of 28 patients from response group 1, five of 30 patients from response group 2, and 14 of 33
patients from response group 3. The difference in relapse-free survival was highly significant for
CETC (hazard ratio ϭ 4.407; 95% CI, 1.739 to 9.418; P Ͻ .001) between patients with decreasing
cell numbers and those with marginal changes and between patients with marginal changes and
those with an increase of more than 10-fold (linear Cox regression model).
Conclusion
These results show that peripherally circulating tumor cells are influenced by systemic chemotherapy
and that an increase (even after initial response to therapy) of 10-fold or more at the end of therapy is
a strong predictor of relapse and a surrogate marker for the aggressiveness of the tumor cells.
J Clin Oncol 26:1208-1215. © 2008 by American Society of Clinical Oncology
INTRODUCTION
Solid malignant tumors of the breast are the most
frequent cause of death in women in the devel-
oped world. Although early detection, precise sur-
gery with wide margins, and adjuvant therapy
have improved results,1
relapse is not infrequent.
In premenopausal women, a first narrow peak
occurs approximately 8 to 10 months after mas-
tectomy, and a second peak occurs at 28 to 30
months. Postmenopausal patients display a peak
at approximately 18 to 20 months.2
After diagno-
sis of metastatic disease, the outcome is fatal. To
date, there is no tool to monitor the effect of
adjuvant treatment apart from statistical analy-
ses3
; however, prediction for the individual pa-
tient is restricted.
Solid tumors can seed tumor cells into the pe-
ripheral blood, which may, even after complete re-
section of the tumor, eventually grow into
metastases.Detectionofsuchcirculatingtumorcells
has been reported in patients with primary4-6
and
metastatic7
breast cancer, with a shorter survival in
patients with cells in bone marrow8
or in patients
with metastatic disease with higher cell numbers in
blood.7
In metastatic disease, the clinical conse-
quence of this result is questionable because there is
no indication that treatment will lead to improved
survivalinpatientswithpoorprognosis.9
Inpatients
with primary tumor, only 40% of patients carrying
isolated tumor cells in bone marrow experience
recurrence,6
indicating that a portion of the circu-
lating epithelial tumor cells (CETCs) may be bio-
logically irrelevant and tumor cells may differ in
JOURNAL OF CLINICAL ONCOLOGY O R I G I N A L R E P O R T
VOLUME 26 ⅐ NUMBER 8 ⅐ MARCH 10 2008
1208
Copyright © 2008 by the American Society of Clinical Oncology. All rights reserved.
Information downloaded from jco.ascopubs.org and provided by F Delbanco on March 9, 2008 from 141.35.226.179.
J Clin Oncol 2008, 26 (8):
1208-1215
Monitoring the Response of
Circulating Epithelial Tumor
Cells (CETC) to Adjuvant
Chemotherapy in Breast
Cancer Allows Detection of
Patients at Risk of Early
Relapse

8. Good prognosis
Pachmann
et
al,
J
Clin
Oncol,
2008,
26
(8):
1208-­‐1215
Decrease
of the cell
number
more than
10 fold
good
prognosis

9. c)
Pachmann
et
al,
J
Clin
Oncol,
2008,
26
(8):
1208-­‐1215
No change
of the cell number
Medium prognosis
(could be better)

10. Pachmann
et
al,
J
Clin
Oncol,
2008,
26
(8):
1208-­‐1215
Increase of the cell
number more than
10fold
bad
prognosis
Bad prognosis

11. 150012009006003000
days
1,0
0,8
0,6
0,4
0,2
0,0
cum.survival
marginal change-
censored
increase>10fold-
censored
decrease>10fold-
censored
marginal change
increase>10fold
decrease>10fold
relapse free survival
Clinical outcome
Increasing
cell numbers
correlate highly
significant with
a poor prognosis
log rank p < 0.001
K
.
Pachmann,
et
al.
J
Clin
Oncol
2008,
26
(8):
1208-­‐1215

12. Increasing cell numbers
What can you do?

13. Chemo-
sensitivity
identify patients who are
most likely to benefit
from a particular
therapeutic product
(‚Companoin Diagnostics‘: FDA)

14. Chemo-
sensitivity
J Cancer Therapy 2013,
4:597-605
Chemosensitivity Testing of
Circulating Epithelial Tumor
Cells (CETC) in Vitro:
Correlation to in Vivo
Sensitivity and Clinical
Outcome.

15. Chemo-
sensitivity
• Exposing the blood sample
to different drugs and
concentrations
• Determin the rate of dying
circulating epithelial tumor
cells to identify the most
effective drug for the patient

16. Typical pictures
Epithelial cells
(green staining),
with red nucleus
as marker for
beginning
cell death.

17. Cells dying under drug effect

18. Highly
effective
Low
effective
N.
Rüdiger
et
al,
J
Cancer
Therapy,
2013,
4,
597-­‐605
no drug
1 /10 concentration
blood equivalent
10 fold concentration

19. Relapse free survial of patients
with ovarian cancer
N.
Rüdiger
et
al,
J
Cancer
Therapy,
2013,
4,
597-­‐605
log rank p≤0.007
Sensitive vs.
resistant tumor
cells to standard
therapy
(carboplatin and
paclitaxel)

20. 10
100
1000
10000
100000
1000000
21.06.10
10.08.10
29.09.10
18.11.10
07.01.11
cells/mlblood
Carbo
Pac
Caelyx/ Doxo
Pro-
gress
0
10
20
30
40
50
60
70
80
90
100
Jun 10 Jul 10 Aug 10 Sep 10 Okt 10 Nov 10 Dez 10 Jan 11
%ofcellsdyingoff
Carbo
Pac
Doxo
Guideline
therapy
Effect following
change of
therapy

21. Diagnosis
Surgery
maintrac
cell counting
1. cycle guidelines
2. cycle guidelines
maintrac
cell counting
Increase in cell numbers
maintrac chemosensitivity
3. cycle adapted
4. cycle adapted
maintrac
cell couning
5. cycle adapted
6. cycle adapted
maintrac
cell counting
Decrease in cell numbers
3. cycle guidelines
4. cycle guidelines
maintrac
cell counting
Increase in cell numbers
maintrac chemosensitivity
5. cycle adapted
6. cycle adapted
maintrac
cell counting
Decrease in cell numbers
5. cycle guidelines
6. cycle guidelines
maintrac
cell counting
OPTIONAL:
maintrac chemosensitivity
D-ML-13345-01-00-
If cell numbers are
increasing,
chemosensitivity
testing can be
considered to
determine the drug with
the best efficiency.

22. Maintenance therapy

23. Good prognosis
Gradual decrease
in cell numbers
during Tamoxifen
therapy
10
100
1.000
10.000
100.000
1.000.000
-­‐400 -­‐200 0 200 400 600 800 1000 1200 1400
cells/ml
days
Tamoxifen

24. Bad prognosis
Continious
increase
during Tamoxifen
therapy
may proceed to
recurrence 10
100
1.000
10.000
100.000
1.000.000
-­‐400 -­‐200 0 200 400 600 800 1000 1200 1400
cells/ml
days
Tamoxifen
Relapse
after 849d
Relapse after
1112d
Relapse after
627d

25. K.
Pachmann
et
al,
J
Cancer
Res
Clin
Oncol
2011,
137:821-­‐828
Patients with
increasing cell
numbers have
a higher risk
of recurrence
Clinical outcome

26. maintenance
therapy
maintrac
cell counting
every 3 month
Increase
in cell numbers
take
change of therapy
into consideration
maintrac
cell counting
every 3 month
Decrease
in cell numbers
go on with therapy
maintrac
cell counting
every 3 month
If cell numbers
increase,
change of therapy
may be considered
monitor every
3 months

27. Long term
surveillance

28. Good prognosis

29. Bad prognosis

30. Long term surveillance

31. Clinical Outcome
P=0,029
Patients with
increasing cell
numbers
after the end of
maintenance
therapy
have an increased
risk of recurrence

32. Discussion
There is already a
discussion going
on, if it would be
better taking
tamoxifen 10
years instead of
stopping after
5 years.
hWp://am.asco.org/
extending-­‐adjuvant-­‐tamoxifen-­‐reduces-­‐breast-­‐cancer-­‐recurrence-­‐mortality

33. Effect of therapy switch
10
100
1000
10000
100000
1000000
08.05.04
20.09.05
02.02.07
16.06.08
29.10.09
13.03.11
AnzahlZellenpromLBlut
4 4
R
Tam Letr
Zol
OP

34. after end
of therapy
maintrac
cell counting
after 3 month
Increase
in cell numbers
restart
maintenance herapy
Decreasing
or stable
cell numbers
maintrac
cell counting
after 3 month
Decreasing
or stable
cell numbers
maintrac
cell counting
every 6 month
If cell numbers
increase,
restart therapy
and monitor

35. Thank you
for your attention

36. Association Transfusion Medicine Center in Bayreuth - TZB
SIMFO Specialized Immunology Science + Development GmbH &
Laboratory Dr. Ulrich Pachmann
Peter Pachmann Laboratory Dr. Pachmann
Kattjahren 8 Kurpromenade 2
22359 Hamburg 95448 Bayreuth
Germany Germany
www.maintrac.de