This document summarizes a study on monitoring the response of circulating epithelial tumor cells (CETCs) to adjuvant chemotherapy in breast cancer patients. The study found that CETCs can be quantified from blood samples of patients before, during, and after chemotherapy. Three typical response patterns were observed: a decrease of more than 10-fold in CETC numbers correlated with a good prognosis; marginal changes in CETC numbers correlated with a medium prognosis; and an increase of more than 10-fold in CETC numbers, even after an initial decrease, correlated with a poor prognosis and high risk of early relapse. Patients with increasing CETC numbers had a significantly worse relapse-free survival compared to the other groups. Therefore,

1. Maintrac®
Liquid biopsy
circulating epithelial tumor cells
Transfusion Medicine Center Bayreuth TZB, Germany

3. CETC
S Carcinomas are from
epithelial origin (tissue)
S Carcinomas dissiminate
epithelial cells
⇒ CETCs
(circulating epithelial
tumor cells)

4. Maintrac®: „Liquid Biopsy“
15 ml EDTA Discrimination
live/ dead
Testing effectiveness
(before treatment)
Monitoring
therapy

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

6. 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 !

7. Living and dead cells
Epithelial cells
(green staining),
with red nucleus
as marker for
beginning
cell death.

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

9. 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.

10. 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

11. Good prognosiscellspermlblood
10
100
1.000
10.000
100.000
250
0
50
100
150
200
FEC
TaxEC
days
Decrease in cell numbers more than tenfold
Pachmann et al, J Clin Oncol, 2008, 26 (8): 1208-1215

12. Medium prognosis
(could be better)cellspermlblood
10
100
1.000
10.000
100.000
250
0
50
100
150
200
CMF
TaxEC
days
marginal changes in cell numbers
Pachmann et al, J Clin Oncol, 2008, 26 (8): 1208-1215

13. Poor prognosiscellspermlblood
10
100
1.000
10.000
100.000
250
0
50
100
150
200
FEC
TaxEC
days
Increase in cell numbers more than tenfold
Pachmann et al, J Clin Oncol, 2008, 26 (8): 1208-1215

14. cum.relapsefreesurvival
0.0
0.2
0.4
0.6
1.0
0.8
9000 300 600 1,200 1,500
days
Adjuvant therapy breast cancer
K . Pachmann et al, J Clin Oncol 2008, 26 (8): 1208-1215
increase > 10fold-censored
decrease > 10fold-censored
marginal change
increase > 10fold
decrease > 10fold
marginal change-censored
log rank p < 0.001
88 Patients were
included in this study
Clinical outcome
Increasing
cell numbers
correlate
highly
significant
with a
poor prognosis

15. Thanks for your attention!
Prof. Katharina Pachmann
kpachmann@laborpachmann.de
+49 921 850200

16. SIMFO Specialized Immunology Research + Development GmbH
& Laboratory Dr. Ulrich Pachmann
Kurpromenade 2
95448 Bayreuth
Germany
www.maintrac.com