Lack of Effect of Adjuvant Chemotherapy on the Elimination of

Lack of Effect of Adjuvant Chemotherapy on the
Elimination of Single Dormant Tumor Cells in Bone
Marrow of High-Risk Breast Cancer Patients
By Stephan Braun, Christina Kentenich, Wolfgang Janni, Florian Hepp, Johann de Waal, Fritz Willgeroth,
Harald Sommer, and Klaus Pantel
Purpose: There is an urgent need for markers that
can predict the efficacy of adjuvant chemotherapy in
patients with solid tumors. This study was designed to
evaluate whether monitoring of micrometastases in
bone marrow can predict the response to systemic
chemotherapy in breast cancer.
Patients and Methods: Bone marrow aspirates of 59
newly diagnosed breast cancer patients with either
inflammatory (n 5 23) or advanced (> four nodes
involved) disease (n 5 36) were examined immunocytochemically with the monoclonal anticytokeratin (CK)
antibody A45-B/B3 (murine immunoglobulin G1; Micromet, Munich, Germany) before and after chemotherapy with taxanes and anthracyclines.
Results: Of 59 patients, 29 (49.2%) and 26 (44.1%)
presented with CK-positive tumor cells in bone marrow
before and after chemotherapy, respectively. After chemotherapy, less than half of the previously CK-positive
patients (14 of 29 patients; 48.3%) had a CK-negative
bone marrow finding, and 11 (36.7%) of 30 previously
CK-negative patients were CK-positive. At a median
follow-up of 19 months (range, 6 to 39 months),
Kaplan-Meier analysis of 55 assessable patients revealed a significantly reduced overall survival (P 5
.011; log-rank test) if CK-positive cells were detected
after chemotherapy. In multivariate analysis, the presence of CK-positive tumor cells in bone marrow after
chemotherapy was an independent predictor for reduced overall survival (relative risk 5 5.0; P 5 .016).
Conclusion: The cytotoxic agents currently used for
chemotherapy in high-risk breast cancer patients do
not completely eliminate CK-positive tumor cells in bone
marrow. The presence of these tumor cells after chemotherapy is associated with poor prognosis. Thus, bone
marrow monitoring might help predict the response to
systemic chemotherapy.
J Clin Oncol 18:80-86. © 2000 by American
Society of Clinical Oncology.
N ADVANCED BREAST cancer, the rationale for adjuvant therapy is based on the assumption that clinically
undetectable hematogenous dissemination of viable tumor
cells has already occurred as indicated by a number of risk
factors, including tumor size greater than 2 cm, cutaneous
lymphangioitis carcinomatosa, and regional lymph node
metastases. This rather indirect extrapolation from locoregional to distant level is necessary because early dissemination of single tumor cells is usually missed by procedures
currently used for tumor staging. The development of
monoclonal antibodies directed toward epithelial differentiation proteins, however, has now opened a diagnostic
window to more directly detect hematogenously disseminated carcinoma cells. Most studies on this issue have used
bone marrow as an indicator organ for the presence of
extrinsic carcinoma cells because the mesenchymal organ is
normally devoid of epithelial cells. This, therefore, allows
detection of early tumor-cell dissemination. Positive findings in bone marrow aspirates were correlated with a poor
prognosis.1-5
As we have previously shown, however, few of these
isolated disseminated tumor cells are in the state of cellular
proliferation. Most of the cells seem to be resting in the G0
phase of the cell cycle, as demonstrated by the absence of
Ki-67 positivity.6 In addition, frequent overexpression of
the erbB-2 proto-oncogene seems to be a common feature of
these cells (Braun S, Heumos I, et al, manuscript submitted
for publication).6,7 For this reason, disseminated tumor cells
in bone marrow might be fairly resistant to cytotoxic
therapeutic agents.
In the present study, we evaluated whether primary and
adjuvant chemotherapy in patients with inflammatory or
node-positive breast cancer can eliminate minimal residual
disease as determined by the detection of single cytokeratin
(CK)-positive tumor cells in bone marrow. Further, we
investigated whether CK-positive tumor cells in bone marrow after chemotherapy have an independent prognostic
influence on overall survival.
Our data demonstrate that CK-positive tumor cells frequently escape aggressive chemotherapy, suggesting that
I
From the I. Frauenklinik der Ludwig-Maximilians-Universität, Munich; Abteilung für Gynäkologie, Kreiskrankenhaus, Dachau; and
Frauenklinik, Universitätsklinikum Eppendorf, Hamburg, Germany.
Submitted March 22, 1999; accepted August 16, 1999.
Supported by grants from ı̀Freunde der Maistrasseı̂, Curt-Bohnewand-Foundation, and Friedrich-Baur-Foundation, Munich, Germany.
Address reprint requests to Stephan Braun, MD, I. Frauenklinik,
Klinikum Innenstadt, Ludwig-Maximilians-Universität, Maistrasse 11,
D-80337 München, Germany; email [email protected].
© 2000 by American Society of Clinical Oncology.
0732-183X/00/1801-80
80
Journal of Clinical Oncology, Vol 18, No 1 (January), 2000: pp 80-86
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81
THERAPY-RESISTENT TUMOR CELLS IN BONE MARROW
these cells are resistant to the applied cytotoxic agents.
Further, we were able to show that bone marrow CK
positivity after chemotherapy is an independent predictor of
reduced overall survival. Therefore, the elimination or
survival of disseminated tumor cells in bone marrow may be
considered a predictive factor in adjuvant breast cancer
therapy.
PATIENTS AND METHODS
Patients and Treatment
Fifty-nine consecutive patients, admitted to the I. Frauenklinik,
Ludwig-Maximilians University (Munich, Germany) between January
1995 and December 1998 with either inflammatory or node-positive
breast cancer but without distant metastatic disease, were included in
this study. After providing written informed consent, patients underwent bone marrow aspiration from both upper iliac crests before and 3
weeks after completion of treatment. The trial was approved by the
institutional ethical board. Tumor stage and grading were classified
according to the 4th edition of the tumor-node-metastasis classification
of the International Union Against Cancer.8 Investigators were unaware
of the immunocytochemical findings in the bone marrow. Likewise,
immunocytochemical bone marrow screening was performed without
knowledge of the individual histopathologic results.
The primary surgical treatment consisted of either breast conservation or modified radical mastectomy, leading to R0 resection in all
reported cases. Routine axillary dissection included levels I to III for
the high-risk study population. In all patients treated with breast
conservation, telecobalt radiation therapy was administered. The median absorbed dose in the target volume was either 50.0 Gy given in 25
fractions or 50.4 Gy given in 28 fractions (in cases of concomitant
chemotherapy).
Patients with greater than four involved axillary lymph nodes (n 5
36) received either six cycles of docetaxel 75 mg/m2 body-surface area
plus epirubicin 90 mg/m2 or four courses of epirubicin 90 mg/m2 plus
cyclophosphamide 600 mg/m2, followed by three courses of cyclophosphamide 600 mg/m2, methotrexate 40 mg/m2, and fluorouracil 600
mg/m2 every 21 days. Patients with inflammatory breast cancer (n 5
23) received primary chemotherapy, which consisted of three cycles of
epirubicin and cyclophosphamide (as described above) or paclitaxel
175 mg/m2 plus epirubicin 90 mg/m2, followed by surgery and three
additional cycles of postoperative chemotherapy.
At the time of primary surgery, complete baseline diagnostic
evaluation for distant metastases included plain chest radiography,
(contralateral) mammography, ultrasound of the liver, and whole-body
bone scan. In case of evidence for distant disease, patients were
excluded from the study. Clinical follow-up examinations were performed every 3 months.
Bone Marrow Preparation
The procedure for bone marrow preparation has been described
previously.9 Bilateral bone marrow samples were obtained under either
general or local anesthesia from both upper iliac crests of each patient
through a needle aspiration and collected in heparin. After centrifugation through a Ficoll-Hypaque density gradient (density 1.077 3 g/mol;
Pharmacia Biotech, Uppsala, Sweden) at 900 3 g for 30 minutes,
mononucleated interface cells (MNCs) were washed, and 106 cells
were reproducibly centrifuged onto each glass slide at 150 3 g for 5
minutes.9 The reliability of cytocentrifugation to allow a well-defined
cell transfer (ie, 106 cells per glass slide) has been previously
documented.9 The volumes of all aspirates ranged from 3.5 to 12.0 mL
(mean, 8.5 mL), yielding between 4.8 3 106 and 6.9 3 107 MNCs
(mean, 1.6 3 107 MNCs).
Immunocytochemistry
After overnight air-drying, slides were either stained immediately or
stored at 280°C. For each patient, 2 3 106 cells were screened
manually by bright-field microscopy; for control purpose, an identical
number of cells served for immunoglobulin isotyping. We entirely
omitted morphologic criteria and relied only on the immunocytochemical staining of MNC. Because of the absence of any background
staining, we obtained no indeterminate results. All slides were examined by two independent observers who agreed on the same result in
more than 95% of the specimens. The final consensus decision on
discrepant results required critical re-evaluation by both investigators.
The monoclonal antibody, A45-B/B3 (murine immunoglobulin G1;
Micromet, Munich, Germany), directed toward a common epitope of
CK polypeptides, including the heterodimers CK8/18 and CK8/19,10
was used at 1.0 to 2.0 mg/mL to detect tumor cells in bone marrow
cytospin preparations. The specificity of the antibody reaction was
controlled by an appropriate dilution of the unrelated mouse myeloma
antibody MOPC21 as isotype control on patients’ bone marrow
specimens (Sigma, Deisenhofen, Germany). The breast cancer cell line
BT-20 served as the positive control for CK immunostaining.9 The
specific reaction of the primary antibody was developed with the
alkaline phosphatase antialkaline phosphatase technique combined
with the new fuchsin method to indicate antibody binding,11 as
previously described.9
Statistical Analysis
To ensure data quality, all reported immunocytochemical and histopathologic results, as well as event reports, were verified during
follow-up by re-examination of original data files. The primary end
point was survival, as measured from the date of surgery to the time of
the last follow-up or cancer-related death.
To compare categorical variables, we used the x2 test. Differences
between means of independent samples with continuous variables were
calculated from the t test. The Kaplan-Meier method12 was applied to
estimate overall survival, and these values were compared using the
log-rank test. The Cox proportional hazards regression model was used
for multivariate analysis; variables were entered stepwise in the model
to assess the independent prognostic value of the CK status compared
with other prognostically relevant variables.13 Differences between
groups were considered significant if the P values were less than .05 in
a two-tailed test. For the described statistical analyses, we used the
SPSS 6.1.1 software package (SPSS, Inc, Chicago, IL).
RESULTS
Bone marrow samples in this study were taken from 59
newly diagnosed high-risk breast cancer patients. The applied immunoassay identified disseminated CK-positive
tumor cells in 29 patients (49.2%) at the time of first
diagnosis. Evidence for the reliable specificity of the applied
antibody is available from our previous studies, which
demonstrated the absence of cross-reactivity with autochthonous bone marrow cells.1,14
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82
BRAUN ET AL
Table 1.
Comparison of Patients’ Variables and Frequency of Isolated Breast Cancer Cells in Bone Marrow
Patients With CK-Positive Cells
Variables
Total
Age†
, 49 years
$ 49 years
Menopausal status
Premenopause
Postmenopause
Histologic type
Inflammatory
Other‡
Number of lymph node metastases§
4-9 lymph nodes
. 9 lymph nodes
Chemotherapy
Docetaxel/epirubicin
Paclitaxel/epirubicin
EC/CMF
Before Therapy
After Therapy
No. of
Patients
No.
%
No.
%
P*
59
29
49
26
41
.58
26
33
13
16
50
48
11
15
42
45
.58
.81
23
36
11
18
48
50
9
17
39
47
.55
.81
23
36
15
14
61
39
13
13
57
36
.55
.81
23
36
9
20
39
56
9
17
39
47
.99
.48
18
14
27
8
6
13
44
42
48
8
4
14
44
29
52
.99
.43
.79
Abbreviation: EC/CMF, epirubicin, cyclophosphamide/cyclophosphamide, methotrexate, fluorouracil.
*x2 test for contingency tables; P , .05 was considered statistically significant. The number of patients with CK-positive cells before chemotherapy was compared
with that after chemotherapy.
†Median, 49 years; range, 28 to 72 years.
‡Includes ductal and lobular carcinomas.
§A mean number of 20 lymph nodes (range, 12 to 35 lymph nodes) per patient were analyzed.
The whole study population had metastasis to regional
axillary lymph nodes (pN1-2) but no evidence of manifest
distant metastases (stage M0). Correlations between patients’ clinical variables and detection of CK-positive tumor
cells in bone marrow are listed in Table 1. In patients
analyzed before chemotherapy, isolated CK-positive tumor
cells were found in 15 (65.2%) of 23 inflammatory cases
and 14 (38.9%) of 36 node-positive cases. After chemotherapy, CK-positive bone marrow findings were assessed in 26
(42.4%) of 59 patients; 13 (56.5%) of 23 inflammatory
cases and 13 (36.1%) of 36 node-positive cases. In inflammatory breast cancer, nine (60.0%) of 15 patients yielded
CK-positive bone marrow aspirates before and after chemotherapy, whereas six (40.0%) of 15 previously CK-positive
patients became CK-negative, and four (50.0%) of eight
previously CK-negative patients became CK-positive (Fig
1A). In node-positive patients, five (35.7%) of 14 primarily
CK-positive patients had positive aspirates before and after
chemotherapy; whereas eight (61.5%) of 13 previously
CK-positive patients became CK-negative, and seven
(25.9%) of 27 previously CK-negative patients became
CK-positive (Fig 1B). Moreover, no significant difference
between the mean number of tumor cells before (17 CKpositive cells per 2 3 106 MNC) and after chemotherapy
(12 CK-positive cells per 2 3 106 MNC) was found (P 5
.21; paired t test, t 5 1.3).
The median observation time of the study population was
19 months (range, 6 to 39 months). Table 2 lists a significantly higher relapse and death rate in patients with CKpositive tumor cells after chemotherapy compared with
patients who either remained CK-negative during therapy or
turned CK-negative after therapy. In the Kaplan-Meier
analysis for overall survival, tumor cells detected after
chemotherapy exerted a significant influence on patients’
prognosis (Fig 2). A Cox multiple regression analysis was
performed to see if bone marrow micrometastases after
chemotherapy were a significant predictor of reduced overall survival, independent of age, menopausal status, histology, number of involved lymph node, and the chemotherapy
applied. As indicated in Table 3, only the presence of
micrometastasis in bone marrow after chemotherapy (P 5
.016) and the number of involved lymph nodes (P 5 .039)
were independent predictors of poor survival.
DISCUSSION
Directing the antibody A45-B/B3 against the epithelial
differentiation marker CK, this study on 59 newly diagnosed breast cancer patients examined the influence of
single CK-positive tumor cells on a patient’s prognosis if
detected after systemic chemotherapy. We have previously
shown, by a critical and continuous (throughout the entire
period of patients’ enrollment) evaluation of 165 blinded
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83
THERAPY-RESISTENT TUMOR CELLS IN BONE MARROW
Fig 1. Monitoring of the elimination of CK-positive tumor cells during
chemotherapy in high-risk breast
cancer patients; every symbol represents the number of CK-positive cells
per 2 3 106 MNC. (A) Inflammatory
disease: epirubicin/cyclophosphamide (Œ) or paclitaxel/epirubicin (F)
treatment. (B) Node-positive disease:
epirubicin, cyclophosphamide/cyclophosphamide, methotrexate, fluorouracil (Œ) or docetaxel/epirubicin
(F) treatment.
noncarcinoma control patients, that the antibody A45-B/B3
is highly specific in detecting epitopes found on epithelial
cells but not on autochthonous bone marrow cells.14 This
specificity is, as we and others have pointed out, in contrast
to the considerable rate of false-positive results obtained
with antibodies directed against epithelial membrane antigen, milk fat globule, human epithelial antigen-125, and
other cellular mucins, including tumor-associated glycoprotein-12.9,14-18 Therefore, we consider the described CK
immunoassay as a reliable tool to detect clinically occult
hematogenous tumor-cell dissemination to bone marrow.
Our recent study of more than 500 newly diagnosed
breast cancer patients (stages I to III) revealed that identification of single CK-positive tumor cells in bone marrow is
an independent indicator for poor survival.1 In the present
study, we examined bone marrow aspirates from both
node-positive and inflammatory breast cancer patients be-
fore and after chemotherapy. Applying our validated bone
marrow immunoassay to these high-risk patients, we found
that cytotoxic therapy with taxanes and anthracyclines did
not significantly reduce the number of CK-positive tumor
cells (Fig 1).
Overall, the number of tumor cells identified per patient
using the described immunoassay was rather low, usually
Table 2. Manifestation of Distant Metastases and Death in Patients With
and Without CK-Positive Tumor Cells After Chemotherapy
Bone Marrow Aspirates After Therapy
CK-Negative
Patients
(n 5 32)
CK-Positive
Patients
(n 5 23)
Variables
No.
%
No.
%
P*
Distant metastases
Deaths
13
10
57
43
4
3
13
9
.0005
.0033
*x2 test for contingency tables; P , .05 was considered statistically
significant.
Fig 2. Cumulative overall survival of 55 breast cancer patients at median
follow-up of 19 months (range, 6 to 39 months). Presence (black line) versus
absence (gray line) of CK-positive tumor cells in bone marrow after
chemotherapy (P 5 .011; log-rank test).
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84
Table 3.
BRAUN ET AL
Univariate and Multivariate Analysis of Overall Survival in 55
Breast Cancer Patients
Univariate
Analysis (P )†
Variables*
Age
$ 49 (7/33); , 49 (6/22)
Menopausal status
pre (5/20); post (8/35)
Number of lymph node metastases
4-9 (1/22); . 9 (12/33)
CK status
CK-positive (10/23); CKnegative (3/32)
Histology
Inflammatory (10/21); other
(3/34)
Chemotherapy
Taxanes (6/29); anthracyclines
(7/26)
Multivariate Analysis‡
P
RR
95% CI
.76
–§
–
.58
.94
–
–
.97
.018
8.6
1.1-66.1
.039
.011
5.0
1.4-18.3
.016
.09
–
–
.22
.77
–
–
.91
Abbreviations: RR, relative risk; CI, confidence interval.
*The number of patients (events/total) is given in parenthesis of patients.
†Factors compared by log-rank test.
‡Cox proportional-hazards model fitted for multivariate analysis.
§No RR available because variable not significant with respect to multivariate model.
between 1 3 10-6 and 10 3 10-6 CK-positive cells (Fig 1),
which is in the range of previous results.1,5,19,20 Thus, the
lower detection level of this assay is determined by an
unavoidable sampling error that seems to be an important
issue of the presented approach. Interpretation of the appearance or disappearance of a few cells as either success or
failure of the applied therapy seems to be possible but
should be performed carefully because false-negative bone
marrow results cannot be completely excluded. Based on
the findings of our previous monitoring study on patients
with breast and colorectal cancers,21 we have improved the
sensitivity of our CK assay by increasing the number of
mononucleated cells analyzed before and after therapy from
4 3 105 to 2 3 106. Considering the low frequency of tumor
cells and their presumed heterogeneous distribution in the
skeleton, we evaluated two aspirates from both iliac crests,
a procedure that has been shown to allow the detection of
approximately 90% of patients with positive bone marrow
findings.9 Using these assay conditions, a recent monitoring
study on stage C prostate cancer patients demonstrated that
a therapeutic depletion of CK-positive cells under androgen
deprivation is measurable.22 In addition, we used an antiCK
antibody that has a higher specificity and sensitivity than the
one used in the prostate cancer study.14 Although the new
developments in the enrichment of tumor cells using immunomagnetic beads are promising, the reproducibility of this
new technology is still under investigation.23 In this study,
we used an improved immunoassay, which closely follows
the recent recommendations of the Tumor Cell Detection
Committee of the International Society of Hematotherapy
and Graft Engineering, to minimize the methodologic influence of a sampling error.24
However, the strongest argument against the assumption
that our bone marrow findings are simply the result of a
sampling error is the validation of these findings by the
clinical course of disease. After a median observation time
of 19 months for the appropriate assessment of predictive
information on patients’ prognosis, we determined patients’
clinical outcome based on a CK-positive bone marrow
finding after chemotherapy. According to these survival
statistics (Table 3), the lack of effect of chemotherapy on
the elimination of residual CK-positive tumor cells in bone
marrow seemed to be measurable, whereas the potential
influence of false-negative results on our analysis was not
statistically relevant. Although we were not able to consider
late recurrences within the short observation time of our
study, our analysis at the time of follow-up showed that the
detection of CK-positive cells after adjuvant chemotherapy
is significantly correlated with reduced overall survival.
After some years, additional follow-up bone marrow aspirations might reveal a therapeutic effect of tamoxifen in
estrogen receptor–positive patients; this, however, was not
the scope of the present study. The high rate of metastatic
recurrences (Table 2) leading to the markedly poor prognosis of the study population is explained by the selection of
high-risk patients with inflammatory breast cancer or extensive lymph node involvement. Yet, within this preselected
study population, detection of CK-positive cells after chemotherapy identified a patient’s subgroup with an even
worse prognosis and shorter life-expectancy (Fig 2). Thus,
CK-positivity of bone marrow after chemotherapy may be
suitable to predict the response to systemic chemotherapy.
The extent of axillary lymph node involvement is generally viewed as the predictor of survival in breast cancer
patients. However, the observation that distant metastases
occurred in up to 30% of node-negative patients25 and the
finding that some 40% of node-positive patients survive for
10 years or more26,27 suggest that lymph node metastases
cannot be equalled to hematogenous spread28 and, hence, to
metastasis-related death. Nevertheless, our multivariate statistics demonstrated that both the increased number of
involved lymph nodes and the persistence of micrometastatic breast cancer cells in bone marrow are independent
prognostic factors for poor survival (Table 3). In contrast to
the presence of residual tumor cells, which can be monitored at any time after administration of adjuvant treatment
and may provide information on the kinetics of metastatic
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85
THERAPY-RESISTENT TUMOR CELLS IN BONE MARROW
cells for the individual patient, the predictive value of the
extent of lymph node metastasis is limited to the time of
surgery. Most notably, we have recently shown that early
hematogenous dissemination to bone marrow seems to be a
prognostic factor independent of lymphatic spread.1
Our previous study on breast cancer patients demonstrated that early dissemination of tumor cells to bone
marrow is associated with an increased frequency of bone
and multiple metastases.1 The increased number of tumor
cells in some patients after chemotherapy may indicate that
chemotherapy has mobilized tumor cells to or from the bone
marrow, as suggested in previous studies.29,30 These data
point out that at least a redistribution of the residual tumor
load by chemotherapy-dependent mobilization and therapyinduced elimination are to be considered for the varying
numbers of CK-positive tumor cells in bone marrow after
chemotherapy. However, the observation of the prognostic
impact of CK-positive tumor cells in bone marrow after
chemotherapy supports the notion that biologic factors
influence persistent bone marrow CK positivity in the face
of receiving adjuvant chemotherapy. Among these, dormancy and metastatic growth potential have been identified
as characteristics of disseminated tumor cells in bone
marrow in our previous studies (Braun S, Heumos I, et al,
manuscript submitted for publication).6 Because micrometastatic cells rarely express proliferation-associated
markers, such as Ki-67 and p120,6 they might be fairly
resistant to some chemotherapeutic agents. Together with
the data of our present study, this assumption was further
supported by recent studies in breast cancer patients, showing the persistence of isolated tumor cells in bone marrow
even after high-dose chemotherapy.31,32 Overexpression of
the erbB-2 proto-oncogene might be regarded as another
factor mediating resistance to cytotoxic regimens.33 Moreover, we have shown that overexpression of the erbB-2
proto-oncogene characterized an aggressive subset of micrometastatic breast cancer stem cells, and such overexpression could be correlated with poor survival (Braun S,
Heumos I, et al, manuscript submitted for publication).
According to these data, together with the prognostic
relevance of potentially chemotherapy-resistant CK-positive cells, it may be appropriate to consider combinations of
chemotherapy with cell cycle–independent treatment modalities. Among several options, antibody-based immunotherapy has been recently proposed as effective treatment in
breast34,35 and colorectal36 cancer. By the implementation
of cell cycle–independent therapies, combined antibodychemotherapy strategies may be beneficial in the prevention
of metastatic disease. In the future, the option of a surrogate
marker for immediate monitoring of the efficacy of anticancer therapy against micrometastatic disease with the described bone marrow assay can be hardly underestimated
because it would relieve the burden of using the 5-year
survival count as the sole assessment of therapeutic efficacy.
The present study may be viewed as one of the first steps
toward the implementation of such a surrogate marker.
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