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Monitoring of disseminated tumor cells in bone marrow in high-risk breast cancer patients treated with high-dose chemotherapy
Article first published online: 27 DEC 2005
Copyright © 2005 Wiley-Liss, Inc.
International Journal of Cancer
Volume 118, Issue 11, pages 2877–2881, 1 June 2006
How to Cite
Drageset, V., Nesland, J. M., Erikstein, B., Skovlund, E., Sommer, H., Anker, G., Wist, E., Lundgren, S., Bergh, J. and Kvalheim, G. (2006), Monitoring of disseminated tumor cells in bone marrow in high-risk breast cancer patients treated with high-dose chemotherapy. Int. J. Cancer, 118: 2877–2881. doi: 10.1002/ijc.21709
- Issue published online: 14 MAR 2006
- Article first published online: 27 DEC 2005
- Manuscript Accepted: 4 OCT 2005
- Manuscript Received: 25 JUL 2005
- disseminated tumor cells;
- high-dose chemotherapy;
The present study aimed to investigate the clinical relevance of disseminated tumor cells (DTC) in breast cancer patients before and after high-dose adjuvant chemotherapy with or without progenitor stem-cell support. One hundred and eighteen high-risk stage II breast cancer patients entering the Scandinavian Study Group multicenter trial were randomized to 9 cycles of tailored and dose-escalated FEC (5-flurouracil, epirubicin, cyclophosphamide) or 3 cycles of standard FEC followed by high-dose chemotherapy. Bone marrow (BM) samples at diagnosis and 6 months after completion of chemotherapy were assessed for the presence of cytokeratin positive (CK+) cells. Before treatment, 29% of the patients were CK+ (21% in the dose-escalated group and 36% in the high-dose-group). Six months after treatment, 17% of the patients were CK+ (17 and 16% respectively). Of the 95 patients who were evaluated 6 months after treatment, 60% were consistently CK−. CK+ cells in BM was evaluated as a prognostic and predictive marker and compared to other defined prognostic factors of the primary tumor. Monitoring BM changes at the time of diagnosis and 6 months posttreatment is an independent predictive factor for breast-cancer-specific survival (BCS) (p = 0.001). Those who have consistent CK negative (−) BM findings constitute a group of patients with good prognosis. Our results suggest that changes in CK+ cells in BM before and after chemotherapy can be used clinically as a surrogate maker to predict outcome in breast cancer patients. © 2005 Wiley-Liss, Inc.
High-dose chemotherapy with stem-cell support has been studied in breast cancer patients with both metastatic and early-stage, high-risk disease. Phase II studies1, 2 suggested that the outcome of patients treated with high-dose therapy was superior to standard-dose chemotherapy. To confirm these results, several large randomized studies have been conducted. Even if a longer follow-up is required until final conclusions can be drawn, the results reported show that progenitor stem-cell-supported high-dose therapy does not appear to be sufficient as tumor reductive therapy.3, 4, 5
Several breast cancer studies6, 7 have shown a significant association between the detection of tumor cells in the BM, both at diagnosis and in follow-up, and increased risk of systemic relapse. Both Braun et al.8 and Wiedswang et al.9 reported that presence of cytokeratin (CK) staining cells in the BM after adjuvant conventional chemotherapy of breast cancer patients was associated with distant metastases and cancer-related death.
The Scandinavian Breast Cancer Group (9401 protocol) published their first results of a randomized adjuvant study in high-risk breast cancer in year 2000.4 Patients treated with 9 cycles of tailored and dose-escalated high-dose FEC with granulocyte colony-stimulating factor (G-CSF) support had significantly fewer breast cancer relapses compared with those treated with 3 cycles of FEC plus cyclophosphamide, thiotepa, carboplatin (CTCb) with autologous stem-cell support (p = 0.04). No significant difference in overall survival (OS) was demonstrated, nor in overall health related quality of life.10 From this study, all the Norwegian patients (n = 128) were asked to participate for further analysis. The aim was to analyze the association between DTC, standard prognostic factors and clinical outcome. Of the 128 Norwegian patients, 11 patients were excluded because of lack of consent for this substudy. The remaining 118 patients were examined for the presence of DTC in the BM before and after therapy, using standardized immunocytochemistry (ICC) and immunomagnetic-based enrichment procedures.7, 11, 12 Standard prognostic factors (age, number of positive lymph nodes, hormone receptor status, histologic grading) have been registered. Associations between these findings and DTC in the BM are reported.
Material and methods
525 Scandinavian women with high-risk breast cancer were randomized after surgery to receive one of two regimens of high-dose chemotherapy with or without hematopoietic progenitor cell support.4 The study was initiated in March 1994 and closed in March 1998. Of the 128 women entering the study from Norway, 118 were assessed for the presence of DTC in BM before therapy, and of them, 95 were reexamined 6 months posttreatment. The difference between numbers of subjects at the 2 points in time is due to dropouts and not deaths. The median follow-up at the time of analysis was 68 months.
The study included patients with an expected 5-year relapse-free survival (RFS) of about 30% or less and a life expectancy exceeding 3 months. The clinical inclusion criterion was primary breast cancer with either 8 or more nodes positive or 5 or more metastatic axillary nodes combined with receptor negativity and high nuclear grade. A bone scan was mandatory for inclusion together with bilateral Jamshidi biopsies.4
The subject characteristics are given in Table I. No differences between the study groups with regard to age, number of involved axillary lymph nodes, histology grade or receptor status were observed.
|Mean (min–max)||Mean (min–max)||Mean (min-max)|
|No. of positive lymph nodes||11 (5–26)||11 (5–26)||11 (5–26)|
|Age at the time of diagnosis||48 (34–60)||47 (30–59)||48 (30–60)|
|n (%)||n (%)||n (%)|
|Histologic grade I||4 (8)||1 (2)||5 (5)|
|II||20 (40)||25 (46)||45 (43)|
|III||26 (52)||28 (52)||54 (52)|
|Hormone receptor positive||23 (44)||29 (51)||52 (48)|
|Negative||29 (56)||28 (49)||57 (52)|
|BM at registration|
|CK+||12 (21)||21 (36)||33 (29)|
|CK−||45 (79)||37 (64)||82 (71)|
|BM after 6 months|
|CK+||8 (17)||8 (16)||16 (17)|
|CK−||38 (83)||41 (68)||79 (83)|
After informed consent, the patients were randomized into 2 groups receiving different types of adjuvant high-dose therapy as described in detail by Bergh et al.4 Briefly, 1 group (FEC) received 9 cycles of tailored FEC where the doses of FEC treatment cycles were based on hematological toxicity. The other group (CTCb) was treated with 3 cycles of standard FEC followed by a high-dose regimen, CTCb (6 g/m2 cyclophosphamide, 500 mg/m2 tiotepa, and 800 mg/m2 carboplatin). Peripheral blood progenitor cells (PBPC) were harvested after the third FEC course and infused 3 days after the CTCb chemotherapy. All patients were given G-CSF (filgrastim 5 μg/kg, Amgen/Roche) daily, starting day 1 after transfusion of the hematopoietic progenitor cells. Tamoxifen, 20 mg daily, was given to the patients following chemotherapy for 5 years irrespective of hormone receptor status. After completion of chemotherapy, all patients received standard, locoregional radiotherapy: 2 Gy × 25 toward the breast, including chest wall and regional lymph nodes.
Clinical follow-up was performed at 5 different hospitals and included clinical examination and blood tests. Clinical examinations and blood tests were carried out at each visit. X-ray examinations were based on clinical signs and symptoms. A bone scan was only done when clinical symptoms or elevated blood tests were suspicious. The follow-up was performed every 3rd month, the first two years, every 6th month, year 3 and 4 and once a year from the 5th year onwards. The study is in compliance with the Helsinki Declaration and is approved by the Regional ethical committee.
Immunocytochemical analysis of BM cells
After informed consent, 20-ml BM were aspirated from both sides of the posterior iliac crest at the time of diagnosis and 6, 12 and 24 months after therapy. Because of dropouts, the number of patients analyzed at 12 (n = 74) and 24 (n = 30) months was too small to draw any firm conclusions from the analysis. For each patient, 2 × 107 mononuclear bone marrow cells were studied. To enhance the sensitivity of the detection, depletion of CD45+ cells was done in all patient samples as described previously.13 After cytospin preparation, the cells were stained with anticytokeratin antibodies AE1 AE3 (MAb, Sanbio, Uden, The Netherlands). CK+ cells were scored as tumor cells, according to the ISHAGE EUROPE guidelines.14
BCS was used as primary end-point. Breast cancer RFS (including both locoregional relapse and distant metastases) was estimated and used as secondary end-point. Survival was estimated by the Kaplan–Meier method. Patients still alive were treated as censored. The log-rank test was performed to assess differences between patients with CK+ and CK− BM examinations. A statistical variable for analyzing the impact of changes in bone marrow findings from the time of diagnosis to 6 months posttreatment was created. This variable was named DTC changes. A test for linear trend was used for ordinal variables such as lymph nodes, age, histologic grading and DTC changes. Based on the biological assumption that CK+ cells remaining after therapy are predicting an unfavorable prognosis, the subgroups of DTC changes were placed in the following order: CK+ CK+, CK− CK+, CK+ CK− and CK− CK−. The variable was considered linear. The median survival time for the CK− patients was not possible to calculate because of the limited number of events. We, therefore, present 5-year survival in the tables.
When performing a log-rank test, other confounding factors may influence the result. Therefore, in addition to the log-rank test, independent prognostic effect of several variables was analyzed by Cox proportional hazards model. The variables were selected by backward elimination. Clearly insignificant factors were removed until one/several factors with borderline/significant association were remaining in the model. For all factors, we evaluated the significance at the time of diagnosis and 6 months after treatment. When assessing the predictive value of a factor, 6 months after treatment, survival was measured from this particular point in time. The proportionality assumption was checked by visual inspection of log minus log plots.
Associations between different variables were analyzed by the χ2 test. Spearman's correlation was used when categories were ordinal. A p-value <0.05 was considered statistically significant. SPSS (versions 9.0 and 10.0) were used for statistical analysis.
CK+ cells in the BM at time of diagnosis and prognosis
At the time of diagnosis, 29% of the patients had CK+ cells in the BM, 21% in the dose-escalated and tailored FEC group and 36% in the CTCb group (Table I). Even corrected for this difference in CK+ cells at the time of diagnosis, treatment was not an independent prognostic factor in the Cox multivariate analysis. The significant association between BM findings at the time of diagnosis and BCS by log-rank test (Table II) was not confirmed in the multivariable analysis (Table III) and may, therefore, be the result of confounding factors.
|RFS after 5 years||BCS after 5 years|
|CK+ BM at the time of diagnosis|
|Positive (n = 33)||48||0.15||48||0.04|
|Negative (n = 82)||61||74|
|CK+ BM 6 months posttreatment|
|Positive (n = 16)||44||0.03||47||0.01|
|Negative (n = 79)||68||79|
|CK+ CK+ (n = 7)||43||0.006||43||0.0006|
|CK− CK+ (n = 21)||44||51|
|CK+ CK− (n = 9)||52||56|
|CK− CK− (n = 55)||74||87|
|Age at the time of diagnosis|
|30–39 (n = 19)||58||0.63||58||0.92|
|40–49 (n = 46)||58||73|
|50–60 (n = 53)||56||65|
|No. of positive lymph nodes|
|≤10 (n = 64)||65||0.02||73||0.11|
|11–20 (n = 46)||47||60|
|>20 (n = 5)||40||50|
|Positive (n = 52)||60||0.77||66||0.69|
|Negative (n = 57)||52||66|
|I (n = 5)||80||0.13||100||0.05|
|II (n = 45)||66||77|
|III (n = 54)||53||59|
|CTCb (n = 60)||53||0.37||65||0.29|
|FEC (n = 58)||62||70|
|Positive (n = 11)||45||0.5||61||0.8|
|Negative (n = 46)||54||65|
|At the time of diagnosis|
|CK+ in BM2||0.12||0.58||0.29–1.14|
|CK+ in BM2||0.21||0.67||0.36–1.25|
|6 months posttreatment|
CK+ cells in the BM post-treatment and prognosis
Six months after treatment, 17% of the patients in the FEC group had CK+ cells in the BM and 16% in the stem-cell group (Table I). Of the 33% who had different BM status at diagnosis and 6 months posttreatment, 23% changed from CK+ to CK−, and 10% changed from CK− to CK+. The number of patients changing CK status is shown in Table IV. DTC-changes were an independent prognostic factor of BCS both in multivariate (p = 0.001) (Table III) and in univariate analysis (p = 0.0006) (Fig. 1, Table II). CK+ cells in the BM 6 months posttreatment was significantly associated with clinical outcome in univariate analysis (p = 0.01) (Table II), but this association was not confirmed in multivariate analysis. Subjects with persistent CK− BM after therapy comprise a group that differs from the others and can be identified as patients with favorable prognosis.
|6 Months posttreatment||Total|
|At diagnosis||CK−||55 (86)||9 (14)||64 (100)|
|CK+||21 (75)||7 (25)||28 (100)|
Standard prognostic factors and prognosis
Histological grade was an independent predictive factor of BCS (p = 0.047) and number of positive lymph nodes showed predictive value with respect to RFS (Table III). Type of treatment and other standard prognostic factors studied were not significantly associated with survival (Tables II and III). This was true for the whole group as well as for the 2 subgroups receiving different treatment.
There are 2 important findings from the BM analyses in the present study. First, we show that DTC-changes in BM before and after therapy are of prognostic value for BCS. Second, we identify a subgroup of patients with particularly favorable prognosis, since patients with continuous CK− BM before and 6 months after therapy have significantly increased BCS. To our knowledge, the latter finding has not previously been reported and should be further investigated in larger studies.
Several investigators have shown an association between DTC in the BM and poor BCS.6, 8, 9, 15 Braun et al. reported that CK+ cells in BM at the time of diagnosis significantly predicted cancer-related deaths and distant metastases (p < 0.001), but not locoregional relapse (p = 0.77).6 Recently, we have found similar results when a larger group of patients was investigated.7 In our study, CK+ cells in the BM at the time of diagnosis was not an independent predictor of BCS or RFS. This could be due to the small sample size studied.
Different groups have like us found that remaining DTC in the bone marrow after both standard-dose and high-dose therapy define patients with an unfavorable prognosis.8, 9, 15, 16, 17 Only few of our patients had increasing numbers of CK+ cells in the bone marrow following the therapy, while some patients had unchanged or decreasing numbers (data not shown). Irrespectively of the number of CK+ cells found, positive bone marrow samples after therapy predicted a worse outcome (Table II). Furthermore, in our study, subjects with CK+ cells in the BM at the time of diagnosis and not 6 months after treatment also have a reduced BCS as compared to those who have never been CK+. It is biologically reasonable to hypothesize that CK+ patients who become CK− would have a better prognosis. Since we, in this study, only had satisfactory information about BM findings 6 months after treatment and not later, we cannot exclude that these patients become positive at a later stage. In previous studies, the timing of bone marrow sampling varies between 3 months until 3 years after chemotherapy. In spite of this, most studies find a correlation between DTC and outcome except Hohaus et al. who examined the bone marrow 4–5 months after high-dose therapy.18 Although the present findings and most other studies suggest that the current method used for DTC detection gives sufficient clinical information to consider a more general use in patients with breast cancer, several questions remain to be answered. Among them is to define the optimal timing of bone marrow sampling after therapy and to study the clinical meaning of changes in number of CK+ cells during and after therapy.
Infusion of contaminating tumor cells in the PBPC graft might contribute to tumor recurrence in the patients. In this study, 11 of 56 autografts contained CK+ cells at the time of infusion, but no significant influence on BCS was revealed (p = 0.84) (Table II). We cannot exclude that the lack of clinical correlation and infusion of tumor cells is due the low number of patients. However, similar results have been reported by others.16
The number of positive lymph nodes was an independent predictor of RFS, but not BCS. Histologic grading was associated with both BCS and RFS in multivariable analysis. Other standard prognostic factors, such as age and hormone receptor status, were unable to predict BCS in the present study (Table III).
The sample size of 118 subjects is small and leads to a weak power. The possibility of type II errors where we do not recognize clinically relevant associations cannot be excluded. This may be true for some of the standard prognostic factors that were not significantly associated with BCS. It may also be the case for CK+ cells in BM at the time of diagnosis where the univariate association was not confirmed by multivariable analysis. We have, therefore, not concluded with any positive findings unless there is significance in multivariable analysis. Although the sample size was too small to reveal any association between certain established markers and prognosis, the BM findings did. This strengthens the assumption that BM findings are strong predictors of clinical outcome.
For the whole study population of 525 patients, we have previously reported a statistically significantly improved RFS for the dose-escalated FEC arm (p = 0.04).4 This was not confirmed in the present substudy (p = 0.37) and the discrepancy with regard to statistical significance is probably due to the smaller number of patients included. We observed a trend toward a higher rate of CK+ cells at the time of diagnosis in the patients assigned to CTCb as compared to FEC (36 vs. 21%, p = 0.072). At 6 months posttreatment, the percentage was similar in the 2 treatment arms.
In contrast to those who have CK− BM at both points of time, patients treated with chemotherapy without the clearance of CK+ cells have a poor prognosis. Earlier reports have indicated that the DTC cells found in BM constitute a heterogeneous population of cells with different biological properties.19 Both dormancy of the cancer cells and/or mutations rendering the tumor cells resistant to chemotherapy could, therefore, explain survival of CK+ cells following standard-dose or high-dose chemotherapy.8, 20, 21 Under such circumstances, more efficient chemotherapy regimens or immunotherapy approaches might be required. Several studies are already ongoing or planned to address this issue. In our hospital, breast cancer patients given standard adjuvant chemotherapy with CK+ cells 6 months after therapy are offered second line chemotherapy with 6 cycles of Docetaxel. The protocol is still recruiting patients and it remains to be seen how many patients have clearance of CK+ cells and if this translate into a longer disease-free period and overall survival. The combination of chemotherapy and monoclonal antibody has been reported earlier to efficiently remove DTC from bone marrow in patients with breast cancer.22 Recently, several different monoclonal antibodies have been introduced into the clinic.23 In breast cancer, the monoclonal antibody trastuzumab (Herceptin), in combination with standard adjuvant chemotherapy, has been shown to reduce relapse and prolong disease-free and overall survival. Different vaccine programs are also being tested.24, 25 Investigations addressing the role of DTC in bone marrow when this new type of immunotherapy approaches is being used are under way.
In summary, our study shows that adjuvant high-dose therapy do not eradicate DTC, and that detection of DTC before and after therapy is potential, useful as markers for assessment of the therapeutic effect of novel treatment strategies in breast cancer patients.
We thank Ester Gihlen, Marianne Dyrhaug at the Clinical Stem Cell Laboratory and Anne Renolen and Ellen Hellesylt at the Department of Pathology and MD Astri Andersgaard for their excellent help.
- 10Quality of life in women with breast cancer during the first year after random assignment to adjuvant treatment with marrow-supported high-dose chemotherapy with cyclophosphamide, thiotepa, and carboplatin or tailored therapy with Fluorouracil, epirubicin, and cyclophosphamide: Scandinavian Breast Group Study 9401. J Clin Oncol 2003; 21: 3659–64., , , , , , , , , , .