Annals of Oncology Advance Access originally published online on January 10, 2008
Annals of Oncology 2008 19(3):496-500; doi:10.1093/annonc/mdm507
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breast cancer |
Prognosis of women with stage IV breast cancer depends on detection of circulating tumor cells rather than disseminated tumor cells
1 Department of Medical Oncology
2 Institut National de la Santé et de la Recherche Médicale U612
3 Department of Pathology
4 Hematology Laboratory
5 Department of Translational Research, Institut Curie, Paris
6 University René Descartes, Paris 5, France
* Correspondence to: Dr J.-Y. Pierga, Departement d'Oncologie Médicale, Institut Curie, 26 rue d'Ulm, 75005 Paris, France. Tel: +33-1-44-32-46-71; Fax: +33-1-44-32-46-71; E-mail: jean-yves.pierga{at}curie.net
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Abstract |
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Background: At metastatic relapse, detection of circulating tumor cells (CTC) in peripheral blood is predictive of poor survival of breast cancer patients. Detection of disseminated tumor cells (DTC) in bone marrow (BM) is an independent prognostic factor in early breast cancer. We evaluated the prognostic value of DTC detection in the BM of metastatic breast cancer patients.
Materials and methods: BM aspirates from 138 patients were screened for DTC with the pancytokeratin mAb A45-B/B3, according to the ISHAGE classification. One hundred and ten patients (80%) were enrolled before first-line treatment. Thirty-seven patients were simultaneously screened for CTC in the blood.
Results: DTC detection rate in the BM was 59%. DTC were associated with bone metastasis (P = 0.0001), but not with a poorer overall survival. Adverse significant prognostic factors were hormone receptor negativity (P = 0.0004) and more than one line of chemotherapy (P = 0.002). CTC detection in the subgroup of 37 metastatic patients was associated with shorter survival (P = 0.01).
Conclusions: Detection of CTC but not BM DTC had a prognostic significance in stage IV breast cancer patients. CTC in blood are a more reliable and a less invasive tool to evaluate prognostic and monitor tumor response in this metastatic setting.
Key words: bone marrow, breast cancer, circulating tumor cells, metastasis, micrometastasis
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introduction |
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TopAbstractintroductionmaterials and methodsresultsdiscussionfundingAcknowledgementsReferences |
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Hematogenous metastasis is a complex biological process involving many sequential steps (extravasation, circulation in the blood, specific recognition of a favorable ‘soil’, extravasation and finally growth within the host organ) and its genetic mechanism remains unclear [1, 2]. The tumor metastatic potential could be monitored by the detection of cytokeratin-expressing (CK+) cancer cells in blood [as circulating tumor cells (CTC)] or in bone marrow (BM) [as disseminated tumor cells (DTC)]. A pooled analysis showed that BM CK+ cells are detected in 31% of stage I–III breast cancer patients and have an independent clinical impact on overall survival (OS) [3], although some rare CK+ BM cells are nonmalignant cells (plasma cells) [4]. On the basis of morphological screening of CK+ cells, we recently reported a 15% BM DTC detection rate in the Institut Curie series of 621 early breast cancer patients [5]. In this study in the adjuvant setting, BM DTC had an independent impact on OS, distant metastasis-free survival and local relapse-free survival. In women with metastatic breast cancer, data on the prognostic value of BM DTC are rare [6], whereas blood CTC have been associated with a poorer OS in 177 patients [7, 8]. On treatment of metastatic breast cancer, changes in CTC number have also been associated with treatment response [9, 10]. In this study, we examined the clinical outcome of women with metastatic breast cancer according to their BM DTC and blood CTC status.
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materials and methods |
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From November 1998 to September 2005, 837 women with breast cancer were included in the Breast Cancer Micrometastasis Project in the Institut Curie (Paris, France) [11–13]; 138 of these women had documented metastatic dissemination. Patient characteristics were recorded prospectively on Institut Curie medical files. All samples were obtained with the patient’s written informed consent after approval by the regional ethics committee.
BM and blood sampling, processing and mononuclear cell (MNC) staining have been described previously [11]. Briefly, BM aspirates was carried out from sternum or from both anterior iliac crests, under local or general anesthesia, respectively. Blood samples (7–14 ml) were obtained by venipuncture after discarding the first 5 ml of blood to avoid contamination by epidermal CK+ cells. After separation by density centrifugation, MNC was collected and cytospins were prepared (1 x 106 MNC per slide). Three slides were incubated with the primary pancytokeratin mAb A45-B/B3 (Micromet, Munich, Germany and Chromavision, San Juan Capistrano, CA), which recognizes several cytokeratin epitopes CK 8, CK 18 and CK 19. Negative controls, stained with anti-fluorescein isothiocyanate immunoglobulin G1 mouse antibody (Sigma Immuno Chemicals, Saint Quentin Fallavier, France), were carried out on an equivalent number of cells (i.e. three slides, 3 x 106 MNC) for each patient. Immune complexes formed by secondary anti-mouse antibodies were revealed by the alkaline phosphatase/antialkaline phosphatase reaction, and the slides were counterstained with hematoxylin to study nuclear morphology.
For BM samples, all slides and controls were read manually by trained pathologists for CK+ cells and further classified into three groups: absence of cytokeratin-positive cells (BM–), presence of CK+ cells with atypical cytologic features (ITC) and other CK+ cells (OCK+). Only ITC were considered to be BM DTC. Atypical cytology was defined as large cell size (larger than surrounding hematopoietic cells), a high nuclear/cytoplasmic ratio for isolated cells or the presence of clusters of large cohesive cells. These criteria for evaluation of CK+ cells were on the basis of the results of cooperative groups [14, 15]. Control slides were systematically read and were taken into account to classify positive cases. In doubtful cases, positive and control slides were blind reviewed by another pathologist and a consensus was established. For blood samples, as the CK+ cell detection rate was much lower, all stained cells were considered to be CTC.
Differences between treatment groups were analyzed by chi-square tests for categorical variables. Survival time and disease-free survival time were measured from the date of BM aspiration until the date of death or last follow-up. Survival curves were determined using a Kaplan–Meier product-limit method [16]. Statistical significance between survival curves was assessed using the log-rank test. Multivariate analysis was carried out to assess the relative influence of prognostic factors on OS, using the Cox proportional hazards model in a forward stepwise procedure [17]. Statistical analyses were carried out by Statview software (SAS Institute, Inc., Cary, NC). For all analyses, a P value of <0.05 was considered to be statistically significant.
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The median age of the patients at the time of BM aspiration was 53 years (range: 30–78 years) and 75% were postmenopausal. Primary tumor was a ductal or lobular invasive carcinoma in 77% and 15% of patients, respectively. Hormone (estrogen and/or progesterone) receptors were positive in 67% of cases. HER2 receptor was screened in 48 out of 138 patients and was overexpressed in 8 (16%) cases. One hundred and ten patients (80%) were screened for disseminated cancer cells before first-line treatment. Forty-six patients (33%) had only one metastatic site. Metastatic sites at the time of BM aspiration were as follows: bone (54%), liver (45%), lungs (30%), lymph nodes (25%), skin (19%), pleural cavity (11%), peritoneal cavity (3%), contralateral breast (3%) and other sites (10%). Only a few patients (<5%) had cytologically-proven (myelogram) BM involvement. One hundred and twenty patients (87%) were followed until their cancer-related death, 4 (3%) were lost during follow-up and 14 (10%) were alive at the time of analysis. OS was measured from the date of BM DTC screening until the date of death or last follow-up and mean OS was 24 months (range: 2–94 months).
Patient characteristics and BM DTC detection rates are shown in Table 1. BM DTC were detected in 81 of the 138 patients (59%). Median number of ITC was 9 (range: 1–3000). The frequency of DTC was significantly higher in lobular carcinomas (N = 21) compared with ductal carcinomas (N = 106) (81% versus 57%, P = 0.049, Fisher’s exact test). DTC were significantly associated with bone metastasis (P < 0.0001). Presence or absence of BM DTC did not correlate with the other parameters on univariate analysis and were not associated with a change in OS (Figure 1A). With a cut-off of 10 ITC per BM sample, BM DTC positivity was associated with a poorer OS (P = 0.04). OCK+ and BM– patients had a similar survival (data not shown). Other adverse prognostic factors for OS were hormone receptor negativity (P = 0.0004) and more than one line of chemotherapy (P = 0.002). Liver involvement was not a statistically significant prognostic factor (P = 0.12). All factors which were significant on univariate analysis were analyzed at multivariate analysis on 126 patients. Hormone receptor status [relative risk (RR) = 2.4, P = 0.0001] and number of lines of treatment (RR = 2.3, P = 0.001) were statistically significant. A nonsignificant trend was observed for DTC positivity with a cut-off of >10 cells (RR = 1.4, P = 0.06).
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CTC were screened in a subgroup of 37 patients and were detected in 15 patients (41%). CTC were not associated with the other parameters (including BM MM, P = 0.2) (Table 1), but were a significant prognostic factor for OS (Figure 1B, P = 0.01) at univariate analysis (Table 2). CTC were not included in the multivariate analysis due to the small number of patients.
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discussion |
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The BM DTC detection rate is markedly increased in the metastatic setting (59%) compared with the 15% detection rate in early breast cancer [5, 18]. No significant difference in BM DTC detection rate was observed between patients in first-line (58%, n = 110 patients) and second (or more) lines of treatment (61%, n = 28 patients). For CTC detection, the standard Ficoll technique used in this study was responsible for a lower blood CTC detection rate compared with an epithelial-cell adhesion molecule (Ep-CAM) enrichment method described previously (40% versus 61%) [7, 8, 19]. This lack of sensitivity may be counterbalanced by a higher specificity, i.e. detection of patients with high CTC count, and could explain why CTC detection represented a significant prognostic factor in our study on a subgroup of 37 patients. Due to the small number of patients with parallel BM and blood sampling, this observation should be reinforced by further studies.
Janni et al. [6] previously reported that an increased number of DTC identified in the BM represents an independent prognostic factor in a short series of 33 metastatic breast cancer patients. In our study, including a much larger number of patients, BM DTC detection was of less clinical significance. We explored the prognostic value of this parameter according to the two methods of analysis: presence or absence or by defining a cut-off value for the number of tumor cells. Neither of these analyses was statistically significant for predicting OS in these 138 patients. Several biological studies assessing the persistence of BM DTC after adjuvant treatments have indicated a possible resistance of these cells to chemotherapy [20–22]. BM DTC detection has been shown to be predictive for bone metastases in the early breast cancer setting [18, 23]. In the present study, we showed that the strong correlation between BM DTC and bone metastasis was maintained after metastatic growth. We observed a higher frequency of DTC in patients with lobular carcinoma compared with ductal carcinoma. These observations indicate that the homing of cancer cells to bone and BM may depend on similar molecular determinants [24, 25]. This is in accordance with the more extensive metastatic spread of lobular carcinoma previously reported by our group [26]. In contrast, CTC were not associated with a specific metastatic pattern.
Finally, DTC, detected in the BM (DTC) or in the blood (CTC), can be assessed at both the early and metastatic stages of breast cancer. In our experience and as reported by other teams, BM DTC detection at an early stage appears to be more closely correlated with breast cancer prognosis than CTC [11, 27, 28]. Clinical studies are currently ongoing to define the value of CTC in the adjuvant setting using more sensitive and specific techniques [29]. This study, in the metastatic setting, using a different (and less sensitive) technique, indicates that CTC in the blood could be a more reliable and much less invasive tool than BM DTC to evaluate prognosis and to monitor tumor response in stage IV breast cancer.
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funding |
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Institut Curie micrometastasis incitative research program.
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The authors would like to thank M. Caly and F. Viard for technical assistance.
Received for publication July 16, 2007. Revision received September 29, 2007. Accepted for publication October 1, 2007.
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