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Clinical Cancer Research 14, 3306, June 1, 2008. doi: 10.1158/1078-0432.CCR-07-4749
© 2008 American Association for Cancer Research
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Imaging, Diagnosis, Prognosis

Disseminated Tumor Cells of Breast Cancer Patients: A Strong Prognostic Factor for Distant and Local Relapse

Francois-Clément Bidard1, Anne Vincent-Salomon2, Stéphanie Gomme3, Claude Nos4, Yann de Rycke3, Jean Paul Thiery5, Brigitte Sigal-Zafrani2, Laurent Mignot1, Xavier Sastre-Garau2, and Jean-Yves Pierga1,6 for the Institut Curie Breast Cancer Study Group

Authors' Affiliations: Departments of 1 Medical Oncology, 2 Pathology, 3 Statistics, and 4 Surgery, Institut Curie, Paris, France; 5 IMCB Biopolis, Singapore, Singapore; and 6 Université Paris Descartes, France

Requests for reprints: Jean-Yves Pierga, Département d'Oncologie Médicale, Institut Curie, 26 rue d'Ulm 75005 Paris, France. Phone: 33-1-44-32-46-81; Fax: 33-1-44-32-46-71; E-mail: jean-yves.pierga{at}curie.net.


   Abstract
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Purpose: Clinical significance of disseminated tumor cells (DTC) in bone marrow of early breast cancer patients has been reported, but improvements in detection methods are needed.

Experimental Design: Bone marrow aspirates from 621 patients with stage I to III breast cancer were screened for cytokeratin-positive (CK+) cells. CK+ cells were categorized into DTC only if they had specific morphologic features of tumor cells. Bone marrow status and clinical and pathologic variables of the patients were correlated with clinical outcome after a median follow-up of 56 months.

Results: DTC and non-DTC CK+ cells were detected in 15% and 34% of patients, respectively, with no correlation with clinical and pathologic variables. On univariate analysis, DTC detection was associated with a poorer distant metastasis-free survival (DMFS; P = 0.0013) and overall survival (OS; P = 0.005). Moreover, DTC detection was also associated with local relapse-free survival (P = 0.0009). On multivariate analysis, DTC detection was an independent prognostic factor for DMFS, local relapse-free survival, and OS. There was no significant interaction between DTC detection and hormonal receptors status (P = 0.34). Non-DTC CK+ cells had no clinical significance.

Conclusion: DTC detection is a powerful prognostic marker for DMFS and OS in early breast cancer patients and can be individualized from irrelevant non-DTC CK+ cells by morphologic criteria. Biologically, despite high rates of systemic adjuvant therapy and locoregional irradiation in this series, DTC detection remains a prognostic factor of distant and, more strikingly, of local relapse, in favor of resistance to treatment of locally or distant disseminated cancer cells in DTC-positive patients.

Adjuvant chemotherapy and endocrine therapy are commonly used for operable breast cancer patients to reduce the risk of systemic metastasis (1). This risk is assessed on clinical and pathologic variables, including age, tumor size and lymph node staging, histopathologic subtype and grade, hormonal receptors, and HER2 status (2). Hematogenous-driven metastasis is a complex biological process involving many sequential steps (intravasation, circulation in the blood, extravasation, specific recognition of a favorable "soil," and final growth within the host organ) and its genetic mechanism remains unclear (3, 4). However, in several cancers, single disseminated cancer cells can be detected in various host organs of patients with early-stage disease (5). Cytokeratins are epithelial markers and epithelial cells can thus be detected in the bone marrow at the time of primary surgery for nonmetastatic breast cancer (6, 7). A pooled analysis showed that these cells are detected in 31% of stage I to III breast cancer patients and have an independent clinical impact on overall survival (OS; ref. 8). However, expression of cytokeratins in rare bone marrow cells does not preclude their carcinomatous nature as some hematopoietic progenitors and plasma cells can be immunolabeled by anti-cytokeratin antibodies (9). Therefore, specific morphologic features of malignancy are required to increase the specificity and relevance of micrometastatic cell detection (9). A consensual classification based on morphologic features and control slides has been reported (10), with a major clinical effect of the most atypical cells (classified as "tumor cells") and a smaller effect of less atypical cells ("uninterpretable cells"; refs. 11, 12). We studied the clinical outcome of breast cancer patients according to three categories: no cytokeratin-positive (CK+) cells, disseminated tumor cells (DTC), and non-DTC CK+ cells, that do not exhibit morphologic features of tumor cells, and report their clinical significance.


   Materials and Methods
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Patients. The accrual in the Breast Cancer Micrometastasis Project was open at the Institut Curie from November 1998 to September 2005 (13–15). Patient characteristics were prospectively recorded in the Institut Curie medical files. All samples were obtained with the patient's written informed consent after approval by the regional ethics committee. The result of bone marrow analysis remained unknown to both patients and clinicians. The eligibility criteria for the study were female patients over age 18 with histologically proven adenocarcinoma of the breast, no previous malignancy other than treated in situ carcinoma of the cervix or nonmelanoma skin cancer, no bilateral breast cancer, and no distant metastasis. The routine diagnostic workup included mammography, mammary biopsy, chest X-rays, abdominal ultrasound, bone scan, blood sampling, and clinical examination. Neoadjuvant chemotherapy was allowed. After surgery, radiotherapy and/or adjuvant chemotherapy and/or hormonotherapy was delivered when recommended by the local guidelines. Adjuvant and neoadjuvant were based on anthracycline regimens for all patients (FAC or FEC). High-risk patients received also docetaxel. During follow-up, chest X-rays, abdominal ultrasound, mammography, and blood analyses were carried out at each clinical examination at 6- to 12- month intervals at the Institut Curie or by the patient's gynecologist. Further diagnostic workup was done only when patients presented symptoms or signs of progression. Local recurrence was defined as a relapse in breast (including every ipsilateral tumor), chest wall, or axillary lymph nodes.

Preparation of bone marrow and immunocytochemistry staining. Bone marrow sampling and processing and mononuclear cell staining have been described previously, together with the sensitivity and specificity of our protocol (14). Briefly, bone marrow aspirate was done at diagnosis from sternum or during primary surgery from both anterior iliac crests, under local or general anesthesia, respectively. Sternal aspiration was done under local anesthesia mainly in patient receiving neoadjuvant chemotherapy, whereas patient treated by primary surgery had bone marrow during general anesthesia (anterior iliac crest). After separation by density centrifugation, mononuclear cells were collected and cytospins were prepared (1 x 106 mononuclear cells per slide). Three slides were incubated with the primary pancytokeratin monoclonal antibody A45-B/B3 (Micromet and Chromavision), which recognizes three cytokeratins: CK8, CK18, and CK19. Negative controls, stained with anti-FITC IgG1 mouse antibody (Sigma Immuno Chemicals), were done on an equivalent number of cells (that is, three slides, 3 x 106 mononuclear cells) for each patient. Immune complexes formed by secondary anti-mouse antibody were revealed by the alkaline phosphatase/anti-alkaline phosphatase reaction, and slides were counterstained with hematoxylin to study nuclear morphology.

CK+ cell detection by light microscopy. All slides and controls were screened manually and interpreted by trained pathologists. Bone marrow aspirates were classified into three categories: absence of detected CK+ cells, presence of CK+ cells with atypical cytology features (DTC), and non-DTC CK+ cells. Atypical cytology was defined as large cell size (larger than surrounding hematopoietic cells), a high nuclear/cytoplasm ratio for isolated cells, or the presence of clusters of large cohesive cells. These criteria for evaluation of CK+ cells in bone marrow were adapted from Borgen et al. (10) based on the results of the European Working Group for standardization of tumor cell detection. 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. The criteria used for DTC classification of CK+ cells in this analysis differ from our previous reports: the first (n = 75 nonmetastatic patients) did not take into account control slides nor CK+ cell morphology (14), whereas the second (n = 270 nonmetastatic patients) did not take into account CK+ cell morphology (8).

Blood sampling and circulating tumor cells detection. In 74 patients, blood samples (7-14 mL) were obtained by venipuncture after discarding the first 5 mL blood to avoid contamination by epidermal CK+ cells. The same detection technique was used to detect circulating tumor cells (CTC), but as the CK+ cell detection rate was low, all stained cells were considered to be CTC.

Statistical methods. OS, distant metastasis-free survival (DMFS), and local relapse-free survival (LRFS) time were measured from the date of surgery until the date of death (or last follow-up) or the date of diagnosis of distant metastasis or local relapse, respectively. Differences between categorical variables were analyzed by {chi}2 tests or Fisher's exact test. Differences between means were analyzed by t tests or Mann-Whitney test. Survival curves were plotted according to the Kaplan-Meier method. Statistical significance between survival curves was assessed using the log-rank test. Multivariate analysis and interaction tests were done by the Cox proportional hazards model. For all analyses, P < 0.05 was considered to be statistically significant.

Funding source. The funding source had no role in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.


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Bone marrow aspiration was done in 655 eligible nonmetastatic patients. Thirty-four patients were excluded from the analysis: 16 did not undergo surgery and 18 bone marrow aspirates were technically inconclusive. Finally, 621 patients without metastatic disease at diagnosis were eligible for further statistical evaluation.

Patient characteristics and treatment. Patient characteristics are shown in Table 1 . The median age was 54 years (range, 25-79). Hormonal (estrogen and/or progesterone) receptors were positive in 81% of cases. HER2 receptor was screened in 212 patients and was overexpressed in 39 (18%). Primary tumor size was <2 cm in 321 patients (44%) and 295 (48%) patients were node negative. One hundred and nineteen patients (19%) received neoadjuvant chemotherapy (bone marrow aspiration was done at diagnosis before any chemotherapy). Surgical treatment consisted of radical mastectomy (27%) or breast conserving surgery (73%). Axillary lymph node dissection was done when lymph nodes were clinically involved or after a positive sentinel lymph node biopsy. Standard adjuvant chemotherapy has been administered to 48% of patients and 65% received endocrine therapy. Adjuvant chemotherapy was mostly anthracycline-based (32%) or a combination of anthracycline and docetaxel (5%). High-dose adjuvant chemotherapy with peripheral blood stem cell reinfusion has been administered to 9% of patients. Tamoxifen or aromatase inhibitors after 2003 were used as adjuvant endocrine therapy in hormonal receptor-positive patients according to their menopausal status. More than 96% of patients received adjuvant radiotherapy according to local guidelines.


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  		Table 1. Patient characteristics, DTC detection rates, and prognostic issues

 

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  		Table 2. Multivariate analysis of prognostic factors for LRFS, DMFS, and OS (n = 597 patients)

 
Detection of DTC and univariate survival analysis. One or more DTC or non-DTC CK+ cells were detected within 3 x 106 bone marrow mononuclear cells in 94 (15.1%) and 208 (33.5%) patients, respectively. Importantly, no CK+ cell with DTC features was detected on control slides. CK+ cell detection rates were similar between sternal and iliac crest aspiration (51% versus 47%, nonsignificant). DTC (and non-DTC CK+) detection did not correlate with any of the patient's clinical or pathologic characteristics (Table 1).

The median follow-up was 56 months (range, 1-100) and follow-up was <6 months for 6 patients. One hundred and twenty-three patients experienced disease recurrence: local relapses were diagnosed in 28 patients (4.5%) and distant metastases were diagnosed in 111 patients (18%). Sixteen patients (2.6%) experienced both local relapse and metastases. Seventy-seven deaths (12%) were reported (including 11 breast cancer unrelated deaths) and were included in the OS analysis. As OS was not significantly different for non-DTC CK+ and no CK+ patients (Cox model, P = 0.3), statistical analyses compared the DTC-positive population with the DTC-negative population. In univariate analysis, OS of patients was dependent on their bone marrow DTC status as shown in Fig. 1A (log-rank P = 0.005). Overall 5-year survival was 89% for DTC-negative patients and 77% for DTC-positive patients. Distant metastases were more frequent in the DTC-positive group (P = 0.0013; Fig. 1B). In patients who developed distant metastasis, DTC detection was associated with liver metastasis (P = 0.05) but not with bone metastasis (P = 0.9) as first site of relapse. Strikingly, locoregional relapse was also strongly associated with the presence of DTC cells in bone marrow (P = 0.0009; Fig. 1C).


Figure 1
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  		Fig. 1. OS (A), DMFS (B), and LRFS (C) curves according to bone marrow status [DTC positive versus DTC negative; log-rank P = 0.0049 (A), 0.0013 (B), and 0.0009 (C)].

 
Together with DTC status, clinical stage (according to the tumor-node-metastasis International Union Against Cancer classification), tumor grade, hormonal receptor status, and vascular emboli were also significantly associated on univariate analysis with OS and DMFS. HER2 status and patient age were also associated to DMFS. For LRFS, the other prognostic factors were patient age, hormonal receptor status, HER2 status, and tumor emboli (Table 1). HER2 status was unknown in 66% of patients and was not subsequently tested. In 74 patients screened for CTC, DTC detection, but not CTC detection, was associated with OS as reported previously with a median follow-up of only 28 months (14).

Subgroups and multivariate analyses. Subgroup analyses for DMFS and LRFS, based on nodal status, are shown in Fig. 2 . The difference in DMFS (and OS; data not shown) for DTC patients was significant in node-positive patients (P = 0.002) but was not significant in node-negative patients (P = 0.2; Fig. 2A). However, the interaction test was nonsignificant [P = 0.80; hazard ratio (HR), 0.86; 95% CI (95% CI), 0.28-2.69]. On the contrary, the difference in LRFS for DTC patients was mostly seen in node-negative patients (P = 0.0002), but the interaction test was also nonsignificant (P = 0.24; HR, 0.38; 95% CI, 0.08-1.90). According to a subgroup analysis, DTC prognosis effect on DMFS was not statistically different in patients with hormonal receptor positive or negative primary tumors (interaction test, P = 0.44; HR, 0.70; 95% CI, 0.29-1.72; Fig. 2B).


Figure 2
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  		Fig. 2. A, DMFS curves in node positive (pN+) and node negative (pN-) patients according to bone marrow DTC status. B, DMFS curves in hormonal receptor positive (HR+) and negative (HR-) patients according to bone marrow DTC status. Interaction tests between pN or hormonal receptor status and DTC status were negative.

 
Multivariate analyses categorized DTC detection, clinical stage, tumor grade, hormonal receptor status, vascular emboli and patient age according to their relative independence. Bone marrow DTC was an independent prognostic factor for OS (P = 0.02; HR, 1.92; 95% CI, 1.12-3.48), DMFS (P = 0.006; HR, 1.96; 95% CI, 1.21-3.17), and LRFS (P = 0.0009; HR, 3.73; 95% CI, 1.71-8.13; Table 2 ).


   Discussion
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Bone marrow cytokeratin-expressing cells can correspond to normal hematopoietic cells or disseminated breast cancer cells. Morphologic analysis of bone marrow in breast cancer patients can display several classes of results for CK+ cells: tumor cells, probable tumor cells, hematopoietic cells, artifacts, and no CK+ cells detected (10). In the preliminary analysis including the first 114 patients of our series (14), every CK+ cell was taken into account as a DTC, and we described an incidence for DTC of almost 50% in 75 nonmetastatic patients. The pooled analysis published by Braun et al. also included the first 270 patients of our series and showed the prognostic significance of micrometastases at early diagnosis of breast cancer (8). In this international study, micrometastatic cells were defined as cytokeratin-expressing cells assessed by immunohistochemistry with detection rates ranging from 12% to 38% (38% at the Institut Curie, patients with positive control slides were then considered as being DTC negative; ref. 8). After completion of our breast cancer micrometastasis study, CK+ cells were found in 49% of patients regardless of morphology or control slide (DTC and non-DTC CK+ cells) as reported in our preliminary analysis. The present study, based on a simple classification (DTC positive or negative), confirms that a further pathologic classification lowers the micrometastasis rate to 15% (DTC only) of breast cancer patients, a result similar to the 13% rate reported previously with a similar pathologic methodology (12). The clinical value of restricting the bone marrow analysis to DTC (instead of CK+ cells) is sustained by the lack of prognostic value of non-DTC CK+ cells. However, a few micrometastatic cells may have been misclassified as non-DTC CK+ cells; in the future, molecular analysis techniques (e.g., fluorescence in situ hybridization) might enhance the sensitivity and specificity of DTC detection. The DTC distribution was independent of any standard clinical or pathologic prognostic factors and patient stratification was therefore not required before statistical analysis in this series. Univariate and multivariate analyses showed that DTC detection was a strong independent prognostic factor, leading to poor 5-years survival rates.

Unexpectedly, DTC also constituted an independent prognostic factor for local relapse of breast cancer that has not been reported previously. The local relapse rate (4.5%) in the present study was similar to that reported in the Oslo series (5.9%), with fewer pT1 tumors (40% versus 61%) and more breast-conserving surgery (73% versus 31%; ref. 11). Our cohort was therefore at high risk of local relapse and adjuvant radiotherapy, an effective treatment to prevent local relapse (16), was administered to 96% of our patients (47% in the Oslo series; ref. 11). The association between DTC detection and local relapses may be due to a repopulation of the primary tumor site by DTC after surgery. This migration of cancer cells may be enhanced by wound-associated chemokines and proangiogenic factors (17, 18). Another explanation of the higher risk of local relapse in DTC-positive patients is to consider that locally disseminated cancer cells have a similar clonal origin to bone marrow DTC and that they share the DTC resistance to adjuvant treatments, which has been reported previously (19–21). According to this hypothesis, the high rate of adjuvant local treatments in our study has therefore unmasked, by killing the sensitive non-DTC-associated locally disseminated cancer cells, a previously unreported link between bone marrow DTC and local relapse.

Finally, the prognostic effect of bone marrow DTC detection in stage I to III breast cancer patients appeared to be homogeneous among the different subgroups of patients included in our serial. The Oslo series showed initially that significance of DTC detection was marked and clear in the node-positive group but more restricted in the node-negative not receiving adjuvant treatment group (22). In a subgroup analysis of our study, DMFS and LRFS were significant in node-positive and node-negative patients, respectively. The six deaths registered in the T1N0 population (n = 177) also did not allow statistical analysis. However, interaction tests showed that these results are likely due to a low number of events in the node-negative and node-positive patients, respectively. A recent report also states that DTC detection is associated with different recurrence risk within molecular subtypes of breast cancer, DTC differentially distinguishing clinical outcome in patients with luminal A-type tumors (23). Luminal A-type tumors are mostly characterized by estrogen receptor expression; although we did not analyze the molecular profile of breast cancers, we did not find any prognostic difference between hormonal receptor positive and negative status in our study. In contrary to recent report on CTC in the blood, we did not find a higher rate or a different prognostic value of DTC according to hormonal receptors or HER2 status of primary tumor (24).

Bone marrow micrometastases are a confirmed strong independent prognostic factor in the whole breast cancer patient population; as indicated by this heavily treated cohort study, these micrometastases are still a clinical challenge due to their association with resistance to local and systemic adjuvant therapy. Although micrometastases are a documented clinical entity, only a few biological preclinical studies have focused on the regulatory step between dormant micrometastasis and metastasis (67% of the DTC population remained disease free in our study). Biological studies showed that macrometastases develop under the control of organ-specific molecular determinants (25, 26). In this model, local recurrence or distant metastatic growth in liver or lungs is likely to be derived from other disseminated cancer cells rather than bone marrow DTC. In our study, the presence of DTC in the bone marrow was associated with more frequent liver metastasis but not with a higher rate of bone metastasis. This point is in contrast with other studies reporting more skeletal and liver metastases (22).

In stage IV patients, we recently reported that DTC detection has no prognosis effect (27), whereas CTC in peripheral blood seem to be associated with outcome and response to treatment as it has been shown by Cristofanilli et al. (28, 29). At early stage, clinical relevance of CTC detection remains to be validated with the same level of proof that DTC in bone marrow with standardized methods (6). As shown in our study, DTC, which indicate occult tumor dissemination and a poorer prognosis, can be detected with an increased specificity by using morphologic criteria. Still now, monitoring adjuvant treatment efficacy in primary breast cancer patients would be based on the analysis of repeated bone marrow aspirates (21, 30). The improved methodology we have used should contribute to the adoption of DTC detection as a standard practice in breast cancer management.


   Disclosure of Potential Conflicts of Interest
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No potential conflicts of interest were disclosed.


   Acknowledgments
 
We thank F. Huguet for critical review of the article and M. Caly and F. Viard for technical assistance.


   Footnotes
 
Grant support: Institut Curie micrometastasis incitative research program funded by individual grants.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10/27/07; revised 12/27/07; accepted 1/ 3/08.


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F.-C. Bidard, Y. M. Kirova, A. Vincent-Salomon, S. Alran, Y. de Rycke, B. Sigal-Zafrani, X. Sastre-Garau, L. Mignot, A. Fourquet, and J.-Y. Pierga
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