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© 2001 American Society for Clinical Oncology Comparative Analysis of Micrometastasis to the Bone Marrow and Lymph Nodes of Node-Negative Breast Cancer Patients Receiving No Adjuvant TherapyFrom the I. Frauenklinik and Department of Gynecological Pathology, Klinikum Innenstadt, Ludwig-Maximilians-University, München; Germany. Address reprint requests to Stephan Braun, MD, Frauenklinik & Poliklinik, Klinikum rechts der Isar, Technical Universität, Ismaninger Strasse 22, D-81675 Munich, Germany; email: stephan.braun{at}lrz.tum.de
PURPOSE: In node-negative patients, of whom up to 30% will recur within 5 years after diagnosis, markers are still needed that identify patients at high enough risk to warrant further adjuvant treatment. In the present study we analyzed whether a correlation exists between microscopic tumor cell spread to bone marrow and to lymph nodes and attempted to determine which route is clinically more important. PATIENTS AND METHODS: According to a prospective design, bone marrow aspirates and axillary lymph nodes of level I (n = 1,590) from 150 node-negative patients with stage I or II breast cancer were analyzed immunocytochemically with monoclonal anticytokeratin (CK) antibodies. We investigated associations with prognostic factors and the effect of micrometastasis on patients’ prognosis. RESULTS: CK-positive cells in bone marrow aspirates were present in 44 (29%) of 150 breast cancer patients, whereas only 13 patients (9%) had such positive findings in lymph nodes; simultaneous microdissemination to bone marrow and lymph nodes was seen in merely two patients. No correlation of bone marrow micrometastases with other risk factors was assessed. Reduced 4-year distant disease-free and overall survival were each associated with a positive bone marrow finding (P = .032 and P = .014, respectively) but not with lymph node micrometastasis. Multivariate analysis revealed an independent prognostic effect of bone marrow micrometastasis on survival, with a hazards ratio of 6.1 (95% confidence interval, 1.2 to 31.3) for cancer-related death (P = .031) in our series. CONCLUSION: Immunocytochemical detection of micrometastatic cells in bone marrow but not in lymph nodes is an independent prognostic risk factor in node-negative breast cancer that may have implications for surgery and stratification into adjuvant therapy trials.
APPROXIMATELY 50% of breast cancer patients with small curatively operable tumors have no metastatic involvement of the axillary lymph nodes. Thus having no evidence of residual or metastatic disease after definitive local-regional treatment (eg, breast conservation or modified radical mastectomy), these patients may be viewed as being cured by surgery alone. However, up to 30% of these women will recur with distant metastatic disease within 5 years, which may have arisen from occult metastatic cells being present in secondary organs at the time of diagnosis of breast cancer. Thus the search for residual micrometastatic cells has become an issue of significant interest because clinical manifestation of distant relapse is now recognized as a consequence of early tumor cell dissemination.1-4 Thus far, the axillary nodal status has been established as the prognostic factor in breast cancer to predict the risk of distant disease manifestation. The shortcomings of this staging procedure are, however, demonstrated by the fact that some 40% of node-positive breast cancer patients survive for 10 years or more without any recurrence,5,6 whereas distant metastases will occur in approximately 30% of node-negative patients.7 Because metastatic breast cancer has remained a disease stage not amenable to curative treatment, there is an urgent need for markers indicating the presence or absence of systemically disseminated tumor cells in order to select patients at high enough risk of distant metastasis for appropriate therapy and to identify those with a significantly lower risk who may warrant no further systemic treatment. Among several approaches for an improved assessment of lymphatic tumor cell spread, a more meticulous analysis of lymph nodes—eg, by serial sectioning8-11 and immunocytochemistry12-18—overall led to the conversion of approximately 15% of assumed node-negative patients.19 The underlying supposition of these studies was that regional lymphatic spread is paralleled by hematogenous dissemination of tumor cells as a function of tumor load quantity. However, no such data appear to be available that describe kinetics of these routes of microdissemination. In the present study, we investigated whether immunocytochemically identifiable micrometastases occurred simultaneously in the bone marrow and lymph nodes of node-negative breast cancer patients. We further analyzed the influence of both events on patient prognosis at a median follow-up of 48 months. Because all patients remained without adjuvant therapy, which accounted for the consensus of the Central Cancer Register Munich at the time of study initiation, the influence of minimal amounts of disseminated tumor cells on prognosis could be assessed independently from an intervening therapy. Our data strongly suggest that early lymphatic and hematogenous tumor cell spread are independent events regulated by distinct mechanisms. The clinical prognosis of the patients was determined by hematogenous but not by lymphatic micrometastasis.
Patients From January 1994 to August 1998, 150 consecutive patients admitted to the I. Frauenklinik, Ludwig-Maximilians University München (Germany), were selected with completely resected breast cancer (R0), absence of lymph node metastasis (pN0), and overt metastases (M0). Of these 150 patients, 119 have already been analyzed in our recent study on the effect of the presence of bone marrow micrometastasis on the survival of patients with stage I to III breast cancer.1 Patients underwent bone marrow aspiration from both upper iliac crests after written, informed consent was received and before initial removal of the primary carcinoma, a procedure approved by the institutional ethical boards. Tumor stage and grade were classified according to the fifth edition of the tumor-node-metastasis classification20 by investigators unaware of the immunocytochemical findings in bone marrow. In return, immunocytochemical bone marrow screening was performed without knowledge on the individual histopathologic results. This enabled validation of the present immunoassay by demonstrating the specificity of immunostaining with the applied monoclonal antibody A45-B/B3 on bone marrow preparations from noncarcinoma control patients.1,21 The primary surgical treatment consisted of either breast conservation (n = 90) or (primary or secondary) modified radical mastectomy (n = 60) leading to R0 resection in all reported cases. Routine axillary dissection included lymph nodes of levels I and II, while those of level III would have been excised only in cases with macroscopic metastatic involvement of the lower levels. For the diagnosis of lymph node metastasis, single embedded lymph nodes were screened at three levels. In all 90 patients treated with breast conservation, telecobalt radiation therapy was administered, while no chest wall irradiation after mastectomy was performed. The median absorbed dose in the target volume was 50.0 Gy given in 25 fractions. According to the recommendations of the Central Tumor Center Munich that were in analogy to those of the St. Gallen Consensus Meeting at the time of study initiation,22 no adjuvant systemic treatment was routinely foreseen for node-negative patients. Thus all patients assigned to the present study received no further adjuvant systemic therapy. 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. These preoperative and perioperative examinations showed no evidence of distant metastases in all patients studied. Patients were followed-up with clinical examinations every 3 months and were further tested only if they had symptoms. The follow-up findings were confirmed in all the patients as of August 16, 2000.
Lymph Node Analysis For immunohistochemical staining, we applied a double-labelling technique using the monoclonal antibody NCL-5D3 (ICN Capple, Irvine, CA) directed against CK8/18/1923,24 and the directly peroxidase-conjugated monoclonal antibodies 3B4 and 2B11&PD7/26 directed against vimentin and CD45, respectively (both Dako, Hamburg, Germany). Epithelial cells stained with antibody NCL-5D3 were specifically labeled with the alkaline-phosphatase conjugated avidin-biotin complexes (Ventana Medical Systems, Tucson, AZ). Absence of immunoglobulin cross-reactivity was ensured by incubation with antibody NCL-5D3 and alkaline-phosphatase conjugated avidin-biotin complexes followed by saturation of antimouse epitopes with murine serum (Dako) before incubation with antibodies 3B4 and 2B11&PD7/26. Antibody-bound peroxidase and alkaline phosphatase were developed with 3,3-diamino-bencidine-tetrahydrochloride and Fast Red, respectively, using Vectastain kits (Ventana Medical Systems) as recommended by the manufacturer. The specificity of the antibody reaction was controlled on appropriate control slides obtained from lymph nodes of node-positive patients. Only the presence of tumor cells within the body of the lymph node was accepted as a metastasis. Because there are reports that subcapsular, sinusoidal, or vascular metastasis are associated with parenchymal lymph node metastasis,11,14 we also registered tumor cells in the subcapsular sinus or in any endothelial-lined spaces within the lymph node.
Bone Marrow Analysis After overnight air-drying, slides were either stained immediately or stored at -80°C. Per patient, 2 x106 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 cells. 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 over 95% of specimens. The final consensus decision on discrepant results required critical re-evaluation by both investigators. The monoclonal antibody A45-B/B3 (Micromet, Munich, Germany) directed against a common epitope of CK polypeptides, including the CK heterodimers 8/18 and 8/19,27 was used at 1.0 to 2.0 µg/mL to detect tumor cells in bone marrow cytospin preparations. The detection antibody for bone marrow aspirates and lymph node sections differed because antibody A45-B/B3 cannot be used on paraffin sections but has been validated for its use on bone marrow specimens.27 The specificity of the antibody reaction was controlled by an appropriate dilution of unrelated mouse-myeloma antibody for isotype control on patients’ bone marrow specimens. The breast cancer cell-line BT-20 served as positive control for CK immunostaining.25 The specific reaction of the primary antibody was developed with the alkaline phosphatase antialkaline phosphatase (APAAP) technique combined with the new fuchsin method28 to indicate antibody binding, as previously described.25 In vitro experiments confirmed that the particular assay and antibody reproducibly detect one single tumor cell against the background of 106 bone marrow cells (unpublished data).
Statistical Analysis
Detection of Micrometastasis Bone marrow micrometastases were detected in 44 (29%) of 150 patients by immunocytochemistry, as shown in Fig 1A. Table 1 shows that the presence of such cells in bone marrow was neither correlated to an increased tumor size and high nuclear grading nor to negative hormone receptor status. As Table 1 further indicates that bone marrow micrometastasis is not associated with microdissemination to lymph nodes, it is remarkable to note that only two patients (5%) with lymph node micrometastasis had disseminated tumor cells in bone marrow.
In 1,590 axillary lymph nodes of level I, we identified 17 nodes with a CK-positive cellular staining. The tumor stage and the number of tumor cells found in both the lymph nodes and bone marrow of these patients are listed in Table 2. Although examination of adjacent H&E stainings had failed to assess isolated or micrometastatic tumor cells, subsequent re-evaluation guided by the immunohistochemically stained section helped to identify these cells and allowed to confirm the malignant nature of these CK-positive cells by histopathologic criteria in 15 of 17 cases (Table 2). To address the possibility of registering CK-positive deposits (eg, as a result of phagocytosis), we performed double-labelling of CK-positive cells using an antibody-cocktail directed against the histogenetic markers vimentin and leukocyte common antigen (CD45) to exclude registration of cells of mesenchymal origin. An example of specific detection of CK-positive and vimentin-/CD45-negative tumor cells within the body of a level I axillary lymph node is shown in Fig 1B. By this procedure, another two cases were to be eliminated due to vimentin-/CD45-positive staining of CK-positive typed cells (Table 2). On the basis of histopathologic confirmation and immunohistochemical validation, we identified 13 of 150 originally assumed node-negative patients who carried lymph node micrometastasis. We expected occult disease to be of only microscopic proportions, but we found five patients with a considerable number of carcinoma foci (eg, 34, 35, 46, 52, and 105 partially clustered tumor cells) (Table 2). In all 13 patients, these carcinoma foci, however, were located in a single lymph node only.
Micrometastasis and Prognosis At median follow-up of 48 months (range, 24 to 72 months), a tumor relapse occurred in 26 patients; of these, 17 patients had local-regional relapse, and 12 patients recurred with distant metastasis ( Table 3). Whereas local-regional relapses were attributed to neither a certain bone marrow status (hazards ratio = 0.5; 95% confidence interval [CI], 0.2 to 1.9; P = .33) nor to the presence of lymph node micrometastasis (hazards ratio 2.3; 95% CI, 0.7 to 7.2; P = .15), manifestation of distant metastasis was significantly associated with bone marrow positivity only ( Fig 2A and 2B). Table 3 demonstrates that patients with bone marrow involvement more frequently presented with distant relapses than patients without such a finding (P = .031). No such prognostic indication was found for patients stratified for histopathologically occult yet immunocytochemically identifiable lymph node micrometastasis (Table 3). A marked increase in the number of bone metastases was observed during follow-up in patients who had bone marrow micrometastases (Table 3), whereas the single patient with lymph node micrometastasis who recurred in bone and lung had bone marrow micrometastasis at the same time.
In 44 patients with evidence of bone marrow micrometastases, five cancer-related deaths (11%) occurred, whereas two cancer-related deaths (2%) were observed in the 106 patients without evidence of hematogenous tumor cell spread (P = .023). As shown in Fig 2C, the overall survival rate was 97% for the patients without bone marrow micrometastases, and 88% for those patients with micrometastases (P = .014; log-rank test). In our series, no such prognostic separation was observed for patients with and without lymph node micrometastasis (Fig 2D). Including the four patients who were originally excluded from survival analyses as being falsely positive revealed no measurable influence on the statistical results (data not shown). A Cox multiple regression analysis was performed to see whether bone marrow micrometastases were a significant predictor of distant disease-free and overall survival independent of age, menopausal status, tumor size, tumor grading, histologic type, hormone receptor expression, and lymph node micrometastasis. Tables 4 demonstrates that in our series, bone marrow micrometastasis was the only independent predictor of both distant-disease recurrence (P = .032) and cancer-related death (P = .031), with hazards ratios of 3.5 (95% CI, 1.1 to 11.1) and 6.1 (95% CI, 1.2 to 31.4), respectively.
With the analysis of regional lymph nodes draining ipsilateral breast carcinoma, the diagnosis of metastatic lymph node involvement is generally accepted to have therapeutic relevance because it is assumed that parallel hematogenous spread as a function of tumor load quantity has occurred. From animal studies as early as in the late 1960s31 and clinical studies in the 1980s,32,33 it could be suspected, however, that differential biologic mechanisms are required for either lymphatic or hematogenous tumor cell spread. It was shown that disruption of the lymphatic system with or without preliminary venous ligation could not prevent tumor cell invasion and distant metastasis after tumor transplantation.31 Accordingly, it has been an interesting though not entirely new supposition that lymphatic tumor cell spread may be rather a crude indicator of an individual tumor’s capacity for systemic dissemination.32 Ménard et al33 reported that the histologic nodal status gave no reliable prognostic information in biologically defined subgroups of node-positive patients, implying that lymph node metastasis cannot be equalled to hematogenous spread. Clinical long-term studies further have shown that up to 40% of node-positive patients survive for 10 years or more,5,6 whereas distant metastases occur in approximately one third of node-negative patients.7 The results of our study based on simultaneous analysis of the bone marrow and lymph nodes indicate that hematogenous and lymphatic tumor cell dissemination are rarely associated with each other. In addition, the hematogenous route of tumor cell dissemination seems to provide more a reliable prognostic indication of the risk of tumor relapse for node-negative patients without adjuvant systemic treatment than the presence of minor amounts of lymphatically spread tumor cells. The numerous studies conducted so far on advanced analyses of axillary lymph nodes from breast cancer reported on increased detection of very variable degree, but they disagreed about the prognostic significance.8-18,34,35 Because the percentages of patients converting from negative to positive remained consistently below those reported to recur with metastases within the 5 years after surgery,18,19 uncertainties prevailed about the clinical significance of lymph node micrometastasis. Because our number of patients studied is rather low and the length of follow-up is relatively short, the presented statistical data may require a cautionary note. Our study nevertheless differed from previous studies in that (1) it was prospectively planned, (2) concerns of cross-reactivity of the anti-CK detection antibody (eg, with extrafollicular and histiocytic reticulum cells) have been addressed, and (3) information on the occurrence and prognostic relevance not only of lymphatic tumor cell spread but also of directly assessable hematogenous micrometastases has been analyzed. Previous studies,36-38 including ours,1,25,39 reported on advantages of anti-CK antibodies over antibodies directed against polymorphic epithelial mucins2,40 for the detection of epithelial tumor cells in mesenchymal organs, because of significantly less false-positive labelling of nonepithelial cells. We therefore studied both bone marrow and lymph nodes using anti-CK antibodies. To maintain the well-accepted specificity of CK staining also for the analysis of lymph node sections, we assessed the constitutive coexpression of vimentin and CD45 on reticulum cells in lymph nodes.23,41 This restriction of CK-immunoreactivity excluded CK-positive, yet nonepithelial cells, such as extrafollicular and histiocytic reticulum cells with CK expression due to pathologic conditions, or simply after phagocytosis of destroyed epithelial cells.41 This procedure contributed to a relatively low frequency of occult lymph node micrometastases (9% in our series, as compared with 15% or higher in recent studies).13,17,18,42,43 This issue though crucial in terms of assay specificity has not been addressed in previous comparable studies,13,17,18,42,43 which may thus have included false-positive results into their analyses. Another explanation for the relatively low percentage of lymph node micrometastases in our series might be seen in the already advanced original histopathologic evaluation consisting of three different levels of every single embedded lymph node. The efficacy of such elaborate original H&E is supported by our findings that in all 13 patients, lymph node micrometastases were only present in a single lymph node, implying that this is the sentinel lymph node. In the present study, we used an improved immunoassay that closely follows the recent recommendations of the Tumor Cell Detection Committee of the International Society of Hematotherapy and Graft Engineering to ensure high reproducibility of results.44 For the identification of blood-borne micrometastases in bone marrow, the novel monoclonal antibody A45-B/B327 was used that recognizes CK8, CK18, and CK19 expressed in normal and transformed epithelial cells but not in bone marrow cells.1,25,39 Evidence for the malignant nature of CK-stained cells derived from recent reports now demonstrating unequivocally that CK-positive cells in bone marrow display multiple chromosomal aberrations specific for tumor cells.45-48 Using this validated immunocytochemical approach for our present study, we were able to confirm our recent findings1 in a different population of node-negative patients. Thus there is emerging evidence that bone marrow micrometastasis is an independent and powerful predictor of poor prognosis in patients with tumor-free lymph nodes (Table 4). With the removal of lymph nodes at primary surgery, no tissue has thus far been available to measure treatment effects in the setting of adjuvant therapy. In contrast, bone marrow micrometastasis has been successfully introduced as a surrogate model for treatment efficacy in previous studies monitoring the effect of cytotoxic,49 hormonal50 and immunologic therapeutics51,52 on isolated tumor cells in bone marrow. None of the members of the study population have received adjuvant therapy (eg, neither cytotoxic nor hormonal treatment). Thus we were able to study the unbiased effect of micrometastasis on prognosis. Nevertheless, the information on disappearance versus presence of bone marrow micrometastases in the face of chemotherapy might be of considerable importance, as the failure of the applied cytotoxic regimen to eliminate those cells might confer a substantially increased risk of clinical tumor relapse.49 The absence of such remnant cells may provide reasons for prognostic optimism, which would relieve the burden to await 5-year survival counts of a distinct therapy regimen to confirm its efficacy. Because this is an interesting option, bone marrow screening according to the presented immunoassay should be considered for implementation into appropriate phase II clinical trials for monitoring of therapeutic efficacy as well as stratification of more homogeneous patient subgroups with respect to their risk of distant disease and cancer-related death.
Supported by the Foundation "Freunde der Maistrasse," the Curt-Bohnewand-Foundation, and Friedrich-Baur-Foundation, München, Germany. We gratefully acknowledge the excellent technical assistance by Beate Zill (München) and thank all of our colleagues at the Department of Gynecology in Munich (Head: Prof Günter Kindermann) for the help in recruiting and following the patients of the present study. We thank Prof Klaus Pantel (Frauenklinik, Eppendorf University, Hamburg; Germany) for constructive critique and discussion of the manuscript. In part, this study contains material analyzed by Banu Semeni Cevatli in preparation of her thesis to achieve the degree of MD at Ludwig-Maximilians University, Medical Faculty, in Munich, Germany.
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Braun S, Hepp F, Kentenich CRM, et al: Monoclonal antibody therapy with edrecolomab in breast cancer patients: Monitoring of elimination of disseminated cytokeratin-positive tumor cells in bone marrow. Clin Cancer Res 5: 3999-4004, 1999 Submitted May 23, 2000; accepted November 2, 2000. This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
2 test; differences of means of independent samples with continuous variables were calculated from the Mann-Whitney U test. 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 for MacIntosh (SPSS, Inc, Chicago, IL). 
