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© 2001 American Society for Clinical Oncology Simultaneous Immunohistochemical Detection of Tumor Cells in Lymph Nodes and Bone Marrow Aspirates in Breast Cancer and Its Correlation With Other Prognostic FactorsFrom the Department of Obstetrics and Gynecology, Department of Pathology, and Institute of Medical Informatics and Biometry, University of Rostock, Germany. Address reprint requests to Bernd Gerber, PhD, Department of Obstetrics and Gynecology, University of Rostock, P.O. Box 10 08 88, 18055 Rostock, Germany; email: bernd.gerber{at}med.uni-rostock.de
PURPOSE: We studied the prognostic and predictive value of immunohistochemically detected occult tumor cells (OTCs) in lymph nodes and bone marrow aspirates obtained from node-negative breast cancer patients. All were classified as distant metastases-free using conventional staging methods. PATIENTS AND METHODS: A total of 484 patients with pT1-2N0M0 breast cancer and 70 with pT1-2N1M0 breast cancer and a single affected lymph node participated in our trial. Ipsilateral axillary lymph nodes and intraoperatively aspirated bone marrow were examined. All samples were examined for OTCs using monoclonal antibodies to cytokeratins 8, 18, 19. Immunohistological findings were correlated with other prognostic factors. The mean follow-up was 54 ± 24 months. RESULTS: OTCs were detected in 180 (37.2%) of 484 pT1-2N0M0 patients: in the bone marrow of 126 patients (26.0%), in the lymph nodes of 31 patients (6.4%), and in bone marrow and lymph nodes of 23 (4.8%) patients. Of the 70 patients with pT1-2N1MO breast cancer and a single involved lymph node, OTCs were identified in the bone marrow of 26 (37.1%). The ability to detect tumor cells increased with the following tumor features: larger size, poor differentiation, and higher proliferation. Tumors of patients with OTCs more frequently demonstrated lymph node invasion, blood vessel invasion, higher urokinase-type plasminogen activator levels, and increased PAI-1 concentrations. Patients with detected OTCs showed reduced disease-free survival (DFS) and overall survival (OAS) rates that were comparable to those observed in patients who had one positive lymph node. Multivariate analysis of prognostic factors revealed that OTCs, histological grading, and tumor size are significant predictors of DFS; OTCs and grading of OAS. CONCLUSION: OTCs detected by simultaneous immunohistochemical analysis of axillary lymph nodes and bone marrow demonstrate independent metastatic pathways. Although OTCs were significantly more frequent in patients with other unfavorable prognostic factors, they were confirmed as an independent prognostic factor for pT1-2N0M0, R0 breast cancer patients.
MANY CASES OF breast cancer are detected at an early stage. Although the disease may seem to be restricted solely to the breast, it may recur elsewhere when only local treatment is applied. Due to breast screening programs and greater public awareness, 60% to 70% of all new cases of breast cancer are without axillary lymph node involvement.1 Although the recurrence rate in node-negative breast cancer patients was reported to be 25% to 30%,2-7 it is possible to reduce this through the use of systemic adjuvant therapy.8,9 However, such therapy is usually associated with attendant side effects such as reduced life quality and increased morbidity and mortality. To date, no valid parameters have been established to identify node-negative patients who may benefit from systemic adjuvant therapy. For this reason, current recommendation for adjuvant treatment includes all patients except those with a tumor size  1.0 cm, grade 1 tumor, positive hormone receptor status, or who are older than 35.10 Lymph node involvement or confirmation of distant metastases in breast cancer always necessitates systemic treatment. Because the detection of occult tumor cells (OTCs) could be a strong indicator of the tumor’s ability to metastasize, many authors have tried to detect them in lymph nodes or bone marrow aspirates. However, simultaneous examination of bone marrow and lymph nodes by equally suitable methods has not yet been reported. Although, several authors11-17 have reviewed literature regarding OTCs or micrometastases, the independent prognostic significance of OTCs detected in regional lymph nodes or other distant sites such as blood or bone marrow remains difficult to assess. To enable comparisons of treatment results and to avoid variation in staging, the presence of OTCs presently should not be considered in tumor-node-metastasis staging and residual tumor classifications.17 For future evaluations of the prognostic importance of OTCs, larger studies with longer follow-ups are urgently needed. Here we report for the first time the simultaneous immunohistochemical investigation of OTCs in lymph nodes and bone marrow aspirates from node-negative breast cancer patients. The aim of this study was to assess the prognostic and therapeutic significance of these detected OTCs and their correlation with other prognostic factors.
Study Design and Patients From 1988 to 1996, we performed immunohistochemical studies on bone marrow aspirates and axillary lymph nodes from 484 assessable, histologically node-negative patients with primary breast cancer. All had undergone surgery at our department. The study was approved by the ethical review board of the Faculty of Medicine of the University of Rostock and informed consent was obtained from all patients. Inclusion criteria were: invasive breast cancers with a histopathologic tumor size of 6 to 50 mm, the examination of at least 10 negative axillary lymph nodes using conventional histology (hematoxylin and eosin, HE), and the absence of distant metastases in chest x-ray, liver ultrasound, and bone scan (pT1b-2 N0 M0). If these criteria were not met, the patient and the corresponding samples were excluded from the study. Bone marrow aspirates from 70 patients with both a tumor size of 6 to 50 mm and a single metastatic axillary lymph node (detected by conventional histology) served as the control group. Patients were classified as postmenopausal when the last menstruation was at least 1 year ago or on the basis of the hormonal status after hysterectomy. There was no evidence of recurrence or metastases within the first 6 months after initial surgery. Based on histologic and immunohistochemical findings in lymph nodes and bone marrow aspirates, as well as on the findings revealed by conventional staging methods, the patients were grouped into the following categories17:
Adjuvant Therapy
Patient Evaluation
Bone Marrow Aspiration and Preparation
Lymph Node Preparation
Antibodies All specimens were evaluated by a pathologist with experience in immunohistochemistry. The classification of the stained cells as either micrometastases or tumor cells also required histomorphologic characteristics of tumor cells. Unspecific reactions or staining of endothelial cells in the marginal sinus of lymph nodes were not considered to be tumor cells.
Prognostic Factors
Statistical Methods
The period of time to first relapse or death was estimated and graphically presented using the Kaplan-Meier method.22 Differences between curves were assessed by Mantel’s log-rank test23 for censored survival data. The Cox proportional hazards model24 was used to assess the independence of tumor cell detection from other prognostic factors. All P values resulted from two-sided statistical tests and P
Patient Characteristics OTCs were detected immunohistochemically in 180 of 484 patients staged as pT1-2 pN0 pM0 using conventional methods. No significant differences were found between the patient groups ( Table 1) with respect to type of surgery, menopausal status, age, number of examined lymph nodes, or amount of aspirated bone marrow. Three hundred ninety-seven (82%) of the 484 node-negative and all 70 node-positive patients received systemic adjuvant treatment. No serious bone marrow aspiration-related complications were seen; with only five patients (1.0%) reporting pain at the puncture site that lasted 1 to 2 days.
Immunohistochemical Findings Tumor cells were immunohistochemically detected in the lymph nodes of 54 node-negative patients (11.2%); the nodes being previously analyzed by conventional histology. OTCs were found in bone aspirates of 149 patients (30.8%) ( Fig 1) and in both lymph nodes and bone marrow of 23 patients (4.8%) ( Table 2). Twenty-six (37.1%) of the 70 patients with a single lymph node macrometastasis had OTCs in bone marrow aspirates.
Patients in whom no tumor cells were detected differ from those with OTCs and single lymph node macrometastases examined by conventional HE staining ( Table 3). Tumors larger than 20 mm were found in 24.5% of patients who had no OTCs at all (pN/M0(i-)), in 48.9% of patients with immunohistochemically detected tumor cells (pN/M0(i+)) and in 48.6% of patients with a single lymph node macrometastasis (pN1) (P < .0001,  2 test). There was a substantial proportion of tumors with a size of 10 mm or smaller in the pN/M0(i+) group (11.7%). Other significantly more unfavorable prognostic factors were identified in patients with OTCs: grading (P = .034), lymph vessel invasion (P = .005), blood vessel invasion (P = .047), ER-ICA (P = .026), extent of proliferation (P < .0001), uPA level (P = .016), and PAI-1 concentration (P = .030). It should be pointed out that the expression of the specified prognostic factors was comparable between patients with single tumor cells and patients with a single lymph node macrometastasis.
Disease-Free Survival and Overall Survival There were 84 disease recurrences: 26 (8.6%) in the pN/M0(i-) group, 43 (23.9%) in the pN/M0(i+) group, and 15 (21.4%) in the pN1 group (P < .0001). There were 50 disease-related deaths: 13 (4.3%) in the pN/M0(i-) group, 26 (14.4%) in the pN/M0(i+) group, and 11 (15.7%) in the pN1 group (P < .0001). No significant correlation was found between the site of the detected OTCs and the location of distant metastases ( Table 4). The number of detected OTCs in bone marrow (1 to 300 per slide) and the number of affected lymph nodes (one to three) did not correlate with the frequency of recurrence (P > .3206).
DFS and OAS rates were observed to be significantly worse in patients with OTCs than without ( Fig 2). The 5- and 10-year DFS rates were: 92.6% and 76.6% (pN/M0(i-)), 71.2% and 54.5% (pN/M0(i+)), and 74.2% and 62.8% (pN1), respectively. Using the log-rank test, statistically significant differences were found between the pN/M0(I-) group and the pN/M0(i+) group (P < .0001), as well as between the pN/M0(i-) group and the pN1 group (P = .0059). Kaplan-Meier curves for patients with OTCs and for those with a single lymph node macrometastasis were virtually identical (P = .4505). The corresponding 5- and 10-year OAS rates were: 95.1% and 87.4% (pN/M0(i-)), 76.6% and 70.7% (pN/M0(i+)), and 77.7% and 73.6% (pN1), respectively. Overall survival of pN/M0(i-) patients was significantly better than that of pN/M0(i+) (P < .0001) and pN1 patients (P = .0024).
Systemic Adjuvant Treatment and Menopausal Status DFS and OAS outcomes as a function of systemic adjuvant treatment and menopausal status were analyzed. Because of the small number of untreated patients in these groups, no statistically significant differences could be revealed (pN/M0(i-): DFS P = .2255, OAS P = 3.519 and pN/M1(i+): DFS P = .3557, OAS P = 3.658). Menopausal status did not significantly affect the outcome of patients without (pN/M0(i-): DFS P = .1455, OAS P =. 5110) or with (pN/M1(i+): DFS P = .1487, OAS P = .1827) OTCs. However, premenopausal patients with a single axillary lymph node metastasis (pN1) showed an adversely affected DFS (P = .0072) and OAS (P = .0125) when compared with postmenopausal patients of the same group.
Univariate and Multivariate Analysis
This study, started in the mid-1980s, was planned to confirm the prognostic and predictive values of immunohistochemically detected OTCs in lymph nodes and bone marrow aspirates from node-negative and distant metastases-free patients (pT1-2N0M0, R0). Initially, systemic treatment of patients with detected OTCs was not planned. However, in 1989, results from several studies25-28 confirmed an increased disease-free survival rate after systemic treatment of node-negative patients. Although it has remained controversial, our local ethical review board has recommended systemic treatment for node-negative patients since this time. Perioperative detection of OTCs may reflect either transient shedding of cells or a possible metastatic potential of the tumor. Because of the possible benefit of adjuvant therapy, it is important to identify these patients.8,9,29,30 To date, it remains unclear whether or not OTCs in bone marrow or lymph nodes of conventional node-negative and distant metastases-free patients represent independent prognostic factors, as suggested by the international cancer committees.31 Recently, Hermanek et al17 have recommended that isolated (disseminated or circulating) tumor cells detected by immunohistochemistry or molecular pathologic methods be distinguished from micrometastases because of their different prognostic value. The detection rate of OTCs in negative lymph nodes was reported to be 7% to 33%14,15 and 2% to 55%11-13,16 in bone marrow. We found OTCs in the lymph nodes of 11.2% and in the bone marrow of 30.8% of the patients examined. Disseminated tumor cells, irrespective of the site, were detected in 37.2% of our 484 node-negative and disease-free patients (pT1-2N0M0). These tumor cells represent potentially metastasizing lesions. Important factors in most studies are small numbers of cases, heterogeneity of included patients, short follow-ups, and treatments that differ. Regarding tumor-node-metastasis stages, some studies that investigated OTCs in bone marrow included T0-2N0-1M0-32,33 or T0-4N0-3M0-patients.34-40 Other studies included patients with distant metastases41 or R2-resection.42 OTC detection rate in patients with T3-4 tumors is 72% as compared with 38% in patients with T1-239 tumors. Furthermore, OTCs were found in 55% of node-positive patients compared with 31% of node-negative patients.40 In our group of 70 patients with a single involved lymph node, tumor cells were found in the bone marrow of 37.1%, compared with 26.0% in node-negative patients. During the process of metastasis, tumor cells change. This is reflected by the heterogeneity of expressed antigens and enzymes.43-47 Funke and Schraut13 subjected various studies of OTCs in bone marrow to a meta-analysis. They reported studies in which mixtures of more than 24 different antibodies were used. However, sensitivity and specificity of some antibodies were insufficiently tested. For example, the carcinoma-associated mucin MUC-1 was expressed in more than 10% of the normal human bone marrow mononuclear cells, as detected by using the anti-MUC-1 monoclonal antibody 2E11.48,49 The detection rate was also affected by site and number of aspirations, blood contamination, and number of bone marrow cells screened per aspiration site.50,51 Comparative immunostaining of bone marrow specimens with the monoclonal antibodies CK2 and A45-B/B3 indicated that downregulation of CK18 in micrometastatic carcinoma cells occurs in approximately 50%, regardless of the primary tumor origin. It is more complicated to "review the dilemma of lymph node micrometastases."14,15 Axillary lymph node micrometastases are generally defined as tumor cell accumulations of up to 2 mm. Lilleng et al52 described three levels of nodal tumor load, each with a different prognosis. The smallest deposits, up to 0.0001 cm2, include embolic growth on the afferent side of the node and are associated with a poor prognosis. In these cases, postoperative prognosis is comparable with that of node-positive patients, node-positive being defined here as having an axillary tumor load of 0.5 cm2 or more. In addition to these, there is a third, intermediate group of patients that represent 40% of the total series. Its prognosis is similar to that of the node-negative patients. Lilleng et al52 suggest that deposits in this intermediate group and deposits with embolic growth that represent a high risk for the development of distant metastases should be termed micrometastases. Larger tumor cell deposits should be classified as node-positive. There is consensus that the chance of identifying a micrometastasis increases with the number of resections studied. Thus, the probability of finding further OTCs after original multiple-level sectioning is low.53 A pathologic examination using immunohistochemical methods permits a more accurate staging than HE staining alone.50,54 Our findings support reports that have shown unfavorable prognoses associated with the detection OTCs in lymph nodes or bone marrow aspirates.11-17 Multivariate analysis in this and other studies38,40,54 confirmed tumor cell detection as an independent factor, although certain reports37 question its relevance. However, because there has been some criticism of the methods used in these studies, eg, large confidence intervals, impressive values of some variables, underestimation of lymph node status, and tumor size, the results of some studies38,40 may not be representative.13 According to Cote et al,54 the presence of occult axillary lymph node micrometastases adversely affected the prognosis of postmenopausal women, but did not influence the prognosis in premenopausal women. Although we could not find significant differences between pre- and postmenopausal patients with OTCs, the prognosis of premenopausal patients with a single lymph node macrometastasis was significantly worse compared with postmenopausal patients of the same group. Although it has been shown that the vast majority of lymph node metastases can be detected by taking two sections located 0.3 mm apart and staining them with monoclonal antibodies,55 extensive serial sectioning and immunohistochemistry for the analysis of all axillary lymph nodes is too expensive and labor intensive to be a suitable method.14,56 Other prognostic tumor factors might be predictors of risk for recurrence. In our study, a correlation between 21 biologic tumor factors and OTCs is reported for the first time. Patients with OTCs were comparable with those with a single lymph node macrometastasis, both showed significantly larger and less differentiated tumors, more frequent lymph and blood vessel invasion, increased frequency of negative estrogen receptors, higher proliferation, and increased uPA- and PAI-1 concentrations, when compared with patients without OTCs. On the other hand, HER2-neu, EGF, p53, cathepsin D, and pS2 did not differ significantly between patients with or without OTCs. Other authors reported a correlation between tumor cell detection and lymph node status,34,36,37,40,57 larger tumors,34,36,37,40,58-60 higher tumor grade,40,54,60,61 vessel invasion,34,36,37,54,60,61 ER,36 c-erb/B-2,62 and laminin receptor.63 On the basis of our results, we conclude that more attention should be paid to OTCs, especially in studies that investigate sentinel lymph node dissection. Micrometastases were immunohistochemically detected in the sentinel lymph node.64-68 Recently, Chu et al69 questioned whether sentinel lymph nodes with micrometastases should be subjected to a complete dissection. In these cases, the risk of systemic tumor cell dissemination is high and systemic treatment can eradicate tumor cells even in their intact lymphatic environment.70 Thus the question remains: What about detection of OTCs in the bone marrow of patients with a negative sentinel lymph node?50 Using different primers, polymerase chain reaction and reverse transcriptase polymerase chain reaction (RT-PCR) have been shown to be more sensitive methods for the detection of axillary lymph node micrometastases than histopathologic examinations, even when serial sectioning and immunohistochemical staining were performed.58,71-76 This also applies to blood samples.59,77 Moreover, RT-PCR analysis is less expensive than currently available histopathologic examination techniques,78 but it fails to distinguish benign from malignant epithelial cells.67 Some cytokeratins (eg, CK-19) seem to have no diagnostic value as mRNA markers for micrometastases; they are also expressed in blood and lymph nodes of healthy controls.79 The illegitimate transcription of tumor-associated or epithelial-specific genes in hematopoetic cells and the deficient expression of the marker gene in OTCs are limiting factors in the detection of tumor cells by RT-PCR.80 The search for OTCs in blood or bone marrow was recommended for monitoring the effectiveness of systemic treatment.77 Mansi et al81 repeated bone marrow aspiration in patients with perioperative detection of tumor cells. Detection rates were 2% and 3%, independent of systemic treatment. It was concluded that many micrometastases in breast cancer patients result from the shedding of cells from the primary carcinoma and that a significant proportion is not viable. Recently, Braun et al82 have shown that in high-risk patients who receive chemotherapy (taxanes and anthracyclines), the difference in OTCs before treatment (49.2%) and after treatment (44.1%) was negligible. Cote et al54 reported that after a mean follow-up of 12 years, patients would benefit from perioperative chemotherapy; specifically for patients in whom no micrometastases were detected, but not for those in whom tumor cells were found. Mansi et al37 found a shorter relapse-free survival in patients with OTCs, even after adjuvant treatment. We were unable to show an influence of OTCs on the effectiveness of adjuvant treatment. In contrast to solid metastases, OTCs are appropriate targets for intravenously applied agents because macromolecules and immunocompetent effector cells should have access to the tumor cells. Because the majority of micrometastatic tumor cells may be nonproliferative (G0 phase), standard cytotoxic chemotherapies directed against proliferating cells may be ineffective, which might partly explain the failure of chemotherapy.11,83 A new promising therapeutic approach represents the murine monoclonal antibody 17-1A (edrecolomab, Panorex; Glaxo Wellcome GmbH, Hamburg, Germany) directed against the epithelial cell adhesion molecule (EpCAM) that is expressed on more than 60% of distant breast cancer tumor cells. Edrecolomab reduced or eliminated tumor cells in a small series of patients with advanced breast cancers.84 After R0 resection, this antibody led to a significant improvement in disease-free survival for colorectal cancer patients.85 Adjuvant treatments, such as antibody-based therapies, are of considerable interest in the treatment of breast cancer, because they could eradicate OTCs before metastatic disease becomes clinically evident. Thus, early detection of OTCs could identify patients who are likely to benefit from adjuvant treatment.
Supported in part by grants from the FORUN project. We thank K. Pantel, MD, PhD, for reviewing the manuscript.
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ABSTRACT
INTRODUCTION
