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Detection of Isolated Tumor Cells in Bone Marrow in Early-Stage Breast Carcinoma Patients

Comparison with Preoperative Clinical Parameters and Primary Tumor Characteristics¹

Departments of Oncology [B. N., B. K.], Pathology [E. B., J. M. N.], and Surgery [H. Q.], The Norwegian Radium Hospital, 0310 Oslo, and Departments of Surgery [R. K.] and Pathology [T. S.], Ullevål Hospital, 0407 Oslo, Norway

	ABSTRACT

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Purpose/Experimental Design: The importance of detection of disseminated isolated tumor cells (ITCs) in bone marrow (BM) is still not settled. BM aspirates from 920 patients with primary breast cancer were analyzed for tumor cells by standardized direct immunocytochemical analysis (ICC) of 2 x 10⁶ mononuclear cells (MNCs) using anticytokeratin monoclonal antibody (AE1/AE3). Samples (637) were analyzed by negative immunomagnetic enrichment (IMS) followed by ICC (10 x 10⁶ MNCs). Analyses of the primary tumor specimens have been performed, including histomorphology, tumor-node-metastasis (TNM) staging, grading, and immunohistochemical analyses.

Results: Of the patients with infiltrating carcinoma, 63% were node negative (N0) and 33%, node positive (N+). The results show the presence of tumor cells in 13.4% of the evaluable patients after direct ICC analysis. The presence of tumor cells correlated to the nodal- and tumor stage, showing BM positivity in 9.9% of the N0 cases and 20.6% in the N+ group (P < 0.0005), 11.2% of the stage T₁ were positive, and 15.0% and 22.6% were positive in the T₂ and T_3/4 groups, respectively (P = 0.013). No correlation between detection of ITC and detection of p53 and cathepsin D expression was found. Vascular invasion and c-erbB2 expression were associated with ITCs in BM (P = 0.045 and P = 0.024, respectively). Node-negative patients with estrogen receptor (ER)+ and/or progesterone receptor (PgR)+ tumors had lower frequency of ITCs than ER-/PgR- (P = 0.004). The use of negative IMS increased the frequency of positive BM by 63% (P < 0.0005).

Conclusions: The direct ICC detection of ITCs in BM correlated with primary tumor stage, nodal stage, vascular invasion, c-erbB2 expression, and ER/PgR status. Analysis of larger BM samples by negative IMS resulted in increased number of ITC-positive patients.

	INTRODUCTION

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During the last decades, strong efforts have been made for earlier detection of breast cancer. The introduction of periodic mammography in women ages >50 years has reduced the tumor size and nodal involvement in the patients and a concordant reduction in mortality has been reported (1 , 2) . Through primary tumor characterization and lymph node staging, adjuvant therapy is decided, and an improved survival in the treated patient groups is observed (3) . However, the current staging does not appropriately identify all of the high-risk patients in the growing group of node-negative patients with small tumors. Although only a small percentage of these early-stage patients are at risk for developing metastases, a high absolute number of them experience a relapse. Furthermore, in those patients receiving systemic therapy, treatment failure can first be noticed when overt metastases have developed, and the disease is uncurable.

The need for additional tools in the selection of early-stage patients at risk for relapse, and for monitoring the effect of systemic therapy, is evident. In addition, techniques for characterization of the disseminated TCs³ are required for prediction of treatment response and for tailored treatment decisions. ICC detection of ITCs in BM and additional analyses of these cells have been introduced as techniques that may fulfil these needs (4, 5, 6, 7, 8, 9, 10, 11) . An increasing number of studies have used this principle for detection of TCs in BM in breast cancer as well as in other carcinomas, and an association with clinical outcome is observed (12, 13, 14, 15, 16, 17, 18, 19) . However, the methods reported thus far have not been properly standardized, and differences in sensitivity and specificity exist. Therefore, The European International Society of Hematotherapy and Graft Engineering group for standardization of TC detection introduced guidelines for analysis and evaluation of ITC by ICC (20) . Furthermore, several tumor-cell enrichment techniques have recently been introduced in an attempt to increase the sensitivity of ITC detection and to improve the ability to quantify and characterize the contaminating TCs (21, 22, 23, 24, 25) . At present, the clinical significance of these techniques, as compared with a standardized direct ICC method, is not known.

The aim of the micrometastasis project in Oslo is: (a) to examine early-stage breast cancer patients for the presence of ITCs in BM by a standardized and controlled ICC technique; (b) to clarify the prognostic value of detection of TCs in BM; (c) to test an immunomagnetic enrichment strategy (negative IMS) for detection of TCs and compare the results with that obtained by standard ICC; and (d) to analyze candidate prognostic factors in the primary tumors by immunohistochemistry. Here, we present the detection of ITCs in BM and compare the primary tumor characteristics and the clinical parameters at the time of diagnosis of 920 patients.

	PATIENTS AND METHODS

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Project and Patients.
From May 1995 to December 1998, 920 patients were referred for surgical treatment of breast cancer to five different hospitals in the Oslo region (Ullevål Hospital, The Norwegian Radium Hospital, Bærum Hospital, Aker Hospital, and Buskerud County Hospital) and were included in the study after giving informed consent. The project was approved by the regional ethics committee. Of the patients, 29.2% had their cancer detected within a mammography screening project.

mAbs.
The AE3 anticytokeratin mAb (Sanbio, Uden, the Netherlands; mouse IgG1) reacts with primarily high molecular weight keratins of the type II basic subfamily (keratin 1–8). The AE1 anticytokeratin mAb (Sanbio; mouse IgG1) reacts with primarily low-and intermediate-type I acidic keratins (keratin 10 and 12–19). mAbs against human ER (clone 6F11, mouse IgG1) and PgR (clone 1A6; mouse IgG1) were purchased from Novocastra (Newcastle upon Tyne, United Kingdom). For detection of mutant p53 protein a mAb reactive against wild-type and mutant p-53 protein (clone DO-1, mouse IgG2a; Santa Cruz Biotechnology, Santa Cruz, CA) was used. Rabbit anti-cathepsin D (whole rabbit antiserum; Zymed Laboratories, San Francisco, CA) reacts against both precursor and activated form of the enzyme. The mAb clone CB11 (mouse IgG1; BioGenex, San Ramon, CA) was used for the detection of c-erbB2 overexpression.

Collection of BM.
A total of 40 ml of BM was aspirated from anterior and posterior iliac crest bilaterally (10 ml/site) under general anesthesia immediately prior to surgery. All of the aspirations were performed in sodium-heparinized tubes and, on the same day, were transported to the laboratory for further processing. In addition, 50 BM samples from normal donors were collected for control purposes.

Preparation of BM MNC Samples and Cytospins.
The BM aspirates were diluted 1:1 in PBS (Life Technologies, Inc., Roskilde, Denmark) and separated by density centrifugation using Lymphoprep (Nycomed, Oslo, Norway). MNCs were collected from the interphase layer, washed twice in PBS (Life Technologies, Inc.) with 10% FCS (Biological Industries, Kibbutz Beit Haemek, Israel). Then, the cells were used in the negative IMS technique and for preparation of direct cytospins. Direct cytospins were prepared by centrifuging the BM MNCs down to polylysine-coated glass slides (5 x 10⁵ MNCs/slide) in a Hettich cytocentrifuge (Tutlingen, Germany). The slides were air-dried overnight and stored at -80°C until immunostaining was performed.

ICC Staining.
Before staining, the cytospins were fixed for 10 min in acetone. Four slides (2 x 10⁶ BM MNCs) were incubated for 30 min in a moist chamber either with the anticytokeratin antibodies (AE1-AE3, 1.1 µg/ml each), and the same number of slides (2 x 10⁶ BM MNCs) were incubated with a negative control mAb of the same isotype (IgG1) in a corresponding concentration [mouse myeloma immunoglobulin (Bionetics, Kensington, MD); mouse anti-FITC (Micromet, Munich, Germany), or MOPC-21 (Sigma Chemical Co., St. Louis, MO)]. This was followed by a 30-min incubation with polyvalent rabbit antimouse Ab (DAKO; Z 259, diluted 1:25 to 1:50) and subsequently preformed complexes of alkaline phosphatase (AP)/monoclonal mouse anti-AP (APAAP-complex; DAKO; D 651, diluted 1:25). The slides were washed twice in Tris-HCl (5 min) between each incubation. The color reaction was developed by 10-min incubation with New Fuchsin solution (0.26%; Aldrich Chemical Company, Milwaukee, WI), containing 0.65% (w/v) naphtol-AS-BI phosphate and 0.45% (w/v) levamisole (Sigma Chemical Co.). The slides were counterstained with hematoxylin for 1 min to visualize nuclear morphology. The slides were subsequently mounted in Kaiser’s glycerine-gelatin (Chroma Gesellschaft Gmbh, Münster, Germany). In all but 231 of the BM samples tested, four slides (2 x 10⁶ MNCs) were stained with AE1-AE3 and four slides with negative control Ab. In the rest, only one slide was available for control staining. Cytospins from 50 normal BM samples were also analyzed (2 x 10⁶ BM MNCs for AE1-AE3 staining and 2 x 10⁶ BM MNCs for negative control staining).

ICC Detection of Positive Cells.
The slides were manually screened by light microscopy using the x10 lens. All of the stained cells were closely evaluated by one of the pathologists (E. B.). Immunopositive cells with a morphology compatible with TCs or lacking hematopoietic characteristics were recorded as positive, according to the recommended guidelines (20) . These guidelines describe criteria for ITC positivity. Examples of occasional cells in which the morphology of hematopoietic cells resemble TCs are also presented (20) . In case of indeterminate cell morphology, a second pathologist (J. M. N.) was consulted and consensus obtained. The presence of morphologically similar, positively stained cells in the corresponding negative control resulted in the exclusion of the patient sample from a diagnostic conclusion. False positive ICC reactions can be caused by hematopoietic cells directly reactive to the exogenous AP added in the APAAP step (26) . The false positive rate in the samples with 1 negative control slide available [11 (4.8%) of 231] was compared with the 64 false positive (9.3%) of 685 samples with 4 negative control slides analyzed. The expected false-positive rate in the evaluable samples (corresponding to the frequency of negative control positive samples coupled with a negative specific test) was estimated at 4% in all of the patient samples analyzed. Four of 50 control BM samples from healthy individuals showed 1 positive cell after staining with AE1-AE3, and 5 of 50 were positive after incubation with isotype-specific control mAb.

Negative IMS Technique.
Negative immunomagnetic technique was performed as described previously (24) . Briefly, samples of 1–2 x 10⁷ BM MNCs were resuspended to 2 x 10⁷ MNCs/ml and incubated with anti-CD45-conjugated M450 Dynabeads (Product 111.19; Dynal, Oslo, Norway). at a bead:cell ratio of 5:1 (10⁸ beads/ml) in polypropylene tubes. After gentle rotation for 45 min, the solution was then transferred to 50-ml tubes, diluted to 30 ml and placed onto a neodynium magnetic particle concentrator (Dynal) for 10–15 min. The non-rosetted cells were collected and centrifuged at 450 x g for 10 min, counted, and resuspended in PBS with 10% FCS to 1 x 10⁶ cells/ml. Cytospins containing 5 x 10⁵ cells/slide were prepared. The negative IMS was considered successful when >70% leukocyte depletion was obtained. The cytospins were analyzed by ICC staining and detection as for direct cytospins (unseparated BM MNCs).

Immunohistochemical Staining of Primary Tumor.
After formalin fixation, selected parts of the primary breast tumor were paraffin embedded and cut into 4–6-µm-thick sections, attached to silane- or chromalun-coated glass slides, and heated at 56°C for 40 min, then at 37°C over night. Before immunostaining, the slides were deparaffinized in xylene and taken through descending alcohol concentrations to water. For the slides submitted to immunostaining for ER, PgR, p53 protein, and cathepsin D, antigen retrieval was performed by boiling in citrate buffer (pH 6) in a microwave oven [Electrolux, household type, 800–850W, 4 x 5 min (ER and PgR) or 2 x 5 min (p-53 and cathepsin D)]. All primary antibodies were incubated for 30 min, in the following dilutions: ER, 1:25; PgR, 1:20–1:30; p-53, 1:400; cathepsin D, 1:50; c-erbB-2, 1:10. Positive and negative controls were performed with satisfactory results. The immunostaining for ER, PgR, p53 protein, and cathepsin D was performed by one of the pathology laboratories (The Norwegian Radium Hospital), using OptiMaxPlus automated cell staining system (BioGenex, San Ramon, CA) and the BioGenex detection system Str.AviGen MultiLink kit. The immunolabeling for c-erbB-2 protein was performed by the other pathology laboratory (Ullevål Hospital), using Ventana ES staining system (Ventana Medical Systems, Tucson, AZ), and the Ventana DAB detection kit.

Immunohistochemical Analysis of Primary Tumor.
The primary tumor was regarded positive for ERs or PgRs if 10% or more of the TC nuclei were immunostained with the respective antibodies. For p53 protein, the threshold for positivity was staining of 5% of the TC nuclei. Tumors having 10% or more carcinoma cells containing well-defined immunolabeled secretory granules were considered cathepsin D positive. The tumor was scored c-erbB2-oncoprotein positive if 10% or more of the TC showed distinct membranous staining. Cases in which the cells revealed only cytoplasmic staining were not considered positive.

Primary Tumor Analysis.
Primary tumors and axillary lymph nodes collected during the surgery were processed on a routine diagnostic basis. Histological tumor type, tumor size, grade, and nodal involvement were analyzed and classified according to the tumor-node-metastasis (TNM) system (WHO). Grading was performed according to recommendations from Elston and Ellis (27) .

Statistical Analysis.
Comparison between groups for the presence of TCs in BM was performed with ² test (Pearson and linear-by-linear). McNemar’s test was used for comparison of direct ICC with negative IMS within groups.

	RESULTS

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Patient Characteristics and Patient Sample Analysis.
A total of 920 patients were included. Median age in the population was 58 years (range, 27–93 years). Clinical and histopathological characteristics of the patients are described in Table 1 . In 29.2% of the patients, breast cancer was detected in an official mammography screening program. The disease was diagnosed in the remaining patients after a patient- or doctor-induced mammography. Forty % had a nonpalpable tumor without symptoms. The study population included 63% node negative patients. Fifty-eight % had T₁ tumor. Forty-five % of the patients were classified as T₁N₀ and the median tumor size was 1.7 cm. In addition to the routine histopathological examinations, immunohistochemical analysis were performed: 74% were positive for ER (74% ER+); 59% PgR+; 24% p53+; and 52% cathepsin D+. The frequency of c-erbB2 positivity was low (7%) and was controlled by repeat analyses of 10% of the samples with the same results (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 1. Frequency distribution of the number of detected TCs in BM-positive patients analyzed by both direct ICC and negative IMS (n = 484). The number of TCs detected in all of the BM-positive cases is presented.

	DISCUSSION

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No association between ITC detection in BM and ER status or PgR status alone was detected, in accordance with previous reports (15 , 19) . However, in the node-negative patients, a markedly higher frequency of ITCs (BM-positive results) was observed among patients with receptor-negative primary tumors. The lower frequency of ITCs in ER+ and/or PgR+ patients could be expected because it has been observed previously (19 , 28 , 29) that early metastatic relapse is less frequent in hormone receptor-positive breast cancer. The overexpression of c-erbB2 and vascular invasion is associated with a more aggressive disease (30 , 31) . This may be consistent with a higher frequency of ITC detection, as presently observed in patients with c-erbB2-positive tumors or tumors with vascular invasion (Table 3) . The low frequency of c-erbB2 expression (Table 1) differs from the results in previous studies (30) . Our results have been extensively controlled, including comparison to Dako’s Herceptest, without altering the frequency of positives. There may be a technical explanation for the low positivity. However, we have not been able to detect any difference between storage time and quality of the slides and the rate of positives.⁴ No statistical difference in TC detection in BM was observed between p53- and p53+ tumors. This is in accordance with the results from a non-small cell lung cancer study (32) .

The reported incidence of ITCs in BM in breast cancer patients has varied considerably (8 , 12, 13, 14, 15 , 19 , 33) . This may be attributable to several factors: the number of MNCs analyzed, the expression of antigen used for the detection of TCs, the specificity of the mAb used, the sensitivity and specificity of the ICC detection, the use of negative controls, the accuracy of the screening process, the evaluation of immunopositive cells, and the stage distribution in the patient series (6 , 20) . These factors make it difficult to compare the results from different studies. An ISHAGE-supported collaborative study among several laboratories in Europe proposes a standardized analysis, evaluation, categorization, and diagnosis of ICC-positive cells (20) . Indeed, a high discordance in the screening results and interpretation of positive findings was revealed between different investigators in the preconsensus analysis (20) . Because the finding of one positive cell among 1–2 x 10⁶ cells categorizes the patient as "BM positive" in most studies, we have made a strong effort to control the specificity of the mAb and the ICC analysis used in our series (24 , 26) . In addition, the consensus guidelines from the European study group have been followed in the present study.

In parallel with the testing of the patient samples in our study, 50 normal donors have been analyzed by ICC after staining with AE1/AE3 and isotype-matched irrelevant control mAb. AE1/AE3-staining resulted in 4 positive BM samples of 50, whereas negative control resulted in 5 of 50 positive samples. This indicates that most of the AE1/AE3 false positivity is caused by unspecific staining unrelated to the primary antibody (Ref. 26 and unpublished results).⁵ The contemporary positivity by AE1/AE3 and negative control mAb staining in three of five of the patient samples in the present study reveal that the majority of the false-positive answers can be controlled by the use of isotype-specific irrelevant mAbs. However, 3–4% false positivity in evaluable patients has to be expected. Most studies published thus far, although not all (18 , 19) , have not reported the use of negative-control testing or have analyzed smaller samples than for the specific tests (6 , 8 , 15, 16, 17 , 34) . Therefore, an uncertainty exists concerning how false-positive reactions influence the results.

Furthermore, results from studies with Abs against tumor-associated glycoprotein 12 (TAG12) have shown a higher frequency of positive BM samples (14 , 15) . Several investigators have argued that TAG12, and also epithelial membrane antigen (EMA), are expressed by a significant proportion of hematopoietic cells, questioning the specificity of the results (6 , 35, 36, 37) . Studies using anticytokeratin mAbs have also tested series of BM samples from non-carcinoma patients with low levels of positive reactions (0–5.3%; Ref. 6 , 8 , 16, 17, 18, 19 , 34 ). The number of cells analyzed varies substantially in these reports (from 3 x 10⁴ to 5 x 10⁶), and this affects the frequency of positive events. Our initial testing indicated that other commonly used mAbs in ICC detection of micrometastases (CK2, A45/B-B3, BerEP4, MOC31) may possess a very low level of cross-reactivity against hematopoietic cells.⁶ This cross-reactivity, nevertheless, can influence the specificity of the ITC detection and is currently investigated in more detail.

The largest studies thus far have shown BM ITC positivity of 36 and 43% in breast cancer (15 , 19) . Both have shown association with clinical outcome, in univariate as well as multivariate analyses. A high frequency of BM-positive patients was reported in the N₀ group. In fact, no difference was observed between the N₀ and the N+ group by Braun et al. (19) . In contrast, the present study reveals a markedly lower frequency of positive findings, and a correlation to both N and T stage is observed. It is possible that the sensitivity by our standard direct ICC technique is lower than in their studies. On the other hand, a similar frequency of positives in N₀ as compared with N+ patients, as shown by Braun et al. (19) , may indicate detection of clinically insignificant positives or false positives in the N₀ group, because N₀ patients reveal less relapse than the N+ group.

A method for detection and further analysis of a higher number of TCs than obtained with the current ICC techniques is desired. This is possible using enrichment strategies like negative IMS, positive IMS, or high-throughput automatic screening of larger volumes of unseparated cells (21, 22, 23, 24, 25 , 38, 39, 40) . In this study, negative IMS increased the frequency of ITC-positive BM samples. The previous testing of this technique showed a 3- to 4-fold higher number of cells detected when 5-fold more cells were analyzed by IMS, in cases in which the Poisson distribution is not affecting the results (positive cell numbers 10; Refs. 23 , 24 ). Accordingly, a 4-fold higher number of positive cells (mean) was detected in the present study, and a close to two-thirds increase in positive BM samples was observed. In most samples, less than three TCs were detected, indicating a very low frequency of such cells in the material analyzed. This indicates that the amount of cells analyzed should be markedly higher to obtain quantitative results (i.e., 5 x 10⁷ cells). The increase in the number of positive samples by negative IMS was highest among the node-negative and T₁/T₂ tumors, reducing the correlation with N and T stage. When follow-up is completed, additional information about the clinically important sensitivity level for ITCs in BM will be achieved. The group with negative IMS positive events, however, did not include all direct ICC positive samples, because one of five of all positive samples (irrespective of the methods used) were positive only by direct ICC. This can be explained by the Poisson distribution effect and an extra manipulation (depletion) step in the negative IMS technique, resulting in some samples of eradication of rare TCs in the end product. An unsatisfactory depletion of MNCs was observed in 14% of the negative IMS cases. The depletion efficiency in peripheral blood is markedly better,⁷ as can be explained by a more homogeneous CD45 expression in mature hematopoietic cells. The use of a mixture of mAbs against different leukocyte antigen instead of only one mAb, should increase the depletion efficiency in the future. For optimal detection of ITCs, both direct ICC and negative IMS should be performed.

This report describes a well-tested, controlled, and standardized detection of ITCs in BM combined with the characterization of the primary tumor in early-stage breast cancer patients. An association between ITC-positivity and established prognostic factors was observed. Together with the forthcoming clinical follow-up the presented analyses will give important information about the usefulness and strength of ITC detection as a prognostic factor.

	ACKNOWLEDGMENTS

 
We acknowledge the skilled processing of BM and sample analysis by the Micrometastasis laboratory at the Norwegian Radium Hospital under the leadership of Anne Renolen, and the c-erbB2 staining that was thoroughly performed by Tove Noren. We are very thankful for statistical support from Professor Eva Skovlund.

	FOOTNOTES

 
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.

¹ Supported by the Norwegian Cancer Society.

² To whom requests for reprints should be addressed, at The Norwegian Radium Hospital, Department of Oncology, 0310 Oslo, Norway. Phone: 47-22934000; Fax: 47-22935011; E-mail: bjorn.naume{at}klinmed.uio.no

³ The abbreviations used are: ICC, immunocytochemical (analysis); BM, bone marrow; Ab, antibody; mAb, monoclonal Ab; TC, tumor cell; ITC, isolated TC; IMS, immunomagnetic separation; MNC, mononuclear cell; APAAP, alkaline phosphatase/anti-alkaline phosphatase (complex); ER, estrogen receptor; PgR, progesterone receptor.

⁴ Unpublished observations.

⁵ Unpublished observations.

⁶ Unpublished observations.

⁷ Unpublished observations.

Received 4/ 9/01; revised 8/30/01; accepted 9/ 4/01.

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