Bone Marrow Micrometastasis In Breast Cancer

Ninety-five percent of patients who present with breast cancer apparently have local disease without evidence of distant metastatic spread on pretreatment staging by conventional methods (1). Despite improvements in surgical techniques, radiotherapy and drug treatment, one third of all patients relapse and die within ten years, and this proportion has not changed significantly. It is accepted that this group of patients has micrometastatic disease at presentation that cannot be detected by current standard methods (2-5). Therefore, indirect prognostic criteria, such as lymph node metastasis, tumor size, and the presence of tumor emboli in lymphatic or vascular spaces associated with the primary tumor, are used in an attempt to identify a group of patients at high risk of developing distant metastases, who would perhaps benefit from adjuvant systemic treatment. Though axillary lymph node status in breast cancer patients remains the single most important predictor of outcomes, our current methods of histopathologic analysis may be inadequate because 30% of node-negative patients recur.

The skeleton is the most common site of distant metastases for breast cancer (6) and is frequently the first site at which distant metastasis is detected. The concept of investigating bone marrow as a site for occult micrometastases has validity from two aspects. First, bone metastases start from bone marrow invasion (7) and second, the lymphovascular function of the marrow represents an ideal location to detect transient cancer cells.

Various methods have been used in an attempt to detect distant metastases at the time of initial diagnosis of the primary tumor (8) but the imaging methods currently available are too insensitive to detect micrometastatic disease. Routine radiological examination of the skeleton is almost always negative in patients with operable breast cancer (9,10). Skeletal bone scanning has a very low incidence of true positive findings in patients with Stage I and II breast cancer (11-18). Conventional techniques of examining bone marrow have a very small likelihood of identifying tumor cells at the time of initial treatment (19-22).

DEVELOPMENT OF AN IMMUNOLOGIC METHOD OF BONE MARROW EVALUATION

A group at the Ludwig Institute for Cancer Research (LICR) in London examined bone marrow aspirates using polyclonal antisera prepared against human epithelial milk-fat-globule membranes. This antigen was termed the epithelial membrane antigen (EMA) (23). Using an indirect immunoperoxidase technique, it was initially shown that EMA has a widespread but highly selective distribution in human tissues. EMA staining was observed in normal breast epithelium, primary mammary carcinomas, carcinoma cells infiltrating bone marrow, xenografts of primary carcinoma in nude mice and the MCF-7 cell line (23). Subsequent more detailed studies of breast cancers showed strong staining (24), and single metastatic breast tumor cells in bone marrow showed intense staining. Bone marrow aspirates from 20 disease-free patients treated for breast cancer 3-5 years previously were negative for EMA stained cells. An additional eight patients with positive nodes were also EMA negative. Eight of 43 (18.6%) patients with metastatic disease and negative routine marrow histology had EMA positive cells in the marrow, and it was concluded that the sampling of paraffin-embedded sections used in this study may be less satisfactory than smears (25). Continued studies using aspiration smears were more satisfactory (26,27). Early data also suggested that multiple sites yielded more information than a single site of bone marrow aspiration (28).

Other investigators have confirmed the observation of immunoreactive cells, using either EMA or anti-cytokeratin (AE1) antibodies in the bone marrow of early stage breast cancer patients (29,30). A highly sensitive immunofluorescent monoclonal antibody method has been developed and used for preliminary studies (31-34). Several mouse monoclonal antibodies specific for epithelial cells have been developed in our laboratory. These antibodies (C26, T16), along with a commercially available monoclonal antibody specific for epithelial cells (anti-cytokeratin intermediate filament antibody AE1, Labsystems, Finland) react with distinct epithelial-specific antigens. All are epithelial-specific and each reacts with most breast cancers tested (35-40). These monoclonal antibodies have not been shown to react with normal marrow components.

METHODS OF DETECTION

Various immunological methods have been used to detect micrometastases of the bone marrow. Usually, these have included a concentration of the nucleated cells in the bone marrow by centrifugation in the separation medium, with the tumour cells migrating with mononuclear population. This can then be removed and resuspended and then cytospun down to prepare slides for immunological detection. Most groups have used Ficoll-Hypaque density gradient separation (25,26, 29,30,36,41,42); to obtain the mononuclear cells. Red cell lysis has been used but has been found to be less effective than density gradient separation (26,43). Cytospin preparations of the nucleated cells obtained are usually fixed with 100% ethanol or cold acetone(29) .

A study of immunochemical staining of bone marrow biopsies, negative for metastases by conventional staining techniques, concluded this technique was not useful; however, only one biopsy was taken and one monoclonal antibody (KLI) was used.(44) Bone marrow biopsy appears to be less efficient than smears because sampling errors may be introduced by examining tissue sections, rather than smears, where the entire specimen can be studied (26).

Immunochemical staining of epithelial cells is obtained using specific polyclonal or monoclonal antibodies singly or in combination, specific for epithelial-derived cell membrane antigens. A combination was suggested to enhance the likelihood of identifying heterogeneous tumor cells and to maximize identification of epithelial cells by their reactivity at two or more cellular sites. Monoclonal antibodies to cytokeratin are superior to other antibodies and there is general agreement that monoclonal antibodies against either CK-18 or optimally CK-19 should be used.

Immunofluorescence by indirect staining has the advantage of easy visual identification of cells exhibiting membrane and cytoplasmic fluorescence in a dark background by phase-contrast microscopy. However, this technique does not permit adequate morphologic examination of fluorescing cells. In addition, no permanent record, other than photo-documentation, is available. Immunocytochemical staining using the alkaline phosphatase anti-alkaline: phosphatase (APAAP) method permits morphologic evaluation of cells stained with Neofuchsin, or Fast Red as well as providing a permanent record. The use of an appropriate positive control cells (MCF-7 cells in bone marrow) is required. Sample size, as defined by the number of bone marrow cells examined, may be an important factor in determining the likelihood of detecting micrometastases. There is considerable variation between studies that have recorded the denominator of cells counted, ranging from 3xl04 to our studies of 4xl06 cells which can be screened in a twenty minute period. The minimum number of cells that should be examined is 1x10 (45).

Table I. Antibodies used for the detection of bone marrow micrometastases in breast cancer

Antibody

Type of Antibody

Target

EMA

Polyclonal

human milk fat globule membrane

HMFG-2

Polyclonal

human milk fat globule

T16

Monoclonal

membrane glycoprotein

C26

Monoclonal

membrane glycoprotein

LICR.LON.M8

Monoclonal

membrane

MBr 1

Monoclonal

membrane glycol i pi d

AE-1

Monoclonal

cy to keratin

CK2

Monoclonal

cy to keratin

35BH11

Monoclonal

cytokeratin

34BE12

Monoclonal

cytokeratin

CAM 5.2

Monoclonal

cytokeratin

DF3

Monoclonal

cytokeratin

PKK 1

Monoclonal

cytokeratin

KLI

Monoclonal

cytokcratin

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