Bi-specific immunomagnetic enrichment of micrometastatic tumour cell clusters from bone marrow of cancer patients

Abstract

Metastasis–the spread of tumour cells from a primary lesion to distant organs–is the main cause of cancer-related death, and bone marrow (BM) is a frequent site for the settlement of disseminated tumour cells. Many BM samples harbour isolated tumour cells, whereas tumour cell clusters, as the potential precursors of solid distant metastases, are rarely detected after current enrichment procedures. We have analysed BM samples from 43 patients with carcinomas of the breast, colon and ovaries; 41 of these patients had no clinical signs of overt metastases (stage M0). Tumour cells in BM were enriched with immunomagnetic beads coupled to monoclonal antibodies against both EpCAM and HER2/neu. After enrichment, tumour cells were identified by immunostaining with the anti-cytokeratin antibody A45-B/B3. In total, 886 CK-positive cells were detected in 16 (35%) samples after immunomagnetic enrichment as compared to 34 cells in 9 (21%) samples using Ficoll density centrifugation previously used as the standard enrichment technique. Most remarkably, clusters of 2 to 10 CK-positive cells were found in 75% of CK-positive samples enriched by immunobeads, whereas no CK-positive cell clusters were detected after Ficoll enrichment. The method described offers an excellent tool for the enrichment of micrometastatic tumour cell clusters; these clusters may represent the initial stage of development from a single disseminated tumour cell towards an overt metastasis.

Introduction

The majority of cancers in industrialised countries are solid tumours derived from epithelial tissues. The fate of patients with completely removed primary tumours largely depends on the question whether overt distant metastases will eventually arise from occult micrometastatic cells that were missed by initial tumour staging procedures. The first phase of the metastatic cascade consists of local tumour cell invasion, followed by either dissemination via the lymphatic vessels to the regional lymph nodes or tumour cell circulation in the blood and homing to secondary distant organs (Chambers et al., 2002). Among the distant organs, BM is a common homing organ for disseminated cancer cells derived from various primary sites including carcinomas of the breast (Braun et al., 2000, Wiedswang et al., 2003a, Woelfle et al., 2003), colon (Lindemann et al., 1992, Soeth et al., 1997) and ovaries (Braun et al., 2001a). Moreover, the presence of these cells predicts the occurrence of overt metastases in bone and other organs (Lindemann et al., 1992, Pantel et al., 1999, Pantel et al., 2003, Braun et al., 2000, Naume et al., 2004). BM seems therefore to be a general indicator organ to screen for the presence of blood-borne disseminated cancer cells throughout the body—even in cancers that rarely form overt metastases in bone such as colon carcinomas.

For epithelial tumours, cytokeratins have become the best marker for sensitive immunocytochemical detection of metastatic cells in BM (Pantel and Brakenhoff, 2004). The specificity of this marker has been sustained by the analysis of large cohorts of non-cancer control patients (Braun et al., 2000). A great deal of our knowledge about the clinical importance of disseminated cells in BM of cancer patients was generated by using Ficoll density gradient centrifugation for tumour cell enrichment (for review, see Pantel et al., 2003). The disadvantage of this enrichment method is the low yield of tumour cells. Most samples contain only one to three tumour cells among 1–2 million MNC (Pantel et al., 1994b, Braun et al., 2000), which is a limiting factor for a reproducible detection and in-depth characterisation of disseminated tumour cells. In particular, micrometastatic cell clusters–as the intermediate stage between single metastatic cells and solid metastases as end points of the metastatic cascade–were rarely found.

To improve the yield of tumour cell enrichment, some new approaches have been introduced over the past 10 years (Myklebust et al., 1993, Naume et al., 2001, Weihrauch et al., 2002, Wiedswang et al., 2003b). The most prominent method is based on the use of immunomagnetic beads coupled with monoclonal antibodies to tumour-associated antigens. In view of the known heterogeneity of epithelial tumour cells, we have employed, for the first time, an antibody cocktail to increase the yield of selected tumour cells. Based on our previous expression analyses on micrometastatic cancer cells of more than 70 breast cancer patients, we selected the epithelial cell adhesion molecule (EpCAM) and the oncogene HER2/neu as targets for immunobead selection (Pantel et al., 1993, Pantel et al., 1999, Braun et al., 1999), because each of these proteins is expressed on 60–70% of CK-positive cells in BM of epithelial cancer patients (Pantel and Brakenhoff, 2004). The choice of HER2/neu was further justified by the previous observation that the expression of this antigen on micrometastatic cells in the BM of breast cancer patients was associated with poor clinical outcome (Braun et al., 2001b), indicating that HER2/neu might be required for the outgrowth of single disseminated cells into overt metastases (Braun et al., 1999). The established method revealed, for the first time, a high yield of micrometastatic tumour cell clusters in CK-positive BM samples from cancer patients.