Research paperBi-specific immunomagnetic enrichment of micrometastatic tumour cell clusters from bone marrow of cancer patients☆
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.
Section snippets
Patients and BM samples
The tumour stages of the cancer patients are presented in Table 1, Table 2. Only 2 cancer patients already had overt distant metastases at primary diagnosis (stage M1); the remaining 41 patients had no clinical signs of overt metastases (stage M0). The initial diagnosis of one patient in the breast cancer group was not confirmed by histopathological analysis and turned out to be a cystic mastopathy, a benign disease of the mammary gland. We excluded this patient from our study. In 3 cases BM
Results
To establish the magnetic enrichment protocol, we carried out a model experiment where breast cancer cell lines were used for the enrichment procedure as the majority of the probes were sampled from breast cancer patients. As demonstrated in Fig. 1 the immunomagnetic beads specifically bound to the outer surface of the cells without causing clustering of cells. We analysed BM samples from 43 patients with various types of primary carcinomas as well as 5 non-tumour control patients. The negative
Discussion
The molecular characterisation of disseminated epithelial tumour cells in cancer patients has been hampered by their extremely low concentration and the rather low yield of common enrichment methods such as Ficoll-based gradient centrifugation (Pantel et al., 1993, Pantel et al., 1999, Braun et al., 1999). Although new enrichment techniques based on positive selection with a specific antibody (e.g. anti-EpCAM)(Racila et al., 1998, Kielhorn et al., 2002) have been developed over recent years,
Acknowledgement
We thank Dr. W. Janni from the Frauenklinik, Ludwig-Maximilians-Universität-München, München, Germany; Dr. A. Dörner from the Krankenhaus Alten Eichen, Hamburg, Germany, and Dr. Ch. Jakisch from the Universitätsfrauenklinik Münster, Münster, Germany for providing additional BM samples. Furthermore, we thank Kathrin Baack and Antje Andreas for technical help.
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Supported by the Deutsche Forschungsgemeinschaft (grant Pa-341-12-1).