Phenotypic and Genetic Characterization of Circulating Tumor Cells by Combining Immunomagnetic Selection and FICTION Techniques
María Campos 1, Celia Prior 1, Fernando Warleta 1, Isabel Zudaire 1, Jesús Ruíz-Mora 1, Raúl Catena 1, Alfonso Calvo 1 and José J. Gaforio 1*
1 Immunology Division, Department of Health Sciences, Faculty of Experimental Sciences, Campus Las Lagunillas, University of Jaén, Jaén, Spain (MC,FW,JR-M,JJG); Division of Oncology, Center for Applied Medical Research (CIMA), Pamplona, Spain (CP,IZ,RC,AC); and Department of Genetics (IZ) and Department of Histology and Pathology (RC,AC), University of Navarra, Pamplona, Spain
* To whom correspondence should be addressed. E-mail: jgaforio{at}ujaen.es .
Submitted on February 22, 2008
Accepted on 27 March 2008
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Abstract |
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The presence of circulating tumor cells (CTCs) in breast cancer patients has been proven to have clinical relevance. Cytogenetic characterization of these cells could have crucial relevance for targeted cancer therapies. We have developed a method that combines an immunomagnetic selection of CTCs from peripheral blood with FICTION technique (Fluorescence immunophenotyping and Interphase Cytogenetics as a Tool for Investigation of Neoplasm). Briefly, peripheral blood (10ml) from healthy donors was spiked with a predetermined number of human breast cancer cells. Nucleated cells were separated by double density gradient centrifugation of blood samples. Tumor cells (TCs) were immunomagnetically isolated with an anti-cytokeratin antibody, and then placed onto slides for FICTION analysis. For immunophenotyping and genetic characterization of TCs, a mixture of primary monoclonal anti-pancytokeratin antibodies was used, followed by fluorescent secondary antibodies, and finally hybridized with a TOP2A/HER-2/CEP17 multi-color probe. Our results demonstrate that TCs can be efficiently isolated from peripheral blood, and characterized by FICTION. Since genetic amplification of TOP2A and ErbB2 (HER-2) in breast cancer correlates with response to anthracyclins and herceptin therapies, respectively, this novel methodology could be useful for a better classification of the patients according to the genetic alterations of CTCs, and for the application of targeted therapies.
Key Words:
breast cancer, circulating tumor cells, cytokeratin expression, FICTION, ERBB2 (HER-2/neu) gene, immunomagnetic selection, TOP2A gene
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