Phenotypic and genetic characterization of circulating tumor cells by combining immunomagnetic selection and FICTION techniques.
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 developed a method that combines an immunomagnetic selection of CTCs from peripheral blood with the fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasm (FICTION) technique. Briefly, peripheral blood (10 ml) 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 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 multicolor probe. Our results show that TCs can be efficiently isolated from peripheral blood and characterized by FICTION. Because genetic amplification of TOP2A and ErbB2 (HER-2) in breast cancer correlates with response to anthracyclines and herceptin therapies, respectively, this novel methodology could be useful for a better classification of patients according to the genetic alterations of CTCs and for the application of targeted therapies.
Campos
María
M
Immunology Division, Department of Health Sciences, Faculty of Experimental Sciences, Campus las Lagunillas, University of Jaén, 23071 Jaén, Spain.
Prior
Celia
C
Warleta
Fernando
F
Zudaire
Isabel
I
Ruíz-Mora
Jesús
J
Catena
Raúl
R
Calvo
Alfonso
A
Gaforio
José J
JJ
D000951:Antigens, Neoplasm / Q000235:genetics; D045744:Cell Line, Tumor; D002886:Chromosomes, Human, Pair 17 / Q000235:genetics; D020732:Cytogenetic Analysis; D004250:DNA Topoisomerases, Type II / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D005240:Feasibility Studies; D018628:Gene Dosage; D006801:Humans; D018189:Immunomagnetic Separation; D016130:Immunophenotyping / Q000379:methods; D009360:Neoplastic Cells, Circulating / Q000473:pathology*; D000075223:Poly-ADP-Ribose Binding Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics
D000951:Antigens, Neoplasm; D004250:DNA Topoisomerases, Type II; D004268:DNA-Binding Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; C000618811:TOP2A protein, human