Phenotypic and genetic characterization of circulating tumor cells by combining immunomagnetic selection and FICTION techniques

Abstract

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.

Figure 1

Peripheral blood from healthy volunteers spiked with different tumor cell lines. Mononuclear and granulocyte cell fractions were processed for positive immunomagnetic tumor cells separation and labeled using immunocytochemistry (A–C) or immunofluorescence (D–F). Tumor cells [MCF-7 (A,D), MDA-MB-231 (B,E), and SK-Br-3 (C,F)] show a solid cytoplasmic cytokeratin (CK) staining pattern, whereas the surrounding hematopoietic cells do not. Bar = 75 μm.

Figure 2

Peripheral blood from healthy volunteers spiked with different tumor cell lines, processed by immunomagnetic enrichment and fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasm (FICTION) techniques. Leukocytes are negative for the CK labeling and were used as positive control for the fluorescence in situ hybridization technique. Tumor cells [MCF-7 (A,D), MDA-MB-231 (B,E), and SK-Br-3 (C,F)] are clearly identified and show hybridization signals for ERBB2 (HER-2/neu) (green), TOP2A (orange), and CEP17 [aqua (light blue)]. (D) MCF-7 cells show three copies of chromosome 17 and two copies of the HER-2 and TOP2A (mean: two copies of both genes/cell). (E) The general pattern found for MDA-MB-231 cells is the presence of three signals for CEP17, HER-2, and TOP2A. (F) SK-Br-3 cells show a high level of HER-2 amplification and a low level of TOP2A amplification. Observe that several signals are also seen for CEP17. Notice that not all signals from genes are in focus at the same time. Bar = 75 μm.