title: A simple multicolor flow cytometry protocol for detection and molecular characterization of circulating tumor cells in epithelial cancers.
abstract: Circulating tumor cells (CTCs) might not only serve as prognostic marker but could also be useful for monitoring treatment efficacy. A multicolor flow cytometry protocol for their detection and molecular characterization in peripheral blood was developed which consisted of erythrocyte lysis followed by staining of cells with fluorochrome-labeled antibodies against CD45 and the epithelial markers EpCam and cytokeratin 7/8. For reducing the number of events acquired by flow cytometry, an electronic threshold for the fluorescent signals from the epithelial markers was applied. After establishment of the protocol by using spiking experiments, its suitability to determine the absolute number of CTCs as well as their expression of epidermal growth factor receptor (EGFR) and its phosphorylated form (phospho-EGFR) in blood samples from patients with squamous cell carcinoma of the head and neck (SCCHN) was validated. Spiking experiments demonstrated an excellent recovery (mean 85%) and a linear performance (R(2) = 0.98) of the protocol. Sensitivity and specificity were comparable to our former protocol using immunomagnetic CTC pre-enrichment. The analysis of 33 SCCHN patient samples revealed the presence of CTCs in 33.3% of cases with a mean ± SD of 1.5 ± 0.5 CTCs per 3.75 ml blood. EGFR was expressed in 100% and phospho-EGFR in 36.4% of the CTC+ cases. We have established a simple and sensitive multicolor flow cytometry protocol for detection of CTCs in patients with epithelial cancers including SCCHN which will allow their detailed molecular characterization.
authors.affiliation: Translational Radiooncology Laboratory, Department of Radiotherapy, Charité Campus Mitte, Berlin, Germany.
authors.forename: Tsvetana
authors.initials: T
authors.lastname: Hristozova
authors.forename: Robert
authors.initials: R
authors.lastname: Konschak
authors.forename: Volker
authors.initials: V
authors.lastname: Budach
authors.forename: Ingeborg
authors.initials: I
authors.lastname: Tinhofer
mesh_terms: D000906:Antibodies / Q000276:immunology; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology; D014408:Biomarkers, Tumor / Q000235:genetics / Q000276:immunology; D002294:Carcinoma, Squamous Cell / Q000175:diagnosis* / Q000276:immunology / Q000473:pathology; D016022:Case-Control Studies; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000276:immunology; D045744:Cell Line, Tumor; D000071858:Epithelial Cell Adhesion Molecule; D066246:ErbB Receptors / Q000235:genetics / Q000276:immunology; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D015870:Gene Expression; D006258:Head and Neck Neoplasms / Q000175:diagnosis* / Q000276:immunology / Q000473:pathology; D006801:Humans; D053552:Keratin-7 / Q000235:genetics / Q000276:immunology; D053533:Keratin-8 / Q000235:genetics / Q000276:immunology; D017493:Leukocyte Common Antigens / Q000235:genetics / Q000276:immunology; D008297:Male; D009360:Neoplastic Cells, Circulating / Q000276:immunology / Q000473:pathology*; D010766:Phosphorylation; D012680:Sensitivity and Specificity; D000077195:Squamous Cell Carcinoma of Head and Neck
chemical_list: D000906:Antibodies; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D005456:Fluorescent Dyes; D053552:Keratin-7; D053533:Keratin-8; D066246:ErbB Receptors; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human