title: Gene expression profiling of circulating tumor cells in breast cancer.
abstract: BACKGROUND
  Determining the transcriptional profile of circulating tumor cells (CTCs) may allow the acquisition of clinically relevant information while overcoming tumor heterogeneity-related biases associated with use of tissue samples for biomarker assessment. However, such molecular characterization is challenging because CTCs are rare and outnumbered by blood cells.
  
  
  METHODS
  Here, we describe a technical protocol to measure the expression of >29 000 genes in CTCs captured from whole blood with magnetic beads linked with antibodies against epithelial cell adhesion molecule (EpCAM) and the carcinoma-associated mucin, MUC1, designed to be used for CTC characterization in clinical samples. Low numbers of cells (5-200) from the MCF7 and MDA-MB-468 breast cancer cell lines were spiked in healthy donor blood samples and isolated with the AdnaTest EMT-1/Stem CellSelect kit. Gene expression profiles (GEPs) were obtained with the WG-DASL HT assay and compared with GEPs obtained from RNA isolated from cultured cell lines and unspiked samples.
  
  
  RESULTS
  GEPs from samples containing 25 or more spiked cells correlated (r = 0.95) with cognate 100-ng RNA input samples, clustered separately from blood control samples, and allowed MCF7 and MDA-MB-468 cells to be distinguished. GEPs with comparable technical quality were also obtained in a preliminary series of clinical samples.
  
  
  CONCLUSIONS
  Our approach allows technically reliable GEPs to be obtained from isolated CTCs for the acquisition of biologically useful information. It is reproducible and suitable for application in prospective studies to assess the clinical utility of CTC GEPs, provided that >25 CTCs can be isolated.
authors.affiliation: Biomarkers Unit, Department of Experimental Oncology and Molecular Medicine.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Fina
authors.affiliation: Biomarkers Unit, Department of Experimental Oncology and Molecular Medicine.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Callari
authors.affiliation: Biomarkers Unit, Department of Experimental Oncology and Molecular Medicine.
authors.forename: Carolina
authors.initials: C
authors.lastname: Reduzzi
authors.affiliation: Biomarkers Unit, Department of Experimental Oncology and Molecular Medicine.
authors.forename: Francesca
authors.initials: F
authors.lastname: D'Aiuto
authors.affiliation: Medical Oncology Unit, and.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Mariani
authors.affiliation: U.O. Multidisciplinare di Patologia Mammaria, U.S. Terapia Molecolare e Farmacogenomica, A.O. Istituti Ospitalieri di Cremona, Cremona, Italy.
authors.forename: Daniele
authors.initials: D
authors.lastname: Generali
authors.affiliation: Scientific Directorate, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;
authors.forename: Marco A
authors.initials: MA
authors.lastname: Pierotti
authors.affiliation: Biomarkers Unit, Department of Experimental Oncology and Molecular Medicine, mariagrazia.daidone@istitutotumori.mi.it.
authors.forename: Maria G
authors.initials: MG
authors.lastname: Daidone
authors.affiliation: Biomarkers Unit, Department of Experimental Oncology and Molecular Medicine.
authors.forename: Vera
authors.initials: V
authors.lastname: Cappelletti
mesh_terms: D001943:Breast Neoplasms / Q000097:blood / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D018929:Cell Culture Techniques; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D063990:Gene Ontology; D055106:Genome-Wide Association Study; D006801:Humans; D061986:MCF-7 Cells; D009360:Neoplastic Cells, Circulating / Q000378:metabolism* / Q000473:pathology; D059467:Transcriptome / Q000235:genetics*