title: Circulating tumour cells escape from EpCAM-based detection due to epithelial-to-mesenchymal transition.
abstract: BACKGROUND
  Circulating tumour cells (CTCs) have shown prognostic relevance in metastatic breast, prostate, colon and pancreatic cancer. For further development of CTCs as a biomarker, we compared the performance of different protocols for CTC detection in murine breast cancer xenograft models (MDA-MB-231, MDA-MB-468 and KPL-4). Blood samples were taken from tumour bearing animals (20 to 200 mm2) and analysed for CTCs using 1. an epithelial marker based enrichment method (AdnaTest), 2. an antibody independent technique, targeting human gene transcripts (qualitative PCR), and 3. an antibody-independent approach, targeting human DNA-sequences (quantitative PCR). Further, gene expression changes associated with epithelial-to-mesenchymal transition (EMT) were determined with an EMT-specific PCR assay.
  
  
  METHODS
  We used the commercially available Adna Test, RT-PCR on human housekeeping genes and a PCR on AluJ sequences to detect CTCs in xenografts models. Phenotypic changes in CTCs were tested with the commercially available "Human Epithelial to Mesenchymal Transition RT-Profiler PCR Array".
  
  
  RESULTS
  Although the AdnaTest detects as few as 1 tumour cell in 1 ml of mouse blood spiking experiments, no CTCs were detectable with this approach in vivo despite visible metastasis formation. The presence of CTCs could, however, be demonstrated by PCR targeting human transcripts or DNA-sequences - without epithelial pre-enrichment. The failure of CTC detection by the AdnaTest resulted from downregulation of EpCAM, whereas mesenchymal markers like Twist and EGFR were upregulated on CTCs. Such a change in the expression profile during metastatic spread of tumour cells has already been reported and was linked to a biological program termed epithelial-mesenchymal transition (EMT).
  
  
  CONCLUSIONS
  The use of EpCAM-based enrichment techniques leads to the failure to detect CTC populations that have undergone EMT. Our findings may explain clinical results where low CTC numbers have been reported even in patients with late metastatic cancers. These results are a starting point for the identification of new markers for detection or capture of CTCs, including the mesenchymal-like subpopulations.
authors.affiliation: Bayer Pharma AG, Muellerstr, 178, Berlin 13353, Germany.
authors.forename: Tobias M
authors.initials: TM
authors.lastname: Gorges
authors.forename: Ingeborg
authors.initials: I
authors.lastname: Tinhofer
authors.forename: Michael
authors.initials: M
authors.lastname: Drosch
authors.forename: Lars
authors.initials: L
authors.lastname: Röse
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Zollner
authors.forename: Thomas
authors.initials: T
authors.lastname: Krahn
authors.forename: Oliver
authors.initials: O
authors.lastname: von Ahsen
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000032:analysis / Q000097:blood*; D014408:Biomarkers, Tumor / Q000032:analysis / Q000097:blood*; D001943:Breast Neoplasms / Q000097:blood* / Q000473:pathology*; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000097:blood*; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods; D000071858:Epithelial Cell Adhesion Molecule; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009362:Neoplasm Metastasis; D009360:Neoplastic Cells, Circulating / Q000737:chemistry / Q000378:metabolism* / Q000473:pathology*; D014183:Transplantation, Heterologous
chemical_list: D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule