title: Heterogeneous detection of circulating tumor cells in patients with colorectal cancer by immunomagnetic enrichment using different EpCAM-specific antibodies.
abstract: BACKGROUND
  Circulating tumor cells (CTC) and disseminated tumor cells (DTC) are thought to be responsible for metastasis, so the detection of CTC may serve as individual prognostic factor in patients suffering from colorectal cancer. Therefore, a series of immunomagnetic enrichment methods for CTC have been developed using a variety of monoclonal antibodies against the Epithelial Cell Adhesion Molecule (EpCAM). However, it remains unclear whether all commercially available EpCAM antibodies show the same sensitivity and specificity. Furthermore, it remains unclear which method of sample preparation and cell extraction is most suitable for immunomagnetic enrichment and detection of CTC. In this study, we aimed to investigate whether the detection of CTC by a cytokeratin 20 reverse transcriptase-polymerase chain reaction (CK20 RT-PCR) may be influenced by the use of various Epithelial Cell Adhesion Molecule (EpCAM) antibodies for immunomagnetic isolation of CTC.
  
  
  RESULTS
  Using both EpCAM antibodies (mAb BerEP4 and mAb KS1/4) for immunomagnetic enrichment in blood samples of 39 patients with colorectal cancer we found heterogenous results in each patient with regard to tumor cell detection. In the tumor cell spiking experiments with whole blood samples the sensitivity of the CK 20 RT-PCR assay was higher using immunomagnetic beads coated with mAb KS1/4 compared to precoated mAb BerEP4 Dynabeads. Extraction of MNC fraction with Ficoll gradient centrifugation prior to immunomagnetic enrichment resulted in a higher sensitivity of the CK 20 RT-PCR assay.
  
  
  CONCLUSIONS
  We concluded that isolation and detection of CTC with immunomagnetic enrichment methods is critically dependent on the used EpCAM clone. Further studies with a larger number of patients should clarify if the enrichment protocol influences the prognostic value of the tumor cell detection protocol.
authors.affiliation: Department of Surgery, University of Heidelberg, Heidelberg, Germany.
authors.forename: Dalibor
authors.initials: D
authors.lastname: Antolovic
authors.forename: Luis
authors.initials: L
authors.lastname: Galindo
authors.forename: Anina
authors.initials: A
authors.lastname: Carstens
authors.forename: Nuh
authors.initials: N
authors.lastname: Rahbari
authors.forename: Markus W
authors.initials: MW
authors.lastname: Büchler
authors.forename: Jürgen
authors.initials: J
authors.lastname: Weitz
authors.forename: Moritz
authors.initials: M
authors.lastname: Koch
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000912:Antibodies, Neoplasm / Q000737:chemistry*; D000951:Antigens, Neoplasm / Q000302:isolation & purification*; D015815:Cell Adhesion Molecules / Q000302:isolation & purification*; D015179:Colorectal Neoplasms / Q000097:blood* / Q000175:diagnosis; D000071858:Epithelial Cell Adhesion Molecule; D005260:Female; D019073:HT29 Cells; D006801:Humans; D018189:Immunomagnetic Separation; D008297:Male; D008875:Middle Aged; D009360:Neoplastic Cells, Circulating; D012680:Sensitivity and Specificity
chemical_list: D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule