title: Comparison of three molecular assays for the detection and molecular characterization of circulating tumor cells in breast cancer.
abstract: BACKGROUND
  Comparison studies between different analytical methodologies for circulating tumor cells (CTC) detection and molecular characterization are urgently needed, since standardization of assays is essential before their use in clinical practice.
  
  
  METHODS
  We compared three different CTC molecular assays. To avoid discrepancies due to pre-analytical errors we used the same cDNAs throughout our study. CTC were isolated using anti-EpCAM and anti-MUC1 coated magnetic beads from 2 × 5 ml of peripheral blood of 254 early and 51 metastatic breast cancer patients and 30 healthy individuals. The same cDNAs were analyzed by: a) singleplex RT-qPCR assay for CK-19; b) multiplex RT-qPCR for CK-19, HER-2, MAGE- A3, and PBGD; and c) a commercially available molecular assay (AdnaTest BreastCancer) for GA733-2, MUC-1, HER-2 and beta-actin.
  
  
  RESULTS
  In early breast cancer, CK-19 RT-qPCR, multiplex RT-qPCR and the AdnaTest, were positive for the presence of CTC in 14.2%, 22.8% and 16.5% subjects, respectively. The concordance between the AdnaTest and CK-19 RT-qPCR was 72.4% while between the AdnaTest and multiplex RT-qPCR was 64.6%. In patients with overt metastasis, CK-19 RT-qPCR, multiplex RT-qPCR and the AdnaTest were positive in 41.2%, 39.2% and 54.9% patients, respectively. The concordance between the AdnaTest and CK-19 RT-qPCR was 70.6% while between the AdnaTest and multiplex RT-qPCR was 68.6%.
  
  
  CONCLUSIONS
  All CTC assays gave similar results in about 70% of cases. Better agreement was found in the metastatic setting, possibly explained by the higher tumor load in this group. Discordances could be attributed to the different gene transcripts used to evaluate CTC positivity. Our results indicate the importance of CTC heterogeneity for their detection by different analytical methodologies.
authors.forename: Areti
authors.initials: A
authors.lastname: Strati
authors.forename: Sabine
authors.initials: S
authors.lastname: Kasimir-Bauer
authors.forename: Athina
authors.initials: A
authors.lastname: Markou
authors.forename: Cleo
authors.initials: C
authors.lastname: Parisi
authors.forename: Evi S
authors.initials: ES
authors.lastname: Lianidou
mesh_terms: D000951:Antigens, Neoplasm / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D014408:Biomarkers, Tumor / Q000378:metabolism*; D001943:Breast Neoplasms / Q000097:blood* / Q000378:metabolism / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000097:blood* / Q000378:metabolism / Q000556:secondary; D018275:Carcinoma, Lobular / Q000097:blood* / Q000378:metabolism / Q000556:secondary; D016022:Case-Control Studies; D015815:Cell Adhesion Molecules / Q000378:metabolism; D000071858:Epithelial Cell Adhesion Molecule; D005260:Female; D005500:Follow-Up Studies; D006801:Humans; D053539:Keratin-19 / Q000378:metabolism; D008207:Lymphatic Metastasis; D008875:Middle Aged; D018396:Mucin-1 / Q000378:metabolism; D060787:Neoplasm Grading; D009367:Neoplasm Staging; D009360:Neoplastic Cells, Circulating / Q000378:metabolism / Q000473:pathology*; D011379:Prognosis; D012333:RNA, Messenger / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
chemical_list: D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D053539:Keratin-19; D018396:Mucin-1; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; C499379:EPCAM protein, human