title: Low-level expression of HER2 and CK19 in normal peripheral blood mononuclear cells: relevance for detection of circulating tumor cells.
abstract: BACKGROUND
  Detection of circulating tumor cells (CTC) in the blood of cancer patients may have prognostic and predictive significance. However, background expression of 'tumor specific markers' in peripheral blood mononuclear cells (PBMC) may confound these studies. The goal of this study was to identify the origin of Cytokeratin 19 (CK19) and HER-2 signal in PBMC and suggest an approach to enhance techniques involved in detection of CTC in breast cancer patients.
  
  
  METHODS
  PBMC from healthy donors were isolated and fractionated into monocytes, lymphocytes, natural killer cells/granulocytes and epithelial populations using immunomagnetic selection and fluorescent cell-sorting for each cell type. RNA isolated from each fraction was analyzed for CK19, HER2 and Beta 2 microglobulin (B2M) using real-time qRT-PCR. Positive selection for epithelial cells and negative selection for NK/granulocytes were used in an attempt to reduce background expression of CK19 and HER2 markers.
  
  
  RESULTS
  In normal PBMC, CK19 was expressed in the lymphocyte population while HER-2 expression was highest in the NK/granulocyte population. Immunomagnetic selection for epithelial cells reduced background CK19 signal to a frequency of <5% in normal donors. Using negative selection, the majority (74-98%) of HER2 signal could be removed from PBMC. Positive selection methods are variably effective at reducing these background signals.
  
  
  CONCLUSIONS
  We present a novel method to improve the specificity of the traditional method of detecting CTC by identifying the source of the background signals and reducing them by negative immunoselection. Further studies are warranted to improve sensitivity and specificity of methods of detecting CTC will prove to be useful tools for clinicians in determining prognosis and monitoring treatment responses of breast cancer patients.
authors.affiliation: Abbott Molecular, Inc, 1300 E, Touhy Avenue, Des Plaines, IL 60018, USA. fangleiyou@yahoo.com
authors.forename: Fanglei
authors.initials: F
authors.lastname: You
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Roberts
authors.forename: S Peter
authors.initials: SP
authors.lastname: Kang
authors.forename: Raquel A
authors.initials: RA
authors.lastname: Nunes
authors.forename: Cinara
authors.initials: C
authors.lastname: Dias
authors.forename: J Dirk
authors.initials: JD
authors.lastname: Iglehart
authors.forename: Natalie A
authors.initials: NA
authors.lastname: Solomon
authors.forename: Paula N
authors.initials: PN
authors.lastname: Friedman
authors.forename: Lyndsay N
authors.initials: LN
authors.lastname: Harris
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000556:secondary; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D053539:Keratin-19 / Q000235:genetics* / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D009360:Neoplastic Cells, Circulating / Q000473:pathology*; D011379:Prognosis; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D001613:beta 2-Microglobulin / Q000235:genetics / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D053539:Keratin-19; D018719:Erb-b2 Receptor Tyrosine Kinases; D001613:beta 2-Microglobulin; C508053:ERBB2 protein, human