title: Detection of HER-2/neu oncogene amplification in flow cytometry-sorted breast ductal cells by competitive polymerase chain reaction.
abstract: BACKGROUND
  The amplification and/or overexpression of the HER-2/neu oncogene has been proposed as an important prognostic marker in breast cancer. However, contradictory results from various groups regarding whether there is statistical significance in HER-2 amplification or overexpression in predicting overall and disease free survival in node positive versus node negative patients exist in the literature. Current assays on quantifying the HER-2 oncogene rely on DNA extracted from homogenized breast tissue. Not only is a large amount of tissue required, but also, the DNA extract is contaminated with DNA from stromal cells and leukocytes, leading to decreased specificity and sensitivity of the HER-2 assay. Improving the specificity (DNA from breast ductal cells) and the sensitivity (competitive polymerase chain reaction [PCR]) of the HER-2 amplification detection assay will help resolve some of these controversies.
  
  
  METHODS
  Using multiparameter flow cytometry (FCM), ductal cells from breast biopsies and fine needle aspirations (FNAs) are identified and selectively sorted using anti-cytokeratin, anti-HER-2 antibody labeling and DNA staining. HER-2 amplification in these sorted cells is then quantified by competitive DNA PCR using a competitive reference standard mutant template that is susceptible to the restriction enzyme Sma-1.
  
  
  RESULTS
  Applying this strategy, SK-BR-3, an HER-2 amplified breast cancer cell line, was found to have approximately 9x baseline HER-2 oncogene copies. In addition, MCF-7, a known HER-2 nonamplified breast cancer cell line, was found to have baseline HER-2 oncogene copies. In the 10 clinical breast samples tested, 4 of the 10 breast cancers were HER-2 amplified using as few as 1000 cells. The cytokeratin positive cells of these cancers, in contrast to the cytokeratin negative cells, have detectably higher HER-2 amplification (7.2 +/- 2.8x versus 3.2 +/- 1.1x, respectively). Hence, HER-2 gene amplification would have been underestimated if unsorted cells were used because of stromal dilution. In the cytokeratin positive cells that were HER-2 oncogene amplified, corresponding HER-2 oncoprotein overexpression was detected by FCM.
  
  
  CONCLUSIONS
  Using FCM, the ductal cell subpopulation of a breast specimen can be successfully sorted from breast biopsy and FNA specimens. Moreover, by applying the technique of competitive PCR, improved specificity and sensitivity in HER-2 oncogene amplification detection is achieved. The entire procedure can be accomplished in 1 day, allowing for a cost-effective assay and rapid turnaround time.
authors.affiliation: Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, New York 14263-0001.
authors.forename: B D
authors.initials: BD
authors.lastname: Li
authors.forename: S P
authors.initials: SP
authors.lastname: Harlow
authors.forename: R M
authors.initials: RM
authors.lastname: Budnick
authors.forename: D L
authors.initials: DL
authors.lastname: Sheedy
authors.forename: C C
authors.initials: CC
authors.lastname: Stewart
mesh_terms: D001483:Base Sequence; D001706:Biopsy; D001943:Breast Neoplasms / Q000235:genetics*; D002460:Cell Line; D002469:Cell Separation; D017931:DNA Primers; D005434:Flow Cytometry; D005784:Gene Amplification; D006801:Humans; D007633:Keratins / Q000032:analysis; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000032:analysis*; D016133:Polymerase Chain Reaction; D018719:Erb-b2 Receptor Tyrosine Kinases; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D007633:Keratins; D009856:Oncogene Proteins, Viral; D018719:Erb-b2 Receptor Tyrosine Kinases