title: A FISH-based method for assessment ofHER-2amplification status in breast cancer circulating tumor cells following CellSearch isolation.
abstract: BACKGROUND
  Amplification of theHER-2/neu(HER-2) proto-oncogene occurs in 10%-15% of primary breast cancer, leading to an activated HER-2 receptor, augmenting growth of cancer cells. Tumor classification is determined in primary tumor tissue and metastatic biopsies. However, malignant cells tend to alter their phenotype during disease progression. Circulating tumor cell (CTC) analysis may serve as an alternative to repeated biopsies. The Food and Drug Administration-approved CellSearch system allows determination of the HER-2 protein, but not of theHER-2gene. The aim of this study was to optimize a fluorescence in situ hybridization (FISH)-based method to quantitatively determineHER-2amplification in breast cancer CTCs following CellSearch-based isolation and verify the method in patient samples.
  
  
  METHODS
  Using healthy donor blood spiked with human epidermal growth factor receptor 2 (HER-2)-positive breast cancer cell lines, SKBr-3 and BT-474, and a corresponding negative control (the HER-2-negative MCF-7 cell line), an in vitro CTC model system was designed. Following isolation in the CellSearch system, CTC samples were further enriched and fixed on microscope slides. Immunocytochemical staining with cytokeratin and 4',6-diamidino-2'-phenylindole dihydrochloride identified CTCs under a fluorescence microscope. A FISH-based procedure was optimized by applying the HER2 IQFISH pharmDx assay for assessment ofHER-2amplification status in breast cancer CTCs.
  
  
  RESULTS
  A method for defining the presence ofHER-2amplification in single breast cancer CTCs after CellSearch isolation was established using cell lines as positive and negative controls. The method was validated in blood from breast cancer patients showing that one out of six patients acquired CTCHER-2amplification during treatment against metastatic disease.
  
  
  CONCLUSIONS
  HER-2amplification status of CTCs can be determined following CellSearch isolation and further enrichment. FISH is superior to protein assessment ofHER-2status in predicting response to HER-2-targeted immunotherapy in breast cancer patients. This assay has the potential of identifying patients with a shift inHER-2status who may benefit from treatment adjustments.
authors.affiliation: Division of Oncology and Pathology.
authors.forename: Henrik
authors.initials: H
authors.lastname: Frithiof
authors.affiliation: Division of Oncology and Pathology.
authors.forename: Kristina
authors.initials: K
authors.lastname: Aaltonen
authors.affiliation: Division of Surgery, Department of Clinical Sciences Lund, Lund University, Lund; Department of Surgery, Skåne University Hospital, Malmö, Sweden.
authors.forename: Lisa
authors.initials: L
authors.lastname: Rydén
keywords: breast neoplasms; circulating; fluorescence; human epidermal growth factor receptor 2; immunomagnetic separation; in situ hybridization; neoplastic cells