title: Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples.
abstract: Determination of HER-2/neu oncogene amplification has become necessary for selection of breast cancer patients for trastuzumab (Herceptin) therapy. Fluorescence in situ hybridization (FISH) is currently regarded as a gold standard method for detecting HER-2/neu amplification, but it is not very practical for routine histopathological laboratories. We evaluated a new modification of in situ hybridization, the chromogenic in situ hybridization (CISH), which enables detection of HER-2/neu gene copies with conventional peroxidase reaction. Archival formalin-fixed paraffin-embedded tumor tissue sections were pretreated (by heating in a microwave oven and using enzyme digestion) and hybridized with a digoxigenin-labeled DNA probe. The probe was detected with anti-digoxigenin fluorescein, anti-fluorescein peroxidase, and diaminobenzidine. Gene copies visualized by CISH could be easily distinguished with a x40 objective in hematoxylin-stained tissue sections. HER-2/neu amplification typically appeared as large peroxidase-positive intranuclear gene copy clusters. CISH and FISH (according to Vysis, made from frozen pulverized tumor samples) correlated well in a series of 157 breast cancers (kappa coefficient, 0.81). The few different classifications were mostly because of low-level amplifications by FISH that were negative by CISH and immunohistochemistry with monoclonal antibody CB-11. We conclude that CISH, using conventional bright-field microscopy in evaluation, is a useful alternative for determination of HER-2/neu amplification in paraffin-embedded tumor samples, especially for confirming the immunohistochemical staining results.
authors.affiliation: Laboratory of Cancer Genetics, Institute of Medical Technology University and University Hospital of Tampere, Tampere, Finland.
authors.forename: M
authors.initials: M
authors.lastname: Tanner
authors.forename: D
authors.initials: D
authors.lastname: Gancberg
authors.forename: A
authors.initials: A
authors.lastname: Di Leo
authors.forename: D
authors.initials: D
authors.lastname: Larsimont
authors.forename: G
authors.initials: G
authors.lastname: Rouas
authors.forename: M J
authors.initials: MJ
authors.lastname: Piccart
authors.forename: J
authors.initials: J
authors.lastname: Isola
mesh_terms: D001109:Archives; D001943:Breast Neoplasms / Q000235:genetics*; D002863:Chromogenic Compounds; D005260:Female; D005784:Gene Amplification; D018734:Genes, erbB-2 / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry / Q000592:standards; D017403:In Situ Hybridization / Q000379:methods* / Q000592:standards; D017404:In Situ Hybridization, Fluorescence / Q000592:standards
chemical_list: D002863:Chromogenic Compounds