title: Real-time reverse transcription-PCR and fluorescence in-situ hybridization are complementary to understand the mechanisms involved in HER-2/neu overexpression in human breast carcinomas.
abstract: OBJECTIVE
  To evaluate the HER-2/neu status at the mRNA and DNA level of breast carcinomas and to compare it with HER-2/neu receptor overexpression by immunohistochemistry (IHC).
  
  
  RESULTS
  In 32 invasive breast carcinomas, frozen tissue was available for real-time detection of HER-2/neu mRNA levels by reverse transcription-polymerase chain reaction (RT-PCR). Corresponding paraffin sections were examined by IHC and fluorescence in-situ hybridization (FISH). Thereby, different IHC and FISH procedures were compared. Using microwave epitope retrieval, all 32 cases scored 3+ on IHC, whereas only 28 out of 32 cases scored IHC 3+ using water bath epitope retrieval. All of these 28 cases showed increased levels of HER-2/neu mRNA. Dual-colour FISH analysis showed corresponding gene amplification in all 28 cases, with two cases showing a peculiar amplification pattern. In the remaining four cases, scoring IHC 2+ using water bath epitope retrieval, mRNA levels were not elevated. Three cases did not have gene amplification and one case showed low-level HER-2/neu gene amplification. All four carcinomas showed chromosome 17 polysomy.
  
  
  CONCLUSIONS
  Real-time RT-PCR is accurate in selecting breast carcinoma cases scoring 3+ by IHC with high-level gene amplification. Results obtained by dual-colour FISH suggest that mechanisms leading to HER-2/neu receptor overexpression may be different between carcinomas scoring 2+ and 3+ on IHC, with polysomy 17 found in the former and gene amplification in the latter.
authors.affiliation: Department of Pathology, University Hospital of K.U. Leuven, and Centre for Human Genetics, K.U. Leuven, Leuven, Belgium. isabelle.vandenbempt@uz.kuleuven.ac.be
authors.forename: I
authors.initials: I
authors.lastname: Vanden Bempt
authors.forename: V
authors.initials: V
authors.lastname: Vanhentenrijk
authors.forename: M
authors.initials: M
authors.lastname: Drijkoningen
authors.forename: I
authors.initials: I
authors.lastname: Wlodarska
authors.forename: P
authors.initials: P
authors.lastname: Vandenberghe
authors.forename: C
authors.initials: C
authors.lastname: De Wolf-Peeters
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011933:Reagent Kits, Diagnostic / Q000592:standards; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
chemical_list: D012333:RNA, Messenger; D011933:Reagent Kits, Diagnostic; D018719:Erb-b2 Receptor Tyrosine Kinases