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

Histopathology. 2005 Apr;46(4):431-41. doi: 10.1111/j.1365-2559.2005.02112.x.


Aims: 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).

Methods and 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.

Publication types

  • Comparative Study

MeSH terms

  • Breast Neoplasms / genetics*
  • Breast Neoplasms / pathology
  • Female
  • Gene Expression Regulation, Neoplastic*
  • Humans
  • In Situ Hybridization, Fluorescence / methods
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Reagent Kits, Diagnostic / standards
  • Receptor, ErbB-2 / genetics*
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction / methods


  • RNA, Messenger
  • Reagent Kits, Diagnostic
  • Receptor, ErbB-2