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

Vanden Bempt, Isabelle; Vanhentenrijk, Vera; Drijkoningen, Maria; Wlodarska, Iwona; Vandenberghe, Peter; Peeters, Christiane

doi:10.1111/j.1365-2559.2005.02112.x

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

Author:

Vanden Bempt, Isabelle

Vanhentenrijk, Vera ; Drijkoningen, Maria ; Wlodarska, Iwona ; Vandenberghe, Peter ; Peeters, Christiane

Keywords:

Breast Neoplasms, Comparative Study, Female, Gene Expression Regulation, Neoplastic, Humans, In Situ Hybridization, Fluorescence, RNA, Messenger, Reagent Kits, Diagnostic, Receptor, erbB-2, Reproducibility of Results, Reverse Transcriptase Polymerase Chain Reaction, Science & Technology, Life Sciences & Biomedicine, Cell Biology, Pathology, breast carcinoma, DNA ploidy, fluorescence in-situ hybridization, HER-2/neu, immunohistochemistry, real-time RT-PCR, POLYMERASE-CHAIN-REACTION, GENE AMPLIFICATION, PROTEIN EXPRESSION, QUANTITATIVE PCR, CANCER, IMMUNOHISTOCHEMISTRY, ONCOGENE, THERAPY, HER2, ANTIBODIES, Receptor, ErbB-2, 1103 Clinical Sciences, 3202 Clinical sciences, 3211 Oncology and carcinogenesis

Abstract:

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.