Copy number analysis of c-erb-B2 (HER-2/neu) and topoisomerase IIalpha genes in breast carcinoma by quantitative real-time polymerase chain reaction using hybridization probes and fluorescence in situ hybridization.
BACKGROUND
The Topoisomerase IIalpha (TOP2A) protein is the target of the anthracycline class of chemotherapeutic agents. TOP2A is frequently coamplified with c-erb-B2 and consequently might be a prognostic and/or predictive factor for breast cancer patients when anthracycline-based chemotherapy is a consideration. A total of 20% to 35% of breast carcinomas show amplification of the erb-B2 gene, some of which also have coamplification of the TOP2A gene. Investigation of the prognostic or predictive significance of these gene amplifications requires a reliable and sensitive method for the measurement of gene copy number in clinical tumor samples.


OBJECTIVE
To assess 2 different assay methods that might allow accurate, reproducible, quantitative, and high-throughput estimation of gene copy number in fresh, frozen, or paraffin-embedded breast cancer specimens.


METHODS
We developed an assay and analyzed the gene copy numbers of the erb-B2 and TOP2A genes in 8 breast cancer cell lines, 6 fresh frozen samples, and 38 paraffin-embedded breast tumor specimens by a novel real-time polymerase chain reaction (PCR) assay using hybridization probes. The results were compared with standard fluorescence in situ hybridization.


RESULTS
We discovered a 100% concordance between assessment of gene copy number of erb-B2 and TOP2A between quantitative PCR and fluorescence in situ hybridization (FISH). Quantitative PCR also had the additional feature of uncovering an erb-B2 gene polymorphism. Finally, we observed that TOP2A amplification only occurred in conjunction with erb-B2 amplification in our paraffin-embedded cases of invasive breast carcinoma and that this event was present in 5 (42%) of 12 erb-B2 amplified cases.


CONCLUSIONS
We conclude that the potentially automatic, real-time PCR analysis using hybridization probes is an efficient method to perform copy number analysis, with results that appear identical to the FISH technique and with the benefit of identifying HER-2 polymorphisms.
Murthy
Sabita K
SK
Department of Pathology, The University of Calgary, Alberta, Canada.
Magliocco
Anthony M
AM
Demetrick
Douglas J
DJ
D000951:Antigens, Neoplasm / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D002277:Carcinoma / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D003199:Computer Systems; D015342:DNA Probes / Q000235:genetics; D004250:DNA Topoisomerases, Type II / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D005784:Gene Amplification / Q000235:genetics*; D018628:Gene Dosage; D018734:Genes, erbB-2 / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D000075223:Poly-ADP-Ribose Binding Proteins; D016133:Polymerase Chain Reaction / Q000379:methods*; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000951:Antigens, Neoplasm; D015342:DNA Probes; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D004250:DNA Topoisomerases, Type II; C000618811:TOP2A protein, human