Affiliation:
1. Laboratoire de Génétique Moléculaire, Faculté des Sciences Pharmaceutiques et Biologiques de Paris, 4 Avenue de l’Observatoire, F-75006 Paris, France
2. Laboratoire d’Oncogénétique, Centre René Huguenin, F-92211 St-Cloud, France
Abstract
Abstract
Background: Gene amplification/overexpression of ERBB2 (HER2, neu) is a major event in human breast tumorigenesis. ERBB2-based therapeutic agents and ERBB2-specific gene therapy are under development. These new perspectives call for a sensitive and accurate method to screen breast cancer patients for ERBB2 alterations.
Methods: We have developed and validated a real-time quantitative reverse transcription (RT)-PCR assay, based on fluorescent TaqMan methodology, to quantify ERBB2 gene expression at the mRNA level in breast tumors. This recently developed method of nucleic acid quantification in homogeneous solutions has the potential for a wide dynamic range, interlaboratory agreement, and high-throughput capacity without tedious post-PCR processing. The ERBB2 mRNA signal was normalized to the signal for TATA box-binding protein mRNA.
Results: The dynamic range was >1000-fold. The relationship between Ct and log starting concentration was linear (r2 ≥0.99). The mean (SD) normalized expression of ERBB2 in healthy breast tissue was 0.95 (0.37). Overexpression (>5 SD above mean for healthy breast) of the ERBB2 gene was observed (at 3.2- to 135-fold) in 23 (17%) of 134 breast tumor RNA samples. As expected, ERBB2 overexpression was present in all tumors with ERBB2 gene amplification but was uncommon and at a low ratio (<5) in breast cancers without gene amplification.
Conclusions: This new simple, rapid, semi-automated assay is a major alternative to fluorescence in situ hybridization and immunochemistry for gene alteration analysis in human tumors and may be a powerful tool for large randomized, prospective cooperative group trials and to support future ERBB2-based biological and gene therapy approaches.
Publisher
Oxford University Press (OUP)
Subject
Biochemistry (medical),Clinical Biochemistry
Cited by
155 articles.
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