Real-Time Reverse Transcription-PCR Assay for Future Management of ERBB2-based Clinical Applications

来自 Semantic Scholar

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100

作者：

B Ivan，O Peter，L Ingrid，O Martine，V Dominique，L Rosette，V Michel

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摘要：

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 TagMan 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 ([r.sup.2] [greater than or equal to] 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.

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关键词：

Immunohistochemistry (Health aspects Purines (Health aspects Tumor proteins (Development and progression Tumor proteins (Health aspects Detectors (Health aspects Oncology, Experimental (Development and progression Oncology, Experimental (Health aspects DNA synthesis (Health aspects Gene expression (Health aspects Ribonuclease (Health aspects