Bièche I, Onody P, Laurendeau I, Olivi M, Vidaud D, Lidereau R, Vidaud M
Laboratoire de Génétique Moléculaire, Faculté des Sciences Pharmaceutiques et Biologiques de Paris, 4 Avenue de l'Observatoire, F-75006 Paris, France.
Clin Chem. 1999 Aug;45(8 Pt 1):1148-56.
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.
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.
The dynamic range was >1000-fold. The relationship between C(t) and log starting concentration was linear (r(2) >/=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.
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.
ERBB2（HER2，neu）基因扩增/过表达是人类乳腺癌发生中的一个主要事件。基于ERBB2的治疗药物和ERBB2特异性基因治疗正在研发中。这些新观点需要一种灵敏且准确的方法来筛选乳腺癌患者的ERBB2改变情况。
我们研发并验证了一种基于荧光TaqMan方法的实时定量逆转录（RT）-PCR检测法，用于在mRNA水平定量检测乳腺肿瘤中ERBB2基因的表达。这种最近在均相溶液中开发的核酸定量方法具有宽动态范围、实验室间一致性以及高通量能力，且无需繁琐的PCR后处理。ERBB2 mRNA信号以TATA盒结合蛋白mRNA的信号进行标准化。
动态范围大于1000倍。C(t)与起始浓度对数之间的关系呈线性（r(2)≥0.99）。健康乳腺组织中ERBB2的平均（标准差）标准化表达为0.95（0.37）。在134份乳腺肿瘤RNA样本中的23份（17%）中观察到ERBB2基因过表达（高于健康乳腺平均值5个标准差以上），过表达倍数为3.2至135倍。正如预期的那样，所有ERBB2基因扩增的肿瘤中均存在ERBB2过表达，但在无基因扩增的乳腺癌中不常见且比例较低（<5）。
这种新的简单、快速、半自动检测法是用于人类肿瘤基因改变分析的荧光原位杂交和免疫化学的主要替代方法，可能是大型随机、前瞻性合作组试验以及支持未来基于ERBB2的生物学和基因治疗方法的有力工具。
