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首页> 外文OA文献 >Detection of Gene Amplification in Archival Breast Cancer Specimens by Laser-Assisted Microdissection and Quantitative Real-Time Polymerase Chain Reaction
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Detection of Gene Amplification in Archival Breast Cancer Specimens by Laser-Assisted Microdissection and Quantitative Real-Time Polymerase Chain Reaction

机译：激光辅助显微切割和定量实时聚合酶链反应检测存档性乳腺癌标本中的基因扩增

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Gene amplification is one of the most important mechanisms leading to deregulated gene expression in cancer. The exact quantitative detection of this frequent genomic alteration in solid tumors is often hampered by an admixture of nonneoplastic bystander and stroma cells. To overcome this obstacle and to develop an objective quantitative method we have combined laser-assisted microdissection of tumor cells with the novel 5′-exonuclease-based real-time polymerase chain reaction (PCR) assay. The latter method enables the highly reproducible exact quantification of minute amounts of nucleic acids. As a model system amplification of c-erbB2/Her-2/neu gene and the adjacent topoisomerase IIα gene was determined in paraffin-embedded breast cancer specimens (n = 23) after immunohistochemical labeling and laser-based microdissection of tumor cells. The high sensitivity of real-time PCR enabled the reliable and objective detection of low-level amplifications in as few as 50 cells from archival tissue sections. Low-level amplifications were shown to escape from detection unless tumor cells were isolated by microdissection. In selected cases intratumor heterogeneity was demonstrated using areas of ∼50 to 100 cells. This novel approach combining immunohistochemistry, laser microdissection, and quantitative kinetic PCR allows morphology-guided studies in archival tissue specimens and will enable the exact quantification of gene copy numbers in even small and precancerous lesions.
机译：基因扩增是导致癌症中基因表达失调的最重要机制之一。实体瘤中这种常见基因组改变的精确定量检测通常受非肿瘤旁观者和基质细胞混合的阻碍。为了克服这一障碍并开发一种客观的定量方法，我们将肿瘤细胞的激光辅助显微切割技术与基于5'-核酸外切酶的实时聚合酶链反应（PCR）实时检测相结合。后一种方法可实现极少量核酸的高度可重复的精确定量。作为模型系统，在免疫组织化学标记和肿瘤细胞激光显微切割后，确定了石蜡包埋的乳腺癌标本（n = 23）中c-erbB2 / Her-2 / neu基因和邻近的拓扑异构酶IIα基因的扩增。实时PCR的高灵敏度使得能够可靠，客观地检测出档案组织切片中多达50个细胞中的低水平扩增。除非通过显微解剖分离出肿瘤细胞，否则低水平的扩增显示无法检测。在某些情况下，使用约50至100个细胞的面积证明了肿瘤内异质性。这种结合了免疫组织化学，激光显微切割和定量动力学PCR的新颖方法，可以在档案组织标本中进行形态学指导的研究，甚至可以精确定量甚至小而癌前病变中的基因拷贝数。

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