Neubauer A, Neubauer B, He M, Effert P, Iglehart D, Frye R A, Liu E
Department of Medicine and Genetics, Lineberger Cancer Research Center, University of North Carolina, Chapel Hill 27599-7295.
Oncogene. 1992 May;7(5):1019-25.
Oncogene amplification is found in many human tumors, and its detection may have important prognostic value. However, analysis of gene amplification may be hampered by inadequate tissue or poor DNA quality. We have previously described a polymerase chain reaction (PCR)-based procedure called differential PCR that can detect variations in gene dosage using miniscule amounts of tumor DNA [Frye, R.A., Benz, C.C. & Liu, E. (1989). Oncogene, 4, 1153-1157]. We now report the optimization of this technique for the analysis of oncogene amplification in paraffin-embedded archival tissues. We find that differential PCR is able to detect amplification of the HER2 (c-erbB-2) and the epidermal growth factor receptor (EGFR) genes and can be used to arrive at a semiquantitative estimate of gene dosage. Furthermore, our approach can determine gene amplification in samples in which the DNA is significantly degraded. Using differential PCR on paraffin-embedded tissues from cases previously investigated by standard DNA extraction and dot-blot procedures, good correlation between the two methods was found. Approaches are described to overcome technical problems posed by factors that affect the differential PCR, including the method of DNA extraction and extreme fragmentation of the DNA (less than 200 base pairs). Furthermore, the resulting analytical algorithm reported herein has proved effective in detecting oncogene amplification in archival breast cancer specimens from standard pathology laboratories. Thus, differential PCR will be particularly helpful in the analysis of tumor specimens that are archived, small in size or rare in occurrence.
癌基因扩增在许多人类肿瘤中都有发现，其检测可能具有重要的预后价值。然而，基因扩增分析可能会因组织不足或DNA质量差而受到阻碍。我们之前描述了一种基于聚合酶链反应（PCR）的方法，称为差异PCR，它可以使用微量肿瘤DNA检测基因剂量的变化[弗莱，R.A.，本兹，C.C.和刘，E.（1989年）。癌基因，4，1153 - 1157]。我们现在报告该技术在石蜡包埋存档组织中癌基因扩增分析方面的优化。我们发现差异PCR能够检测HER2（c-erbB-2）和表皮生长因子受体（EGFR）基因的扩增，并可用于得出基因剂量的半定量估计。此外，我们的方法可以在DNA显著降解的样本中确定基因扩增。对先前通过标准DNA提取和点杂交程序研究的病例的石蜡包埋组织进行差异PCR，发现两种方法之间具有良好的相关性。文中描述了克服影响差异PCR的因素所带来的技术问题的方法，包括DNA提取方法和DNA的极端片段化（小于200个碱基对）。此外，本文报道的所得分析算法已被证明在检测标准病理实验室存档的乳腺癌标本中的癌基因扩增方面有效。因此，差异PCR在分析存档、体积小或罕见肿瘤标本时将特别有用。
