Millson Alison, Suli Arminda, Hartung Leah, Kunitake Steve, Bennett Ann, Nordberg Mary C Lowry, Hanna Wedad, Wittwer Carl T, Seth Arun, Lyon Elaine
Associated Regional and University Pathologists Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA.
J Mol Diagn. 2003 Aug;5(3):184-90. doi: 10.1016/S1525-1578(10)60471-0.
Two quantitative polymerase chain reaction (PCR) methods for HER2/neu gene quantification were evaluated for implementation into a clinical laboratory. Assays were developed using sequence-specific hybridization probes to detect a target (HER2/neu) and a reference gene (beta-globin) simultaneously. One method utilizes real-time quantification while the second uses internal competitors and melting curves to quantify the unknown sample. These two methods were evaluated using three cell lines and 97 breast tumor samples. Two hundred ninety-four samples were subsequently evaluated using the real-time quantification and immunohistochemical (IHC) staining. Real-time PCR gave HER2/neu gene doses of 10 for SKBR3 and 2 for T47D while the competitive PCR gave doses of 11 for SKBR3 and 2.2 for T47D. Both methods produced coefficients of variation (CV) of less than 3% for within-run and less than 6% for between-run analysis. Examination of 97 breast tumors found a correlation of r = 0.974 between the two methods. IHC and PCR results agreed for 234 of the subsequent 294 samples analyzed (79% concordance). A subset of ten discrepant samples was microdissected. After microdissection all ten were positive by PCR, thus resolving the discrepancy. Real-time quantification and microdissection is useful clinically for HER2/neu quantification. Its ease of use and broad dynamic range allows screening for amplification of HER2/neu.
评估了两种用于HER2/neu基因定量的定量聚合酶链反应（PCR）方法，以应用于临床实验室。使用序列特异性杂交探针开发了检测方法，可同时检测靶标（HER2/neu）和参考基因（β-珠蛋白）。一种方法利用实时定量，而另一种方法使用内部竞争物和熔解曲线来定量未知样品。使用三种细胞系和97个乳腺肿瘤样本对这两种方法进行了评估。随后使用实时定量和免疫组织化学（IHC）染色对294个样本进行了评估。实时PCR得出SKBR3的HER2/neu基因剂量为10，T47D为2；而竞争性PCR得出SKBR3的剂量为11，T47D为2.2。两种方法批内变异系数（CV）均小于3%，批间分析变异系数小于6%。对97个乳腺肿瘤的检测发现，两种方法之间的相关性r = 0.974。在随后分析的294个样本中，IHC和PCR结果在234个样本中一致（一致性为79%）。对十个有差异的样本进行了显微切割。显微切割后，所有十个样本经PCR检测均为阳性，从而解决了差异问题。实时定量和显微切割在临床上对于HER2/neu定量很有用。其易用性和宽动态范围允许筛选HER2/neu的扩增情况。
