Gjerdrum Lise Mette, Sorensen Boe Sandahl, Kjeldsen Eigil, Sorensen Flemming Brandt, Nexo Ebba, Hamilton-Dutoit Stephen
Institutes of Pathology, Aarhus University Hospital, Aarhus, Denmark.
J Mol Diagn. 2004 Feb;6(1):42-51. doi: 10.1016/S1525-1578(10)60490-4.
We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods. Study cases (27 carcinomas and 3 ductal breast carcinoma in situ (DCIS) cases) showed varying Her-2 expression as determined by IHC (HercepTest). In carcinomas, there was a good correlation between HER-2 DNA amplification and strong HER-2 protein expression detected by FISH and IHC, respectively. A single DCIS case was amplified in FISH, but not in IHC. Both HER-2 gene amplification and expression could be quantified in microdissected paraffin-embedded tumors using real-time PCR, DNA and RNA being successfully detected in 146 of 150 (97%) and 141 of 150 (94%) samples, respectively. PCR analysis for HER-2 DNA amplification using the LightCycler HER2/neu DNA Quantification kit (Roche Molecular Biochemicals, Mannheim, Germany) correlated fairly well with IHC and FISH. All IHC HER-2 3+ tumors were amplified according to the kit, as was the FISH-amplified DCIS case. DNA-PCR identified five additional tumors as being amplified. Interestingly, all these scored 2+ with the HercepTest, but were negative using FISH. We believe that real-time quantitative PCR analysis of HER-2 DNA amplification following microdissection represents a useful supplementary or perhaps even an alternative technique for establishing HER-2 status in paraffin-embedded tumors.
我们研究了使用实时定量聚合酶链反应（PCR）来测定显微切割的福尔马林固定、石蜡包埋乳腺肿瘤中HER-2 DNA扩增及mRNA表达的可行性，并将其与标准免疫组织化学（IHC）和荧光原位杂交（FISH）方法进行比较。研究病例（27例癌和3例导管原位癌（DCIS））经IHC（HercepTest）检测显示出不同的Her-2表达。在癌组织中，HER-2 DNA扩增与分别通过FISH和IHC检测到的强HER-2蛋白表达之间存在良好的相关性。有1例DCIS病例在FISH检测中呈扩增状态，但在IHC检测中未扩增。使用实时PCR可对显微切割的石蜡包埋肿瘤中的HER-2基因扩增和表达进行定量，在150个样本中分别成功检测到DNA的有146个（97%），检测到RNA的有141个（94%）。使用LightCycler HER2/neu DNA定量试剂盒（德国曼海姆罗氏分子生化公司）对HER-2 DNA扩增进行的PCR分析与IHC和FISH的相关性相当好。根据该试剂盒，所有IHC检测为HER-2 3+的肿瘤均呈扩增状态，FISH检测呈扩增状态的DCIS病例也是如此。DNA-PCR还额外鉴定出5个扩增的肿瘤。有趣的是，所有这些肿瘤在HercepTest检测中评分为2+，但FISH检测为阴性。我们认为，显微切割后对HER-2 DNA扩增进行实时定量PCR分析是一种有用的补充技术，甚至可能是用于确定石蜡包埋肿瘤中HER-2状态的替代技术。
