Medical Innovation, Ventana Medical Systems, Inc, Tucson, AZ, USA.
Diagn Pathol. 2012 May 30;7:60. doi: 10.1186/1746-1596-7-60.
The eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2)-directed therapies is determined by the HER2 gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by in situ hybridization (ISH) or immunohistochemistry (IHC), respectively. Our objective was to combine the US Food and Drug Administration (FDA)-approved HER2 & chromosome 17 centromere (CEN17) brightfield ISH (BISH) and HER2 IHC assays into a single automated HER2 gene-protein assay allowing simultaneous detection of all three targets in a single tissue section.
The HER2 gene-protein assay was optimized using formalin-fixed, paraffin-embedded (FFPE) samples of the xenograft tumors MCF7 [HER2 negative (non-amplified gene, protein negative)] and Calu-3 [HER2 positive (amplified gene, protein positive)]. HER2 IHC was performed using a rabbit monoclonal anti-HER2 antibody (clone 4B5) and a conventional 3,3'-diaminobenzidine IHC detection. The HER2 & CEN17 BISH signals were visualized using horseradish peroxidase-based silver and alkaline phosphatase-based red detection systems, respectively with a cocktail of 2,4-dinitrophenyl-labeled HER2 and digoxigenin-labeled CEN17 probes. The performance of the gene-protein assay on tissue microarray slides containing 189 randomly selected FFPE clinical breast cancer tissue cores was compared to that of the separate HER2 IHC and HER2 & CEN17 BISH assays.
HER2 protein detection was optimal when the HER2 IHC protocol was used before (rather than after) the BISH protocol. The sequential use of HER2 IHC and HER2 & CEN17 BISH detection steps on FFPE xenograft tumor sections appropriately co-localized the HER2 protein, HER2 gene, and CEN17 signals after mitigating the silver background staining by using a naphthol phosphate-containing hybridization buffer for the hybridization step. The HER2 protein and HER2 gene status obtained using the multiplex HER2 gene-protein assay demonstrated high concordance with those obtained using the separate HER2 IHC and HER2 & CEN17 BISH assays, respectively.
We have developed a protocol that allows simultaneous visualization of the HER2 IHC and HER2 & CEN17 BISH targets. This automated protocol facilitated the determination of HER2 protein and HER2 gene status in randomly selected breast cancer samples, particularly in cases that were equivocal or exhibited tumor heterogeneity. The HER2 gene-protein assay produced results virtually equivalent to those of the single FDA-approved HER2 IHC and HER2 & CEN17 BISH assays.
The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2041964038705297.
乳腺癌患者是否有资格接受人表皮生长因子受体 2（HER2）靶向治疗，取决于乳腺肿瘤中 HER2 基因扩增和/或 HER2 蛋白过表达的情况，这分别通过原位杂交（ISH）或免疫组织化学（IHC）来确定。我们的目的是将美国食品和药物管理局（FDA）批准的 HER2 和染色体 17 着丝粒（CEN17）荧光原位杂交（BISH）和 HER2 IHC 检测方法结合到一个单一的自动化 HER2 基因-蛋白检测方法中，允许在一个组织切片中同时检测所有三个靶点。
使用异种移植肿瘤 MCF7（HER2 阴性（基因未扩增，蛋白阴性））和 Calu-3（HER2 阳性（基因扩增，蛋白阳性））的福尔马林固定、石蜡包埋（FFPE）样本对 HER2 基因-蛋白检测方法进行了优化。HER2 IHC 使用兔单克隆抗 HER2 抗体（克隆 4B5）和传统的 3,3'-二氨基联苯胺 IHC 检测方法进行。HER2 和 CEN17 BISH 信号分别使用基于辣根过氧化物酶的银和基于碱性磷酸酶的红色检测系统进行可视化，HER2 和 CEN17 探针的混合物为 2,4-二硝基苯标记的 HER2 和 digoxigenin 标记的 CEN17。在包含 189 个随机选择的 FFPE 临床乳腺癌组织芯的组织微阵列载玻片上，比较了基因-蛋白检测方法与单独的 HER2 IHC 和 HER2 和 CEN17 BISH 检测方法的性能。
当在 BISH 方案之前（而不是之后）使用 HER2 IHC 方案时，HER2 蛋白检测效果最佳。在对 FFPE 异种移植肿瘤切片进行顺序的 HER2 IHC 和 HER2 和 CEN17 BISH 检测步骤后，通过使用含有萘酚磷酸盐的杂交缓冲液进行杂交步骤，可以适当共定位 HER2 蛋白、HER2 基因和 CEN17 信号，从而减轻了银背景染色。使用多聚体 HER2 基因-蛋白检测方法获得的 HER2 蛋白和 HER2 基因状态与单独使用 HER2 IHC 和 HER2 和 CEN17 BISH 检测方法获得的状态高度一致。
我们开发了一种允许同时可视化 HER2 IHC 和 HER2 和 CEN17 BISH 靶点的方案。这种自动化方案促进了随机选择的乳腺癌样本中 HER2 蛋白和 HER2 基因状态的确定，特别是在存在模棱两可或表现出肿瘤异质性的情况下。HER2 基因-蛋白检测方法产生的结果与单个 FDA 批准的 HER2 IHC 和 HER2 和 CEN17 BISH 检测方法的结果几乎相同。
本文的虚拟幻灯片可在此处找到：http://www.diagnosticpathology.diagnomx.eu/vs/2041964038705297。
