Zhu Yazhen, Lu Dan, Lira Maruja E, Xu Qing, Du Yunzhi, Xiong Jianghong, Mao Mao, Chung Hyun Cheol, Zheng Guangjuan
Guangdong Provincial Hospital of Traditional Chinese Medicine (GPHTCM), Guangzhou, China.
Translational Bioscience and Diagnostics, WuXi AppTec Co., Ltd., Waigaoqiao Free Trade Zone, Shanghai, China.
Exp Mol Pathol. 2016 Apr;100(2):287-93. doi: 10.1016/j.yexmp.2015.11.027. Epub 2015 Nov 25.
Human epidermal growth factor receptor 2 (HER2) is a key driver of tumorigenesis, and over-expression as a result of HER2 gene amplification has been observed in a number of solid tumors. Recently HER2 has become an important biomarker for the monoclonal antibody treatment of HER2-positive metastatic breast and advanced gastric cancer. The HER2 targeting antibody trastuzumab treatment requires accurate measurement of HER2 levels for proper diagnosis. Droplet digital PCR (ddPCR) with highly direct, precise and absolute nucleic acid quantification could be used to detect HER2 amplification levels.
Our objective was to evaluate a robust, accurate and less subjective application of ddPCR for HER2 amplification levels and test the assay performance in clinical formalin-fixed paraffin-embedded (FFPE) breast and gastric carcinoma samples.
Genomic DNA from HER2 amplified cell line SK-BR-3 was used to set up the ddPCR assays. The copy number of HER2 was compared to the chromosome 17 centromere reference gene (CEP17), expressed as HER2:CEP17 ratio. Genomic DNAs of FFPE specimens from 145 Asian patients with breast and gastric carcinomas were assayed using both standard methods, immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH), and ddPCR.
Based on 145 clinical breast and gastric carcinoma cases, our study demonstrated a high concordance of ddPCR results to FISH and IHC. In breast cancer specimens, the ddPCR results had high concordance with FISH and IHC defined HER2 status with a sensitivity of 90.9% (30/33) and a specificity of 100% (77/77). In gastric cancer specimens that were concordant in both FISH and IHC, our assay was 95.5% concordant with FISH and IHC (21/22).
ddPCR has the advantage of automation and also allows levels of HER2 amplification to be easily evaluated in large numbers of samples, and presents a potential option to define HER2 status.
人表皮生长因子受体2（HER2）是肿瘤发生的关键驱动因素，在许多实体瘤中均观察到由于HER2基因扩增导致的过表达。最近，HER2已成为HER2阳性转移性乳腺癌和晚期胃癌单克隆抗体治疗的重要生物标志物。HER2靶向抗体曲妥珠单抗治疗需要准确测量HER2水平以进行正确诊断。具有高度直接、精确和绝对核酸定量功能的液滴数字PCR（ddPCR）可用于检测HER2扩增水平。
我们的目的是评估ddPCR在HER2扩增水平检测中的稳健性、准确性和较少主观性的应用，并测试该检测方法在临床福尔马林固定石蜡包埋（FFPE）乳腺癌和胃癌样本中的检测性能。
使用来自HER2扩增细胞系SK-BR-3的基因组DNA建立ddPCR检测方法。将HER2的拷贝数与17号染色体着丝粒参考基因（CEP17）进行比较，以HER2:CEP17比值表示。使用标准方法免疫组织化学（IHC）和/或荧光原位杂交（FISH）以及ddPCR对145例亚洲乳腺癌和胃癌患者的FFPE标本的基因组DNA进行检测。
基于145例临床乳腺癌和胃癌病例，我们的研究表明ddPCR结果与FISH和IHC高度一致。在乳腺癌标本中，ddPCR结果与FISH和IHC定义的HER2状态高度一致，灵敏度为90.9%（30/33），特异性为100%（77/77）。在FISH和IHC结果一致的胃癌标本中，我们的检测方法与FISH和IHC的一致性为95.5%（21/22）。
ddPCR具有自动化优势，还能轻松评估大量样本中的HER2扩增水平，为定义HER2状态提供了一个潜在选择。
