基于qPCR的结直肠癌HER2扩增检测方法的 构建及应用

Construction and Application of Detection Method of HER2 Amplification Based on qPCR for Colorectal Cancer

摘要: 目的：构建基于实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction, qPCR)的结直肠癌HER2扩增检测作为荧光原位杂交(Fluorescence in situ hybridization, FISH)的补充。方法：首先构建基于qPCR平台检测HER2和RPPH1基因拷贝数的流程，然后根据3σ原则并基于30例正常对照样本的检测结果制定临床结直肠癌样本HER2扩增阳性的判定标准，并在临床样本中进行验证。结果：根据30例正常对照样本的检测结果制定了临床样本HER2扩增阳性的判定标准，即RPPH1和HER2的Ct差值大于1.6。27例结直肠癌样本的检测及判定结果显示，qPCR与FISH的检测结果具有良好一致性(Kappa值为0.509)，qPCR检测FISH阳性样本的灵敏度60%，特异性90.9%。此外，qPCR检测FISH阳性或FISH阴性结直肠癌患者的HER2扩增水平均显著高于正常对照人群。结论：通过qPCR技术检测结直肠癌HER2扩增具有可行性，但仍需进一步优化检测流程并在更多临床样本中加以验证。

Abstract: Objective: To construct a quantitative real-time polymerase chain reaction (qPCR) based HER2 amplification detection method for colorectal cancer as a supplement to fluorescence in situ hybridization (FISH). Methods: Firstly, the process of detecting the copy number of HER2 and RPPH1 genes based on qPCR platform was constructed, and then the criterion for judging the amplification of HER2 in clinical samples was established according to the 3σ principle and based on the detection results of 30 normal control samples. Finally, it is verified in colorectal cancer samples. Results: The judgement criteria for HER2 amplification in clinical samples was established, that is, the difference of threshold cycle(Ct) between RPPH1 and HER2 was greater than 1.6. The results of 27 colorectal cancer samples showed that the detection results of qPCR and FISH were in good agreement (Kappa value was 0.509), and the sensitivity of qPCR in detecting FISH positive samples were 60% with specificity of 90.9%. In addition, the amplification level of HER2 in FISH-positive or FISH-negative colorectal cancer patients detected by qPCR was significantly higher than that in normal control population. Conclusion: It is feasible to detect HER2 amplification of colorectal cancer by qPCR technology, but it is still necessary to further optimize the detection process and verify it in more clinical samples.