肝癌血浆游离DNA甲基化谱检测及临床意义

Methylation profile of tumor suppressor genes in the cell-free DNA of plasma in hepatocellular carcinoma

目的：在外周血游离DNA中筛选肝癌特异的甲基化谱。方法：收集55例肝癌和54例慢性肝病患者血浆标本，应用甲基化特异性PCR方法检测血浆游离DNA的12个抑癌基因甲基化状态。结果：在肝癌组中，APC、p16、GSTP1、Cyclin D2、LHX1、TFPI2、DKK2、DKK3、SFRP2、14-3-3 sigma、ppENK、NPTX2基因的甲基化频率分别为78.18%、63.64%、58.18%、49.09%、49.09%、47.27%、40.00%、18.18%、16.36%、9.09%、7.27%和5.45%，而在慢性肝病组中，各基因的甲基化频率分别为27.78%、22.22%、7.41%、3.70%、16.67%、37.04%、37.04%、11.11%、20.37%、7.41%、7.41%和9.26%。APC、Cyclin D2、TFPI2、DKK3和GSTP1基因在肝癌组中的甲基化频率高于慢性肝病组（P〈0.01）。将它们组成5基因甲基化谱，肝癌组甲基化指数（中位值为0.6，IQR 0.4～0.8）显著高于慢性肝病组（中位值为0.2，IQR 0～0.2）。在肝癌组中，甲基化指数与患者年龄具有相关性，年龄大者甲基化指数较高，而甲基化指数与其它临床病理参数未见相关性。甲基化指数与肝癌患者的无瘤生存期及总体生存期均未见相关性。结论：该甲基化谱可能成为肝癌辅助诊断的核酸标志物。

Objective：This study aimed to detect the special methylation profile in peripheral blood for hepatocellular carcinoma （HCC）. Methods：The methylation status of 12 tumor suppressor genes （TSGs） in the plasma of 55 HCCs and 54 chronic liver diseases （CLDs） was tested by methylation-specific PCR （MSP）. Results：In HCC, the methylation frequencies were 78.18%in APC, 63.64%in cyclin D2, 58.18% in TFPI2, 49.09% in DKK3, 49.09% in GSTP1, 47.27% in p16, 40.00% in Sigma 14-3-3, 18.18% in SFRP2, 16.36% in ppENK, 9.09% in DKK2, 7.27% in NPTX2, and 5.45% in LHX1. In CLD, the methylation frequencies were 27.78% in APC, 22.22%in cyclin D2, 7.41%in TFPI2, 3.70%in DKK3, 16.67%in GSTP1, 37.04%in p16, 37.04%in Sigma 14-3-3, 11.11%in SFRP2, 20.37%in ppENK, 7.41%in DKK2, 7.41%in NPTX2, and 9.26%in LHX1. The methylation frequencies of APC, cyclin D2, TFPI2, DKK3, and GSTP1 were higher in HCC than in CLD （P〈0.01）. The methylation index （MI） of the five-gene methylation profile was statistically higher in HCC （median, 0.6;IQR, 0.4-0.8） than CLD （median, 0.2;IQR, 0-0.2） （P〈0.01）. In HCC, MI was statistically related to the patient＇s age. Older patients with HCC had a higher MI. No significant correlation was observed between MI and other clinicopathological data. Moreover, MI was not related to the disease free survival and the overall survival in HCC. Conclusion：This five-gene methylation profile may be a promising biomarker for the assistant diagnosis of HCC.