多基因启动子甲基化诊断乙型肝炎病毒相关肝癌的研究

Promoter methylation of multiple genes for diagnosis of HBV-related hepatocellular carcinoma

摘要:

目的  探讨血清多基因启动子甲基化在乙型肝炎病毒（HBV）相关肝细胞癌（HCC）中的诊断价值。方法  随机收集98例HCC、75例肝硬化（LC）、90例慢性乙型肝炎（CHB）患者以及80例健康者血清各2ml，以血清BVES、APC、RASSF1A、TIMP3、GSTP1和HOXA9基因作为候选靶标。采用磁珠法提取血清DNA，MethyLight法检测基因启动子甲基化。通过构建受试者工作特征曲线（ROC）评价各检测指标的诊断效能。结果  血清各基因启动子甲基化在HCC患者检测阳性率分别为：RASSF1A（52.04%）、APC（36.73%）、BVES（29.59%）、HOXA9（20.41%）、GSTP1（17.35%）和TIMP3（11.22%），其中APC甲基化阳性完全与RASSF1A重叠。血清RASSF1A甲基化从慢性HBV感染患者中诊断HCC的效能最高[敏感性=0.520，特异性=0.915，曲线下面积（AUC）=0.718]，优于血清AFP（敏感性=0.480，特异性=0.739，AUC=0.609）；血清6个基因启动子甲基化联合使用的敏感性、特异性及诊断效能进一步提高，分别为0.806、0.855和0.845。结论  血清BVES、APC、RASSF1A、TIMP3、GSTP1及HOXA9基因启动甲基化联合检测能显著提高高危人群中HCC的诊断能力。

Abstract:

Objective To evaluate the clinical values of promoter methylation of multiple genes in serum for diagnosis of HBV-related hepatocellular carcinoma (HCC). Methods Two milliliters of sera were prepared from each participant including 98 HCC patients, 75 liver cirrhosis (LC) patients, 90 chronic hepatitis B (CHB) patients and 80 healthy controls. BVES, APC, RASSF1A, TIMP3, GSTP1 and HOXA9 were selected as candidate genes. Serum DNA was extracted with magnetic beads and modified by sodium bisulfite treatment. The methylation of the promoters of these genes was measured with MethyLight method. Receiver operating characteristic (ROC) curves were constructed and the areas under the ROC curves (AUCs) were calculated to determine the feasibility of using serum gene methylation as a biomarker for HCC. Results The positive rates of promoter methylation of the 6 genes in serum of the HCC patients were 52.04% for RASSF1A, 36.73% for APC, 29.59% for BVES, 20.41% for HOXA9, 17.35% for GSTP1 and 11.22% for TIMP3. The HCC patients with methylated APC had methylation of RASSF1A as well. The diagnostic performance of serum methylated RASSF1A was superior to that of AFP and the other indicators for the discrimination of the patients with HCC from those with chronic HBV infection. Furthermore, the sensitivity, specificity and AUC of combination of promoter methylation of the 6 genes obviously increased. Conclusions Combined detection of serum BVES, APC, RASSF1A, TIMP3, GSTP1 and HOXA9 promoter methylation could improve the diagnosis of HCC in high-risk population.