METHODS: A three-phase multicentre study was conducted to screen, optimise and validate HCC-specific differentially methylated regions (DMRs) using next-generation sequencing and quantitative methylation-specific PCR (qMSP).
RESULTS: Genome-wide methylation profiling was conducted to identify DMRs distinguishing HCC tumours from peritumoural tissues and healthy plasmas. The twenty most effective DMRs were verified and incorporated into a multilocus qMSP assay (HepaAiQ). The HepaAiQ model was trained to separate 293 HCC patients (Barcelona Clinic Liver Cancer (BCLC) stage 0/A, 224) from 266 controls including chronic hepatitis B (CHB) or liver cirrhosis (LC) (CHB/LC, 96), benign hepatic lesions (BHL, 23), and healthy controls (HC, 147). The model achieved an area under the curve (AUC) of 0.944 with a sensitivity of 86.0% in HCC and a specificity of 92.1% in controls. Blind validation of the HepaAiQ model in a cohort of 523 participants resulted in an AUC of 0.940 with a sensitivity of 84.4% in 205 HCC cases (BCLC stage 0/A, 167) and a specificity of 90.3% in 318 controls (CHB/LC, 100; BHL, 102; HC, 116). When evaluated in an independent test set, the HepaAiQ model exhibited a sensitivity of 70.8% in 65 HCC patients at BCLC stage 0/A and a specificity of 89.5% in 124 patients with CHB/LC. Moreover, HepaAiQ model was assessed in paired pre- and postoperative plasma samples from 103 HCC patients and correlated with 2-year patient outcomes. Patients with high postoperative HepaAiQ score showed a higher recurrence risk (Hazard ratio, 3.33, p < .001).
CONCLUSIONS: HepaAiQ, a noninvasive qMSP assay, was developed to accurately measure HCC-specific DMRs and shows great potential for the diagnosis, detection and prognosis of HCC, benefiting at-risk populations.
方法:进行了一项三阶段多中心研究,使用下一代测序和定量甲基化特异性PCR(qMSP)优化和验证HCC特异性差异甲基化区域(DMRs)。
结果:进行了全基因组甲基化分析,以鉴定DMRs将HCC肿瘤与肿瘤周围组织和健康血浆区分开。验证了20个最有效的DMRs并将其并入多位点qMSP测定(HepaAiQ)中。对HepaAiQ模型进行了训练,以分离293名HCC患者(巴塞罗那诊所肝癌(BCLC)0期/A,224)从266个对照,包括慢性乙型肝炎(CHB)或肝硬化(LC)(CHB/LC,96),良性肝脏病变(BHL,23),和健康对照(HC,147).该模型实现了0.944的曲线下面积(AUC),在HCC中的灵敏度为86.0%,在对照中的特异性为92.1%。在523名参与者的队列中对HepaAiQ模型进行盲验证,在205例HCC病例中,AUC为0.940,灵敏度为84.4%(BCLC分期0/A,167)和318个对照的90.3%的特异性(CHB/LC,100;BHL,102;HC,116).在独立测试集中评估时,HepaAiQ模型在65例BCLC0/A期HCC患者中的敏感性为70.8%,在124例CHB/LC患者中的特异性为89.5%.此外,在来自103例HCC患者的配对的术前和术后血浆样本中评估HepaAiQ模型,并与2年患者预后相关。术后HepaAiQ评分高的患者显示出更高的复发风险(危险比,3.33,p<.001)。
结论:HepaAiQ,非侵入性qMSP测定,是为了准确测量HCC特异性DMRs而开发的,并显示出巨大的诊断潜力,肝癌的检测和预后,使处于危险中的人群受益。