Hu Ran, Tran Benjamin, Li Shuo, Stackpole Mary L, Zeng Weihua, Zhou Yonggang, Melehy Andrew, Sadeghi Saeed, Finn Richard S, Zhou Xianghong Jasmine, Li Wenyuan, Agopian Vatche G
Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California, United States of America.
Bioinformatics Interdepartmental Graduate Program, University of California at Los Angeles, Los Angeles, California, United States of America.
PLoS One. 2025 Apr 25;20(4):e0321736. doi: 10.1371/journal.pone.0321736. eCollection 2025.
The current noninvasive prognostic evaluation methods for hepatocellular carcinoma (HCC), which are largely reliant on radiographic imaging features and serum biomarkers such as alpha-fetoprotein (AFP), have limited effectiveness in discriminating patient outcomes. Identification of new prognostic biomarkers is a critical unmet need to improve treatment decision-making. Epigenetic changes in cell-free DNA (cfDNA) have shown promise in early cancer diagnosis and prognosis. Thus, we aim to evaluate the potential of cfDNA methylation as a noninvasive predictor for prognostication in patients with active, radiographically viable HCC.
Using Illumina HumanMethylation450 array data of 377 HCC tumors and 50 adjacent normal tissues obtained from The Cancer Genome Atlas (TCGA), we identified 158 HCC-related DNA methylation markers associated with overall survival (OS). This signature was further validated in 29 HCC tumor tissue samples. Subsequently, we applied the signature to an independent cohort of 52 patients with plasma cfDNA samples by calculating the cfDNA methylation-based risk score (methRisk) via random survival forest models with 10-fold cross-validation for the prognostication of OS.
The cfDNA-based methRisk showed strong discriminatory power when evaluated as a single predictor for OS (3-year AUC = 0.81, 95% CI: 0.68-0.94). Integrating the methRisk with existing risk indices like Barcelona clinic liver cancer (BCLC) staging significantly improved the noninvasive prognostic assessments for OS (3-year AUC = 0.91, 95% CI: 0.80-1), and methRisk remained an independent predictor of survival in the multivariate Cox model (P = 0.007).
Our study serves as a pilot study demonstrating that cfDNA methylation biomarkers assessed from a peripheral blood draw can stratify HCC patients into clinically meaningful risk groups. These findings indicate that cfDNA methylation is a promising noninvasive prognostic biomarker for HCC, providing a proof-of-concept for its potential clinical utility and laying the groundwork for broader applications.
目前肝细胞癌（HCC）的非侵入性预后评估方法在很大程度上依赖于影像学特征和血清生物标志物，如甲胎蛋白（AFP），在区分患者预后方面效果有限。识别新的预后生物标志物是改善治疗决策的关键未满足需求。游离DNA（cfDNA）的表观遗传变化在癌症早期诊断和预后方面已显示出前景。因此，我们旨在评估cfDNA甲基化作为活动性、影像学上可存活的HCC患者预后的非侵入性预测指标的潜力。
利用从癌症基因组图谱（TCGA）获得的377个HCC肿瘤和50个相邻正常组织的Illumina HumanMethylation450芯片数据，我们鉴定出158个与总生存期（OS）相关的HCC相关DNA甲基化标记物。该特征在29个HCC肿瘤组织样本中进一步得到验证。随后，我们通过随机生存森林模型并进行10倍交叉验证来计算基于cfDNA甲基化的风险评分（methRisk），将该特征应用于52例有血浆cfDNA样本的独立队列患者，以预测OS。
当将基于cfDNA的methRisk作为OS的单一预测指标进行评估时，显示出强大的区分能力（3年AUC = 0.81，95% CI：0.68 - 0.94）。将methRisk与现有的风险指数如巴塞罗那临床肝癌（BCLC）分期相结合，显著改善了对OS的非侵入性预后评估（3年AUC = 0.91，95% CI：0.80 - 1），并且在多变量Cox模型中，methRisk仍然是生存的独立预测指标（P = 0.007）。
我们的研究作为一项初步研究表明，从外周血检测评估的cfDNA甲基化生物标志物可将HCC患者分层为具有临床意义的风险组。这些发现表明，cfDNA甲基化是一种有前景的HCC非侵入性预后生物标志物，为其潜在的临床应用提供了概念验证，并为更广泛的应用奠定了基础。
