Fu Siyu, Boers Ruben G, Boers Joachim B, van der Meeren Pam E, Helmijr Jean, de Weerd Vanja, Doukas Michail, Jansen Maurice, Hansen Bettina E, de Wilde Roeland F, Sprengers Dave, Gribnau Joost, Wilting Saskia M, Debes José D, Boonstra Andre
Department of Gastroenterology and Hepatology, Erasmus University Medical Center, Wytemaweg 80, 3015 CN, Rotterdam, The Netherlands.
Department of Developmental Biology, Erasmus University Medical Center, Rotterdam, The Netherlands.
J Exp Clin Cancer Res. 2025 May 15;44(1):144. doi: 10.1186/s13046-025-03412-9.
Hepatocellular carcinoma (HCC) is associated with a poor 5-year survival mainly due to detection at late stages. Better non-invasive surveillance methods are needed to improve early detection and maximize survival. We performed a strict assessment of DNA methylation markers (DMMs) for HCC detection.
A total of 385 samples from liver tissues and blood were analyzed. Genome-wide Methylated DNA sequencing (MeD-seq) was initially performed on 46 liver tissues, followed by the validation using quantitative methylation-specific PCR (qMSP) on 175 liver tissues. The selected DMMs with and without ASAP/GAAD score were further evaluated in 180 blood samples. Additionally, MeD-seq was performed to validate the results on blood.
MeD-seq revealed a substantial number of differentially methylated regions (DMRs) in HCC tissues compared to non-HCC controls. By qMSP, the top 5 DMMs demonstrated strong performance in distinguishing cirrhotic HCC from cirrhosis controls in tissue (AUC 0.842 to 0.957). However, evaluation of these DMMs in blood showed lower performance in early HCC detection compared to cirrhosis in both the training (sensitivity 26.7-43.3%, 81.3% specificity) and validation cohorts (sensitivity 16.2-43.2%, 85.7% specificity). The addition of DMMs to the ASAP/GAAD score only provided an additional 5.4% sensitivity in the validation cohort compared to the ASAP/GAAD score alone. These findings were confirmed using MeD-seq analysis in blood samples, which revealed no detectable DMRs between cirrhotic HCC and cirrhosis controls. Interestingly, DNA methylation patterns in blood of healthy individuals differed strongly from both groups (cirrhosis and cirrhotic HCC).
DNA methylation patterns in liver tissue were distinctly different between HCC and controls. In blood, DMMs contributed minimally to early-stage HCC detection compared to cirrhosis, whether used alone or in combination with the ASAP/GAAD score. It is likely that high baseline DNA methylation related to cirrhosis and possibly the low input of tumor-related DNA impacts the use of DMMs in early HCC detection in blood.
肝细胞癌（HCC）患者的5年生存率较低，主要原因是其在疾病晚期才被发现。因此，需要更好的非侵入性监测方法来改善早期检测并最大限度地提高生存率。我们对用于HCC检测的DNA甲基化标志物（DMM）进行了严格评估。
共分析了385份来自肝脏组织和血液的样本。最初对46份肝脏组织进行了全基因组甲基化DNA测序（MeD-seq），随后在175份肝脏组织上使用定量甲基化特异性PCR（qMSP）进行验证。在180份血液样本中进一步评估了具有和不具有ASAP/GAAD评分的选定DMM。此外，进行MeD-seq以验证血液样本的结果。
与非HCC对照相比，MeD-seq显示HCC组织中存在大量差异甲基化区域（DMR）。通过qMSP，前5个DMM在区分肝硬化HCC与肝硬化对照组织方面表现出强大性能（AUC为0.842至0.957）。然而，在血液中对这些DMM的评估显示，与肝硬化相比，其在早期HCC检测中的性能在训练队列（敏感性26.7 - 43.3%，特异性81.3%）和验证队列（敏感性16.2 - 43.2%，特异性85.7%）中均较低。与单独使用ASAP/GAAD评分相比，在验证队列中，将DMM添加到ASAP/GAAD评分中仅额外提高了5.4%的敏感性。在血液样本中使用MeD-seq分析证实了这些发现，该分析显示肝硬化HCC和肝硬化对照之间未检测到DMR。有趣的是，健康个体血液中的DNA甲基化模式与两组（肝硬化和肝硬化HCC）有很大差异。
HCC与对照之间肝脏组织中的DNA甲基化模式明显不同。在血液中，无论单独使用还是与ASAP/GAAD评分联合使用，DMM对早期HCC检测的贡献相对于肝硬化都很小。可能是与肝硬化相关的高基线DNA甲基化以及可能较低的肿瘤相关DNA输入影响了DMM在血液中早期HCC检测的应用。
