Cell-free DNA in hepatocellular carcinoma: Biology to treatment response

Table 1 Diagnostic performance of cell-free DNA-based biomarkers for hepatocellular carcinoma detection

Ref.	Patients (HCC cohort)	cfDNA characteristics	Sensitivity	Specificity	AUROC
Xu et al[6], 2017	1098 (training + validation)	HCC bisulphite methylation marker	86%	94%	0.97 (0.96-0.98)
Cai et al[7], 2019	1204 tests	5-hMC signatures	79%	91%	0.92 (0.88-0.92)
Cai et al[8], 2021	135 (training + tests)	5-hMC, AFP and PIVKA2	79%	91%	0.92 (0.88-0.92)
Chen et al[9], 2021	350 (training + validation)	HIFI score = 4 cfDNA genomic features	96%	95%	0.995 (0.99-1.000)
Luo et al[10], 2022	187 (training + validation)	cfDNA methylation	86%	98%	0.98 (0.97-0.99)
Pös et al[11], 2020	159 (training)	cfDNA fragmentomic profiles	97%	99%	0.995
Kim et al[22], 2023	313 tests	Methylation markers of RNF135 and LDHB	57%	94%	0.80 (0.76-0.83)
Guo et al[41], 2024	498 (training + validation)	DMRs HepaAiQ: 20 best DMRs	86%	92%	0.94 (0.93-0.96)
Chen et al[44], 2024	510 (stage1)	PreCar score = 5 cfDNA genomic features	94%	95%	NA

Summarizes index tests, cohort structures (training, validation, and test where applicable), and corresponding sensitivity, specificity, and area under the receiver operating characteristic curve as reported by the original studies, enabling side-by-side appraisal of cell-free DNA methylation, hydroxymethylation, fragmentomics, and integrated multi-feature models for hepatocellular carcinoma detection in at-risk populations and mixed clinical cohorts. HCC: Hepatocellular carcinoma; cfDNA: Cell-free DNA; AUROC: Area under the receiver operating characteristic curve; 5-hmC: 5-hydroxymethylcytosine motif; AFP: Alpha-fetoprotein; PIVKA2: Protein induced by vitamin K absence or antagonist-II; HIFI: Integrated model of 5-hydroxymethylcytosine motif/fragmentation/nucleosome footprint; RNF135: Ring finger protein 135; LDHB: Lactate dehydrogenase B; DMR: Differentially methylated region; HepaAiQ: Hepatic artificial intelligence quantitative; NA: Not available.

Table 2 Summary of cell-free DNA/circulating tumor DNA biomarker studies in hepatocellular carcinoma evaluating prognostic and therapeutic response

Ref.	Patients	Therapy	Biomarker	Methodology	Prognostic impact/therapy response	Strength
Wang et al[31], 2021	117 HBV-HCC patients	Radical treatments (resection, ablation)	cfDNA CNVs (TFx, P-score, S-score)	Low-coverage WGS; CNV profiling at genome, chromosomal-arm, and 1-Mb bin levels	High scores (TFx ≥ 0.02, P ≥ 0.74, S ≥ 0.04) predicted worse OS and RFS	Multi-level cfDNA CNV profiling; bin-level CNV shown as superior prognostic marker
Fu et al[18], 2022	258 HCC patients undergoing curative liver resection	Surgery (curative resection)	ctDNA mutation profiles (high-risk gene set)	ctDNA mutation detection; RNA sequencing for tumor immune infiltration	Higher number of mutant genes linked to early recurrence (HR = 2.2); high-risk mutations worsened RFS (HR = 13)	Largest prospective study on preoperative ctDNA predicting recurrence; ctDNA-guided risk stratification
Dong et al[23], 2022	64 HCC patients	TACE	Plasma cfDNA; CNV profiling; TFx	Low-depth WGS for CNV detection; TFx quantification; plasma centrifugation protocol	Changes in TFx pre- vs post-TACE predict TACE response; CNVs and NQO1 amplification associated with efficacy; lipiodol deposition correlated with CNVs	cfDNA CNV profiling via low-depth WGS provides noninvasive, cost-effective real-time monitoring of tumor burden and TACE response
Muraoka et al[29], 2021	32 TACE patients, 35 TKI patients with available plasma (advanced HCC)	TACE (epirubicin/cisplatin) and TKI (sorafenib or lenvatinib)	Plasma cfDNA (hTERT promoter mutations)	dPCR quantification of hTERT promoter mutant and wild-type cfDNA	Changes in mutant cfDNA during TACE/TKI reflect therapy response; wild-type cfDNA reflects liver damage	First study to show short-term cfDNA dynamics for monitoring therapy response; noninvasive marker of tumor necrosis
Nakatsuka et al[32], 2021	100 HCC patients	TACE (32), RFA (33), MTAs (35) (lenvatinib, sorafenib, regorafenib)	Plasma cfDNA; TERT promoter mutations; post-treatment cfDNA used for ultra-deep sequencing	ddPCR for TERT; targeted ultra-deep sequencing (22000 × coverage)	cfDNA level post-treatment correlated with therapy response; TERT mutation alone not prognostic	Ultra-deep sequencing of post-treatment cfDNA; analysis of therapy response via liquid biopsy
Li et al[33], 2022	60 patients with primary HCC	Primary liver resection	TP53 mutations in exosomal cfDNA	exoDNA, TP53 mutation	High-frequency TP53 mutations in circulating exoDNA associated with poor prognosis, independent of other clinicopathologic features	80% of patients had detectable TP53 mutations in exoDNA; exoDNA considered more stable and robust than cfDNA
Campani et al[5], 2024	173 HCC patients and 56 chronic liver disease controls	Locoregional (percutaneous ablation, TACE) and systemic (atezolizumab-bevacizumab, others)	ctDNA mutations (TERT, TP53, CTNNB1, PIK3CA, NFE2 L2)	ddPCR and MiSeq sequencing	Persistence of ctDNA mutations under atezolizumab-bevacizumab associated with radiological progression; disappearance linked to response	Longitudinal design; paired tumour-plasma analysis; dynamic ctDNA assessment across therapies

Highlights various assay types, genomic targets, and their clinical correlations across surgical, locoregional, and systemic therapies. Tumor fraction, P-score, and S-score were derived from low-coverage whole-genome sequencing-based copy number variation profiling. cfDNA: Cell-free DNA; ctDNA: Circulating tumor DNA; exoDNA: Exosomal DNA; CNV: Copy number variation; TFx: Tumor fraction; OS: Overall survival; RFS: Recurrence-free survival; WGS: Whole-genome sequencing; ddPCR: Droplet digital polymerase chain reaction; dPCR: Digital polymerase chain reaction; RFA: Radiofrequency ablation; TACE: Transarterial chemoembolization; TKI: Tyrosine kinase inhibitor; MTA: Molecular-targeted agent; HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; CLD: Chronic liver disease; HR: Hazard ratio; NQO1: NAD(P)H quinone dehydrogenase 1; TERT: Telomerase reverse transcriptase; CTNNB1: Catenin beta 1; PIK3CA: Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; NFE2 L2: Nuclear factor erythroid 2-like 2; hTERT: Human telomerase reverse transcriptase.