Table 2.

cfDNA mutations and epigenetic markers with prognostic value in hepatocellular carcinoma (HCC).

	Publication (n)	Methods	Tested Markers	Findings	Additional Information
Mutations	Oversoe et al., 2020 (95) [34]	Digital droplet PCR (ddPCR)	TERT (C228T)	TERT mutation conferred increased mortality (HR 2.16 (1.20–3.88), p = 0.01) when detected in plasma (adjusted HR 2.16 (1.20–3.88), p = 0.010), but not in tumor (adjusted HR 1.11 (0.35–3.56), p = 0.860).	In treatment-naïve HCC, mortality was more pronounced (HR 4.11 (1.73–9.76) p= 0.001) Detectable plasma TERT mutation was correlated with advanced TNM stage and vascular invasion (p < 0.0005) Non-concordance was associated with an early TNM stage.
	Jiao et al., 2018 (215) [85]	ddPCR	TERT (C228T and C250T)	Mutation-positive tumors with cirrhosis have worse prognosis (p = 0.0042). This association was significant in MVA	Mutations associated with worse OS (p = 0.0062) for all HCC patients (with and without cirrhosis). This association was not significant on MVA
	Yang et al., 2011 (60) [70]	Quantitative fluorescent polymerase chain reaction (FQ-PCR)	TERT	Plasma TERT DNA levels were closely related to tumor size (<5 vs. 5–10 vs. >10 cm), PVTT and TNM (I-II vs. III-IV) stage (p = 0.013, p = 0.010, and p = 0.029, respectively)	Cutoff–variable Survival analysis is not available.
	Youssef et al., 2023 (100) [86]	PCR	BCL9 and RPS6KB1	BCL9 gain and BCL9 + RPS6KB1 gain led to higher mortality rates and reduced survival times.	Prevalence of CNV gain in BCL9 and RPS6KB1 genes was detected in 14% and 24% of patients, respectively. Gain in both genes showed a high risk of HCC with elevated liver enzymes, Schistosomiasis, BCLC C, and PS > 1
	He et al., 2019 (29) [31].	Next Gen Sequencing (NGS)	2800 COSMIC hotspots in 50 high-frequency mutations in tumor genes.	MAF in the TP53, CTNNB1, PIK3CA, and CDKN2A increase the risk of larger tumors (>5 cm) MAF in the TP53, RET, FGFR3 and APC associated with multiple tumors or distant metastasis	35 mutant genes/50 tested were noted in tumors. TP53 was detected in 50% of tumors. Cut-off for MAF is >1%
	Kim et al., 2020 (107) [44]	NGS	2924 SNVs in 69 genes	MLH1 SNV, in combination with an increased ctDNA level (≥5.77 ng/mL), predicted poor overall survival among	OS was worse in HCC with high ctDNA (vs. low, p = 0.0014); High ctDNA with MLH1 mutation vs. high ctDNA + MLH1 witld type (WT) vs. low ctDNA + MLH1 WT
Epigenetic markers	Tangkijvanich et al., 2007 (85) [88]	Combined bisulfite restriction analysis PCR	Serum hypomethylation status of LINE-1 repetitive sequences	High serum LINE-1 hypomethylation associated with worse OS (10.5 vs. 5.5 m, p = 0.012)	High levels are associated with larger (>5 cm) and advanced (based on CLIP scoring). Cut off- 53.17 ± 7.74%,
	Huang et al., 2011 (72) [57]	MSRE-qPCR	APC, GSTP1, RASSF1A, and SFRP1	High levels of APC or RASSF1 methylation have worse outcomes. GSTP1 has a trend towards worst outcome (p = 0.062)	Methylated RASSF1A alone was independent risk factor for OS (hazard ratio = 3.262, 95% CI: 1.476–7.209, p = 0.003).
	Sun et al., 2013 (43) [59]	PCR	TFPI2 methylation percentage	Higher stage is associated with increased TFPI2 methylation percentage	Higher percentage with stage—39, 38, 64, 75, from Stages I–IV, respectively (percentage = methylated/unmethylated)
	Li et al., 2014 (136) [87]	PCR	IGFBP7	Methylation is associated with vascular invasion (84% versus 60%, p = 0.010).	Frequency of serum IGFBP7 promoter methylation in HCC patients was 65% (89/136)
	Xu et al., 2017 (1098) [55]	Deep sequencing of bis-DNA target-captured with molecular-inversion	BMPR1A PSD ARHGAP25 KLF3 PLAC8 ATXN1 Chr 6:170 Chr 6:3 ATAD2 Chr 8:20	Higher combined diagnostic score (cd-score) was associated with advanced TNM stage, poor response, early recurrence, and tumor burden	cd-score also has a diagnostic value.
	Xu et al., 2017 (1098) [55]	Deep sequencing of bis-DNA target-captured with molecular-inversion	SH3PXD2A C11orf9 Chr 17:78 PPFIA1 SERPINB5 NOTCH3 TMEM8B GRHL2	High combined prognosis score (cp-score) was associated with poor survival. Cp-score and TNM staging better than individual alone.	Cut-off for cp-score was >0.24. Cp-score was validated by MVA (HR: 2.405; 95%; p < 0.00)

HR—hazard ratio; OS—overall survival; BCLC—Barcelona Clinic Liver Cancer; MVA—macrovascualr invasion; PVTT—portal vein tumor thrombosis; MAF—Mutant allele frequency; PS—performance status; SNV—single nucleotide variants; CNV—gene copy number variants; MSRE—methylation-sensitive restriction enzymes-based quantitative.