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Page 4 of 10 Banini et al. Hepatoma Res 2019;5:34 I http://dx.doi.org/10.20517/2394-5079.2019.30

Figure 1. Liquid biopsy for hepatocellular carcinoma. Tumors release a number of molecules into circulation including tumor cells, cell-
free DNA, different circulating RNA classes, proteins and extracellular vesicles including exosomes and microvesicles. Cell-free DNA can
be isolated from blood or other body fluids and analyzed to determine genetic and epigenetic changes present in circulation which are
reflective of changes occurring in tissues, potentially avoiding the need for invasive tissue sampling. cfDNA: cell free DNA; cfmiRNA: cell
free microRNA; CTC: circulating tumor cell; HCC: hepatocellular carcinoma

[41]
recurrence [34,38-40] . A recent manuscript by Yan et al. analyzed cfDNA and AFP levels from 24 patients
with HCC and 62 patients with chronic hepatitis B with varying degrees of fibrosis (F0 to F6). Using
multivariate analysis, the authors found that age and cfDNA were independent predictors of HCC, while
AFP was not an independent predictor. They developed a combination model including cfDNA level, age
and AFP, collectively referred to as HCC index, for HCC diagnosis by backward logistic regression analysis.
The HCC index showed an area under the receiver operating characteristic curve (AUROC) of 0.98 (95%
confidence interval 0.92-1.00), a sensitivity of 87% and specificity of 100% for the diagnosis of HCC at a cut-
[41]
[41]
off value of 0.61 , proving superior to cfDNA alone or AFP alone in the diagnosis of HCC . As shown
[41]
by Yan et al. , combination of cfDNA level with other protein or genetic biomarkers holds promise as a
liquid-biopsy based clinical tool in the early diagnosis of HCC.

HCC-ASSOCIATED QUALITATIVE CHANGES IN CELL-FREE DNA
Genetic or epigenetic alterations in cfDNA in association with HCC are detectable by liquid biopsy and
are reliable indicators of changes occurring in tumor tissues. In general, these changes are grouped into
single nucleotide mutations [35,42] , variations in DNA copy number [35,36] , chromosomal rearrangements, loss
[37]
of heterozygosity, microsatellite instability, and changes in methylation pattern .
Single nucleotide mutations
Indepth genomic analysis of HCC tumor tissue to the base pair level has shown that no two tumors carry
[43]
the same cadre of somatic mutations . There is considerable variability in the number of mutations even
among patients with advanced stage HCC, as demonstrated by analysis of three patients with advanced
[43]
HCC which showed 7.2, 15 and 7,910 mutant fragments per 5 mL of plasma . Tumor-specific somatic
mutations in several genes have been identified in the peripheral blood of HCC patients, including TP53 ,
[44]
[45]
[46]
HCK and TERT .
The three most frequent somatic mutations in HCC are TERT promoter activating mutations which are
found in 40%-60% of HCC patients; and the mutually exclusive TP53 and CTNNB1 mutations which are
found in 30-50% of HCC cases [47-49] . Digital droplet PCR to interrogate the single nucleotide mutations
TERT c.-124C>T, TP53 c.747G>T (p.R249S), CTNNB1 c.121A>G (p.T41A) and c.133T>C (p.S45A) in the