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Guerriero et al. Hepatoma Res 2019;5:6 I http://dx.doi.org/10.20517/2394-5079.2018.108 Page 5 of 22

[23]
For prognostic assessment, Wong et al. evaluated the methylation status of p15 and p16 in tumor tissues,
plasma, serum and buffy coat samples from HCC patients, non-HCC controls and healthy individuals and
found promoter methylation in plasma or serum of 40%-50% HCC patients. Most of patients associated
with gene methylation exhibited a poorer prognosis in comparison with patients negative for aberrant
[23]
methylation . The detection of methylation of APC and RASSF1A promoters was also associated with
shorter OS in HCC patients and RASSF1A methylation was demonstrated to be an independent prognostic
[21]
factor . Very recently, another gene whose aberrant methylation detected in plasma was associated with
[24]
patients’ poorer prognosis was SOCS3 .
The analysis of panels of aberrantly methylated genes through the use of next generation sequencing (NGS)
is expected to further improve sensitivity and specificity of aberrant methylation biomarkers. For example,
targeted deep-sequencing of plasma DNA after bisulfite treatment could be used to simultaneously assess the
[25]
methylation status of several targets. While Holmila et al. identified two genes (VIM and FBLN1) whose
promoters were differentially methylated in HCC using this approach, these areas of study have not been
thoroughly investigated.

Compared to cfDNA abundance in plasma or serum, the detection of aberrant DNA methylations in
cfDNA provides a more specific tumor biomarker, especially if a combination of multiple genes is employed.
However, it still contains a limitation. Considering that aberrant methylation generally affects tumor
suppressor genes, the analysis cannot reveal alterations in oncogenes potentially targets of specific therapies.
With the development of more sophisticated approaches, the identification of tumor-specific genetic
alterations has become feasible and has been applied to HCC.

Cancer gene mutations in plasma or serum cfDNA
An analysis of cancer gene mutations in serum or plasma of HCC patients was investigated in the diagnostic,
prognostic and predictive settings.

In the diagnostic setting, R249S mutation of the TP53 gene is hallmark of aflatoxin B1 exposure, one of the
major causes of HCC in certain geographic areas. Using droplet digital PCR the authors identified a higher
prevalence of this mutation in plasma cfDNAs of HCC in Cameroonian and Central African patients in
comparison with control subjects with and without liver disease (almost 25% of patients with HCC and 3%-9%
of non-HCC subjects were R249S carriers), suggesting a potential use of this biomarker as an early risk
[26]
factor for HCC in individuals exposed to aflatoxin B1 . Targeted deep sequencing was used to investigate
several cancer genes involved in HCC. For example, the ultra-deep sequencing analysis of 58 cancer genes
performed in 8 HCC tissues and paired plasma/serum samples revealed that 15 of the 21 somatic tumor
[27]
mutations (71%) could also be detected in plasma/serum cfDNA , thus indicating the translational
potential of this approach for HCC diagnosis. In another recent study, cancer alterations in four hot-spot
regions of TP53, CTNNB1 and TERT genes were investigated in plasma cfDNA and corresponding tumor
DNA from 48 HCC patients. Interestingly, the authors found that many gene alterations found in plasma
[28]
DNA were different from those found in tumor tissues, an evidence of tumor heterogeneity . Confirming
tumor heterogeneity in HCC, a recent study analysed plasma cfDNA from 26 HCC patients for the presence
of mutations in a large set of genes. Authors found tumor heterogeneity and evolution over time by tracking
[29]
circulating mutation pattern in a patient who developed progression after capecitabine treatment .

The identification of gene mutations offers the possibility of identifying potential actionable alterations,
useful for guiding treatment choice. It has been demonstrated that HCC harbouring mutant RAS exhibited
[30]
a better clinical response to refametinib plus sorafenib, compared to wild-type RAS tumors . Notably, in
the course of the study aimed at detecting KRAS or NRAS mutations in plasma cfDNA of a large cohort of
HCC patients, authors found other actionable mutations in EGFR, JAK2, BRAF, FLT3, PIK3CA, and cKIT,
suggesting that available target therapies could potentially be effective in defined, albeit small, subsets of