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Banini et al. Hepatoma Res 2019;5:34 I http://dx.doi.org/10.20517/2394-5079.2019.30 Page 3 of 10
[10]
The first report of cfNA derived from human peripheral blood was published by Mandel and Metais in
1948, however its significance was not realized until several decades later in 1977 when it was discovered
that serum and plasma from cancer patients carry higher concentrations of cfDNA compared to healthy
[11]
individuals . About a decade later, Vasioukhin showed that cfDNA can have cancer characteristics,
[12]
suggesting that cancer cells can release DNA into peripheral blood . This notion was soon confirmed by
other investigators [13,14] , and cfDNA released by cancer cells into circulation has been subsequently referred
to as circulating tumor DNA (ctDNA).

Analysis of plasma and serum cfRNA is limited by the very small quantities present in circulation as well
as degradation by ribonuclease (RNase). Incorporation of cfRNA into extracellular vesicles protects them
from degradation. Over the past decade, several groups have shown that cfRNA can potentially be applied
in HCC detection and monitoring [15-17] . A recent study by Xu et al. showed that serum mRNA levels of
[18]
exosomal hnRNPH1 in patients with primarily HBV-associated HCC were significantly higher than in
[18]
patients with chronic hepatitis B, liver cirrhosis, or healthy control . Exosomal hnRNPH1 levels also
associated with TNM stage, Child-Pugh classification, portal vein embolism and lymph node metastasis.

Non-coding RNA, especially cfmiRNAs were first demonstrated as a promising biomarker in patients with
solid cancers in 2008 . Since then, there have been several studies on non-coding RNAs in different types
[19]
[20]
of cancer including HCC . A recent article mapped the differential expression of non-coding RNAs in
normal liver tissue and in various stages of liver disease leading to HCC; each liver phenotype was found
[21]
to demonstrate a unique RNA signature . Induction of exosomal miR-21 and miR-10b in HCC was found
to promote cancer cell proliferation and metastasis, potentially serving in prognostication and therapy for
[22]
[23]
HCC . Several other cfmiRNAs have been studied, including miR-1 , miR-16 [24-26] , and miR-122 [23,27-29] .
An in-depth review of circulating miRNA signatures in HCC is beyond the scope of this article, and the
[30]
reader is referred to a recent article by Mirzaei et al. for further information.
Liquid biopsy analysis in HCC has significantly expanded over the past decade, providing substantial
information on different HCC tumors and their microenvironment, and the potential application of such
information to disease diagnosis and monitoring.

Isolation of cell-free DNA in liquid biopsy samples
There are several challenges in the isolation of cfDNA in general and ctDNA in particular, including DNA
lysis as a results of blood clotting in collection tubes, DNA contamination during processing or DNA loss
during isolation. Thus, the right sample collection tube and optimal processing methods are crucial to the
success of isolation and to the accuracy of the sample obtained. A 1 mL volume of blood typically yields 10 ng
of cfDNA, and in cancer patients, about 0.01% to 1% of cfDNA comprises ctDNA. Several methods have
been used in the isolation of ctDNA, including targeted methods involving polymerase chain reaction (PCR)
based on known genetic mutations, for instance digital PCR; bead, emulsion, amplification and magnetics
(BEAMing) PCR; and amplification-refractory mutation system-PCR. Alternatively, a variety of untargeted
methods can be employed to sequence millions of DNA fragments, including Sanger sequencing and
next-generation sequencing techniques such as targeted amplification sequencing or targeted capture
sequencing [31,32] .

HCC-ASSOCIATED QUANTITATIVE CHANGES IN CELL-FREE DNA
Cancer is associated with both quantitative and qualitative changes in cfDNA detectable by liquid
biopsy [33-37] [Figure 1]. In patients with HCC, total cfDNA concentration is significantly higher than in
those without HCC [33,34] . Although non-specific, cfDNA increase in association with HCC has potential
utility in screening for HCC, as well as in monitoring of treatment response and in predicting HCC