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Page 2 of 10 Banini et al. Hepatoma Res 2019;5:34 I http://dx.doi.org/10.20517/2394-5079.2019.30
[2]
one-year survival for HCC in the United States is less than 50%, while the five-year survival is 10% . With
the advent of potent therapy for chronic hepatitis C virus (HCV) infection, the overall global incidence of
HCC may plateau or decrease as a result of decreased HCV-associated HCC, however these gains appear to
be threatened by the increasing incidence of nonalcoholic fatty liver disease (NAFLD)-associated HCC and
the persistently high levels of hepatitis B virus (HBV)-associated HCC.

Diagnosis of HCC can be made by imaging studies such as multiphasic computed tomography scan
or magnetic resonance imaging. However, tissue biopsy remains the gold-standard for HCC diagnosis
especially in non-cirrhotic patients or those with nonspecific imaging studies. The risks of biopsy include
procedure-related complications such as pain, bleeding, and perforation of adjacent organs, as well as
tumor seeding along the needle track and sampling errors resulting in false negative results. Aside from
these risks, technological advancements over the past few decades have led to a better understanding of
the high heterogeneity and dynamic evolution of HCC tumor cells, and a time- and location-constrained
tissue biopsy is inadequate in reflecting these dynamic changes. These shortcomings have led to increased
interest in the application of liquid biopsy analysis to HCC. Although the concept of liquid biopsy has
been in existence for several decades, the term gained traction in the early 2000’s, with one of its first
uses pertaining to the capture of circulating tumor cells (CTCs) for biomarker analysis in breast cancer
[3]
patients .

Liquid biopsy generally refers to the analysis of blood or other body fluids to obtain genetic or epigenetic
information which can be applied in screening, diagnosis, prognostication, treatment monitoring or disease
[4]
recurrence . The major advantage of liquid biopsy is non-invasiveness which makes it attractive for frequent
analysis to track mutations and other molecular changes over time. The most commonly used HCC serum
biomarker is serum alpha-fetoprotein (AFP), together with its fucosylated glycoform (AFP-L3). AFP is
normally produced during gestation by the fetal liver and yolk sac, and levels decline rapidly after birth.
Regeneration of liver cells leads to AFP production, as can be seen in chronic liver disease and in HCC.
Other types of malignancy, for instance testicular or ovarian cancer, can also cause AFP elevation. The
AFP-L3 glycoform, named for its ability to bind Lens culinaris agglutinin, is a relatively new test developed
[5]
in 1992 that is more specific for HCC, compared to AFP . Serum AFP concentration can be normal even
[6]
in advanced HCC . In two studies of approximately 1800 patients, AFP was found to have about 60%
[7,8]
sensitivity and 80% specificity in detecting HCC using a cut off level between 10 to 20 ng/mL . Higher serum
AFP levels are associated with greater specificity and less sensitivity, for instance AFP > 400 ng/mL implies
[9]
HCC until proven otherwise. However, fewer than 20% of HCC cases have such elevated AFP levels .
Serum and plasma biomarkers detectable through liquid biopsy show promise in the early detection
of HCC either alone or in combination with AFP. These markers have the potential to be adjunctive
or superior to conventional methods of HCC diagnosis. Several of these markers, however, are still in
preclinical development and testing and none of them has of yet been recommended for HCC diagnosis.
Here, we provide an updated summary of cell-free nucleic acid (cfNA) analysis in the diagnosis HCC, with
emphasis on cell-free DNA (cfDNA).

LIQUID BIOPSY FOR HCC
Liquid biopsy specimens contain genetic information in CTCs or in the form of cfNAs released by
apoptotic cells or living cells. cfNA can be found circulating freely in body fluids or are taken up by
extracellular vesicles such as exosomes and microvesicles. The various types of cfNA include cfDNA,
mRNA (cfRNA), and noncoding RNAs including miRNAs (cfmiRNA). Other noncoding RNAs including
long noncoding RNA, small nuclear RNA, small nucleolar RNA, and piwi-interacting RNA may also be
present in liquid biopsy specimens and could potentially serve as biomarkers although there are currently
very few studies on these subtypes.