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Page 16 of 22 Guerriero et al. Hepatoma Res 2019;5:6 I http://dx.doi.org/10.20517/2394-5079.2018.108

Although there are small amounts of nucleic acids in the circulation and only a fraction originate from
tumor cells, the current technologies allow to pinpoint the changes induced by the presence of a tumor both
qualitatively and quantitatively. Deep sequencing-based approaches for high-throughput and quantitative
PCR analyses for targeted investigations demonstrated the appropriate ability to highlight such decisive
traces of disease. However, it is clear that there are differences deriving from the analysis of DNA or RNA in
the circulation.

From this point of view, DNA analysis is simpler and provides a straightforward interpretation: the
presence, even in traces, of genetic or epigenetic alterations typically associated with a neoplastic disease
provides a tangible sign of the presence of tumor cells. However, ctDNA analysis can also provide
additional information. In fact, the technologies either based on NGS or ddPCR are quantitative and
therefore allow to monitor the quantitative evolution of genetic or epigenetic alterations over time, thereby
providing an assessment of therapy effectiveness. Furthermore, it is possible that distinct tumor clones may
differentially respond to therapy. The mutational analysis of ctDNA offers the possibility of highlighting
tumor heterogeneity, that tissue biopsy does not allow, thus making possible the detection of tumor clones
differentially responsive to therapy. In this regard, identification of mutations in specific “actionable” genes
offers the clinician the possibility of using targeted therapies if a molecular target is spotted. At present,
the situation in HCC is limited, but auspiciously in evolution. Although sorafenib and regorafenib were
the only few available options in advanced HCC until now, two new kinase inhibitors, namely lenvatinib
and cabozantinib, have been recently approved by FDA for first-line and second-line treatment [108,109] .
Unfortunately, an important limitation is that none of these drugs is associated with specific molecular
alterations. As seen in other tumor types, it is however reasonable to expect that molecular subgroups of
HCCs might be recognized for their differential responsiveness to specific targeted therapies. From these
considerations, it is therefore clear that ctDNA analysis, which widely surpasses AFP and DCP circulating
biomarkers in many aspects, can find clinical application for the management of HCC patients. It possibly
presents a limitation, namely the possibility of detecting the presence of gene mutations in the early stages of
disease, which can be difficult due to the very little amount of DNA released by small tumors. Overcoming
this current limit will certainly be a goal of future research. Another limitation in HCC is the availability of
a limited number of effective drugs against the most frequently mutated genes in HCC, such as catenin, as
currently there are no target drugs capable of acting against the encoded oncoproteins.

The results from circulating RNA studies have probably a different conceptual meaning and so far the
produced outcomes are not yet ready for clinical use. From a practical point of view, unlike genetic or
epigenetic DNA-based tests that display characteristics intrinsically distinctive of neoplastic cells, in the
case of circulating RNAs, they are also present in circulation of unaffected individuals and differences with
cancer patients are exclusively quantitative. This indicates that for clinical application it will be necessary to
define significance thresholds and appropriate methods to obtain reliable data. Quantitative PCR methods
can be easily applied to diagnostic applications and, at present, ddPCR emerged as a robust method to
quantify circulating miRNAs [37,39,40,110,111] .

In summary, the analysis of circulating miRNA/lncRNA is potentially useful in different phases of the
disease including early stages and the ddPCR method is optimally effective for performing the analysis.
However, the approach is at present immature for clinical use both for methodological and scientific issues
that need to be solved. Both pre-analytical and analytical procedures need to be standardized to guarantee
solid results from independent laboratories [112] . On the scientific side, the levels of circulating RNAs show
a normal variability among individuals. To diagnose HCC is essential to reaching an understanding of
the variability in circulating miRNA/lncRNA levels not only in healthy individuals, but also and more
importantly in patients affected by chronic liver diseases, such as chronic HBV/HCV infection, alcoholic and
non-alcoholic fatty liver disease, steatohepatitis and cirrhosis. To this end, large study cohorts are needed [113] .