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

[31]
HCC patients . These results highlight the usefulness of cfDNA analysis for identifying actionable targets
and for stratifying patients according to the potentially most appropriate therapeutic approach.

The approach has also been employed to monitor therapy efficacy over time. The mutation analysis of 574
cancer genes applied to plasma cfDNA and matched HCC from four patients, demonstrated that 97% of
tumor alterations were present in the blood and that it was possible to assess tumor progression, to track
the possible sites of recurrence and understand tumor clonal dynamics in relation to sequential therapies.
The possibility to track tumor dynamics from plasma analysis provides a valuable strategy for monitoring
[32]
therapy efficacy and infer clinical outcomes , helping clinician modulate therapeutic approaches in a more
rational and proper direction.

MIRNAS
miRNAs are 20-24 nucleotides long RNAs. By interacting with homologous target mRNAs, they act by fine-
tuning gene expression through a post-transcriptional mechanism. Each tissue exhibits a unique profile of
miRNAs, which is altered in pathological conditions. In tumor tissues, miRNAs are aberrantly regulated
and it has been demonstrated that some deregulated miRNAs can act as oncogenes and others as tumor
[33]
suppressors . The interest in circulating miRNAs as non-invasive tumor biomarkers surfaced when their
presence was reported as stable molecules in serum or plasma of healthy individuals and cancer patients [34,35] .

Approaches for detection and quantification of circulating miRNAs
The most common technologies employed to measure miRNA expression in biological samples include
microarray, NGS, quantitative real-time PCR (RT-qPCR) and droplet digital PCR (ddPCR) [36-38] . Microarray
and NGS technologies are suitable for screening and discovery purposes, qPCR and ddPCR remain the
choices for validation and clinical tests development. Both microarrays and NGS provide high throughput
analysis of miRNA expression profiles. Microarrays can quantify all the known miRNAs. NGS can also
identify new miRNA species and differentiate closely related sequences. NGS can also detect miRNA length
[38]
variation (isoforms of miRNA) . qPCR and ddPCR are not high throughput technologies, but technology
is relatively inexpensive, available in most laboratories and can offer higher sensitivity by exploiting
amplification steps.

Among quantitative PCR approaches, ddPCR was shown to be superior to conventional real time qPCR
for quantifying circulating miRNAs, as it allowed an easier absolute quantification of circulating RNAs
without requiring an internal standard for normalization. Furthermore, ddPCR proved to be more tolerant
[39]
than real time qPCR to the presence of inhibitors . Finally, ddPCR generally exhibits a higher precision
and reproducibility than real time qPCR, thus allowing an easier discrimination between cases and
controls [37,40,41] .

Circulating miRNAs for HCC diagnosis
Circulating miRNAs have been tested for their ability of discriminating HCC patients from control
individuals [Table 2]. As shown in Table 2, however, it is evident that published studies are heterogeneous as
they often differ for technical characteristics and experimental design. This heterogeneity makes it difficult
to compare results and limits their transferability into applications of clinical interest.

A first source of heterogeneity is given by the use of serum or plasma for measuring circulating miRNA
levels. Albeit early studies reported that composition and levels of miRNAs in serum and plasma are
[42]
[35]
similar , there are several examples that subsequently contradicted this idea. Heegaard et al. tested
miRNA levels in paired serum and plasma samples of lung cancer patients and they concluded that these
apparently similar sources of circulating miRNAs exhibit very different miRNA levels. Supporting this
conclusion in liver cancer patients, miR-223-3p was found consistently low in plasma of HCC patients [43,44]
but the same miRNA was high in the serum [45,46] ; miR-21 was found low in serum of HCC patients in