Page 64 - Read Online

Page 8 of 17 Rojas et al. Hepatoma Res 2018;4:31 I http://dx.doi.org/10.20517/2394-5079.2018.60

cancer liver tissue of HCC, in plasma from healthy people, indicating that this mutation is not exclusive of
HCC [84,85] . In addition, rs894151 or rs12428080 were found significantly associated with a decreased overall
survival and time to recurrence after liver transplantation [86,87] . Using digital droplet PCR, ctDNA mutations
in TERT promoter, CTNNB1 and TP53 could be detected in a higher rate (56%) compared to the use of NGS
(20%). A recent pilot study of ultra-deep targeted sequencing of plasma DNA identifies driver mutations and
it demonstrates how ultra- deep targeted sequencing of cfDNA in the plasma of HCC patients is a feasible,
reliable and minimally invasive approach to interrogate HCC genetics and emerges as a promising tool for
[88]
predictive biomarker development in HCC .

DNA methylation is also an important epigenetic aberration found in ctDNA with a great application in
the diagnosis, prognosis, and effective evaluation of HCC. Another tumor suppressor gene, Ras association
domain family protein 1A (RASSF1A) was found hypermethylated in 93% HCC patients compared to
healthy people being this frequency similar to the one found in RASSF1A hypermethylation in HCC tumor
[89]
tissues . Moreover, the combination of several methylations has been postulated in order to improve the
[90]
specificity and efficacy for the early diagnosis . The plasma methylation levels of APC, GSTP1, RASSF1A,
and SFRP1 were significantly higher in HCCs than those in normal or benign controls (P < 0.05). The
combination of these four genes resulted in an increased AUC of 0.933 with 92.7% sensitivity and 81.9%
specificity in discriminating HCC from normal control and GSTP1 hypermethylation was significantly
correlated with elevated serum AFP levels (P = 0.026).

Finally, the clinical significance of ctDNA as a diagnostic and predictive biomarker in HCC patients should
be further evaluated. ctDNA may be quite low and therefore below the limit of detection, especially in early‐
stage and indolent tumors. The improvement of different technics such as digital PCR and sequencing
technologies provide us an effective way for the discovery of additional ctDNA markers [91,92] .

Circulating non-coding RNA
In the context of liquid biopsy, non-coding RNA (ncRNA) is also included. The number of ncRNA genes
is increasing due to the development of high-throughput RNA sequencing technology. They have a role
in several physiological and pathological processes such as cancer. The main feature is their lack to codify
for proteins. Depending on the length, ncRNAs could be classified into short or long ncRNAs with an
arbitrary size cut-off at 200 bases of length, being the most known (a) the long-ncRNA (lncRNA) and (b) the
microRNA (miRNA).

lncRNAs function take place by different molecular mechanisms such as interactions with DNA, RNA and
proteins and can be classified into oncogenic or tumor suppressive genes [93-98] . Besides lncRNAs, miRNAs
are endogenous small RNAs molecules with 20-25 bases of length. They are involved in multiple activities
of mammalian cells like lncRNA but its function is to regulate gene expression through their binding
to the 3´UTR of mRNAs and consequently degradation or translational suppression of targeted gene
[99]
transcript . Both lncRNAs and miRNAs are often deregulated in liver cancer and it has been reported the
existence of circulating particle shape ncRNA in the peripheral blood. A group of ncRNA is packaged into
small membrane vesicles called exosomes, binding to lipoprotein or other proteins in order to increase its
stability [100-103] . In fact, the possibility that circulating ncRNA could be useful as a biomarker in the context
of HCC is raising. Interestingly, modified circulating levels of these RNAs were repeatedly found in HCC
patients.

Circulating lncRNAs
lncRNAs have emerged as important regulators of gene expression in many types of cancer including
HCC [104,105] . Alterations in expression of several lncRNAs have been recently reported in HCC.

Serum levels of lncRNA-uc003wbd and lncRNA-AF085935 were found upregulated in HCC and HBV