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Page 6 of 10 Banini et al. Hepatoma Res 2019;5:34 I http://dx.doi.org/10.20517/2394-5079.2019.30

further investigation and may have clinical utility in the diagnosis or monitoring of HCC either alone or in
combination with other biomarkers.

Loss of heterozygosity and microsatellite instability
[62]
Pang et al. used three high-polymorphic microsatellite markers located on chromosome 8p, D8S277,
D8S298 and D8S1771 to examine loss of heterozygosity (LOH) and microsatellite instability. By analyzing
plasma cfDNA and tumor tissues from 62 HCC patients, they examined the features of these aberrations
in peripheral blood and determined their concordance with tumor tissue. LOH in one or more of the
three examined loci was identified in about 58% of patients, occurring at a higher rate in those with
[62]
metastatic HCC (63%) compared to those with non-metastatic disease (26%) . Majority of patients
carried microsatellite instability in plasma samples at the same loci as their corresponding HCC tissues,
[63]
with a concordance rate of about 73% . Their findings suggest that LOH and microsatellite alterations
may potentially serve in non-invasive diagnosis of HCC, however these alterations generally occur less
commonly than the other genetic alterations discussed, and studies are needed to delineate the clinical
applicability of these observations.

Alterations in DNA methylation
DNA methylation, one of the earliest known and well-studied epigenetic modifications, confers changes in
chromatin structure, DNA stability and DNA-protein interactions to modify gene expression. Methylation
events occur very early in carcinogenesis hence are often detected in precancerous states. To date, several
studies have showed that altered DNA methylation at several genes are associated with the initiation
[68]
[69]
[67]
[64]
[65]
[66]
and progression of HCC, including p15 and p16 , APC , SPINT2 , SFRP1 , TFP12 , GSTP1 and
[70]
RASSF1A . NAFLD-related HCC is associated with hypermethylation of the glycine N-methyltransferase
[71]
(GNMT) promoter, resulting in reduced gene expression . Differential DNA hypomethylation has also
been seen in HCC. DNA hypomethylation is known to induce several processes leading to transposon
activation, chromosomal instability, and the generation of copy number variations. Hypomethylation of
repetitive DNA sequences by way of long interspersed nucleotide elements 1 (LINE-1) has been detected in
[72]
the plasma of patients with HCC . Concordance in the methylation profile of several tumor suppressor
genes between HCC plasma and tumor tissue has been demonstrated by several studies.
[37]
Wong et al. showed that 25% of patients with p15 methylation in tissue also demonstrated methylated p15
in blood samples, and nearly all patients with p15 and p16 methylation in tissues demonstrated methylation
abnormalities in blood samples. Importantly, patients with p15 and p16 methylation developed HCC
metastasis or recurrence after treatment, suggesting that analysis of p15/p16 methylation in cfDNA derived
from peripheral blood can serve as a biomarker for predicting the metastasis or recurrence of HCC.

[65]
Iyer et al. analyzed the tumor methylation profile of several tumor suppressor genes including APC,
FHIT and E-cadherin through analysis of plasma and corresponding tumor DNA from 28 HCC patients,
as well as plasma DNA from age and sex-matched controls. The analysis showed a statistically significant
concordance in methylation profile between plasma and corresponding tumor DNA for all genes analyzed.
The concordance for APC methylation in plasma DNA vs. HCC tumor tissue was almost 82%, with
sensitivity and specificity of 78% and 90%. For FHIT, the concordance, sensitivity and specificity were all
approximately 86%. For E-cadherin, concordance was 79%, with sensitivity and specificity of 68% and 100%.
As in other studies, p15 and p16 methylation patterns were also found to be concordant with sensitivities
ranging from 50%-60% and specificities in the 85%-95% range.

RASSF1A, a member of the Ras association domain family protein is a tumor suppressor frequently
silenced in malignancy by hypermethylation. Serum analysis showed that 90% of HCC patients and 62.5%
[73]
of HCV patients demonstrate RASSF1A hypermethylation, compared to 10% in healthy serum .