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

peripheral blood of patients with predominantly HBV-positive and BCLC stage A showed that 56% of
[46]
the patients harbored ctDNA containing these mutations . TERT promoter mutations, however, have
been observed in both HCC patients as well as non-HCC cirrhotic patients, suggesting limited utility as a
biomarker for HCC [50-52] . On the other hand, the TP53 p.R249S mutation appears specific for HCC and has
been identified in plasma, serum and urine samples obtained from cancer patients [53-57] . The TP53 p.R249S
mutation is more common in HBV- and aflatoxin-associated HCC, compared to HCC associated with
other etiologies. ctDNA in 14 patients with advanced HCC using next generation sequencing showed that
somatic CTNNB1 mutations were the second most common mutation and occurred in 29% of the patients
[58]
studied .

The significant heterogeneity of HCC genetics in association with different etiologies (for instance alcohol
related liver disease vs. HBV vs. HCV vs. NAFLD) has posed a major challenge to the development of a
universal biomarker panel for detecting HCC. This challenge necessitates the integration of multiple genes
and multiple loci within a given gene, as well as combining a vast array of protein and genetic biomarkers.
CancerSEEK is a recently developed blood test which detects eight tumor-associated protein biomarkers
[59]
and mutations (including single base substitutions) in 1933 distinct genomic positions . The test was used
to query peripheral blood derived from 812 healthy controls and 192 non-metastatic cancers of the breast,
colorectum, esophagus, liver, lung, ovary, pancreas and stomach . Among 44 patients with HCC, the test
[59]
a showed 98% sensitivity and 99% specificity in cancer detection. Overall, the test detected five cancer types
with sensitivities ranging from 69% to 98%, and with over 99% specificity. The performance of CancerSEEK
in differentiating HCC patients from other high risk patients, for instance those with advanced fibrosis or
cirrhosis, is yet to be studied.

Chromosomal rearrangements
[60]
Genomic sequencing has identified a number of chromosomal rearrangements in HCC. Ono et al.
determined cancer-associated genomic rearrangements in HCC tumors through whole-genome
sequencing. Subsequently, they validated some of these rearrangements by means of PCR using ctDNA
isolated pre-operatively from peripheral blood of HCC patients and primers designed to detect the
breakpoints of chromosomal rearrangements seen in tumor tissue. The authors found that pre-operative
ctDNA from 7 HCC patients showed several deletions, inversions, tandem duplications and translocations
[60]
seen in HCC tumor tissue . Chromosomal rearrangements can lead to copy number variations and other
genetic aberrations, potentially serving as an early noninvasive marker for HCC.

Copy number variations
Shotgun massively parallel sequencing (MPS) was used to determine tumor-associated copy number
variations in the tumor tissue of 4 HCC patients, and in their plasma pre- and post-resection of tumor,
[35]
compared to 16 healthy controls . Characteristic copy number variations in tumor tissue were reflected
in pre-resection plasma samples, and were missing almost entirely in post-resection plasma samples.
The pre-resection plasma samples detected approximately 10% to 100% of tumor-associated copy
number aberrations seen in their corresponding tumor tissue, with detectability of plasma copy number
[35]
aberrations strongly correlating with plasma ctDNA concentration . In another study, MPS analysis of
plasma ctDNA size in 90 HCC patients compared to patients with chronic hepatitis B (n = 67), hepatitis
B-associated cirrhosis (n = 36), and healthy controls (n = 32) showed that HCC plasma carried high levels
[36]
of aberrantly short and long DNA . The short ctDNA preferentially carried tumor-associated copy
number aberrations. Among the 90 HCC patients, 76 (84%) had at least one chromosomal arm-level copy
number aberration on chromosomes 1 or 8. In addition, plasma derived from HCC patients contained
[36]
high levels of mitochondrial DNA albeit much shorter than nuclear DNA . The observation that cfDNA
in HCC patients are shorter and more fragmented than in patients without liver disease or with non-
malignant liver processes has been made by several other investigators [36,61] . This observation is worthy of