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Rojas et al. Hepatoma Res 2018;4:31 I http://dx.doi.org/10.20517/2394-5079.2018.60 Page 7 of 17

In addition, these authors showed that most of the patients with an elevated CTC level at the time of disease
imaging reassessment showed disease progression after TACE or radiotherapy, whereas patients with stable
[65]
or deceasing CTC levels showed tumor remission or stable disease .
Due to this fact, researchers suggested a combination of antibodies against a variety of surface markers on
CTCs in order to avoid the loss of CTCs during the isolation. For this reason, the CTC-chip, based on the
microfluidic procedure with higher sensitivity and specificity in CTCs purification (99.1% and specificity
100% of the CTC-chip across all five cancers; metastatic lung, prostate, pancreatic, breast and colon cancer),
[66]
is standing out intensely in this field .

CTCs detection in HCC patients has been reported in several studies [67,68] . The numbers of CTCs were
closely correlated with portal vein thrombosis, tumor infiltration, prognosis and Child-Pugh grade [61,64,69] .
Most of the studies have shown that before liver resection or transplantation, tumor cells from the primary
lesions were detached and threw into the blood being the early event of HCC metastases. Fan et al [69,70]
reported that the tumor recurrence after resection was associated with the number of CTCs detected, maybe
because CTCs were still surviving into the blood [70-72] . However, the role of CTCs and HCC recurrence
require further investigations. The enumeration and characterization of CTCs may become an indispensable
biomarker for monitoring the efficacy of HCC treatments however, clinical application of CTC assay in HCC
remains in the initial stage, especially in the field of early diagnosis.

Cell-free tumor associated DNA
Circulating cell-free DNA (cfDNA) is defined as extracellular DNA present in plasma or serum samples.
cfDNA is released into circulation from cells that undergo metabolic secretion, apoptosis or necrosis. Cells
are phagocytized by macrophages releasing digested DNA into the circulating system. Tumor cells are
considered to be the major source of tumor-related cfDNA in blood of cancer patients [73,74] . cfDNA is also
detected in healthy patients but in patients suffering from cancer cfDNA carries tumor-specific genetic or
epigenetic alterations, such as mutations, copy number variations, chromosomal rearrangements or DNA
[54]
methylations among others . Compared to tissue biopsy, circulating tumor DNA (ctDNA) may represent
the entire molecular biology of the tumor and its qualitative and quantitative analysis might help to assess
the biological characteristics of the tumor. Recently, several studies have demonstrated that ctDNA could be
a non-invasive potential biomarker [54,75] . ctDNA is highly specific and could be detected easier compared to
CTCs purification, thus it could be an ideal source for the early diagnosis or as recurrence biomarker [76,77] .

To study the ctDNA in plasma or serum two strategies are implemented: (a) measuring the quantity or (b)
detecting tumor - specific genetic aberrations.

Several studies have shown that HCC patients have large amounts of cfDNA being these associated with
[77]
the degree of malignancy (poorer prognosis) and size of the tumor size [77,78] . Huang et al. showed that
plasma DNA detection was able to discriminate HCC from normal controls with 90.2% sensitivity and 90.3%
specificity and AUC was 0.949 (95% CI) (measured by real-time quantitative PCR method). Moreover, plasma
DNA and serum AFP revealed an elevated AUC of 0.974 with 95.1% sensitivity and 94.4% specificity in
discriminating HCC from normal controls. Furthermore, the plasma DNA levels were positively associated
[77]
with tumor size and vascular invasion (P = 0.012 and P = 0.035 respectively) . However further studies are
needed due to the controversial results related to the methodology used.

Changes of DNA mutations could play an important role in the carcinogenesis process [79-82] . By now, several
studies confirmed that TP53, EFGR, KRAS and APC are genes with common tumor specific mutations.
The proportion of HCC patients with detectable ctDNA varies wildly between studies. Tumor suppressive
[83]
gene TP53 mutations such as Ser249, were found present in 50% of HCC patients exposed to aflatoxin .
However, Ser249 of TP53, one of the most reported mutations in HCC patients, was also detected in non-