Hindawi Publishing Corporation

International Journal of Hepatology

Volume 2012, Article ID 859076, 7 pages
doi:10.1155/2012/859076

Review Article
Biomarkers for Hepatocellular Carcinoma

Tara Behne1 and M. Sitki Copur1, 2

1 College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198, USA
2 Saint Francis Cancer Treatment Center, Grand Island, NE 68803, USA

Correspondence should be addressed to M. Sitki Copur, [email protected]

Received 16 October 2011; Accepted 27 February 2012

Academic Editor: Neil Guha

Copyright © 2012 T. Behne and M. S. Copur. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

The hepatocellular carcinoma (HCC) is one of the most common malignant tumors and carries a poor survival rate. The
management of patients at risk for developing HCC remains challenging. Increased understanding of cancer biology and
technological advances have enabled identification of a multitude of pathological, genetic, and molecular events that drive
hepatocarcinogenesis leading to discovery of numerous potential biomarkers in this disease. They are currently being aggressively
evaluated to establish their value in early diagnosis, optimization of therapy, reducing the emergence of new tumors, and
preventing the recurrence after surgical resection or liver transplantation. These markers not only help in prediction of prognosis or
recurrence but may also assist in deciding appropriate modality of therapy and may represent novel potential targets for therapeutic
interventions. In this paper, a summary of most relevant available data from published papers reporting various tissue and serum
biomarkers involved in hepatocellular carcinoma was presented.

1. Introduction HCC is the fifth most common malignant tumor and

the third leading cause of cancer-related deaths. Worldwide,
As molecular indicators of biological status, biomarkers, there are about 626,000 new HCC cases and nearly 600,000
detectable in blood, urine, or tissue, can be useful for the clin- HCC-related deaths each year with an incidence equal to the
ical management of various disease states. Threshold concen- death rate [1, 2]. Although the molecular mechanisms by
trations can be utilized to identify the presence of various which HCC develops remain largely unclear, a multitude of
diseases. Concentration fluctuations have the potential to pathological, genetic, and molecular events that drive hepa-
guide therapy in disease progression. Numerous biomarkers tocellular carcinogenesis has been identified.
have been identified for various disease states. Research is Current gold standard and most commonly used bio-
ongoing to fully understand and evaluate the clinical signifi- markers for patients at risk for HCC, alpha-fetoprotein
cance of utilizing biomarkers. Time and money can be (AFP) along with ultrasound every 6 to 12 months, is far
saved by avoiding empiric or broad treatment approaches from perfect. Serum AFP levels of more than 400 ng/mL
to diseases of particular organs or systems, and ideally, bio- are considered diagnostic; however, such high values are
markers could serve as a measurement tool to detect dis- observed only in a small percentage of patients with HCC.
ease presence and progression and to guide more targeted Ultrasound surveillance even performed at every three
therapy. Many disease states, especially various types of can- monthly intervals cannot improve detection of small HCC
cer, can be better understood by the utilization of tumor bio- because of limitations in recall procedures [3, 4].
markers. Hepatocellular carcinoma (HCC) is one such can- With advances in understanding of tumor biology, along
cer that can benefit from tumor biomarkers’ diagnostic, ther- with the development of cellular and molecular techniques,
apeutic, and prognostic capabilities. the role of biomarkers related to early detection, invasiveness,
2 International Journal of Hepatology

metastasis, and recurrence has attracted great deal of research Table 1: Diagnostic values of HCC serum markers [12–14].
interest resulting in discovery and utilization of several
Sensitivity Specificity
novel markers in this disease. In this paper we try to give Type of test
(%) (%)
an overview of available data on this burgeoning area of
research. AFP-L3 61.6 92.0
DCP 72.7 90.0
AFP 67.7 71.0
2. Biomarkers for Liver Cancer AFP-L3 + DCP 84.8 97.8
AFP-L3 + AFP 73.7 86.6
2.1. Oncofetal and Glycoprotein Antigens
DCP + AFP 84.8 90.2
2.1.1. Alpha-Fetoprotein (AFP). The first serologic assay for AFP-L3 + DCP + AFP 85.9 59.0
detection and clinical followup of patients with hepatocellu-
lar carcinoma was alpha-fetoprotein (AFP) which has been
the standard tumor biomarker for HCC for many years. It is or moderately differentiated HCC, and the simultaneous
a glycoprotein produced by the fetal liver and yolk sac during determination of both markers improves overall sensitivity
pregnancy. Serum AFP levels are often elevated in HCC, from 50% to 72%. Recently, a study compared the survival
but this is not always the case. AFP levels may be elevated rate between the GPC3-positive and GPC3-negative HCC
initially in the early stages of HCC and then drop or even patients. GPC3 positivity correlated with poor prognosis and
normalize before rising again as disease progression occurs identified as an independent prognostic factor for the overall
[5]. Additionally, AFP elevation has also been recognized survival on multivariate analysis [11].
in the presence of acute and chronic viral hepatitis as well
as in patients with cirrhosis caused by hepatitis C. Given
2.2. Enzymes and Isoenzymes
the multiple indications that present with elevated AFP
levels, it is necessary to evaluate the significance of serum 2.2.1. Des-Gamma-Carboxy (Abnormal) Prothrombin (DCP).
concentrations. In general, consistently elevated serum AFP DCP is produced by the malignant hepatocyte and appears
levels greater than 500 ng/mL are indicative of HCC. Lower to result from an acquired posttranslational defect in the
serum concentrations which are only transient in nature are vitamin-K-dependent carboxylase system. DCP production
more often present in benign liver disease [6]. If a patient has is independent of vitamin K deficiency, although phar-
known risk factors for HCC, such as the presence of cirrhosis, macological doses of vitamin K can transiently suppress
increasing levels of AFP have been shown to correlate with DCP production in some tumors. DCP levels greater than
the development of HCC [6]. Unfortunately, AFP serum 0.1 AU/mL (100 ng/mL) on ELISA are highly suggestive of
concentrations do not correlate well with the prognostic val- HCC or tumor recurrence. Normalization of DCP levels
ues of HCC such as tumor size, stage, or disease progression, correlates well with successful tumor resection and appears
and ethnic variability may also exist. Furthermore, in some to be an excellent marker of tumor activity. It is thought that
cases of HCC, AFP elevations are not apparent at all [7]. Total the combination of AFP and DCP assays will increase the
AFP can be divided into three different glycoforms, AFP-L1, sensitivity of testing. The correlation between tumor size and
AFP-L2, and AFP-L3-based on their binding capability to DCP levels is not yet clearly defined. It appears that there
lectin Lens culinaris agglutinin (LCA). High percentage is a correlation in DCP levels and large tumors; however,
of AFP-L3 has been shown to be associated with poor dif- the same is not the case in small tumors (<3 cm) [15].
ferentiation and biologically malignant characteristics, worse A cross-sectional case control study involving 207 patients
liver function, and larger tumor mass [8]. determined that DCP is more sensitive and specific than AFP
for differentiating HCC from nonmalignant liver disease. In
2.1.2. Glypican-3. Glypican-3 (GPC3), a membrane-an- this study there were 4 groups studied: normal healthy sub-
chored heparin sulfate proteoglycan, has been demonstrated jects; patients with noncirrhotic chronic hepatitis, patients
to interact with growth factors and modulate their activities. with compensated cirrhosis, and patients with histologically
It binds to the cell membrane through the glycosylphos- proven HCC. Both DCP and AFP levels increased among the
phatidylinositol anchors. GPC3 mRNA was upregulated sig- groups as disease severity increased (from normal to HCC),
nificantly in tumor tissues of HCC compared to paraneoplas- but DCP values had less overlap among the groups than AFP.
tic liver tissue, liver tissues of healthy adults, and liver tissues Study results concluded that a DCP value of 125 mAU/mL
of patients with nonmalignant hepatopathy. The expression yielded the best sensitivity and specificity for differentiating
of GPC3 (at both mRNA and protein levels) in the serum of patients with HCC from those with cirrhosis and chronic
HCC patients was significantly higher than that in the serum hepatitis [16]. Sensitivity and specifity of total AFP, AFP
of healthy adults or patients with nonmalignant disease. It glycoforms, DCP, and combinations of both markers have
can be detected in 40–53% of HCC patients and 33% of been summarized in Table 1.
HCC patients seronegative for both AFP and Des-gamma-
carboxyprothrombin (DCP) [9, 10]. It has been shown 2.2.2. Gamma-Glutamyl Transferase. Serum gamma-gluta-
that soluble GPC3 (sGPC3), the NH2 -terminal portion of myl transferase (GGT) in healthy adults is mainly secreted
GPC3, is superior to AFP in the sensitivity of detecting well by hepatic Kupffer cell and endothelial cell of bile duct,
International Journal of Hepatology 3

and its activity increases in HCC tissues. Total GGT can be [24]. Serum TGF-beta level has been found to be elevated in
divided into 13 isoenzymes by using polymer acrylamide HCC patients compared to healthy adults or patients with
gradient gel electrophoresis, and some of them can only nonmalignant liver disease [25–27].
be detected in the serum of HCC patients. Sensitivities of
GGTII have been reported to be 74.0% in detecting large 2.3.2. Tumor-Specific Growth Factor (TSGF). Malignant tu-
HCC and 43.8% in detecting small HCC. Sensitivity can be mors release tumor-specific growth factor (TSGF) into
significantly improved with the simultaneous determination peripheral blood during their growing period. Serum levels
of GGTII, DCP, and AFP [17]. of TSGF may reflect the existence of tumor. TSGF can be used
as a diagnostic marker in detecting HCC, and its sensitivity
2.2.3. Serum Alpha-1-Fucosidase. Alpha-l-fucosidase (AFU) can reach 82% at the cut-off value of 62 U/mL and may have
is a lysosomal enzyme found in all mammalian cells with a higher accuracy with the simultaneous determination of
a function to hydrolyze fucose glycosidic linkages of gly- other tumor markers. The simultaneous determination of
coprotein and glycolipids. Its activity increases in the se- TSGF (at the cut-off value of 65 U/mL), AFP (at the cut-off
rum of HCC patients (1418.62 ± 575.76 nmol/mL/h) com- value of 25 ng/mL), and serum ferritin (at the cut-off value
pared with that in the serum of healthy adults (504.18 ± of 240 ng/mL) can reach a sensitivity and specificity of 98.4%
121.88 nmol/mL/h, P < 0.05), patients with cirrhosis and 99%, respectively [26].
(831.25 ± 261.13 nmol/mL/h), and patients with chronic
hepatitis (717.71 ± 205.86 nmol/mL/h). It has been reported 2.3.3. Epidermal Growth Factor Receptor Family. The epider-
that the sensitivity and specificity of AFU at the cut-off mal growth factor receptor (EGFR) family consists of four
value of 870 nmol/mL/h were 81.7% and 70.7%, respectively closely related transmembrane tyrosine kinase receptors:
[18]. AFU measurement is useful in association with AFP EGFR (erbB-1), c-erb-2 (Her-2/neu), c-erb-3 (HER-3), and
in early diagnosis of HCC and could serve as a valuable c-erb-4 (HER-4). These bind with ligands of the EGF
supplementary to AFP. It has been indicated that HCC will family, including EGF, TGF-alpha, and heparin-binding EGF.
develop within few years in 82% of patients with liver cir- High levels of EGFR expression have been associated with
rhosis, if their serum AFU activity exceeds 700 nmol/mL/h. early recurrence and reduced disease-free survival following
The activity of AFU was reported to be elevated in 85% of resection of hepatocellular carcinoma [27].
patients at least 6 months before the detection of HCC by
ultrasonography [19]. 2.3.4. Hepatocyte Growth Factor/Scatter Factor. Hepatocyte
growth factor/scatter factor (HGF/SF) is a cytokine with a
2.2.4. Human Carbonyl Reductase 2. This enzyme expressed wide range of effects from embryonic development and liver
in the human liver and kidney is important in detoxification regeneration. It is associated with molecular mechanisms
of the reactive alpha-dicarbonyl compounds and reactive of hepatocarcinogenesis via paracrine system involving its
oxygen species deriving from oxidative stress in HCC. The cellular receptor, c-met. High c-met expression has been
human carbonyl reductase 2 levels have been shown to be shown in invasive-type HCC and has been associated with
inversely correlated to the pathological grading of HCC [20]. metastasis and reduced overall survival [28, 29].

2.2.5. Golgi Phosphoprotein 2. Golgi phosphoprotein 2 2.3.5. Basic Fibroblast Growth Factor. This is a soluble hepa-
(GOLPH2), a Golgi-apparatus-associated protein, has been rin-binding polypeptide with a potent mitogenic effect on
shown to have a higher sensitivity than AFP in the detection endothelial cells. Elevated levels above the median of
of HCC [21]. A recent study found that GOLPH2 protein >10.8 pg/mL have been shown to predict decreased disease-
was highly expressed in tissues of HCC (71%) and bile free survival [30]. Recent preliminary data with targeted
duct carcinoma (85%) patients. GOLPH2 protein levels were therapy lenalidomide which inhibits fibroblast growth factor
detectable and quantifiable in sera by ELISA. In patients (FGF) showed promising and in some patients dramatic
with hepatitis C, serial ELISA measurements in the course of activity in HCC patients [31].
the disease appear to be a promising complimentary serum
marker in the surveillance of HCC [22].
2.4. Molecular Markers

2.3. Growth Factors and Their Receptors 2.4.1. Circulating Nucleic Acids: mRNAs. The analysis of cir-
culating nucleic acids in plasma offers another avenue for
2.3.1. Transforming Growth Factor-Beta (TGF-Beta). Belong- noninvasive monitoring of a variety of physiological and
ing to a superfamily of polypeptide signaling molecules in- pathologic conditions [30, 31]. Numerous applications based
volved in regulating cell growth, differentiation, angiogen- on the detection of circulating cell-free nucleic acids in
esis, invasion, and immune function, TGF-beta is a predomi- human plasma have been reported for the management of
nant form of growth factor family in humans. Its mRNA malignancies. The fundamental principle underlying these
and protein are overexpressed in HCC compared with sur- applications relates to the detection in plasma of extracel-
rounding liver tissues, especially in small and well-differen- lular nucleic acid molecules derived from diseased organs.
tiated HCCs [23]. However, no relationship has been shown Analysis of cell-free plasma RNA offers an opportunity for
between TGF-beta expression and posthepatectomy survival the development of pathology-related markers [32–34].
4 International Journal of Hepatology

Alpha-Fetoprotein mRNA (AFP mRNA). Matsumura et al. than cell-based assays (circulating tumor cells) in detection
first reported that single HCC cell could be detected in cir- of preneoplastic lesions and micrometastases as plasma levels
culation by means of reverse-transcription polymerase chain of circulating cancer-derived nucleic acid are higher than
reaction (RT-PCR), targeting AFP mRNA [35]. This led to the levels of circulating cancer cells and are less prone
further reports of the value of AFP mRNA as a predictor for to sampling errors. Cheung and colleagues studied the
HCC recurrence. Rather controversial results were attributed preoperative plasma samples obtained from 72 HCC patients
to the blood borne dispersion of both tumor cells and normal who had undergone liver transplantation and found that
liver cells and the mistranscription of mRNA encoding patients with plasma albumin mRNA level (>14.6) had a
AFP by peripheral mononuclear cells. The recurrence-free significantly higher recurrence rate on multivariate analysis.
interval of HCC patients with postoperative serum AFP High plasma albumin mRNA level predicted the 2-year
mRNA positivity has been reported to be significantly shorter recurrence rate with sensitivity and specificity of 73% and
than that of HCC patients with postoperative negativity 70%, respectively [43].
(53% versus 88% at 1 year, 37% versus 60% at 2 years,
P = 0.014) [34] and (52.6% versus 81.8% at 1 year, 15.6%
versus 54.5% at 2 years, and 0% versus 29.2% at 3 years, MicroRNAs (miRNAs). MicroRNAs (miRNAs) are a family
P < 0.001) [36]. A meta-analysis showed that the expression of endogenous, small (21–23 nucleotides), noncoding but
of AFP mRNA one week after surgery was correlated with the functional RNAs, which have been found in worms, flies,
recurrence of HCC [37]. and mammals including human beings [44]. It is estimated
that there are about 1,000 miRNA genes in the human
Gamma-Glutamyl Transferase mRNA (GGT mRNA). Similar genome with approximately 500 miRNA genes being already
to AFP, GGT mRNA can be detected in the serum and liver identified [45]. Similar to mRNA, HCC-associated miRNAs
tissues of healthy adults, patients with liver disease, benign could be used as diagnostic and prognostic biomarkers of
liver tumor, HCC, and secondary tumors of the liver [38]. HCC with a potential for even greater accuracy. MiRNAs
The two types of GGT mRNA, type A and type B, have been can accurately predict whether liver cancer will spread and
identified. Type B is the predominant one in cancerous tissue whether liver cancer patients will have shorter or longer
suggesting that changes in the expression of hepatic GGT survival. MicroRNAs regulate gene expression by binding to
mRNA may be related to the development of HCC [39]. specific messenger RNAs and prevent their translation into
Patients with HCC harboring type B GGT mRNA both in protein. Because each type of miRNA is able to downregulate
cancer and in noncancerous tissue had a worse outcome, hundreds of genes at a time, they can control entire tran-
earlier recurrence, and more recurrence-related mortality. scriptional programs that determine fundamental cellular
The presence of type B GGT mRNA in cancerous tissue was properties and behavior. Accordingly, miRNA profiling has
statistically correlated with high serum level of AFP, daughter emerged as an extremely valuable method for phenotyping
nodules, higher postresection recurrence rate than those and subclassifying tumors [44]. Compared to conventional
without it (63.6% versus 14.3%), and lower postrecurrence gene expression profiling (in which protein-coding, mes-
survival. The presence of type B GGT mRNA in non- senger RNAs are examined), miRNA analysis has several
cancerous liver tissue was significantly correlated with hep- advantages. Due to the stability of miRNAs, formalin-fixed
atitis C infection, high serum level of AFP, absence of infil- samples (rather than frozen tissue) can be used. Further-
tration of capsule, vascular permeation, daughter nodules, more, the interrogation of hundreds of miRNAs (and often
postresection recurrence, and postrecurrence survival [40]. significantly fewer) yields as much information as might be
gleaned from examining thousands of messenger RNAs.
Many independent groups have conducted compre-
Insulin-Like Growth Factor II (IGF-II) mRNA. Abnormal hensive analyses of miRNAs in HCC, and a plethora of
expression of IGF-II mRNA can be a useful tumor marker information on miRNA markers has been identified. Many of
for diagnosis, differentiation, extrahepatic metastasis, and these miRNA signatures correlate with important biological
monitoring of postoperative recurrence in HCC. The deter- parameters, such as metastasis [46–48], differentiation [49–
mination of serum insulin-like growth factor-II (IGF-II) (at 51], HBV or HCV infection [52, 53], tumor recurrence
the cut-off value of 4.1 mg/g, prealbumin) has a sensitivity [54], and patient survival [55, 56]. Some miRNAs are
of 63%, specificity of 90%, and accuracy of 70% in the involved in HCC carcinogenesis by promoting cancer stem
diagnosis of small HCC [41]. It can be a complementary cell and by controlling cell proliferation and apoptosis;
tumor marker to AFP for diagnosis of small HCC. The others are associated with HCC progression by controlling
simultaneous determination of IGF-II and AFP (at the cut- cell migration and invasion. These HCC-associated miRNAs
off value of 50 ng/mL) can improve the sensitivity to 80% and not only provide new insights into the molecular basis
accuracy to 88% [42]. of HCC but also serve as new tools for HCC diagnosis
and prognosis. Currently a few miRNA signatures, however,
Albumin mRNA. Albumin is the most abundant protein could potentially be used in this area. Some miRNAs have
in the body synthesized by the liver. mRNA of albumin is been validated in an independent cohort, paving the way for
detectable in human plasma and could be a diagnostically clinically useful platforms to assess HCC risk and outcome.
sensitive marker for liver pathologies. Extracellular-based This promising area of research awaits further validation in
assays (circulating DNA/RNA) have been found to be better prospective studies [57].
International Journal of Hepatology 5

Table 2: Various HCC biomarkers and their clinical use.

HCC marker Clinical use
Alpha-fetoprotein Early diagnosis, monitoring, and recurrence
Lens culinaris agglutinin reactive AFP (AFP-L3%) Early diagnosis and prognosis, vascular invasion
Des-gamma-carboxy prothrombin (DCP) Early diagnosis and prognosis, portal vein invasion and metastasis
Gamma-glutamyl transferase Early diagnosis complementary to other markers
Alpha-l-fucosidase Early diagnosis
Glypican-3 Early diagnosis
Human carbonyl reductase 2 Prognosis
Golgi phosphoprotein 2 Tumor aggressiveness
Transforming growth factor beta Tumor invasiveness
Hepatocyte growth factor (HGF) Prognosis and disease recurrence
Transforming growth factor-b (TGF-b) Prognosis invasiveness
Tumor specific growth factor Diagnosis complementary to other markers
Epidermal growth factor receptor family Early recurrence
Hepatocyte growth factor Metastasis reduced survival
Micro RNAs Tumor spread and survival

2.5. Pathological Biomarkers. Finally there have been reports research favors the circulating hepatoma-specific AFP sub-
of pathological biomarkers of HCC for diagnosis and pro- fraction AFP-L3 and DCP over AFP alone in differentiating
gnosis. Some of these diagnostic biomarkers focus on immu- HCC from nonmalignant hepatopathy and detecting small
nochemical staining patterns to distinguish high-grade dys- HCC. Furthermore, some other tumor markers, such as
plastic nodules and well-differentiated HCC. The best type GPC3, GGT II, AFU, have been shown to be supplementary
of immunostaining for this difficult condition has been to AFP and DCP in the detection of HCC. Some of them
reported to be the combination of heat-shock protein 70 even can be detected in HCC patients seronegative for
(HSP70), glypican-3 (GPC3), and glutamine synthetase both AFP and DCP, thus indicating that the simultaneous
(GS). For prognostic use a number of histological and immu- determination of these markers may improve the accuracy.
no-histochemical markers such as markers of cell prolifera- However, most exciting and promising area of research
tion (Ki67), apoptosis or cell survival (survivin), cell adhe- in this disease has been the identification of a new group of
sion molecules (E-cadherin), neoangiogeneis (VEGF), and molecules called miRNAs. MiRNAs have been discovered to
more have been looked in small studies showing promise; be aberrantly expressed in HCC, and some of them are func-
however, most of these markers have not been validated in tionally involved in HCC carcinogenesis and progression.
large studies [57]. Various HCC biomarkers and their clinical Furthermore, certain microRNAs are associated with HCC
use have been summarized in Table 2. or related to HCC subtypes, implicating the potential use of
microRNAs in HCC patient stratification of diagnosis and
prognosis. Some of these HCC-associated miRNAs have been
3. Discussion validated in independent cohorts. This brings the possibility
of developing clinically useful platforms to develop HCC
Hepatocarcinogenesis is a complex multistate process usually diagnosis, risk assessment, and patient risk stratification with
occurring after many years of chronic exposure to sev- the ultimate goal of personalized therapy.
eral mitogenic and mutagenic environments precipitating
random genetic alterations. Recent evidence suggest that
intrinsic biologic characteristics of the tumor in terms 4. Conclusion
of proliferation and invasiveness are probably related to
different composition and activity of the microenvironment, Research into the molecular biology of hepatocarcinogenesis
leading to very different clinical outcomes. HCC is rather has identified numerous biomarkers which could provide
unique with its ability to synthesize various tumor-related additional information for HCC biologic behavior metastasis
proteins rendering itself more suitable to biomarker-related and recurrence to that gained from traditional histopatho-
research than other tumors. Because of the large multitude of logical features. A large number of biomarkers have been
biomarkers reported in this disease, selecting the biomarkers shown to have potential predictive significance. However,
which would be most useful in clinical practice has been most of them have been studied retrospectively. Efforts
more than challenging. In this rather brief overview, we tried should be directed towards prospective clinical trials in eval-
to focus on most widely used and accepted biomarkers. uating the prognostic significance of these markers. These
Despite its limitations, serum AFP still remains the molecules not only help in prediction of prognosis for pa-
most widely used tumor marker in clinical practice. Recent tients with HCC but may also assist in deciding appropriate
6 International Journal of Hepatology

modality of therapy and represent novel targets for therapeu- [17] R. Cui, J. He, F. Zhang et al., “Diagnostic value of protein
tic interventions. induced by vitamin K absence (PIVKAII) and hepatoma-
specific band of serum gamma-glutamyl transferase (GGTII)
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