Serum markers of hepatocellular carcinoma.
ABSTRACT The hepatocellular carcinoma is one of the most common malignant tumors and carries a poor survival rate. The management of patients at risk for developing HCC remains intricate.
A literature search identified potential markers for hepatocellular carcinoma. These markers were analysed and justification was provided for these factors' inclusion to (or exclusion from) the markers of hepatocellular carcinoma (HCC). A search of the literature was made using cancer literature and the PubMed database for the following keywords: "markers and HCC," "Lens culinaris agglutinin reactive AFP (AFP-L3) and HCC," "Des-γ-carboxy prothrombin (DCP) and HCC," "Glypican-3 and HCC," "Chromogranin A and HCC," "Transforming growth factor β1(TGF) and HCC," "α-l-fucosidase (AFU) and HCC," "Golgi protein-73 (GP73) and HCC," "Hepatocyte growth factor (HGF) and HCC," "Nervous growth factor (NGF) and HCC."
Despite the large number of studies devoted to the immunohistochemistry of HCC, at the present time, the absolute positive and negative markers for HCC are still lacking, and even those characterized by very high sensitivity and specificity do not have an universal diagnostic usefulness. Given the poor response to current therapies, a better understanding of the molecular pathways active in this disease could potentially provide new targets for therapy. However, AFP shows a low sensitivity, therefore other biomarkers have been developed to make an early diagnosis and improve patients' prognosis.
- SourceAvailable from: Carla Viegas[Show abstract] [Hide abstract]
ABSTRACT: Gla-rich protein (GRP) was described in sturgeon as a new vitamin-K-dependent protein (VKDP) with a high density of Gla residues and associated with ectopic calcifications in humans. Although VKDPs function has been related with íµí»¾-carboxylation, the Gla status of GRP in humans is still unknown. Here, we investigated the expression of recently identified GRP spliced transcripts, the íµí»¾-carboxylation status, and its association with ectopic calcifications, in skin basal cell and breast carcinomas. GRP-F1 was identified as the predominant splice variant expressed in healthy and cancer tissues. Patterns of íµí»¾-carboxylated GRP (cGRP)/undercarboxylated GRP (ucGRP) accumulation in healthy and cancer tissues were determined by immunohistochemistry, using newly developed conformation-specific antibodies. Both GRP protein forms were found colocalized in healthy tissues, while ucGRP was the predominant form associated with tumor cells. Both cGRP and ucGRP found at sites of microcalcifications were shown to have in vitro calcium mineral-binding capacity. The decreased levels of cGRP and predominance of ucGRP in tumor cells suggest that GRP may represent a new target for the anticancer potential of vitamin K. Also, the direct interaction of cGRP and ucGRP with BCP crystals provides a possible mechanism explaining GRP association with pathological mineralization.BioMed Research International 04/2014; 2014. · 2.88 Impact Factor
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ABSTRACT: Background:Preoperative alpha-L-fucosidase (AFU) has been used as a diagnostic biomarker for hepatocellular carcinoma (HCC), but its role as a prognostic predictor after partial hepatectomy has not been well defined. The study aimed to investigate the prognostic significance of preoperative serum AFU for HCC patients after hepatic resection.Methods:A retrospective training data set and a prospective validation data set were used to evaluate the prognosis of HCC after partial hepatectomy. A total of 669 patients with histopathologically confirmed HCC were enrolled. Univariate and multivariate analyses were used to identify the prognostic significance of preoperative serum AFU.Results:The retrospective training data set showed a preoperative AFU>35 u l(-1) should be used. The prospective validation data set showed preoperative AFU was an independent prognostic factor of overall survival (OS) (P=0.008; hazard ratio: 2.333; 95% confidence interval: 1.249-4.369). Patients with a preoperative AFU>35 u l(-1) had a lower recurrence-free survival rate and an OS rate than those with AFU35 u l(-1), and they have a higher tendency to form macrovascular invasion. Furthermore, the prognostic significance of AFU>35 u l(-1) could also be applied to patients with alpha-fetoprotein levels of 400 ng ml(-1).Conclusions:Preoperative serum AFU is a prognostic predictor of HCC.British Journal of Cancer advance online publication, 25 February 2014; doi:10.1038/bjc.2014.102 www.bjcancer.com.British Journal of Cancer 02/2014; · 5.08 Impact Factor
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ABSTRACT: Recently, a relationship between platelets and cancer metastasis has been reported. The aim of this study is to elucidate the risk factors for extra-hepatic metastasis (EHM), with emphasis on association with platelets in patients, with hepatocellular carcinoma (HCC). We examined risk factors for EHM in 1613 consecutive, newly diagnosed HCC patients by logistic regression analysis (case-control study). We also examined the factors by Cox proportional hazard model in a retrospective cohort fashion in 803 patients who received non-curative treatment for HCC. In the case-control study, multivariate analysis revealed that high platelet counts [odds ratio (OR)=4.84; 95% confidence interval (CI)=1.29－29.54; p=0.01], high tumor number, and the presence of macroscopic vascular invasion were significantly associated with EHM. In the cohort study, EHM was diagnosed in 71 patients during the study period (mean observation time=23.3 months). On multivariate analysis, high tumor number, high des-gamma-carboxy prothrombin (DCP), and Child-Pugh class A were significantly correlated with EHM, and the patients with high platelet counts tended to develop EHM (OR=1.73; 95% CI=0.99－3.14; p=0.055). HCC patients with high platelet counts, as well as large numbers of tumors, high serum DCP, and Child-Pugh class A, are at risk for EHM.Hepatology Research 02/2014; · 2.07 Impact Factor
Serum Markers of Hepatocellular Carcinoma
Giulia Malaguarnera•Maria Giordano•
Isabella Paladina•Massimiliano Berretta•
Alessandro Cappellani•Mariano Malaguarnera
Received: 18 December 2009/Accepted: 25 February 2010
? Springer Science+Business Media, LLC 2010
most common malignant tumors and carries a poor survival
rate. The management of patients at risk for developing
HCC remains intricate.
A literature search identified potential markers
for hepatocellular carcinoma. These markers were analysed
and justification was provided for these factors’ inclusion to
(or exclusion from) the markers of hepatocellular carci-
noma (HCC). A search of the literature was made using
cancer literature and the PubMed database for the following
keywords: ‘‘markers and HCC,’’ ‘‘Lens culinaris agglutinin
reactive AFP (AFP-L3) and HCC,’’ ‘‘Des-c-carboxy pro-
thrombin (DCP) and HCC,’’ ‘‘Glypican-3 and HCC,’’
‘‘Chromogranin A and HCC,’’ ‘‘Transforminggrowthfactor
b1(TGF) and HCC,’’ ‘‘a-l-fucosidase (AFU) and HCC,’’
‘‘Golgi protein-73 (GP73) and HCC,’’ ‘‘Hepatocyte growth
factor (HGF) and HCC,’’ ‘‘Nervous growth factor (NGF)
The hepatocellular carcinoma is one of the
to the immunohistochemistry of HCC, at the present time,
the absolute positive and negative markers for HCC are
still lacking, and even those characterized by very high
sensitivity and specificity do not have an universal diag-
nostic usefulness. Given the poor response to current
therapies, a better understanding of the molecular pathways
active in this disease could potentially provide new targets
for therapy. However, AFP shows a low sensitivity,
therefore other biomarkers have been developed to make
an early diagnosis and improve patients’ prognosis.
Despite the large number of studies devoted
(AFP) ? Lens culinaris agglutinin reactive AFP (AFP-L3) ?
Des-c-carboxy prothrombin (DCP) ? Glypican-3 (GPC3) ?
Chromogranin-A(CgA) ? Transforming growth factor b1
(TGF-b1) ? Alfa-l-fucosidase (AFU) ? Golgi protein 73
Hepatocellular carcinoma ? Alpha-fetoprotein
Hepatocellular carcinoma (HCC) is one of the most com-
mon cancers worldwide and the third most common cause
of cancer-related death . The most important risk factors
for HCC are chronic hepatitis B or C infection, cirrhosis,
non-alcoholic fatty liver disease (NAFLD), alcohol-
induced liver disease (ALD), and exposure to aflatoxin and
other carcinogens [2–7]. The clinical manifestations of
HCC include abdominal pain in the right hypochondrium,
hepatomegaly, and weight loss. The diagnosis of HCC is
usually based on the atypical histopathology combined
with the laboratory screening including index of hepatic
damage (alanine aminotransferase and aspartate amino-
transferase), the index of cholestasis (alkaline phosphatase
G. Malaguarnera (&)
Department of Biomedical Science, University of Catania,
Via Androne 83, 95124 Catania, Italy
M. Giordano ? I. Paladina ? M. Berretta ? M. Malaguarnera
Senescence, Urological, and Neurological Sciences, University
of Catania, Via Messina 829, 95126, Catania, Italy
Department of Medical Oncology, National Cancer Institute,
Via Franco Gallini 2, 33081 Aviano (PN), Italy
Section of General Surgery and Oncology, Department of
General Surgery, University Medical School of Catania,
Ospedale Vittorio Emanuele Via Plebiscito 628,
95100 Catania, Italy
Dig Dis Sci
and gamma-glutamyl transpeptidase), the index of hepatic
synthesis (albumin, prothrombin time, bilirubin) and
finally, tumor markers and instrumental tests, which
include hepatic ultrasonography, computed tomography
(CT), nuclear magnetic resonance (NMR), and angiogra-
phy. The best therapy for HCC is surgical hepatic resec-
tion, but when this is not possible, other treatments may be
utilized such as systemic chemotherapy, hepatic intra-
embolization and chemoembolization (TACE), percutane-
ous ethanol injection (PEI), and hormone treatment . So
far, alpha-fetoprotein is the most common marker used in
clinical practice, in conjunction with hepatic ultrasonog-
raphy, to detect HCC in cirrhosis patients. An early diag-
nosis of HCC is extremely important in improving the
survival of patients. The identification of biological
markers of HCC recurrence and metastasis is indispensable
for the proper management of HCC.
In this review, we attempt to collect the wide-ranging
body of existing literature on this subject. The motivation
behind this effort is that each existing marker alone is
poorly specific to predict this disease. Most markers are not
related to each other. False-negative results may signifi-
cantly contribute to an incorrect diagnosis and using more
than one marker at a time should greatly reduce the chance
of errors from false-negative results.
Alpha-fetoprotein is a glycoprotein with a molecular
weight of about 70 kDa. Under physiological conditions,
AFP is synthesized by the embryonic liver cells of the
vitelline sac and fetal intestinal tract in the first trimester of
The AFP gene is expressed in hepatocytes and endo-
dermal cells of the yolk sac during fetal life. Its expression
is reduced after birth. The elevation of AFP occurs
in hepatocyte regeneration, hepatocarcinogenesis, and
The biological function of AFP is still not well identi-
fied. Since AFP is similar to albumin, it is possible that
AFP function as a carrier for several ligands such as bili-
rubin, fatty acids, steroids, heavy metals, flavonoids, phy-
toestrogens, dioxin, and various drugs [9, 10]. The increase
of AFP levels [500 ng/ml is correlated with the tumor
size: 80% of small HCC show no increase of AFP con-
centration. Furthermore, sensitivity of AFP decreases from
52 to 25% when tumor diameter is [3 and \3 cm,
respectively . Some patients with cirrhosis and/or
hepatic inflammation can have an elevated AFP without the
presence of tumor. The clinical use of AFP has been
indicated principally (1) to execute the screening and
diagnosis of HCC in patients at risk of developing HCC. In
this case the measurement of AFP level is accompanied by
hepatic ultrasonography; (2) as a marker for detecting
tumor progression in patients with AFP-producing HCC.
After treatment of the tumor, complete response is likely if
the pre-treatment-elevated AFP levels decline to normal
levels during subsequent follow-up measurements; (3) In
staging: one of the most important staging systems for
HCC is the CLIP (Cancer of the liver Italian program)
staging system. The CLIP system assigns a score to the
following four independent factors:
AFP concentration: (higher or lower than 400 ng/ml)
Portal vein thrombosis
The CLIP system was used to define the parameters of
liver function and tumor characteristics to establish a
prognosis for HCC patients, and patients are followed-up to
monitor the response to treatment. The measurement of
AFP serum concentration during the follow-up of patients
after treatment is a helpful test in conjunction with com-
puted tomography or magnetic resonance imaging . A
decrease of AFP levels less than 10 ng/ml within 30 days is
sufficientto establish treatment
Reduction of AFP levels after palliative treatment, such as
with transarterial chemoembolization, indicates a favorable
response to treatment. However, the evaluation of serum
AFP concentration is clinically significant when AFP is
elevated before the therapy.
Lens Culinaris Agglutinin Reactive AFP
There are several AFP glycoforms that differ in the binding
affinity to lectins such as Lens culinaris agglutinin (LCA).
The AFP glycoforms include: AFP-L1 or LCA no reactive
is the principal AFP isoform in patients’ serum with
chronic hepatitis and liver cirrhosis; AFP-L2 presented
intermediate affinity to LCA. It is produced by yolk sac
tumors and could also be detected in maternal serum during
pregnancy; Lens culinaris agglutinin reactive AFP (AFP-
L3%) or Lens culinaris agglutinin reactive fraction of AFP,
has an elevated affinity to LCA. The latter isoform has 1–6
fucose residues attaching at reducing terminus of N-ace-
tylglucosamine and is derived only by cancer cells, so it
has been reported to be a more specific marker for HCC
[14, 15]. AFP-L3% should be used as a supplemental test
in those patients with elevated total AFP. However, the
clinical utility of AFP-L3% and the ratio of AFP-L3% to
total AFP remain unclear. AFP-L3% levels have been
found to be related to progression from moderately
Dig Dis Sci
differentiated to poorly differentiated tumors . The
cut-off for AFP-L3% is set up[10% of total serum AFP.
AFP-L3% measurement for HCC has a specificity [95%
[17, 18] and a sensitivity of approximately 51%. Therefore,
it may be used as an early diagnosis of HCC when the
tumor diameter is \2 cm. The sensitivity of AFP-L3%
changes with HCC along with clinical stages: in small
HCC (diameter\2 cm) AFP-L3% shows a sensitivity of
around 35–45%, while its sensitivity reaches 80–90% when
the tumoral diameter is[5 cm . Since AFP-L3%-
positive patients develop early vascular invasion and
intrahepatic metastasis, AFP-L3% is also considered as a
marker for the aggressiveness of HCC. In this regard, it
was suggested that AFP-L3% expression is connected with
increased nuclear expression of Ki67 (an indicator of the
aggressive nature of cancer) and with decreased expression of
a-catenin, which is associated with distant metastasis .
Moreover, there is a relationship between AFP-L3%
levels and histological grade [20, 21] underscored by evi-
dences that AFP-L3%-positive patients show poorly dif-
ferentiated tumor. AFP-L3% is used not only for
prognostic information [22, 23] but also in the patients’
follow-up after initial treatment . In fact, it is an
indicator of poor prognosis for HCC and of metastasis .
Moreover, patients positive for AFP-L3% after therapy
show a shorter survival than those who are AFP-L3%-
Des-c-carboxy prothrombin (DCP) or prothrombin induced
by vitamin K absence (PIVKA) is an abnormal prothrom-
bin derived by an acquired defect in the post-translational
carboxylation of the prothrombin precursor in HCC cells
. DCP derived by reduction ccarboxylase activity that
resulted in a lack of c-carboxylation of the glutamic-acid
residues. The reduced activity of c-carboxylase was
attributed to defective gene expression in HCC patients
. There are various differences between DCP and total
AFP. First of all, DCP is a more specific HCC marker than
AFP because other liver diseases don’t cause an increase of
DCP serum levels. DCP measurement for HCC has a
sensitivity of 48–62% and a specificity of 81–98% .
The accuracy of DCP is decreased in prolonged obstructive
jaundice, intrahepatic cholestasis with vitamin k defi-
ciency, and intake of warfarin. Furthermore, DCP serum
half-life (of around 40–72 h) is shorter than AFP serum
half-life (of around 5–7 days), so DCP allows valuing the
therapeutic efficacy of HCC in a timelier manner. DCP
measurement in HCC patients is connected with the
prognosis. In fact DCP high levels are associated with a
poorer prognosis . Lastly, there is no correlation
between DCP levels and total AFP levels.
DCP- positive patients frequently develop portal vein
invasion, intrahepatic metastasis, hepatic vein thrombosis,
and capsular infiltration . Additionally, DCP is con-
sidered a clinical marker for the development of portal vein
invasion which leads to intrahepatic metastasis [31, 32].
DCP is involved in tumoral angiogenesis: recent studies
have shown that DCP is able to augment the proliferation
and migration of human vascular endothelial cells  and
there is a correlation between the cell proliferation marker
as PCNA and DCP tissue expression in HCC . In fact,
not only does DCP function as a growth factor, it is also
able to increase genic expression of angiogenic factors
such as EGF-R, VEGF, and MMP-2.
Glypican-3 (GPC3) is one of the members of heparan
sulphate proteoglycans . It binds to the cell membrane
through the glycosil-phosphatidylinositol anchors. GPC3
interacts with several growth factors  and this inter-
action regulates positively or negatively (depending on the
specific growth factors) the growth factor activity .
Usually, GPC3 has a role in regulating cell proliferation
and survival during embryonic development by modulating
the activity of various growth factors. It also acts as a
tumor suppressor . GPC3 is mutated in patients with
Simpson-Golabi-Behmel syndrome, an X-linked disease
Recent studies have shown that GPC3 levels are
increased in HCC patients [39, 40]. GPC3 is able to dif-
ferentiate between malignant and benign hepatic lesions
; in fact, GPC3 levels are undetectable in healthy
subjects and in benign hepatic disease patients (such as
dysplastic or cirrhotic nodules). When GPC3 is over-
expressed, it acquires a new function that lacks in normal
tissues [41, 42]. Since the heparin sulphate chains of GPC3
interacts with heparin-binding growth factors and other
growth factors such as HGF and VEGF, can contribute to
the development of hepatic cancer.
P-aPKC-i, E-Cadherin, b-Catenin
P-aPKC-i, E-cadherin, and b-catenin play an important
role in tight-junctions formation among tumor cells.
P-aPKC-i is a member of the family of serine-threonine
kinases (PKC) that play an important role in cellular pro-
liferation and differentiation . P-aPKC-i is very
important for apicobasal maintenance and cellular junction
formation . Recent studies have shown that atypical
PKC-i is highly expressed in some malignant tumors and
its expression level is correlated to the genesis, develop-
ment, and prognosis of cancer [45, 46]. The P-aPKC-i
expression is increased in HCC and is higher in
Dig Dis Sci
undifferentiated cancer than in well-differentiated cancer.
In normal liver tissue, P-aPKC-i is localized at the apical
membrane, while in HCC tissues it is localized at the basal
membrane and in cytoplasm . The high expression of
aPKc-i caused the loss of cell polarity and cellular junction
that lead to metastasis.
E-cadherin and b-catenin-mediating intercellular adhe-
sion are involved in invasion and metastasis of the cancer
. E-cadherin is a transmembrane glycoprotein and its
intracellular domain is connected, through b-catenin and
other catenins, to the acting cytoskeleton. E-cadherin is
more expressed in well-differentiated tumors than in poorly
differentiated cancers that have lost intercellular adhesion
and have developed metastasis . E-cadherin is con-
sidered a marker of tumor differentiation . The reduced
expression of E-cadherin, through the inhibition of the
formation of a tight junction among tumoral cells, is cor-
related to insufficient tumoral differentiation and devel-
opment of metastasis.
Regarding b-catenin, its cytoplasmatic overexpression
in HCC tissues is involved in activation of the WNT sig-
naling pathway. Additionally, b-catenin induces the gene
expression of c-myc, cyclin D, VEGF, and other genes that
increase cell proliferation.
Human Carbonyl Reductase 2
Human carbonyl reductase 2 (HCR2) gene encodes a
cytosolic enzyme that is expressed in the human liver and
kidney. This enzyme is important in detoxification of the
reactive a-dicarbonyl compounds and reactive oxygen
species (ROS) deriving from oxidative stress. In HCC, the
antioxidant defense system including HCR2 and glutathi-
one-S transferase (GSH) is repressed. This altered detoxi-
fication system is involved in HCC progression .
Therefore, the decreased expression of HCR2 in HCC tis-
sues contributes to cancer growth because it increases the
cellular damage induced by ROS and other carcinogens.
The HCR2 levels are inversely correlated to the patho-
logical grading of HCC: lower HCR2 expression is
detected in advanced lesions .
a-l-fucosidase (AFU) is a lysosomal enzyme found in all
mammalian cells and is linked to the degradation of a
variety of fucose containing fuco glycoconjugates . Its
activity is higher in HCC patients than in healthy individ-
uals and in chronic hepatic disease patients. The cut-off
value is set to 870 nmol/l. AFU shows a sensitivity of
81.7% and a specificity of 70.7%. There is no correlation
between AFU serum concentration and AFP levels or
alanine aminotransferase (ALT) activity. So, the increased
AFU levels in HCC patients is not related to liver regen-
eration or necrosis but probably associated with an
increased synthesis of protein that leads to an increase in
fucose turnover . Nevertheless, this explanation is not
supported by recent studies that show a decrease of AFU
expression in tumoral liver tissues compared to normal
tissues . AFU measurement is useful in association
with AFP in the early diagnosis of HCC . Moreover,
there is a positive correlation between AFU levels and
tumor size in HCC patients . The AFU increase has
been observed in non-cancerous extrahepatic disease such
as diabetes, pancreatitis, and hypothyroidism.
Vascular Endothelial Growth Factor
The development of solid tumors is strictly correlated with
angiogenesis. Vascular endothelial growth factor (VEGF)
plays an important role in angiogenesis: it stimulates the
proliferation and migration of endothelial cells and
increases vascular permeability. VEGF is highly expressed
in various human cancers [57–59]. HCC shows an elevated
expression of VEGF [60, 61], and particularly increased
VEGF expression is present in advanced HCC compared to
early HCC. Moreover, VEGF levels are higher in HCC
patients than in chronic hepatic disease patients. VEGF is
produced by HCC cells but the plasma VEGF elevation in
advanced HCC suggests that other mechanisms are
involved in the increase of VEGF levels. Vascular damage
and invasion by cancer cells are fundamental for distant
metastasis. Vascular injury causes the agglutination and
platelet activation. Platelets, activated by vascular invasion
of HCC cells, release VEGF . As consequence, the
increased vascular permeability induced by VEGF makes
easier the VEGF passage into circulation. Therefore, VEGF
is considered a possible tumor marker for the metastasis of
HCC. High serum VEGF is associated with portal vein
emboli, poorly encapsulated tumors, microscopic vein
invasion, and recurrence in HCC patients . VEGF is a
predictor of tumor aggressiveness, disease-free survival,
and overall survival in patients who underwent HCC
Squamous Cell Carcinoma Antigen (SCCA)
SCCA belongs to the high-molecular-weight family of
serin protease inhibitors (serpins) . There are two dif-
ferent isoforms that are expressed in the suprabasal layer of
multi-stratified squamous epithelium . SCCA expres-
sion, as well as AFP production, could be the consequence
of the dedifferentiation often observed in HCC. Since there
is an important difference between SCCA expression in
Dig Dis Sci
HCC and in peritumoral tissues in the same patients, it can
be used in immunohistochemical diagnosis of HCC or to
explore micrometastasis . HCC patients show higher
SCCA serum levels than cirrhotic patients . There is no
clear correlation between SCCA expression tissue and
SCCA serological levels because SCCA is expressed in the
cytosol and is not associated with the cellular membrane.
Conceivably, circulating SCCA is not secreted by cells, but
derived by cellular lysis . SCCA may be used for HCC
diagnosis as it shows a sensitivity of 84.2% and a speci-
ficity of 48.9%. Given that SCCA is inversely correlated
with tumor size, it is helpful for early HCC diagnosis and
in screening of chronic hepatic disease patients.
Chromogranin A (CgA) is an acidic glycoprotein contained
in secretory granules of neuroendocrine cells . Many
studies show high serum Cg-A concentration in patients
with HCC, suggesting that CgA might represent a useful
marker for HCC . CgA levels are increased in other
tumors such as pancreatic and prostate cancer [70, 71].
Spadaro et al.  report that the determination of CgA
serum values is useful in monitoring cirrhosis patients for
the early detection of an increase or decrease of HCC Cg-A
levels according to the degree of neuroendocrine differ-
entiation of HCC.
Moreover, CgA degradation is decreased because of
progressive hepatocellular failure. The correlation between
circulating CgA levels and histological stage of fibrosis
suggests that CgA may be involved in hepatic fibrogenesis.
Since CgA levels increase in both HCC patients and in
cirrhotic patients, it shows a low diagnostic specificity.
However, CgA concentration is a useful indicator for
assessing neuroendocrine differentiation in connection with
the stage of HCC. Patients with a higher CgA serum con-
centration show a poorer outcome than those with lower
CgA levels . Moreover, CgA serum concentration is
increased in patients with neuroendocrine tumors that have
metastasized to the liver . In these patients, a positive
correlation between the tumor size and CgA serum levels
has been reported . In contrast, CgA serum concen-
tration is rarely increased in patients with small neuroen-
docrine tumors. Additionally, CgA can be utilized in HCC
Transforming Growth Factor b1
Transforming growth factor b1 (TGF-b1) is a negative
factor in tumor growth: it arrests the cell cycle in the G1
phase, inducing inhibition of cell proliferation and trig-
gering apoptosis . In normal liver tissues, TGF-b1 is
produced only by nonparenchymal cells (Kupffer cells,
storing cells, and endothelial cells). Many studies report an
up-regulated expression of hepatic TGF-b1 in tumor cells,
including HCC. Recent studies show that TGF-b1 serum
levels are increased in HCC patients . TGF-b1 is
secreted by HCC cells and there is an over-expression of
the TGF-b1 gene in HCC cells . The increased
expression of TGF-b1 in HCC is correlated with hepato-
carcinogenesis, since it not only inhibits the recognition of
tumor by immunological system and the immune-mediated
cytolysis but also promotes tumor angiogenesis [79, 80].
The expression of TGF-b1 mRNA tends to be higher in the
patients with increased AFP and ALT levels while
decreased TGF-b1 mRNA expression is correlated with the
change of platelets count. The levels of TGF-b1 mRNA are
higher in patients with advancing histological aggressive-
ness: in general, in the larger tumor the TGF-b1 mRNA
expression is higher. It is important to mention that
TGF-b1 induces growth inhibition in epithelial cells
through a reduction of cyclin D expression in several tis-
sues . HCC cells show resistance to TGF-b1 growth
inhibition because in tumoral cells there is an overexpres-
sion of cyclin D1 correlated with the dysregulation of the
cell cycle and tumor progression [82, 83].
Golgi protein-73 (GP73) is a resident Golgi glycoprotein
expressed in epithelial human cells . Physiologically,
GP73 is expressed in biliary epithelial cells but not in
hepatocytes. In liver disease, GP73 expression is increased
in hepatic cells . Moreover, Gp73 serum levels are
increased in chronic liver disease patients, particularly,
GP73 values are higher in early HCC patients than in cir-
rhotic patients . GP73 is considered a possible marker
for HCC, in fact it shows a specificity of 75% and a sen-
sitivity of 69%.
Since GP73 is a Golgi-resident protein, its presence in
circulation is surprising. A possible explanation of the
detection of GP73 serum can be that this protein is able to
arrive to the plasma membrane and pass into the circula-
tion. There are several isoforms of GP73 correlated with
different levels of glycosylation . Therefore, some
isoforms are more specific for HCC. Further studies are
needed to confirm the role of GP73 in HCC diagnosis.
Hepatocyte Growth Factor
Hepatocyte growth factor (HGF) is a cytokine having a
wide range of effects, from embryonic development and
liver regeneration to protection and/or repair of various
organs, including kidney, lung, and cardiovascular system
[88, 89]. The principal and most successful therapy for
HCC is hepatic resection when the patient maintains good
Dig Dis Sci
liver function . The pre-operative evaluation of hepatic
function is very important to avoid liver failure . Since
good liver function prolongs the survival of patients that
can receive further therapies, the liver function examina-
tion is very useful in predicting post-operative complica-
tions and survival after surgery. HGF stimulates hepatocyte
proliferation including HCC cells  through expression
of its receptor, the c-met receptor.
Hepatocyte growth factor (HGF) is detected in the
serum of hepatic chronic disease patients. There is a cor-
relation between HGF serum values and a worsening of
liver disease .
The increase of HGF serum levels in cirrhotic patients is
an indicator of HCC development . HGF serum levels
higher than or equal to 1.0 ng/ml have been correlated with
poor survival. Therefore, pre-operative high HGF levels are
related to development of post-operative complications,
such as liver failure  and a poor survival. HGF can be
helpful in assessing hepatic function before surgery and for
predicting a patient’s prognosis. Moreover, elevated HGF
serum levels, after surgery, is able to predict early tumor
recurrence and metastasis .
The p53 gene is an onco-suppressor gene encoding a
nuclear phosphoprotein (p53 protein) that inhibits cellular
proliferation and transformation . Mutations of the p53
gene have been reported in several human cancers. P53
alterations occur at the late stages of hepatocarcinogenesis.
Therefore, p53 alteration is not an early event in HCC and
it is connected with the prognosis and survival of HCC
patients. Mutated p53 proteins for its prolonged half-life
are liable to accumulate in tumoral cells . In fact, there
is a correlation between p53 gene mutations and protein
accumulated . This correlation makes possible the use
of simple immunologic methods for p53 detection. P53
mutations are correlated with poorly differentiated cancer
and shorter survival of patients with HCC . Mutant p53
proteins can be released in the serum by tumor cells;
therefore, antibodies to p53 protein have been detected in
HCC and in other tumors such as breast cancer , lung
cancer , prostate cancer, leukemia, B-cell lymphoma,
thyroid cancer, and pancreas cancer . P53 alterations
are detected in 30–50% of HCC patients  and these
abnormalities are associated with a poor prognosis of HCC
Nervous Growth Factor
Nervous growth factor (NGF) is involved in aspects of
tumor biology such as growth invasion and metastasis, in
addition to its role in differentiation and survival of
neuronal cells. NGF can interact with two types of cell
membrane receptors: TrkA NGF and p75NGF . TrkA
is a high-affinity receptor with tyrosine kinase activity and
binding results in intracellular signaling through the mito-
gen-activated protein kinase and phosphatidylinositol-
3-kinase cascades . p75NGF is a low-affinity glyco-
protein receptor. p75NGF structurally resembles members
of the p55 tumor necrosis factor receptor family and has no
tyrosine kinase activity and binding of NGF stimulates
recruitment of cytoplasmic factors to the intracellular
domain of the receptor that may lead to either apoptosis or
cell survival [106, 107]. Various studies show that NGF is
over-expressed in approximately 60% of human HCC tis-
sues compared to the surrounding liver tissue with cirrhosis
and chronic hepatitis, suggesting a role for NGF in the
progression of HCC . In fact, hepatic stellate cells
express neurotrophins and their receptors are increased
during hepatic regeneration [109, 110]. NGF and its related
receptors play an important role in modulating the phys-
iopathology of the intrahepatic biliary epithelium in the
course of liver tissue remodeling processes and HCC pro-
gression. The mechanism of NGF involvement in liver
tissue remodeling processes and HCC remains unclear.
Rasi et al. , defining NGF distribution both inside the
liver and in the intracellular compartments (in the cyto-
plasmic vesicle and in the endoplasmic reticulum), dem-
onstrated that NGF can function in a paracrine and
autocrine manner as a messenger molecule in the cross-talk
between different cell types. An interesting perspective for
the possible use of NGF is not only as a marker of pro-
gression and transformation but also as an attractive target
for future therapeutic approaches .
Serum proteomics, through the study of serum protein
profiling, is useful in the detection of new biomarkers for
early HCC diagnosis. Serum proteomics aims to identify
the changes in protein expression, structure, and post-
translational modifications. Some of these modifications
are connected to HCC development. Recent studies have
detected serum protein profile derived from patients with or
without HCC. The serum of these patients is depleted of
the most abundant protein as it has been shown using the
proteomic analysis applying the method of surface-
enhanced laser desorption ionization time-of-flight mass
spectrometry (SELDY-TOF MS) protein Chip system.
Through this approach, 30 peaks have been detected and
the levels of these were different according to the presence
or absence of HCC. Particularly, a combination of six of
these peaks distinguished HCC and non-HCC patients. The
fragment C-terminal of vitronectin was identified as the
highest discriminating peak (8,900 Da). Vitronectin is a
Dig Dis Sci
glycoprotein that is produced by hepatocytes and plays an
important role in cell adhesion, migration, and matrix
remodeling of cancer cells . Since vitronectin gene
expression is downregulated in HCC tissues, its increase is
correlated to its own degradation. Additionally, in HCC
there is an increase of metalloprotease-2 (MMP-2) gene
expression and activity  that is involved in vitronectin
catabolism. Therefore, in the serum protein profiling of
HCC patients, the 8,900-Da biomarker may reflect tumor
aggressiveness. A correlation was found between the
8,900-Da peak and tumor size. So, in the serum protein
profiling of HCC patients, the 8,900-Da biomarker may
reflect tumor aggressiveness.
increased in different chronic inflammatory and tumor
diseases [114, 115]. The b2MG production by hepatocytes
is associated with chronic inflammation correlated to viral
hepatitis (hepatitis B and C virus) . HCC cells show a
higher expression of class I HLA antigens than normal
hepatocytes, so tumor cells avoid immunological response
The b2MG serum concentration, which is increased in
HCC, is correlated with class I HLA antigen expression
levels. There is a positive correlation between b2MG
serum concentration and interleukin-6 levels . It
seems that Il-6 is able to reduce immunological response
and to induce the enhanced expression of b2MG in HCC
cells. Moreover, b2MG serum levels are correlated with
tumor size . Therefore, it is considered a useful
(b2MG) serum concentrationis
marker for indicating HCC progression. Glycylproline
dipeptidyl aminopeptidase (GPDA) is consistently positive
in patients with HCC . This marker is particularly
useful for HCC diagnosis in patients with non-AFP-pro-
ducing HCC. Further studies are needed to establish the
utility of these markers in clinical practice .
Summary and Perspective
The question of which molecular markers will prove to be
the most useful for selecting treatment for individual
patients with HCC and which will be validated remains
unanswered. In this review, we have summarized the
prognostic and predictive factors of these markers
Furthermore, we still do not know whether the molec-
ular profile of a tumor changes at the time of disease
recurrence after surgery, or even after therapy for more
advanced disease. There is little information as to whether
primary and metastatic tumors always share the same
molecular profile, although there is some evidence for
molecular discordance between early and metastatic dis-
ease. If this finding is shown to be a frequent occurrence,
repeat biopsy with molecular profiling of fresh tissue might
be required when treatments change, especially if the new
treatments have a specific molecular target [123–126].
In summary, serological markers specific for HCC play
important roles in this disease in the following aspects:
Table 1 Usefulness of principal hepatocellular carcinoma
HCC marker Principal use
Alpha-fetoprotein HCC early diagnosis, monitoring, and recurrence
Lens culinaris agglutinin reactive AFP (AFP-L3%)
Des-c-carboxy prothrombin (DCP)
P-aPKCi, E-chaderin, b-catenin
Human carbonyl reductase (HCR2)
HCC early diagnosis and prognosis (vascular invasion and intrahepatic metastasis)
HCC early diagnosis and prognosis (early portal vein invasion and metastasis)
HCC early diagnosis
HCC early diagnosis
Squamous cell carcinoma antigen (SCCA)HCC early diagnosis
Serum proteomics HCC early diagnosis
Golgi protein 73 HCC early diagnosis
Chromogranin A (CgA) HCC prognosis and possible therapeutic treatment
Vascular endothelial growth factor (VEGF)HCC prognosis (metastasis development)
Hepatocyte growth factor (HGF)
Transforming growth factor-b (TGF-b)
HCC prognosis and disease recurrence
HCC prognosis (poor differentiation)
Nervous growth factor (NGF)HCC prognosis and progression
Dig Dis Sci
Screening for early malignancy: a-feto protein is a
unique marker that is used in clinical practice in combi-
nation with hepatic echography in the screening of cirrhotic
patients to discover HCC, but other markers have been
studied to reach an earlier diagnosis. Moreover, cirrhotic
patients can show a transient AFP elevation that is asso-
ciated with hepatocyte regeneration as a consequence of
liver necroinflammation [127, 128]. Persistent AFP eleva-
tion is found in some of these patients. In this case Lens
culinaris agglutinin reactive AFP (AFP-L3%), measure-
ment may be of help in the HCC diagnosis. AFP-L3% is
the product of a-1-6 fucosyltransferase; this enzyme is
higher in HCC tissues than in peritumoral tissues .
Therefore, AFP-L3% is considered more specific than AFP
in HCC diagnosis.
Des-gamma-carboxyprothrombin is a useful marker for
detecting HCC in conjunction with AFP and ultrasonog-
Acting as a diagnostic aid for HCC: In the HCC diag-
nosis, other AFP and AFP-L3%, other markers can be used.
Des-gamma-carboxyprothrombin is an abnormal pro-
thrombin identified as a biomarker for HCC diagnosis.
Squamous cell carcinoma antigen (SCCA) expression is
more increased in premalignant dysplastic nodules than in
HCC . Smaller HCC show a higher SCCA expression
than larger ones: decreased SCCA expression is correlated
with progression of tumor size while increased SCCA
expression in surrounding non-tumoral tissues of larger
HCC is a marker for neoplastic transformation. Serum
proteomics is used for the serologic recognition of protein
profiles associated with cancer. Proteomic approach can
accurately identify clinical HCC in cirrhotic patients. Golgi
Protein 73 is considered a possible marker for HCC; in
fact, it shows a specificity of 75% and a sensitivity of 69%.
Determining prognosis in HCC: Des-gamma-carbox-
yprothrombin is increased in advanced HCC with portal
vein invasion. It is considered a prognostic indicator able to
predict rapid tumor progression and poorer prognosis.
Glypican-3 expression is less frequently observed in well-
differentiated HCC than in moderately and poorly differ-
GPC3-positive patients show a lower survival than
Vascular endothelial growth factor regulates positively
expression of VEGF have a lower survival rate.
The increase of P-aPKC-i expression is correlated with
more aggressive tumoral behavior: it is considered a prog-
A is used to evaluate neuroendocrine differentiation of HCC
and it may be of help in the therapeutic approach.
AFP-L3% expression is correlated with infiltrative
growth type and poorly differentiated cancer while DCP
expression is connected to intrahepatic metastasis and
vascular invasion. The over-expression of hepatic trans-
forming growth factor b1 is found in HCC and is correlated
with carcinogenesis, progression, and prognosis of HCC.
Maintaining surveillance following surgical removal of
the primary tumor: Since HCC patients are prone to
develop a second liver tumor, other markers other than
AFP are proposed for the patients’ follow-up. AFP-L3%
measurement after treatment can be useful for under-
standing the prognosis and recurrence of HCC. VEGF is a
possible tumor marker for metastasis in HCC.
Monitoring therapy in advanced HCC: Hepatocyte
growth factor is considered a useful marker for evaluating
the possible complications arising after curative hepatic
Serum anti-p53 positivity is correlated with a poor
prognosis and a shorter survival. It is used in the planning
of HCC therapy . E-cadherin and b-catenin are
reduced in poorly differentiated cancer and their expression
is correlated with metastasis development.
Technical issues are also important in this argument.
At present, very few routine clinical laboratories have
access to sophisticated molecular techniques, such as
qRT-PCR, mutational analysis, FISH, and microarray,
although most can do immunohistochemistry. However,
standardized, optimized protocols and antibodies need to
be applied in order to validate prospective validation;
these technologies will also need optimization and stan-
dardization before being generally accepted as a valid
Microarray is also an exciting technique, but probably it
is not ready for entry into routine clinical practice until
relevant validation studies have been done in many centers.
Ultimately, the most promising biomarkers of prediction
and response require prospective validation in carefully
designed randomized clinical trials using standardized
protocols. This will require cooperation across borders and
expert assistant in the preparation and correction of the manuscript.
The authors thank Miss Paola Favetta, for her
Conflict of interest statement
No potential conflicts of interest
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