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.
- [show abstract] [hide abstract]
ABSTRACT: Methylation of gene promoter CpG islands is an important early event in hepatocellular carcinoma (HCC), and detection of cell-free tumor-specific DNA methylation is becoming a useful noninvasive method for HCC. This study was aimed at determining the diagnostic value of serum insulin-like growth factor-binding protein 7 (IGFBP7) promoter methylation in hepatitis B virus-associated HCC. A total of 217 subjects, including 136 HCC patients, 46 patients with chronic hepatitis B (CHB), and 35 healthy controls (HCs), were included. The methylation status of the serum IGFBP7 gene promoter was determined using methylation-specific PCR. The frequency of serum IGFBP7 promoter methylation in HCC patients (89/136, 65%) was significantly higher than that in CHB patients (8/46, 17%; X(2) = 31.883, P < 0.001) and HCs (5/35, 14%; X(2) = 29.429, P < 0.001). Moreover, elevated IGFBP7 methylation frequency was also observed in HCC patients with vascular invasion compared with those without vascular invasion (84 versus 60%, X(2) = 6.633, P = 0.010). The sensitivities of serum IGFBP7 methylation and alpha-fetoprotein (AFP) in detecting HCC were 65 and 57%, respectively. Of note, the combination of IGFBP7 methylation and AFP showed 85% for sensitivity. These results suggest that methylation of the serum IGFBP7 gene promoter may serve as a useful noninvasive biomarker for HCC diagnosis. © 2013 Wiley Periodicals, Inc.Genes Chromosomes and Cancer 10/2013; · 3.55 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Hepatocellular carcinoma (HCC) is the commonest primary malignant cancer of the liver in the world. This study was conducted to investigate the serum levels of hepatocyte growth factor (HGF)in HCC patients and the relationship with tumor progression and known prognostic parameters. Fifty-four patients with HCC were investigated. Pretreatment HGF levels were employed the quantitative sandwich enzyme immunoassay technique (ELISA). Age and sex matched 20 healthy controls were included in the analysis. The median age of the patients was 60 years (range 36-77 years); where males consistituted of majority of the group (88.8 %). All of patients had cirrhotic history. Fourty-six percent (n = 25) of patients had Child-Pugh Score A, 30 % (n = 16) had Score B or C. All of the patients were treated with local therapies but none of them received sorafenib. The baseline serum HGF levels were significantly higher in patients with HCC than in the control group (p < 0.001). Male patients had higher serum HGF levels compared with female patients (p = 0.01). Serum HGF levels were significantly higher in the patients with elevated serum ALT levels than others with normal serum ALT levels (p = 0.05). Poor performance status (p < 0.001), viral etiology of cirrhosis (p = 0.03), larger tumor size (p = 0.01), lower serum hemogloblin levels (p = 0.03), and not be treated for HCC (p = 0.001) related to worse survival. However, serum HGF did not have significantly adverse effect on survival (p = 0.58). Despite serum HGF levels were found diagnostic value, serum HGF levels had no prognostic value in patients with HCC.Tumor Biology 10/2013; · 2.52 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Many individual studies have evaluated the diagnostic efficiency of serum glypican-3 (GPC-3) for diagnosing hepatocellular carcinoma (HCC), but the results have been inconsistent. The aim of present study was to meta-analyze the overall diagnostic accuracy of serum GPC-3 for diagnosing HCC. English language studies which evaluated the diagnostic performance of GPC-3 and published before March 22, 2013 were retrieved. The quality of the studies was assessed by revised QUADAS tools. The performance characteristics were pooled and determined by random-effects models. Twelve studies with a total of 898 HCC patients and 835 non-HCC patients were included. For the studies in which the majority of reference participants had HBV or HCV infections, the overall diagnostic sensitivity and specificity were 0.53 (95% CI: 0.49-0.57) and 0.77 (95% CI: 0.74-0.81), respectively. The area under summary receiver operating characteristic (sROC) curves (AUC) was 0.82. The major design deficiencies of included studies were differential verification bias, and a lack of clear exclusion and inclusion criteria. GPC-3 has moderate diagnostic accuracy for HCC. Due to the design limitations, results in published studies should be carefully interpreted. In addition, more well-designed studies with large sample sizes should be performed to rigorously evaluate the diagnostic value the GPC-3.Clinical biochemistry 12/2013; · 2.02 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
1. Bosch FX, Ribes J, Cleries R, et al. Epidemiology of hepato-
cellular carcinoma. Clin Liver Dis. 2005;9:191–211.
2. Buck J, Miller RH, Kew MC, Purcell R. Hepatitis C virus RNA
in southern African blacks with hepatocellular carcinoma. Proc
Natl Acad Sci USA. 1993;90:1848–1851.
3. Malaguarnera M, Di Fazio I, Laurino A, Pistone G, Restuccia S,
Trovato BA. Decrease of interferon gamma serum levels in
Dig Dis Sci
patients with chronic hepatitis C. Biomed Pharmacother.
4. Malaguarnera M, Di Rosa M, Nicoletti F, Malaguarnera L.
Molecular mechanisms involved in NAFLD progression. J Mol
5. Malaguarnera L, Madeddu R, Palio E, Arena N, Malaguarnera
M. Heme oxygenase-1 levels and oxidative stress-related
parameters in non-alcoholic fatty liver disease patients. J Hep-
6. Malaguarnera L, Rosa MD, Zambito AM, dell’Ombra N, Marco
RD, Malaguarnera M. Potential role of chitotriosidase gene in
non-alcoholic fatty liver disease evolution. Am J Gastroenterol.
7. Malaguarnera L, Di Rosa M, Zambito AM, dell’Ombra N,
Nicoletti F, Malaguarnera M. Chitotriosidase gene expression in
Kupffer cells from patients with non-alcoholic fatty liver dis-
ease. Gut. 2006;55:1313–1320.
8. Malaguarnera M, Trovato G, Restuccia S, et al. Treatment of
nonresectable hepatocellular carcinoma: review of the literature
and meta-analysis. Adv Therapy. 1994;11:303–319.
9. Terentiev AA, Moldogazieva NT. Structural and functional
mapping of alpha-fetoprotein. Biochemistry (Mosc). 2006;71:
10. Mizejewski GJ. Biological role of alpha-fetoprotein in cancer:
prospects for anticancer therapy. Expert Rev Anticancer Ther.
11. Saffroy R, Pham P, Reffas M, Takka M, Lemoine A, Debuire B.
New perspectives and strategy research biomarkers for hepato-
cellular carcinoma. Clin Chem Lab Med. 2007;45:1169–1179.
12. Bruix J, Sherman M, Llovet JM, et al. Clinical management of
hepatocellular carcinoma. Conclusions of the Barcelona-2000
EASL conference. European association for the study of the
liver. J Hepatol. 2001;35:421–430.
13. Han SJ, Yoo S, Choi SH, Hwang EH. Actual half-life of alpha-
fetoprotein as a prognostic tool in pediatric malignant tumors.
Pediatr Surg Int. 1997;12:599–602.
14. Oka H, Saito A, Ito K, et al. Multicenter prospective analysis of
newly diagnosed hepatocellular carcinoma with respect to the
percentage of Lens culinaris agglutinin-reactive alpha-fetopro-
tein. J Gastroenterol Hepatol. 2001;16:1378–1383.
15. Sato Y, Nakata K, Kato Y, et al. Early recognition of hepato-
cellular carcinoma based on altered profiles of alpha-fetoprotein.
N Engl J Med. 1993;328:1802–1806.
16. Miyaaki H, Nakashima O, Kurogi M, Eguchi K, Kojiro M. Lens
induced by vitamin K absence II are potential indicators of a
poor prognosis: a histopathological study of surgically resected
hepatocellular carcinoma. J Gastroenterol. 2007;42:962–968.
17. Aoyagi Y, Suzuki Y, Isemura M, et al. The fucosylation index of
alpha-fetoprotein and its usefulness in the early diagnosis of
hepatocellular carcinoma. Cancer. 1988;61:769–774.
18. Taketa K. Alpha-fetoprotein: reevaluation in hepatology. Hepa-
19. Sassa T, Kumada T, Nakano S, Uematsu T. Clinical utility of
carboxy prothrombin and Lens culinaris agglutinin A-reactive
Eur J Gastroenterol Hepatol. 1999;11:1387–1392.
20. Yamashita F, Tanaka M, Satomura S, Tanikawa K. Prognostic
significance of Lens culinaris agglutinin A-reactive alpha-feto-
protein in small hepatocellular carcinomas. Gastroenterology.
21. Kuromatsu R, Tanaka M, Tanikawa K. Serum alpha-fetoprotein
and lens culinaris agglutinin-reactive fraction of alpha-fetopro-
tein in patients with hepatocellular carcinoma. Liver. 1993;13:
22. Hayashi K, Kumada T, Nakano S, et al. Usefulness of mea-
surement of Lens culinaris agglutinin-reactive fraction of alpha-
fetoprotein as a marker of prognosis and recurrence of small
hepatocellular carcinoma. Am J Gastroenterol. 1999;94:3028–
23. Yamashita F, Tanaka M, Satomura S, Tanikawa K. Monitoring
of lectin-reactive alpha-fetoproteins in patients with hepatocel-
lular carcinoma treated using transcatheter arterial embolization.
Eur J Gastroenterol Hepatol. 1995;7:627–633.
24. Yamashiki N, Seki T, Wakabayashi M, et al. Usefulness of Lens
culinaris agglutinin A-reactive fraction of alpha-fetoprotein
(AFP-L3) as a marker of distant metastasis from hepatocellular
carcinoma. Oncol Rep. 1999;6:1229–1232.
25. Yamashita F, Tanaka M, Satomura S, Tanikawa K. Prognostic
significance of Lens culinaris agglutinin A-reactive alpha-feto-
protein in small hepatocellular carcinoma. Gastroenterology.
26. Ono M, Ohat H, Ohhira M, et al. Measurement of immunore-
active prothrombin precursor and vitamin-K-dependent gamma-
carboxylation in human hepatocellular tissues: decreased
carboxylation of prothrombin precursor as a cause of des-
gamma-carboxyprothrombin synthesis. Tumour Biol. 1990;11(6):
27. Grizzi F, Franceschini B, Hamrick C, Frezza EE, Cobos E,
Chiriva-Internati M. Usefulness of cancer-testis antigens as
biomarkers for the diagnosis and treatment of hepatocellular
carcinoma. J Transl Med. 2007;5:3.
28. Nakagawa T, Seki T, Shiro T, et al. Clinicopathologic signifi-
cance of protein induced by vitamin k absence or antagonistic II
and alpha-fetoprotein in hepatocellular carcinoma. Int J Oncol.
29. Fujiyama S, Tanaka M, Maeda S, et al. Tumormarkers in early
diagnosis, follow-up and management of patients with hepato-
cellular carcinoma. Oncology. 2002;62:57–63.
30. Suehiro T, Sugimachi K, Matsumata T, Itasaka H, Taketomi A,
Maeda T. Protein induced by vitamin K absence or antagonist II
(PIVKA-II) as a prognostic marker in hepatocellular carcinoma:
comparison with a-fetoprotein. Cancer. 1994;73:2464–2471.
31. Toyosaka A, Okamoto E, Mitsunobu M, Oriyama T, Nakao N,
Miura K. Intrahepatic metastases in hepatocellular carcinoma:
evidence for spread via the portal vein as an efferent vessel. Am
J Gastroenterol. 1996;91:1610–1615.
32. Mitsunobu M, Toyosaka A, Oriyama T, Okamoto E, Nakao N.
Intrahepatic metastases in hepatocellular carcinoma: the role of
the portal vein as an efferent vessel. Clin Exp Metastasis.
33. Fujikawa T, Shiraha H, Ueda N, et al. Des-gamma-carboxyl
prothrombin-promoted vascular endothelial cell proliferation
and migration. J Biol Chem. 2007;282:8741–8748.
34. Suzuki M, Shiraha H, Fujikawa T, et al. Des-gamma-carboxyl
prothrombin is a potential autologous growth factor for hepa-
tocellular carcinoma. J Biol Chem. 2005;280:6409–6415.
35. Bernfield M, Go ¨tte M, Park PW, et al. Functions of cell surface
heparan sulfate proteoglycans. Annu Rev Biochem. 1999;68:
36. Song HH, Shi W, Filmus J. OCI-5/rat glypican-3 binds to
fibroblast growth factor-2 but not to insulin-like growth factor-2.
J Biol Chem. 1997;272:7574–7577.
37. Reich-Slotky R, Bonneh-Barkay D, Shaoul E, Bluma B, Svahn
CM, Ron D. Differential effect of cell-associated heparan sul-
fates on the binding of keratinocyte growth factor (KGF) and
acidic fibroblast growth factor to the KGF receptor. J Biol
38. Pilia G, Hughes-Benzie RM, MacKenzie A, et al. Mutations in
GPC3, a glypican gene, cause the Simpson-Golabi-Behmel
overgrowth syndrome. Nat Genet. 1996;12:241.
Dig Dis Sci
39. Hsue HC, Cheng W, Pl Lai. Cloning and expression of a
developmentally regulated transcripts MXR7 in hepatocellular
carcinoma: biological significance and temporospatial distribu-
tion. Cancer Res. 1997;57:5179–5184.
40. Zhu ZW, Friess H, Wang L, et al. Enhanced glypican-3
expression differentiates the majority of hepatocellular carci-
nomas from benign hepatic disorders. Gut. 2001;48:558–564.
41. Hagihara K, Watanabe K, Yamaguchi J. Glypican-4 is an FGF2-
binding heparan sulfate proteoglycans expressed in neural pre-
cursor cells. Dev Dyn. 2000;219:353–367.
42. Gengrinovitch S, Berman B, David G, Witte L, Neufeld G, Ron
D. Glypican-1 is a VEGF165 binding proteoglycan that acts as
an extracellular chaperone for VEGF 165. J Biol Chem.
43. Knapp LT, Klann E. Superoxide- induced stimulation of protein
kinase C via thiol modification and modulation of zinc content.
J Biol Chem. 2000;275:24136–24145.
44. Suzuki A, Hirata M, Kamimura K, et al. aPKC acts upstream
of PAR-1b in both the establishment and maintenance
of mammalian epithelial polarity. Curr Biol. 2004;14:1425–
45. Eder AM, Sui X, Rosen DG, et al. Atypical PKCiota contributes
to poor prognosis through loss of apical–basal polarity and
cyclin E overexpression in ovarian cancer. Proc Natl Acad Sci
46. Regala RP, Weems C, Jamieson L, Copland JA, Thompson EA,
Fields AP. Atypical protein kinase C iota plays a critical role in
human lung cancer cell growth and tumorigenicity. J Biol Chem.
47. Ikeguchi M, Makino M, Kaibara N. Clinical significance of
E-cadherin-catenin complex expression in metastatic foci of
colorectal carcinoma. J Surg Oncol. 2001;77:201–207.
48. Wijnhoven BP, Dinjens WN, Pignatelli M. E-cadherin-catenin
cell-cell adhesion complex and human cancer. Br J Surg. 2000;
49. Shiozaki H, Oka H, Inoue M, Tamura S, Monden M. E-cadherin
mediated adhesion system in cancer cells. Cancer. 1996;77:
50. Hsu IC, Metcalf RA, Sun T, Welsh JA, Wang NJ, Harris CC.
Mutational hotspots in the p53 gene in human hepatocellular
carcinomas. Nature. 1991;350:427–428.
51. Liu S, Ma L, Huang W, et al. Decreased expression of the
human carbonyl reductase 2 Gene HCR2 in hepatocellular car-
cinoma. Cell Mol Biol Lett. 2006;11:230–241.
52. Haidon GH, Hayes PC. Screening for hepatocellular carcinoma.
Eur J Gastroenterol Hepatol. 1996;8:856–860.
53. Deugnier Y, David V, Bressot P, et al. Serum a-L-fucosidase: a
new marker for the diagnosis of primary hepatic carcinoma?
54. Leray G, Deugnier Y, Jouanolle AM, et al. Biochemical aspects
of a-L-fucosidase in hepatocellular carcinoma. Hepatology.
55. Giardina MG, Matarazzo M, Varriale A, Morante R, Napoli A,
Martino R. Serum alpha-L-fucosidase. A useful marker in the
diagnosis of hepatocellular carcinoma. Cancer. 1992;70:1044–
56. Ishizuka H, Nakayama T, Matsuoka S, et al. Prediction of the
development of hepato-cellular-carcinoma in patients with liver
cirrhosis by the serial determinations of serum alpha-L-fucosi-
dase activity. Intern Med. 1999;38:927–931.
57. Mattern J, Koomagi R, Volm M. Association of vascular
endothelial growth factor expression with intratumoural micro-
vessel density and tumor cell proliferation in human epidermoid
lung carcinoma. Br J Cancer. 1996;73:931–934.
58. Brown LF, Berse B, Jackman RW, et al. Expression of vascular
permeability factor (vascular endothelial growth factor) and its
receptors in adenocarcinoma of gastrointestinal tract. Cancer
59. Toi M, Hoshina S, Takayanagi T, et al. Association of vascular
endothelial growth factor expression with tumour angiogenesis
and early relapse in primary breast cancer. Jpn J Cancer Res.
60. Suzuki K, Hayashi M, Miyamaoto Y, et al. Expression of vas-
cular permeability factor/vascular endothelial growth factor in
human hepatocellular carcinoma. Cancer Res. 1996;56:3004–
61. Mise M, Arii S, Higashituji H, Furutani M, et al. Clinical sig-
nificance of vascular endothelial growth factor and basic fibro-
blast growth factor gene expression in liver tumor. Hepatology.
62. Mohle R, Green D, Moore MAS, et al. Constitutive production
and thrombin-induced release of vascular endothelial growth
factor by human megakaryocytes and platelets. Proc Natl Acad
Sci USA. 1997;94:663–668.
63. Li XM, Tang ZY, Qin LX, Zhou J, Sun HC. Serum vascular
endothelial growth factor is a predictor of invasion and metas-
tasis in hepatocellular carcinoma. J Exp Clin Cancer Res.
64. Suminami Y, Kishi F, Sekiguchi K, Kato H. Squamous cell
carcinoma antigen is a new member of the serine protease
inhibitors. Biochem Biophys Res Commun. 1991;181:51–58.
65. Kato H, Suehiro Y, Morioka H, et al. Heterogeneous distribution
of acidic TA-4 in cervical squamous cell carcinoma: immuno-
histochemical demonstration with monoclonal antibodies. Jpn J
Cancer Res. 1987;78:1246–1250.
66. Giannelli G, Marinosci F, Sgarra C, Lupo L, Dentico P, Anto-
naci S. Clinical role of tissue and serum levels of SCCA antigen
in hepatocellular carcinoma. Int J Cancer. 2005;10(116):
67. Uemura Y, Pak SC, Luke C, Cataltepe S, Tsu C, Schick C,
Kamachi Y, Pomeroy SL, Perlmutter DH, Silverman GA.
Circulating serpin tumor markers SCCA1 and SCCA2 are not
actively secreted but reside in the cytosol of squamous carci-
noma cells. Int J Cancer. 2000;89:368–377.
68. Deftos LJ. Chromogranin A: its role in endocrine function and
as an endocrine and neuroendocrine tumor marker. Endocr Rev.
69. Leone N, Pellicano R, Brunello F, Rizzetto M, Ponzetto A.
Elevated serum chromogranin A in patients with hepatocellular
carcinoma. Clin Exp Med. 2002;2:119–123.
70. Ranno S, Motta M, Rampello E, Risino C, Bennati E, Mala-
guarnera M. The chromogranin-A(CgA) in prostate cancer. Arch
Gerontol Geriatr. 2006;43:117–126.
71. Malaguarnera M, Cristaldi E, Cammalleri L, et al. Elevated
chromogranin A (CgA) serum levels in the patients with
advanced pancreatic cancer. Arch Gerontol Geriatr. 2009;48:
72. Spadaro A, Ajello A, Morace C, et al. Serum chromogranin-A in
hepatocellular carcinoma: diagnostic utility and limits. World J
73. Malaguarnera M, Vacante M, Fichera R, Cappellani A, Cristaldi
E, Motta M. Chromogranin A (CgA) serum level as a marker
of progression in hepatocellular carcinoma (HCC) of elderly
74. Wilander E, Lundqvist M, Oberg K. Gastrointestinal carcinoid
tumours. Histogenetic, histochemical, immunohistochemical,
clinical and therapeutic aspects. Prog Histochem Cytochem.
75. Hsiao RJ, Parmer RJ, Takiyyuddin MA, O’Connor DT. Chro-
mogranin A storage and secretion: sensitivity and specificity for
the diagnosis of pheochromocytoma. Medicine. 1991;70:33–45.
Dig Dis Sci
76. Malaguarnera L, Pignatelli S, Simpore ` J, Malaguarnera M,
Musumeci S. Plasma levels of interleukin-12 (IL-12), interleu-
kin-18 (IL-18) and transforming growth factor beta (TGF-beta)
in Plasmodium falciparum malaria. Eur Cytokine Netw. 2002;
77. Bedossa P, Peltier E, Terries B, Franco D, Poynard T. Trans-
forming growth factor -b1 (TGF-b1) and TGF-b1 receptors in
normal, cirrhotic and neoplastic human livers. Hepatology.
78. Ito N, Kawata S, Tamura S, et al. Expression of transforming
growth factor b1 mRNA in human hepatocellular carcinoma.
Jpn J Cancer Res. 1990;81:1202–1205.
79. Grizzi F, Franceschini B, Hamrick C, et al. Usefulness of can-
cer-testis antigens as biomarkers for the diagnosis and treatment
of hepatocellular carcinoma. J Transl Med. 2007;5:3.
80. Mann CD, Neal CP, Garcea G, et al. Prognostic molecular
markers in hepatocellular carcinoma: a systematic review. Eur J
81. Ko TC, Tu W, Sakai T, et al. TGF-b1 effects on proliferation of
rat intestinal epithelial cells are due to inhibition of cyclin D1
expression. Oncogene. 1998;16:3445–3454.
82. Izzo JG, Papadimitrakopoulou VA, Li XQ, et al. Dysregulated
cyclin D1 expression early in head and neck tumorigenesis: in
vivo evidence for an association with subsequent gene amplifi-
cation. Oncogene. 1998;17:2313–2322.
83. Seewaldt VL, Kim JH, Parker MB, Dietze EC, Vasan KV,
Caldwell LE. Dysregulated expression of cyclin D1 in normal
human mammary epithelial cells inhibits all-trans-retinoic acid-
mediated G0/G1-phase arrest and differentiation in vitro. Exp
Cell Res. 1999;249:70–85.
84. Kladney RD, Bulla GA, Guo L, et al. GP73, a novel Golgi-
85. Kladney RD, Cui X, Bulla GA, Brunt EM, Fimmel CJ.
Expression of GP73, a resident membrane protein, in viral and
non-viral liver disease. Hepatology. 2002;35:1431–1440.
86. Block TM, Comunale MA, Lowman M, et al. Use of targeted
glycoproteins that correlated with liver cancer in woodchucks
and humans. Proc Natl Acad Sci USA. 2005;102:779–784.
87. Comunale MA, Mattu TS, Lowman MA, et al. Comparative
proteomic analysis of de-N-glycosylated serum from hepatitis B
carriers reveals polypeptides that correlate with disease status.
88. Nakamura T. Hepatocyte growth factor as mitogen, motogen
and morphogen and its roles in organ regeneration. Princess
Takamatsu Symp. 1994;24:195–213.
89. Birchmeier C, Gherardi E. Development roles of HGF/SF and
its receptor c-Met tyrosine kinase. Trends Cell Biol. 1998;8:
90. El-Serag HB, Mason AC. Rising incidence of hepatocellular
carcinoma in the United States. N Engl J Med. 1999;340:
91. Schneider PD. Preoperative assessment of liver function. Surg
Clin North Am. 2004;84:355–373.
92. Breuhan K, Longerich T, Schirmacher P. Dysregulation of
growth factor signalling in human hepatocellular carcinoma.
93. Yamagamim H, Moriyana M, Matsumura H, et al. Serum con-
centrations of human hepatocyte growth factor is a useful
indicator for predicting the occurrence of hepatocellular carci-
nomas in C-viral chronic liver diseases. Cancer. 2002;95:
94. Mizuguchi T, Katsuramachi T, Nobuoka T, et al. Serum hyal-
uronate level for predicting subclinical liver dysfunction after
hepatectomy. World J Surg. 2004;28:971–976.
95. Wu FS, Zheng SS, Wu LJ, et al. Study on the prognostic value
of hepatocyte growth factor and c-met for patients with hepa-
tocellular carcinoma. Zhongua Wai Ke Za Zhi. 2006;44:
96. Vogelstein B, Kinzler KW. p53 function and dysfunction. Cell.
97. Gannon JV, Greaves R, Iggo R, Lane DP. Activating mutations
in p53 produce a common conformational effect-a monoclonal
antibody specific for the mutant form. EMBO J. 1990;9:1595–
98. Hsu H-C, Tseng H-J, Lai P-L, Lee P-H, Peng S-Y. Expression of
p53 gene in 184 unifocal hepatocellular carcinoma: association
with tumor growth and invasiveness. Cancer Res. 1993;53:
99. Hayashi H, Sugio K, Matsumata T, Adachi E, Takenaka K,
Sugimachi K. The clinical significance of p53 gene mutation in
hepatocellular carcinomas from Japan. Hepatology. 1995;22:
100. Crawford LV, Pim DC, Bulbrook RD. Detection of antibodies
against cellular protein p53 in sera from patients with breast
cancer. Int J Cancer. 1982;30:403–408.
101. Winter SF, Minna JD, Johnson BE, Takahashi T, Gazdar AF,
Carbone DP. Development of antibodies against p53 in lung
cancer patients appears to be dependent on the type of p53
mutation. Cancer Res. 1992;52:4168–4174.
102. Schlichtholz RLB, Bengoufa D, Zalcman BG, et al. Analysis of
p53 antibodies in patients with various cancer define B-cell
epitopes of human p53: distribution on primary structure and
exposure on protein surface. Cancer Res. 1993;53:5872–5876.
103. Bressac B, Kew M, Wands J, Ozturk M. Selective G to muta-
tions of p53 gene in HCC from southern Africa. Nature.
104. Bothwell M. Functional interactions of neurotrophins and neu-
rotrophin receptors. Annu Rev Neurosci. 1995;18:223–253.
105. Gregor LM, McCune BK, Graff JR, et al. Roles of trk family
neurotrophin receptors in medullary thyroid carcinoma devel-
opment and progression. Proc Natl Acad Sci USA. 1990;96:
106. Roux PP, Barker PA. Neurotrophin signaling through the p75
neurotrophin receptor. Prog Neurobiol. 2002;67:203–233.
107. Chapman BS. A region of the 75-kDa neurotrophin receptor
homologous to the death domains of TNFR-I and Fas. FEBS
108. Tokusashi Y, Asai K, Tamakawa S, et al. Expression of NGF in
hepatocellular carcinoma cells with its receptors in non-tumor
cell components. Int J Cancer. 2005;114:39–45.
109. Trim N, Morgan S, Evans M, et al. Hepatic stellate cells express
the low affinity nerve growth factor receptor p75 and undergo
apoptosis in response to nerve growth factor stimulation. Am J
110. Cassiman D, Roskams TJ. Beauty is in the eye of the beholder:
emerging concepts and pitfalls in hepatic stellate cell research.
111. Rasi G, Serafino A, Bellis L, et al. Nerve growth factor
involvement in liver cirrhosis and hepatocellular carcinoma.
World J Gastroenterol. 2007;13:4986–4995.
112. Preissner KT, Jenne D. Vitronectin: a new molecular connection
in haemostasis. Thromb Haemost. 1991;66:189–194.
113. Musso O, Theret N, Campion SP, et al. In situ detection of
matrix metalloproteinase-2 (MMP2) and metalloproteinase
inhibitor TIMP2 transcripts in human primary hepatocellular
carcinoma and in liver metastasis. J Hepatol. 1997;26:593–605.
114. Malaguarnera L, Ferlito L, Di Mauro S, Imbesi RM, Scalia G,
Malaguarnera M. Immunosenescence and cancer: a review. Arch
Gerontol Geriatrics. 2001;32:77–93.
Dig Dis Sci
115. Evrin PE, Wibell L. Serum b-2 microglobulin in various dis-
orders. Clin Chim Acta. 1973;43:183–186.
116. Weistal R, Norkrans G, Weiland O, et al. Lymphocyte subsets
and b2-microglobulin expression in chronic hepatitis C/non-A.
non-B. Effects of interferon-alpha treatment. Clin Exp Immunol.
117. Malaguarnera M, Restuccia S, Di Fazio I, Zoccolo AM, Trovato
BA, Pistone G. Serum beta-2 microglobulin in chronic hepatitis
C. Dig Dis Sci. 1997;42:762–766.
118. Motta M, Giugno I, Ruello P, Pistone G, Di Fazio I, Mala-
guarnera M. Lipoprotein (a) behaviour in patients with hepato-
cellular carcinoma. Minerva Medica. 2001;92:301–305.
119. Malaguarnera M, Di Fazio I, Laurino A, Motta M, Giugno I,
Trovato B. A Ro `le de interleukine-6 dans le carcinome he `pa-
tocellulaire. Bull Cancer. 1996;83:379–384.
120. Malaguarnera M, Di Fazio I, Ferlito L, et al. Increase of serum
b-2 microglobulin in patients affected by HCV correlated
hepatocellular carcinoma. Eur J Gastroenterol Hepatol. 2000;
121. Ni RZ, Huang JF, Xiao MB, et al. Glycylproline dipeptidyl
aminopeptidase isoenzyme in diagnosis of primary hepatocel-
lular carcinoma. World J Gastroenterol. 2003;9:710–713.
122. Vinci E, Rampello E, Zanoli L, Oreste G, Pistone G, Mala-
guarnera M. Serum carnitine levels in patients with tumoral
cachexia. Eur J Intern Med. 2005;16:419–423.
123. Malaguarnera M, Laurino A, Di Mauro S, Motta M, Di Fazio I,
Maugeri D. The comorbidities of elderly oncologic patients.
Arch Gerontol Geriatr. 2000;30:237–244.
124. Motta M, Ferlito L, Malaguarnera L, et al. Alterations of the
lymphocytic set-up in elderly patients with cancer. Arch Ger-
ontol Geriatr. 2003;36:7–14.
125. Motta M, Pistone G, Franzone AM, et al. Antibodies against ox-
LDL serum levels in patients with hepatocellular carcinoma.
Panminerva Med. 2003;45:69–73.
126. Malaguarnera L, Cristaldi E, Malaguarnera M (2009) The role
of immunity in elderly cancer. Crit Rev Oncol Hematol. PMID
127. Liaw YF, Tai DI, Chen TJ, Chu CM, Huang MJ. Alpha-feto-
protein changes in the course of chronic hepatitis: relation to
bridging hepatic necrosis and hepatocellular carcinoma. Liver.
128. Malaguarnera M, Gargante MP, Fricia T, Rampello E, Risino C,
Romano M. Hepatitis C virus in elderly cancer patients. Eur J
Intern Med. 2006;17:325–329.
129. Noda K, Miyoshi E, Uozumi N, et al. Gene expression of
alpha1–6 fucosyltransferase in human hepatoma tissues: a pos-
sible implication for increased fucosylation of alpha-fetoprotein.
130. Guido M, Roskams T, Pontisso P, et al. Squamous cell carci-
noma antigen in human liver carcinogenesis. J Clin Pathol.
131. Wu TT, Hsieh YH, Wu CC, Hsieh YS, Huang CY, Liu JY.
Overexpression of protein kinase C alpha mRNA in human
hepatocellular carcinoma: a potential marker of disease prog-
nosis. Clin Chim Acta. 2007;382:54–58.
Dig Dis Sci