Introduction
Hepatocellular carcinoma (HCC) is the primary type
of liver cancer; both age-adjusted incidence and mortality
of HCC have steadily increased in recent years.
The prognosis of advanced HCC is very poor. When
diagnosed, most cases have already exceeded the limits of tumor treatment. The major risk factors for HCC
are viral (chronic hepatitis B and C), toxic (alcohol
and aflatoxins), metabolic (diabetes and nonalcoholic
fatty liver disease, hereditary haemochromatosis), and
immune-related diseases (primary biliary cirrhosis and
autoimmune hepatitis).[]
International consensus on a diagnostic pathway biochemisexists
because there is no ideal screening modality.
Serum alpha fetoprotein (AFP) level is the most commonly
used serological test in conjunction with hepatic
ultrasonography to detect HCC in cirrhotic patients.
A number of serum markers [e.g., AFP, hepatocyte
growth factor (HGF), transforming growth factor beta
1 (TGFB-1), and serum proteomics] have been proposed,
and several of these markers have been promising
for detecting HCC in the clinical setting. Future
research may show more specific and sensitive markers
using proteomic or metabolomic approaches to screen
blood or other biological fluids, such as urine.[]
Glypicans (GPCs) are released from the cell surface
by a lipase to regulate the signaling of wnts, hedgehogs,
fibroblast growth factors, and bone morphogenetic proteins.[-] In mammals, the GPC family comprises six
members.[] It has been detected in the placenta and
fetal liver, but not in other adult organs. GPC3 belongs
to a group of heparan sulfate proteoglycans bound to
the outer surface of the cell membrane through a glycosylphosphatidylinositol
anchor.[] 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
gene. GPC3 can differentiate between malignant
and benign hepatic lesions. Therefore, it seems to be
a potential biomarker for the early diagnosis of HCC.
[,]
Angiogenesis, defined as the formation of new
blood vessels from the existing vasculature, is an important
process regulating the growth and development
of malignancies, including HCC.[] The extensive
hypervascularity associated with HCC is thought
to be partly driven by the proangiogenic factor vascular
endothelial growth factor (VEGF). VEGF is an
endothelial cell mitogen that initiates and promotes
neovascularization and endothelial cell proliferation,
and it was initially identified as a vascular permeability
factor. VEGF has a major effect in regulating angiogenesis,
and its expression has been shown to correlate
with carcinogenesis. Furthermore, the invasiveness of
certain HCC lesions has recently been associated with
high levels of VEGF, thereby leading several authors to
conclude that an important association exists between
VEGF and prognosis for HCC.[,]
Interleukin-6 (IL-6) is one of the major inflammatory
cytokines, and in several types of target cells,
it affects a variety of biological responses, including
changes in cell differentiation, growth, and apoptosis
and induction of acute-phase responses.[] IL-6
expression is induced in various cell types, including endothelial cells, hepatocytes, and Kupffer cells. An
increasing body of evidence indicates a key role of
the pleiotropic cytokine IL-6 in liver damage process
(chronic hepatitis B, cirrhosis) and carcinogenesis [].
IL-6 is also implicated in tumor growth, progression,
metastasis, and immune evasion, suggesting the future
possibility of treating cancers via the modulation of the
IL-6 pathway.[,]
Early diagnosis of HCC with a simple blood test detecting
the increased levels of GPC3, VEGF, and IL-6
would likely be effective for the prognosis and treatment
of the disease. In the present study, we investigated
whether these serum markers can be useful for
clinicians in deciding the diagnosis of HCC. We aimed
to investigate the variance of these parameters in cirrhotic
patients as it is known that 90% of patients with
HCC have liver cirrhosis on the ground, and liver failure
affects the prognosis.
Methods
This study comprised 54 patients with HCC and 30
with liver cirrhosis who were referred from Istanbul
Medical Faculty Department of Gastroenterology and
21 healthy controls. The median age at diagnosis was
60 years (range, 36-77), where males constituted the
majority of the group (89%). All the patients had history
of cirrhosis without vascular invasion, portal vein
thrombosis, and extrahepatic disease. According to
AASLD guideline 2011, liver biopsy for HCC diagnosis
is not necessary in cirrhotic patients whose liver nodules
(>1 cm) fulfill the contrast-enhanced imaging criteria
(hypervascular in the arterial phase with washout
in the portal venosus or delayed phase) or whose AFP
levels are >200 ng/mL. Liver biopsy was performed for
only nine patients. Patients were staged according to
the Child-Pugh score, Barcelona Clinic Liver Cancer
(BCLC) staging system, and model for end-stage liver
disease (MELD). Patients with HCC were divided
into two groups: early stage (Child-Pugh and BCLC,
A; MELD, <11) and advanced stage (Child-Pugh and
BCLC, B or C; MELD, >11). Patients with HCC underwent
various imaging modalities, such as computed tomography
(CT), magnetic resonance imaging (MRI),
and magnetic resonance cholangiopancreatography
(MRCP). Blood samples of the patients were collected
before any treatment was administered. The pretreatment
evaluation included detailed clinical history and
physical examination with biochemistry tests and complete
blood cell counts. Outpatients with ECOG performance
status ?2 and appropriate blood biochemistry tests received ablative treatment (hepatic resection
and/or radiofrequency ablation) or palliative treatment,
such as 90Y radioembolization and/or transarterial
chemoembolization. None of the patients received
sorafenib. The response to treatment was evaluated according
to internal criteria. Treatment was continued
until disease progression or unacceptable toxicity.
For comparison of serum GPC3, VEGF, and IL-6
levels, 21 age- and sex-matched healthy controls were
included in the analysis. Our study was approved by
the Istanbul University Ethics Committee (2556-24).
The protocol was consistent with the Declaration of
Helsinki (1989). Informed consents were obtained
from all the study participants.
Blood samples of the patient and control groups
were obtained by venipuncture and clotted at room
temperature. The sera were collected following centrifugation
and immediately frozen at -20ºC until analysis.
Measurement of serum GPC3, VEGF, and IL-6 levels
GPC3 enzyme-linked immunosorbent assay (ELISA;
USCN Life Science Inc., Wuhan P.R., China) is a sandwich
enzyme immunoassay employing monoclonal
antibodies. An antibody specific for human GPC3 was
immobilized onto the surface. The sample and biotinylated
detector monoclonal antibody were pipetted into
the wells and allowed to incubate for 2 h. GPC3 binds
to the capture and detection of antibodies. Unbound
GPC3 was washed away. Next, horseradish peroxidaseconjugated
streptavidin was added, which bound to the
detector antibody. Horseradish peroxidase catalyzes
the conversion of the chromogenic substrate tetramethylbenzidine
(TMB) from a colorless to blue solution
(or yellow after the addition of stopping reagent),
the intensity of which is proportional to the amount of
GPC3 in the sample. The colored reaction product was
measured using an automated ELISA reader (Rayto,
RT-1904C Chemistry Analyzer, Atlanta GA, USA). The
results were expressed as ng/mL.
VEGF assay (Invitrogen Corporation, Camarillo,
USA) employs the quantitative sandwich enzyme immunoassay
technique. A monoclonal antibody specific
for VEGF was precoated onto a microplate. Standards
and samples were pipetted into the wells, and any
VEGF present was bound by the immobilized antibody.
After washing any unbound VEGF, an enzymelinked
polyclonal antibody for VEGF was added to the
wells. Following a wash for removing any unbound
antibody-enzyme-reagent, a substrate solution was
added to the wells. Color development is proportional to the amount of VEGF bound in the initial step.
Color development was stopped, and the intensity of
the color was measured using an ELISA reader (Rayto,
RT-1904C Chemistry Analyzer). The results were expressed
as ng/mL.
IL-6 (Invitrogen Corporation) levels were determined
using specific ELISA. IL-6 anti- monoclonal
coating antibody was adsorbed onto the microwells.
IL-6 present in the sample or standard bound to antibodies
adsorbed onto the microwells. Next, an FITCconjugated
monoclonal anti-IL-6 antibody was added,
and IL-6 bound to the IL-6 adsorbed onto the first antibody.
Following incubation, anti-IL-6 was washed, and
HRP-conjugated antibody was added and immobilized
by the sandwich. Following incubation, unbound anti-
FITC-HRP was washed, and substrate solution reactive
with HRP was added to the wells. A colored product
was formed proportional to the amount of IL-6 present
in the sample. The reaction was terminated by the addition of a stop solution, and absorbance was measured
using an ELISA reader (Rayto, RT-1904C Chemistry
Analyzer).The results were expressed as pg/mL.
Statistical analysis
SPSS software (version 16; SPSS, Chicago, IL) was used
for statistical analysis. The data did not show a normal
distribution; therefore, the nonparametric Mann?
Whitney U test was used to evaluate the differences
between patients and normal controls. A two-tailed pvalue
of <0.05 was considered statistically significant.
The report design was adopted from the Standards
for Reporting Diagnostic Accuracy (STARD) group
(Bossuyt et al. 2004). Survival was calculated from the
date of first hospital admission to death resulting from
any cause or to last contact with the patient or any family
member. The Kaplan?Meier method was used for
the estimation of survival distribution and log-rank
statistics for differences in survival. A p-value of <0.05
was considered statistically significant. The sensitivity
and specificity of the tests were calculated using receiver
operating characteristics (ROC) curves.
Results
Histopathological characteristics and demographic
features of patients are listed in Tables 1 and 2, respectively.
A total of 46% (n=25) of patients had Child-
Pugh score A, 30% (n=16) had B or C, and scores for
24% (n=13) of patients were unknown. The levels of
serum GPC3, VEGF, IL-6, and AFP in patients with
HCC, those with cirrhosis, and healthy controls are
shown in Table 3. The baseline serum GPC3 levels
were significantly higher in patients with HCC (6.1 vs.
1.9 ng/mL; p<0.001) and those with cirrhosis (5.9 vs
1.9 ng/mL; p=0.001) than in controls (Fig. 1a). The serum
VEGF levels were significantly higher in patients
with HCC (213.6 vs 24.4 ng/mL; p<0.001) and those
with cirrhosis (118.3 vs 24.4 ng/mL; p=0.003) than in
controls (Fig. 1b). The serum IL-6 levels were significantly
higher in patients with HCC (5.8 vs 0.2 pg/mL;
p=0.02) and those with cirrhosis (2.3 vs 0.2 pg/ mL;
p=0.04) than in the controls (Fig. 1c). The serum AFP
levels were significantly higher in patients with HCC
(p=0.001) and those with cirrhosis (p=0.002) than in
controls. The correlation between GPC3 and VEGF
levels was statistically significant (rs=0.269, n=54,
p=0.02; Fig. 2a). The correlation between GPC3 and
IL-6 levels was statistically significant (rs=0.257, n=54,
p=0.02; Fig. 2b). The correlation between VEGF and
IL-6 levels was not statistically significant (rs=0.416,
n=54, p>0.05; Spearman"s correlation; Fig. 2c).
The median follow-up duration was 14 months (range,
0-53 months). At the end of the observation period, 31 patients (57.4%) were dead. The median survival of
all the patients was 19.0±8.6 months (95% CI=1.9?36.0
months), where the 1-year overall survival rate was
63% (95% CI=49.7-76.3). Poor performance status
(p<0.001), viral etiology of cirrhosis (p=0.03), larger
tumor size (p=0.01), lower serum hemoglobin levels
(p=0.03), and not treated for HCC (p=0.001) were
related to worse survivals (Tables 4a and 4b). Serum
GPC3, VEGF, and IL-6 levels were not found to have
significant adverse effects on survival (p=0.37, p=0.30,
and p=0.40; Figures 3, 4, and 5, respectively).
To determine the cut-off values and sensitivity and
specificity of the tests, we used the ROC curves (Fig. 6).
The cut-off values were chosen according to the ROC curve coordinate points, and cut-off points for serum
VEGF, GPC3, and IL-6 were equal to their mean values.
The cut-off levels (x+2SD) for VEGF, GPC-3, and
IL-6 were 58.24 pg/mL, 5.27 pg/mL, and 5.96 ng/mL,
respectively.
Fig 1: (a) The values of serum GPC3 assays in patients
with HCC and healthy controls (p<0.001). (b)
The values of serum VEGF assays in patients with
HCC and healthy controls (p<0.001). (c) The values
of serum IL-6 assays in patients with HCC
and healthy controls (p=0.02).
Fig 2: (a) Correlation between serum GPC3 and VEGF
levels in patients with HCC (rs=0.269, n=54,
p=0.02; Spearman"s correlation). (b) Correlation
between serum GPC3 and IL-6 levels in patients
with HCC (rs=0.257, n=54, p=0.02; Spearman"s
correlation). (c) Correlation between serum VEGF
and IL-6 levels in patients with HCC (rs=0.416,
n=54, p<0.001; Spearman"s correlation).
Fig 3: Survival curves in patients with HCC according
to serum GPC3 levels (p=0.37).
Fig 4: Survival curves in patients with HCC according
to serum VEGF levels (p=0.30).
Fig 5: Survival curves in patients with HCC according
to serum IL-6 levels (p=0.40).
Fig 6: ROC curve.
Table 1: HCC patients and disease characteristics
Table 2: Cirrhotic patients characteristics
Table 3: The values of serum marker levels in patients with HCC, those with cirrhosis, and healthy controls
Table 4a: Survival analyses of clinical characteristics
Table 4b: Survival analyses of laboratory parameters
The cut-off values were calculated using the ROC
curve, which plots the ROC curve corresponding to
the sensitivity and specificity of the test. The sensitivity
and specificity values were as follows: VEGF, 100%
and 92.6%; IL-6, 95.2% and 46.3%; GPC3, 90.5% and
51.9%, respectively.
Discussion
The major diagnostic parameters for HCC include serum
markers, various imaging modalities, and histological
analysis. A number of serum markers have been
proposed, and several are currently used in common
clinical practice for detecting HCC.[] However, new
markers for early diagnosis and better prediction of
prognosis are required. In the present study, we evaluated
the serum levels of GPC3, VEGF, and IL-6 in patients
with HCC.
It was recently reported that GPC3 is present only
in HCC cells but not in benign liver tissues and thus
can be used as a potential biomarker for the early diagnosis
of HCC. However, no correlation was observed
between AFP and GPC3 levels, and only 53% of patients
with HCC had significantly elevated serum levels
of GPC3. A subsequent study confirmed the presence
of significantly increased serum GPC3 levels in patients
with HCC.[,] Consistent with these results,
serum GPC3 levels of the HCC group were significantly higher than those of the control group in this study.
Most studies have revealed that GPC3 is an important tumor marker in the diagnosis of HCC. Taken
together, our data demonstrated that GPC3 may be a
more effective diagnostic marker than AFP for HCC
because more frequent upregulation of GPC3 than of
AFP was detected in patients with HCC.[] To extend
these findings, a recent large-scale study showed the
potential of GPC3 in distinguishing liver adenoma and
other benign hepatocellular conditions from well-differentiated
HCC. Also, researchers have revealed that
GPC3 could be used as an early marker for hepatic carcinogenesis
as significantly increased levels of GPC3
were found in high-grade dysplastic or early HCC
than in benign or low-grade dysplastic macronodules.
Moreover, serum GPC3 levels measured using ELISA
could be a useful tool in monitoring patients with HCC
during follow-ups.[] As a result, it can be concluded
that GPC3 is an important marker for liver cancer, and
recent data have proved its role in the diagnosis of hepatocellular
neoplasms.[,]
High VEGF levels have been observed in patients
with HCC with lesions of advanced pathological stage,
vascular invasion, lack of capsule formation, thereby
suggesting a strong relationship between VEGF and
HCC prognosis.[] In a recent study investigating
the usefulness of serum VEGF level as an indirect
marker for tumor levels using immunohistochemical
staining of 60 specimens of resected HCC, it was
found that VEGF level in hepatic tissue was in correlation
with that in the platelets in circulation.[] In the present study, the serum VEGF levels of patients
with HCC were significantly higher than those in the
control group, indicating a relationship between VEGF
and HCC diagnosis. Our observations were supported
by related recent studies which evaluated the impact of
VEGF as a biomarker for HCC diagnosis. We found
that VEGF had a sensitivity of 78% and specificity of
85% for HCC diagnosis, and significantly poor outcome
was observed in patients with higher levels of serum
VEGF. Interestingly, in a recent study, it was also
reported that patients with higher levels of both angiogenic
and anti-angiogenic factors had poorer survival.
[] The most noticeable factor associated with high
levels of serum VEGF in patients with advanced HCC
was hypoalbuminea, which is also known to be an independent
risk factor in patients with HCC.
The level of serum IL-6 has been reported to be
significantly elevated in patients with cholangiocarcinoma,
hepatocellular carcinoma, and metastatic
colorectal cancer compared with healthy controls and
those with benign biliary diseases.[] On the other
hand, IL-6 may be responsible for liver inflammation
and regeneration in chronic liver disease. IL-6 is also
shown to induce the expression of mitogenic, motogenic,
morphogenic, and pro-neoangiogenic scatter
factors and HGF, which are commonly expressed at
high levels in HCC.[] Moreover, it is implicated in
tumor growth, progression, metastasis, and invasion;
therefore, the IL-6 pathway may be a possible target for
cancer treatment in the future.[,]
Our results, which were in substantial agreement
with some previous literature, indicated a potential role
for IL-6 as a tumor marker for HCC. In particular, the
diagnostic value of the test was significantly increased
when it was used in association with AFP. Combining
the two markers provided a new perspective in the diagnosis
of HCC. Although the combined use of IL-6
and other serum biological markers was not found to
have any prognostic value in a large cohort of patients
with HCC, further trials are warranted to investigate its
diagnostic and especially prognostic values to confirm
its clinical usefulness for diagnosis and monitoring of
patients with HCC.[]
In conclusion, we evaluated the serum levels of
GPC3, VEGF, and IL-6 in HCC patients of various
clinical stages. We found that these markers had diagnostic
values but could not identify any prognostic
value. We determined higher serum levels of GPC3
in patients with HCC who had increased serum levels
of AFP. In addition, GPC3 acts as an oncofetal protein,
such as AFP; therefore, combined use of serum GPC3 and AFP levels may be an important marker
for the diagnosis of HCC. We believe that GPC3 can
be an important marker when used with FAP in HCC
diagnosis. Moreover, in patients who have another accompanying
disease that causes elevated AFP levels, we
suggest that the evaluation of GPC3 in association with
AFP may help in the diagnosis of HCC in the clinical
practice. The determination of serum GPC-3 levels can
be combined with radiodiagnostic methods.
Further studies with larger sample sizes are needed
to determine the potential clinical significance of these
markers in HCC diagnosis. Furthermore, additional
studies are required to investigate if their roles can be
extended to design targeted therapies for cancer.
Conclusion
In conclusion, we evaluated the serum levels of GPC3,
VEGF, and IL-6 in HCC patients of various clinical
stages. We found that these markers had diagnostic
values but could not identify any prognostic value. We
determined higher serum levels of GPC3 in patients
with HCC who had increased serum levels of AFP. In
addition, GPC3 acts as an oncofetal protein, such as
AFP; therefore, combined use of serum GPC3 and AFP
levels may be an important marker for the diagnosis
of HCC. We believe that GPC3 can be an important
marker when used with FAP in HCC diagnosis. Moreover,
in patients who have another accompanying disease
that causes elevated AFP levels, we suggest that the
evaluation of GPC3 in association with AFP may help
in the diagnosis of HCC in the clinical practice. The
determination of serum GPC-3 levels can be combined
with radiodiagnostic methods. Further studies with
larger sample sizes are needed to determine the potential
clinical significance of these markers in HCC diagnosis.
Furthermore, additional studies are required
to investigate if their roles can be extended to design
targeted therapies for cancer.
Acknowledgment: This study was supported by I. U. Research
Fund (4973) (Local Ethical Committee, Number:
2556-24).
This article was reviewed by a biostatistician from I.U. Oncology
Institute, Preventive Oncology, Biostatistics and Epidemiology
Department.
Peer-review: Externally peer-reviewed.
Conflict of Interest: The authors declare that they have no
conflicts of interest.
Authorship contributions: Concept - M.S.; Design - M.S.,
S.K.; Supervision - D.D., V.Y.; Materials - A.Ö.Ç.; Data collection
&/or processing - F.A., S.K., A.Ö.Ç.; Analysis and/
or interpretation - S.K., D.D.; Literature search - M.S., D.D.;
Writing - M.S.; Critical review - D.D., F.A.
List of abbreviations:
HCC: Hepatocellular carcinoma
GPC3: Gypican-3
VEGF: Vascular endothelial growth factor
IL-6: Interleukin-6
AFP: Alpha fetoprotein
HGF: Hepatocyte growth factor
TGFB-1: Transforming growth factor beta 1
BCLC: Barcelona Clinic Liver Cancer
MELD: Model For End-Stage Liver Disease
AASLD: American Association for the Study of Liver Diseases.
CT: Computed tomography
MRI: Magnetic resonance imaging
MRCP: Magnetic resonance cholangiopancreatography
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