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Contents Alexandria Journal of Hepatogastroenterology, Volume IIIX ( III ), December 2013
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Disclaimer: The Publisher, the Egyptian Society of
Hepatology Gastroenterology and Infectious Diseases in
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Egyptian Society of Hepatology Gastroenterology &
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the Publisher, society, and editors of the products advertised.
Original Article:
The Beneficial Effects of Resveratrol in A Murine Model
of Schistsoma Mansoni-Induced Liver Fibrosis.
Nihal M. El Habachi1, Gehan Yassin Soliman 1, Gihan M.
Sharara2, Abderrhman AM Ismeil3
1Department of Medical Physiology, Faculty of Medicine,
Alexandria University, Egypt., 2Department of Medical
Biochemistry, Faculty of Medicine, Alexandria University, Egypt. 3Department of Physiology, Faculty of Medicine,
Sinnar University, Sudan.
-------------------------------------------
Original Article:
Clinical Utility of Alfa- Fetoprotein-L3 in Hepatocellular
Carcinoma Patients
Ayman Mohamed Shamseya (1), Amal El Mahdi Mohamed Shafaey (2), Khaled Mustapha Belal (2) & Waleed Said
Mohamed Moftah (2)1) Department of internal medicine,
Faculty of medicine-Alexandria University, (2) Department of Clinical and Chemical Pathology, Faculty of Medicine-
Benha University
-------------------------------------------
Original Article
Study of Hepatic Neo-Angiogensis in Different Stages of
Chronic Hepatitis C Related Liver Diseases
Mohamed Yousri Taher Rashed1, El Said Hassan Ibrahim1,
Nahed Mohamed Baddour2, Amany Saleh Abd EL Aziz El
Yamany1, Mohamed Saed Said Shater1
Hepatobiliary Unit Alexandria University Internal Medicine department1, pathology department2, Faculty of Medicine,
University of Alexandria1,2 Egypt
------------------------------------------- Original Article
Tenascin C: A new promising Marker for Liver
Inflammation and Fibrosis in Hepatitis C Patients
Amal Sobhy El Sedfy1 and Marwa A. Madkour2 1Department of Pathology; 2Department of Clinical and
Experimental Internal Medicine, Medical Research Institute, University of Alexandria.
-------------------------------------------
2
14
26
40
Original Article
The Beneficial Effects of Resveratrol in A Murine Model of Schistsoma
Mansoni-Induced Liver Fibrosis.
Nihal M. El Habachi1, Gehan Yassin Soliman 1, Gihan M. Sharara2, Abderrhman AM Ismeil3
1Department of Medical Physiology, Faculty of Medicine, Alexandria University, Egypt., 2Department of Medical
Biochemistry, Faculty of Medicine, Alexandria University, Egypt. 3Department of Physiology, Faculty of Medicine,
Sinnar University, Sudan.
ABSTRACT
In Schistosomiasis, reactive oxygen species generation can result in stellate cells activation and consequently, liver
fibrosis. Resveratrol, a natural polyphenol, has been shown to possess antioxidant and anti-inflammatory
properties. However, studies of its protective effects against Schistosoma mansoni-induced liver fibrosis are limited.
Aim of the works: The present study was designed to explore the beneficial effects of resveratrol in a murine model of
Schistosoma mansoni-induced liver fibrosis. Material and methods: Sixty male albino mice were divided into four
groups of 15 mice as follows: normal resveratrol-untreated, normal resveratrol-treated, Schistosoma mansoni-infected
resveratrol-untreated and schistosoma mansoni-infected resveratrol-treated. At the end of the experimental period,
blood samples were collected to measure serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and
tumour necrosis factor-α (TNF-α). Liver tissue was collected for malondialdehyde (MDA) measurement,
histopathological examination and fibronectin gene expression analysis. Results: AST, ALT, TNF-α, and MDA levels
were significantly increased in the infected resveratrol-untreated group compared to normal resveratrol-untreated group
(all, P < 0.05). However, their levels were significantly decreased in the infected resveratrol-treated group compared to
infected resveratrol-untreated group (all, P < 0.05). In addition, fibronectin gene expression was highly up-regulated in
the infected resveratrol-untreated group compared to normal resveratrol-untreated group (P < 0.05).Furthermore, the
infected resveratrol-treated group have significantly lower fibronectin compared to infected resveratrol-untreated group
(P < 0.05). Conclusion: Results of the study indicate that resveratrol can attenuate Shistosoma mansoni-induced liver
fibrosis via mechanisms involving its anti-oxidant, anti-inflammatory and anti-fibrotic properties. Therefore, further
researches are needed to validate its use as a therapeutic modality in fibrotic liver conditions.
Introduction
Hepatic fibrosis represents the response of the
liver to different chronic insults and is
associated with significant morbidity and
mortality (1). A variety of stimuli such as
parasitic infections, toxins, viruses and
hypoxia can trigger fibrogenesis(2). Schisto-
somiasis, a disease caused by parasitic worms
of the genus Schistosoma, is one of the major
causes of morbidity and mortality in the
developing world. Worldwide, almost 240
million individuals are chronically infected
with Schistosomes. [3,4] . Liver fibrosis,
resulting from Shistosoma mansoni (S.
mansoni) infection, is of clinical importance
among other chronic liver diseases [5]. Liver
fibrosis is initiated by a periportal granu-
lomatous inflammation around parasite eggs,
which reach the liver through the portal
circulation [6]. Experimental shistosome
infections in laboratory animals, particularly
mice, are used frequently to model the
pathophysiological features of the infection in
humans. These models have contributed to the
understanding of the immunopathology of
infection, particularly the mechanisms
associated with granuloma formation and
fibrotic scaring which are not easily
investigated in humans due to ethical and
operational reasons.(7). However, several
considerations must be taken into account
when interpreting results from those models
because in some aspects, the infection is not
similar to the clinical situation in endemic
regions (8). Murine models of S.mansoni
infection indicate that eggs which are trapped
in hepatic sinusoids, released antigens
resulting in granulomatous inflammatory
response which activate hepatic stellate cells
(HSCs). (9) Upon activation, HSCs proliferate
and transform into myofibroblast like cells
that deposit and secrete large amounts of
extracellular matrix components (ECM)
including collagen, proteoglycans and gly-
coproteins. Synthesis and secretion of these
components of ECM by activated HSCs play
a major role in the pathogenesis of hepatic
fibrosis (10,11). Fibronectin (FN) is multi-
functional glycoprotein and a prominent
component of ECM and plasma. Two types of
fibronectin can be distinguished, soluble
plasma fibronectin and insoluble cellular
fibronectin produced by hepatocytes and
fibroblasts, respectively (12). Cytokines are
important regulators of immuno- inflamm-
atory responses and have been shown to
modulate hepatic fibrosis in vivo and in
vitro.(13) Tumour necrosis factor –alpha (TNF-
α) is an proinflammatory cytokine which its
level has been shown to be elevated in
chronic liver injuries including infection,
alcoholic liver, ischemia and biliary
obstruction. (14) In addition to its role as proin-
flammatory cytokine, TNF-α also mediates
caspase-independent death via formation of
reactive oxygen species (ROS). (14,15).
Oxidative stress plays a crucial role in liver
fibrosis .(16) Damaged hepatocytes ,activated
kupffer cells, neutrophils and activated HSCs
serve as the major source of ROS in liver
fibrosis.(16,17) Lipid peroxidation is considered
a hallmark of oxidative stress, in which ROS
interact with the polyunsaturated fatty acids
(PUFAs) in the cell membrane forming, a
variety of lipid products such as MDA ,this
will affect the membrane integrity and the
homeostatic environment of the cell. MDA
has been frequently used as a marker of
oxidative stress and the most common method
for its assessment is the thiobarbituric acid
reactive substances (TBARS).(18,19). Resvera-
trol is a natural phytoalexin found mainly in
grape skin, has been reported to have a wide
range of biological properties and multiple
molecular targets [20]. It possesses potent
antioxidant, anti-proliferative and anti-
inflammatory properties [20,21]. Studies invest-
igating the beneficial effects of resveratrol in
Schistosoma-induced liver fibrosis and the
possible mechanisms are scarce. Therefore,
the aims of the present study were to
investigate the potential antioxidant anti-
inflammatory and anti-fibrotic effects of
resveratrol in a murine model of S. Mansoni
induced liver fibrosis in order to explore new
therapeutic strategy for this condition.
Materials and Methods
Experimental animals: This study was carried
out on 60 Male CD1 Swiss albino mice
weighing 18-22 gm . Mice were fed on
standard diet containing 24% protein, 4% fat
and about 4-5% fibre and water adlibitum.
Animals were maintained under standard
conditions of temperature about 22-25◦C with
regular 12 h light/dark cycle for a week prior
to the study. All procedures involving the
animals were conducted in accordance to the
ethical guidelines of both Theodor Bilharz
Research Institute ,Cairo and Faculty of
Medicine, Alexandria University. Experime-
ntal Design: Mice were divided randomly into
four main groups as follows: Group I :
Normal resveratrol-untreated (NU): Consisted
of 15 mice as a control group, received
distilled water as placebo infection
subcutaneously in the first day of the
experiment and also distilled water as placebo
treatment intraperitoneally. Group II: Normal
resveratrol-treated (NT): Consisted of 15 mice
injected with placebo infection and then
treated with resveratrol (Sigma-Aldrich
Chemie; Steinheim, Germany). Resveratrol
was injected intraperitoneal (I.P) in a dose of
20 mg/kg body weight, twice/week. The
treatment was started after 4 weeks from
beginning of the experiment and continued till
the end of the 10th week(end of the study).(22).
Group III : Infected resveratrol-untreated (IU)
group: Consisted of 15 mice, infected with an
Egyptian strain of S. mansoni cercariae. The
strain was maintained by a laboratory passage
in an Egyptian strain of Biomphalaria
Alexandrina snails. Infection was done by
subcutaneous injection of each mouse with ±
80 S. mansoni cercariae suspended in 0.2 ml
dechlorinated water. Mice received distilled
water as a placebo treatment after 4 weeks as
in Group I.(23). Group IV: Infected resveratrol-
treated (IT): It consisted of 15 mice. Each
mouse was infected with S. mansoni cercariae
as previously mentioned in Group III prior to
treatment with Resveratrol I.P at a dose of 20
mg/kg, twice/week as in group II. Laboratory
Investigations: At the end of the experiment,
blood samples were collected from the retro-
orbital venous plexus of the mouse by a
capillary haematocrit tube under light ether
anaesthesia (24). Blood was collected into a
clean dry non-heparinized Wassermann tubes
for separation of serum. The serum was
separated by centrifugation at 3000 rpm for
15 minutes and was aliquot into 2 samples
and stored at -80 ºC until assayed for
biochemical estimation of AST and ALT
colorimetrically (NS Biotec) (25), and TNF-α
(RayBio®, Mouse TNF-α ELISA Enzyme
Immunoassay).(26). Measurement of liver
malondialdehyde (MDA) content: After blood
sample collection, the mice were sacrificed
with decapitation, the anterior abdomen were
incised to expose the liver. The liver from
each mouse was excised immediately after
perfusion and rinsed with ice-cold saline
solution. For hepatic MDA determination, 25
mg of tissue was weighed and 250 μl of RIPA
buffer with protease inhibitors were added.
The mixture was sonicated for 15 seconds at
40 V over ice and centrifuged at 1.600 × g for
10 minutes at 4°C. We used the supernatant
for analysis. MDA was quantified using the
thiobarbituric acid reaction as described by
Ohkawa et al, and levels were expressed in
μM/g tissue (27). Quantification of fibronectin
gene expression (28). DNA extraction. DNA
was purified from liver tissue by using Gene
JET™ Genomic DNA Purification Kits.
Samples were digested with proteinase K in
the supplied Digestion or Lysis Solution.
RNA was removed by treating the samples
with RNase A. The lysate was then mixed
with ethanol and loaded on the purification
column where the DNA binds to the silica
membrane. Impurities were effectively
removed by washing the column with the
prepared wash buffers. Genomic DNA was
then eluted under low ionic strength
conditions with the Elution Buffer. PCR
amplification of fibronectin gene.
DreamTaq™ Green PCR Master Mix
(Fermentas Life Sciences) was a ready to use
solution containing DreamTaq™ DNA
polymerase, optimized DreamTaq™ Green
buffer, MgCl2 and dNTPs. The master mix
was supplemented with two tracking dyes and
a density reagent that allow for direct loading
of the PCR product on a gel. For a total 25μl
reaction volume, 12.5 μl DreamTaq™ Green
PCR Master Mix (2X), 1 μl forward and
reverse primers (F: 5'-GTGTCCTCCTTCCA
TCTTC-3' and R: 5'-CAGACTGTCGGTACT
CACG-3, respectively; Fermentas Life
Sciences.), 5 μl extracted DNA and 5.5 μl
nuclease-free water were mixed in PCR tubes.
Tubes were transferred to a thermal cycler
(Biometra) where the PCR was performed
according to the following protocol: Initial
denaturation at 95°C for 5 minutes, 35 cycles
of denaturation at 94°C for 1 minute,
annealing at 55°C for 1 minute, extension at
72°C for 1 minute, final extension at 72°C for
15 minutes. After PCR amplification, gel
electrophoresis was performed using 12 µl of
the DNA product, 8 µl of the TE buffer 2 µl
of the 50 bp ladder + 2 µl of loading dye 6X
(Amresco). A 302 nm ultraviolet transi-
lluminator was used for visualization of the
DNA bands. Gel electrophoresis images were
captured with a Kodak DC120 digital camera
and agarose gel analysis was performed to
determine the approximate amount of DNA in
the samples using a marker of 1-kb DNA
ladder. Liver histopatholology: The ventral
median lobe of the liver was fixed in 10%
neutral buffered formalin for histo-
pathological study to confirm liver fibrosis.
After fixation, liver samples were embedded
in paraffin wax, sectioned and stained with
hematoxylin and eosin (H&E) and/or
Masson’s trichrome. At least two different
sections were examined from each mouse.
The tissues were examined under microscope
for histopathological assessment. Photo-
micrographs were taken to compare between
the groups.
Statistical Analysis
The obtained data was expressed as mean ±
standard deviation (SD). Statistical
comparisons between groups were performed
using analysis of variance (ANOVA) and
unpaired Student’s t test. P < 0.05 was
considered statistically significant. Statistical
analyses were carried out using Graphpad
Prism version 5.0 (GraphPad Software, San
Diego, CA, USA).
Results
Resveratrol attenuates Schistosoma
mansoni-induced liver enzymes elevation:
Serum AST levels increased significantly in
infected untreated (IU) group compared to
both normal untreated (NU) and normal
treated (NT), (34.24 ± 3.95 vs.7.73 ± 1.39 and
8.90 ± 2.30 IU/L, respectively, p < 0.001;
Figure 1). Serum AST levels was
significantly decreased in the infected
Resveratrol treated (IT) compared to infected
untreated (IU) group (34.24 ± 3.95 vs. 10.55
± 1.73 IU/L, p < 0.001; Figure 1). Similarly,
ALT levels were significantly increased in
serum of ALT in IU group compared to NT
and IT groups (40.88 ± 4.09 vs. 16.05 ± 5.71,
15.99 ± 6.82 and 15.95 ± 6.41 IU/L,
respectively, P < 0.001; Figure 2). In contrast,
IT group had significantly lowered ALT
compared to IU group (15.95 ± 6.41 vs.
40.88 ± 4.09 IU / L p < 0.001; Figure 2).
Resveratrol attenuates Schistosoma mansoni-
induced increase in serum TNF-α level. To
investigate the anti- inflammatory effect of
the Resveratrol, TNF-α level was quantified
in serum using ELISA. While TNF-α levels in
IU group significantly increased compared to
the NU group (249.76±122.65 vs. 78.86 ±
10.39 pg/ml; P < 0.001; Figure 3), IT group
had significant decreased serum TNF-α levels
compared to the IU group (80.50 ± 32.56 vs.
249.76±122.65 pg/ml; P < 0.001; Figure 3).
Resveratrol attenuates S. mansoni-induced
lipid peroxidation: Next, we investigated
whether resveratrol could protect liver from
lipid peroxidation by measuring MDA. There
was significant increase in liver MDA levels
in the IU group as compared to the NU group
(10.08 ± 0.52 vs. 3.34 ± 0.68 μM/gm; P <
0.05, Figure 4). IT had significantly
decreased MDA levels compared to IU group
(3.71 ± 0.56 vs. 10.08 ± 0.52; P < 0.05, Figure
4). Resveratrol down-regulates S.mansoni-
induced Fibronectin gene expression:
Fibronectin expression significantly increased
in infected untreated (IU) group, as indicated
by DNA band densities, compared to normal
untreated (NU) control (5.38 ± 0.79 vs. 1.0 ±
0.0; P < 0.05; /5 Figure 5C). In contrast, IT
group had a significant decrease in fibronectin
DNA band densities compared to IU group
(1.69 ± 0.59 in vs. 5.38 ± 0.79; P < 0.05; /5
Figure 5C). Resveratrol attenuates S.mansoni-
induced histopathological changes :
Hematoxylin and Eosin (HE)-stained liver
sections of the IU group showed multiple
granulomas composed of central ova
surrounded by laminated layers of fibrous
tissue associated with inflammatory cells at
the periphery with sever necrosis of the
hepatic tissue (Figure 6C). However, in the IT
group, granulomas were seen as a concentric
focus of mononuclear and polymorphonuclear
cells around the egg and the laminated layers
of fibrous tissue nearly disappeared. In
addition, minimal microvascular changes and
no hepatocyte necrosis were noticed
compared to IU group (Figure 6D). Masson̕s
trichrome staining of liver sections of IU
group . showed expanded portal tracts with
fibrous tissue with occasional bridging
fibrosis as well as scattered periovular
granulomas compared to NU group (Figure 6a
and 6c). Resveratrol treatment improved S.
mansoni-induced liver damage in IT group as
it reduced the fibrosis compared to IU group
(Figure 6c and 6d).
Figure 1: The effect of resveratrol on serum AST in S.
mansoni-infected mice. NU= normal resveratrol-
untreated, NT= normal resveratrol-treated, IU= S.
mansoni-infected resveratrol-untreated and IT= S.
mansoni-infected resveratrol-treated. * P < 0.001.
Figure 2: The effect of resveratrol on serum ALT in S.
mansoni-infected mice. NU= normal resveratrol-
untreated, NT= normal resveratrol-treated, IU= S.
mansoni-infected resveratrol-untreated and IT= S.
mansoni-infected resveratrol-treated. * P < 0.05.
Figure 3: The effect of resveratrol on serum TNF-α level
in S. mansoni-infected mice. NU= normal resveratrol-
untreated, NT= normal resveratrol-treated, IU= S.
mansoni-infected resveratrol-untreated and IT= S.
mansoni-infected resveratrol-treated. * P < 0.001.
Figure 4: The effect of resveratrol on levels of
malondialdehyde (MDA) in μM/gm tissue in liver of S.
mansoni-infected mice. NU= normal resveratrol-
untreated, NT= normal resveratrol-treated, IU= S.
mansoni-infected resveratrol-untreated and IT= S.
mansoni-infected resveratrol-treated. * P < 0.05.
Figure 5: The effect of resveratrol on fibronectin gene expression in liver of S. mansoni-infected mice. NU= normal
resveratrol-untreated, NT= normal resveratrol-treated, IU= S. mansoni-infected resveratrol-untreated and IT= S. mansoni-
infected resveratrol-treated. A. Fibronectin DNA band densities in liver tissue of control and infected untreated group. B.
Fibronectin DNA band densities in liver tissue of infected- resveratrol untreated and infected-resveratrol treated group. C.
Fold change in fibronectin gene expression, * P < 0.05.
Figure 6: The effect of resveratrol on on liver histopathology in S. mansoni-infected mice. [A, a] NU= normal resveratrol-
untreated, [B, b] NT= normal resveratrol-treated, [C, c] IU= S. mansoni-infected resveratrol-untreated and [D, d] IT= S.
mansoni-infected resveratrol-treated. (A-D) stained with H&E staining and (a-d) stained with Masson’s trichrome stain.
Discussion
The results of the present study demonstrate
that resveratrol can attenuate S.mansoni-
induced AST and ALT elevation, TNF-α
production, and lipid peroxidation. Further-
more, resveratrol may prevent the develop-
pment of liver fibrosis by down-regulating
fibronectin gene expression in a murine
model of Schistosoma Mansoni ( S.mansoni
infection). Most chronic morbity in
Shistosomiasis is not due to the adult worms
but is related to the T-cell-dependent immune
response of the host,which is directed against
S. eggs trapped in tissues mainly in liver in
the case of S.mansoni.The trapped egg secrete
a range of molecules leading to
granulomatous reaction and collagen
deposition leading to fibrosis.(29). The present
study was conducted to explore the beneficial
effects of Resveratrol in S. mansoni induced
hepatic fibrosis in mice. Resveratrol is a
natural anti-oxidant compound belongs to the
phytoalexin polyphenols and its beneficial
effects are increasingly investigated (30). Our
data have shown elevated AST and ALT
levels in infected untreated mice. The
relatively high intensity of infection in the
experimental model may produce disturbance
in hepatic cell wall; immunological reactions,
inflammation and necrosis. Intraperitoneal
administration of resveratrol to the infected
mice remarkably attenuated the increased
levels of liver enzymes almost towards the
basal levels. These findings are in line with
Fan et al., who investigated the effects of
resveratrol against carbon tetrachloride
(CCL4) induced hepatic injury and reported
increased AST and ALT levels could be
normalized by resveratrol . (31). In that study, a
single dose of resveratrol was used pointing
to the effectiveness of resveratrol in reversing
the toxicity of CCl4 which is much more
damaging to the liver than Schistosomiasis .
Similarly, Schmatz et al, observed an increase
in AST, ALT levels induced by experimental
diabetes in rats and the administration of
resveratrol was able to decrease AST and
ALT levels .(32). It has been shown that
granulomatous reactions which occur in
Schistosomiasis is mediated by
proinflammatory cytokines such as TNF-α
and interleukin one beta (IL-1 β) which are
involved in part of multi-factorial defense
system that defend the infected liver against
inflammatory agents and injury.(33) In this
study, TNF-α serum levels were increased in
the infected untreated mice and could be
attenuated by resveratrol administration. Our
findings are comparable to previous data
where resveratrol has been demonstrated to
down-regulate TNF-α expression through
mechanisms involving inhibition of the
nuclear factor Kappa β (NF-kB) transcription
factor [34]. Muriel et al. have defined more
specifically that NF-kB regulates expression
of array of genes encoding cytokines such as
TNFα. In damaged hepatocytes, elevated
TNFα can induce NF-kB activation resulting
in more TNFα production which may result in
more destruction in the hepatic parenchyma
and impaired liver cell function. (35). In
contrast, Bujanda and colleagues have
demonstrated in mice that resveratrol can
decrease IL-1 with no effect on the high level
of the TNFα induced by alcohol administer-
ation [36]. This discrepancy might be explained
by the different methods that were used to
induce liver fibrosis. Oxidative stress plays a
crucial role in liver fibrosis .Damaged
hepatocytes, activated Kupffer cells and
neutrophils serve as major sources of ROS at
the initial stages of liver fibrosis. ROS
stimulate HSCs resulting in increased
collagen production (17). MDA has longer
half-life than ROS and is used as an indicator
of lipid peroxidation. (37)In the present study,
infection of mice with S.mansoni resulted in
increase in lipid peroxidation as measured by
MDA level in liver homogenates. Our data is
in accordance with Czeczot et al. (38), who
found increased MDA level and changes in
antioxidant enzyme activity in patients with
liver fibrosis. Similarly, Wu et al. [39],
observed that Thioacetamide-induced liver
fibrosis in rats was associated with a
significant increase in hepatic MDA level,
suggesting the impairment of hepatic anti-
oxidant capacities. Our data have
demonstrated that administration of res-
veratrol to the infected mice can decrease
MDA levels. Resveratrol is known to have
strong antioxidant effects, including inhi-
bition of free radical formation and/or lipid
peroxidation propagation, keeping the
structural integrity of the membrane and
preventing cellular damages. This data is in
line with data obtained by Ara et al. . (40), who
reported that resveratrol can decrease MDA
levels in liver tissues induced by bile duct
ligation. However, it has been reported that
resveratrol administration can result in an
increased brain MDA levels . (41) Similar data
has been reported in renal tissue [42]. The
differences in organ responsiveness towards
various harmful agents and resveratrol could
explain such discrepancies. Interestingly, a
significant decrease in hepatic MDA levels
was noted in the NT mice compared with the
NU pointing to resveratrol potent anti-oxidant
effects. These findings are in agreement with
a previous study showing that resveratrol can
up-regulate antioxidant enzymes mRNA
expression and consequently, lipid pero-
xidation prevention . (43) In addition, res-
veratrol has been demonstrated to inhibit
oxidative stress generation through
mechanisms involving superoxide dismutase,
glutathione peroxidase, and catalase up-
regulation . (44) The anti-oxidant capacity of
resveratrol might be attributed to a hydrogen-
electron donation from its hydroxyl groups
resulting in ROS scavenging [45]. The
scavenge of ROS can ultimately protect
against DNA damage and lipid peroxidation
in cell membrane [46]. Our data showed a
significant up-regulation in fibronectin
expression in the liver of infected untreated
group compared to the normal group. It has
been demonstrated that cytokines and
granulomatous reactions that occur in
Shistosomiasis induce macrophages to
produce transforming growth-factor-beta-one
(TGF β1) and activate HSCs playing an
important role in the pathogenesis of hepatic
fibrosis by triggering the expression of
fibronectin and collagens.(47) In our study,
fibronectin expression was remarkably down-
regulated in resveratrol treated group
indicating its strong anti-fibrotic effects. This
beneficial effect of resveratrol as antifibrotic
agent has been reported in other animal
models of liver fibrosis.(48,49) . Liver biopsy is
considered the gold-standard method for the
assessment of liver fibrosis because there is
no serological test which can diagnose
accurately this condition. (50) In the current
study, histopathological examination of H&E
stained liver sections of infected untreated
mice showed multiple granulomas associated
with inflammatory cells infiltration and focal
necrosis as compared to the normal liver
architecture in normal control group
confirming our biochemical results. These
hepatic changes are in agreement with the
previous study of El-Agamy et al., (51) which
demonstrated that S.mansomi infection can
result in enlarged fibrotic granulomas around
the eggs with marked increase in the amount
of collagen fibers, and focal necrosis .(51)
There is accumulating experimental evidence
that resveratrol might be of therapeutic value
in different hepatic disorders characterized by
liver fibrosis ,however limited reports have
investigated the role of this agent in S.model
of liver fibrosis.(52) In the present study, the
concentric granulomas and the laminated
layers of the fibrous tissue nearly disappeared
by the administration of resveratrol to the
infected mice, pointing to its beneficial effect
in suppressing the development of liver
fibrosis. These findings confirm further
previous data reporting that resveratrol can
reduce live fibrosis in S.mansoni-infected
mice [51]. In conclusion, Schistosomiasis can
lead to hepatic fibrosis. Resveratrol possesses
potent anti-inflammatory, antioxidant and
antifibrotic activity reversing S. Mansoni-
induced liver fibrosis in our model. Therefore,
resveratrol may be of therapeutic value in
hepatic disorders characterized by excessive
hepatic fibrosis. However, further clinical
investigations are still needed.
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Original Article
Clinical Utility of Alfa- Fetoprotein-L3 in Hepatocellular Carcinoma Patients
Ayman Mohamed Shamseya (1), Amal El Mahdi Mohamed Shafaey (2), Khaled Mustapha Belal (2) & Waleed Said
Mohamed Moftah (2)1) Department of internal medicine, Faculty of medicine-Alexandria University, (2) Department of
Clinical and Chemical Pathology, Faculty of Medicine- Benha University
ABSTRACT
The present study included 46 patients with HCC regardless of the underlying etiology. The pathological control group
included 20 patients with benign hepatic disorders such as chronic hepatitis and liver cirrhosis. Aim of the work: was
to evaluate AFP-L3 as a promising marker for diagnosis of HCC especially when combined with AFP. Patients and
methods: 20 sex- and age- matched apparent healthy individuals were serving as a normal control group. All studied
cases of HCC were submitted to clinical examination and radiological investigations, routine liver function tests and
serum AFP and AFP-L3. Results : In this study, it was found that the median value of both serum AFP-L3 and AFP-
L3/AFP ratio was significantly higher in HCC patients group when compared to chronic liver disease patients and
normal control individuals. There was a significant positive correlation between each of serum AFP, AFP-L3, with each
of Child Pough classification, tumor size and number of lesions , among HCC patients. A significant correlation was
found between AFP and AFP-L3/AFP ratio among HCC patients. Receiver operator characteristics (ROC) curves were
constructed for serum AFP, AFP-L3 and AFP-L3/AFP ratio as predictors of HCC. Serum AFP-L3 had the largest area
under the curve (AUC) when compared to serum AFP and AFP-L3/AFP ratio. The best cut-off point for AFP as
predictor of HCC was 62 ng/mL (sensitivity 83%, specificity 80%, PPV 91%, NPV 67% and efficacy 82%). The best
cut off point for AFP-L3 as predictor of HCC was 15 ng/mL (sensitivity 83% specificity 85%, PPV 93%, NPV 68% and
efficacy 83%). The best cut-off point for AFP-L3/AFP ratio was 20 % (sensitivity 70%, specificity 75%, PPV 87%,
NPV 52% and efficacy 71%). Combination of AFP and AFP-L3 (subjects considered having HCC when being positive
for AFP and-or AFP-L3) revealed best cut-off of AFP at 100 ng/mL and AFP-L3 at 4 ng/mL giving sensitivity of 100%,
specificity of 85%, PPV 94%, NPV100% and efficacy 96%. In conclusion AFP-L3 is a promising marker for diagnosis
of HCC especially when combined with AFP as the diagnostic sensitivity was optimum so both markers can be used in
the screening of HCC. The increasing incidence of HCC, in addition to the fact that the majority of these tumors are
diagnosed at a late stage when curative treatments are not possible, implicate that performing regular surveillance of
high risk individuals is recommended.
Introduction
Diagnosis of HCC depends on clinical
evaluation, laboratory diagnosis, imaging
techniques and histopathological techniques.
The patient may be completely asymptomatic
with no physical signs other than those of
cirrhosis. Therefore the laboratory markers of
HCC are very important in early diagnosis for
better prognosis (1). The conventional and the
most commonly used marker for HCC is
AFP, but it has low specificity and unsatis-
factory sensitivity in the diagnosis of early
HCC. Thus, there is need for supplementary
markers for AFP to increase the sensitivity in
early diagnosis of HCC as well as the spec-
ificity in differentiation between HCC and
benign lesions (2). Total AFP can be divided
into three different glycoforms, namely AFP-
L1, AFP-L2 and AFP-L3, according to their
binding capability to lectin lens culinaris
agglutin. Some clinical researches have
indicated that the high percentage of AFP-L3
is closely related to poor differentiation and
biologically malignant characteristics, specia-
lly portal vein invasion as it is produced only
from malignant liver cells of HCC (3). The
most used serological marker for the
diagnosis of HCC is AFP. However, the
sensitivity of this marker is still limited
especially in small, well-differentiated HCC.
False-negative or positive rates with AFP are
as high as 30% and 40% respectively for
patient with small HCC carcinoma, so that a
complementary serum marker has been
clinically required (2). Over the past decades,
researches have shown that total AFP is a
collection of heterogeneous glycoproteins
consisting of three different glycoforms. AFP
from benign chronic liver diseases, such as
chronic hepatitis and liver cirrhosis, and from
HCC displayed differential affinities to a
lectin, the lens culinaris agglutinin (LCA).
The total AFP can be separated into three
subspecies, AFP-L1 to AFP-L3, based on its
reactivity to LCA on affinity electrophoresis.
AFP-L3 has LCA-binding activity. It appears
to be produced only by cancer cells. Clinical
research has suggested AFP-L3 as a marker of
HCC (4). The aim of this study was to assess
the diagnostic value of AFP-L3 in HCC
patients and its role in prognosis through its
relation to different HCC stages. The results
of AFP- L3 were compared to the routinely
done AFP in order to explore whether it is
superior or not in the diagnosis and prognosis
of HCC patients.
Subjects and Methods
Subjects: This study was conducted at Alex.
University Hospital. It included eighty six
(86) subjects whose were classified into two
groups: I) Patient's groups: 1) Group 1:
Hepatocellular Carcinoma Patients (n=46).
This included 46 patients with primary HCC
regardless of the underlying etiology. The
diagnosis was confirmed by spiral CT. They
were 40 males and 6 females, whose ages
ranged from 41 to 58 years (mean 49.1 ± 4.4).
According to (BCLC) staging system, they
were divided into: Stage A subgroup (n=20):
Single tumor < 5 cm. This included 16 males
and 4 females, with a mean age of 49.8 ± 4.6.
Stage B subgroup (n=17): Large multifocal
tumor, Child-Pugh A-B. This included 15
males and 2 females, with a mean age of 48 ±
4.5. Stage C subgroup (n=9): In which there is
vascular invasion, extrahepatic manifest-
tations, Child - Pugh A - B. This included 9
patients all were males, mean age of 49.6±
3.6. 2) Group 2: Pathological Control Patients
(n=20). This included another 20 patients
with benign hepatic disorders such as chronic
hepatitis and liver cirrhosis. The diagnosis
was based on the clinical picture, ultra-
sonography, elevated AST, low albumin and
prolonged prothrombin time (PT). They were
18 males and 2 females, whose age ranged
from 42 to 56 years (mean 48.5 ± 4.2). II)
Control group: 3) Group 3: Healthy Control
(n=20). This included 20, sex and age
matched healthy individuals. They were 17
males and 3 females, whose age ranged from
41 to 58 years (mean 49.1 ± 4.3).
Table (1): Child-Pugh classification (Dourand, 2005).
Parameter Points assigned
1 2 3
Ascites Absent Slight Moderate
Bilirubin, mg/dl ≤ 2 2-3 >3
Albumin, g/dl >3.5 2.8-3.5 <2.8
Prothrombin time
*Seconds over control
* INR
1-3
<1.8
4-6
1.8-2.3
>6
>2.3
Encephalopathy None Grade 1-2 Grade 3-4
Grade Points
A:well-compensated disease 5-6
B:Significant functional compromise 7-9
C:Decompensated 10-15
Sampling: Freshly drawn 6mL blood was
divided to 1.8 ml, collected on citrated tube
for prothrombin time, and the rest was
collected on sterile vaccutainer. Serum was
separated by centrifugation at 1000 xg for 10
minutes. One milliliter serum was used for
immediate assay of AST, ALT, albumin, total
bilirubin. The rest was stored at freezer or at –
20 °C till assay of AFP and AFP- L3.
Results
The current study was conducted at Alex.
University Hospitals during the period
between January 2012 and March 2012. The
results of the present study are presented in
tables (2) to (10).
Table (2) Descriptive data of demographic and clinical parameters in the different studied groups.
Parameter
Group I
(HCC Group)
[n=46]
Group II
(Chronic Liver
Disease Group)
[n=20]
Group III
(Control Group)
[n=20]
Age (years) 49.1 ± 4.4 48.7 ± 4.2 49.1 ± 4.6
Sex [n (%)]
Male:
Female:
M: 40 (87%)
F: 6 (13%)
M: 18 (90%)
F: 2 (10%)
M: 17 (85%)
F: 3 (15%)
Jaundice
[n (%)] 12(26.1%) 2(10%) -
Edema
[n (%)] 24(52.2%) 5 (25%) -
Ascites
[n (%)] 22 (47.8%) 2 (10%) -
Tumor size(cm2) :
<5 [n (%)]
>5 [n (%)]
13(28.3%)
33(71.7%)
-
-
Tumor number:
Single [n (%)]
Multiple [n (%)]
34(73.9%)
12(26.1%)
- -
Child –Pugh score:
[n(%)]A
[n(%)]B
[n(%)]C
30(65.2%)
15(32.6%)
1(2.2%)
17 (85%)
2 (10%)
1 (5%)
-
Table (3) Statistical comparison of the clinical parameters between group I and II using modified Chi-Squared test.
Parameter Group I
(n = 46)
Group II
(n = 20) X2 P
Presence of Jaundice [n (%)] 12(26.1%) 2(10%) 2.2 >0.05
Presence of edema [n (%)] 24(52.2%) 5 (25%) 4.2 <0.05
Presence of ascites [n (%)] 22 (47.8%) 2 (10%) 8.6 <0.01
HCV positive cases [No. (%)] 44 (95.7%) 16 (80%) 4.1 <0.05
HBV positive cases [No. (%)] 2 (4.3%) 4 (20%) 4.1 <0.05
Sex
Male 40(87%) 18(90%)
0.12 >0.05
Female 6(13%) 2(10%)
Child score
A 30(65.2%) 17(85%)
3.9 >0.05 B 15(32.6%) 2(10%)
C 1(2.2%) 1(5%)
Comparative statistical analysis of studied
groups revealed that: 1- The incidence of
ascites and edema was significantly higher in
group I in comparison to group II (p< 0.01)
and (p< 0.05) respectively. 2- There was a
significantly higher proportion of cases
positive for HCV and significantly lower
proportion of cases positive for HBV in group
I when compared to group II (p<0.05).
Table (4): Descriptive and comparative data of laboratory parameters in the different studied groups.
Parameter
Group I
(HCC
Group)
[n=46]
Group II
(Chronic Liver
Disease Group)
[n=20]
Group III
(Control
Group)
[n=20]
H p
I.N.R.
Median 1.4 1.0 0.9
42.2 <0.01 IQR (1.2 – 1.6) (0.9 – 1.2) (0.9 – 1)
ALT (IU/L)
Median 61.5 46.5 6.0 50.1 <0.01
IQR (41.5 – 86.75) (25 - 59) (5 - 7)
AST (IU/L)
Median 59.5 45.0 8.0 49.6 <0.01
IQR (48 – 83.75) (26 – 69.75) (7 – 9)
Albumin(g/mL)
Mean 2.95 3.8 4.0 *15.1 <0.01
SD± 0.72 0.76 0.21
T.Bilirubin (mg/dL) Median 1.75 0.75 0.6
32.2 <0.01 IQR (1.08 – 2.5) (0.5 – 0.95) (0.4 – 0.76)
AFP (ng/mL)
Median 115.0 46.5 2.5 57.5 <0.01
IQR (80–422) (10 – 61.5) (2 – 4)
AFP-L3 (ng/mL) Median 59.0 3.0 0
57.5 <0.01 IQR (20–113.7) (0 – 11.5) (0 – 0)
AFP-L3/AFP ratio Median 0.33 0.12 0
45.9 <0.01 IQR (0.14–0.55) (0 – 0.22) (0 – 0)
Table shows: 1- INR results median and
(IQR) were significantly higher in group I
when compared to group II (p<0.01) and
when compared to group III (p<0.01), but
there was no significant difference between
group II and III (p>0.05). 2- ALT and AST
results median and (IQR) showed a highly
significant difference between groups I and
III (p<0. 01). 3- There was significant
difference in the levels of ALT and AST
between groups II and III (p<0.01).
Furthermore, ALT and AST showed a highly
significant difference between group I and II
(p<0.01). 4- Serum albumin was significantly
lower in group I than group III (p<0.01) and
in group I than group II (p<0.05). It also
showed no significant difference between
groups II and III. 5- Total bilirubin median
and (IQR), higher levels were found in group
I as compared to groups II and III, yielding a
highly significant difference (p<0.01) but
showed no significance difference when
compared between group II and III (p>0.05)
Table (5): Statistical comparison of laboratory parameters in the different studied groups using Mann Whitney U test.
This revealed that median serum levels of
AFP-L3 showed a highly significant
difference in group I when compared to group
II (p<0.01) and when compared to group III
(p<0.01), and significant difference in group
II when compared to group III (p<0.05).
Concerning the median value of AFP-L3/AFP
ratio, it gave significantly higher results in
cases of group I when compared to cases of
group II and group III (p<0.01), significant
difference when compared between group II
and III (P<0.05)
Parameter Group I vs. Group II Group I vs. Group III Group II vs. Group III
I.N.R Z +4.35 +5.93 +0.95
P <0.01 <0.01 >0.05
ALT
Z +2.77 +6.42 +5.42
P
<0.01
<0.01
<0.01
AST
Z +2.63 +6.41 +5.42
P
<0.01
<0.01
<0.01
Albumin
t -2.43 -4.96 -1.81
P
<0.05
<0.01
>0.05
T. Bilirubin
Z -3.81 -5.04 -1.83
P
<0.01
<0.01
>0.05
AFP
Z +4.53 +6.43 +5.39
P
<0.01
<0.01
<0.01
AFP-L3
Z +5.36 +6.47 +2.86
P
<0.01
<0.01
<0.05
AFP-L3/AFP
Ratio
Z +4.15 +6.17 +2.80
P <0.01 <0.01 <0.05
Table (6): Statistical comparison of AFP (ng/mL), AFP-L3 (ng/mL), AFP-L3/AFP ratio among the 3 different stages of the
HCC cases using Kruskall Wallis test.
Parameter
Stage A
(n =20)
Median (IQR)
Stage B
(n = 17)
Median (IQR)
Stage C
(n = 9)
Median (IQR)
H
P
AFP (ng/mL) 85 (82.5 – 159.8) 110 (90 – 619) 367 (150 – 784) 7.31 <0.05
AFP-L3 (ng/mL) 35 (20 – 97.5) 50 (15 – 112.5) 130 (111 – 136) 12.0 <0.01
AFP-L3/AFP ratio 0.33 (0.23 – 0.56) 0.22 (0.1 – 0.53) 0.37 (0.14 – 0.86) 2.2 >0.05
As regard the prognostic role of AFP and AFP-L3, the median values of them were significantly
increasing from stage A to C
Table (7): Comparisons of AFP, AFP-L3 and AFP-L3/AFP among the 3 stages of HCC cases using Mann Whitney U test.
Parameter Stage
A vs. B
Stage
A vs. C
Stage
B vs. C
AFP Z -1.6 -2.45 -1.5
P >0.05 >0.05 >0.05
AFP-L3 Z -0.6 -3.47 -2.64
P >0.05 >0.01 >0.01
AFP-L3/AFP Z +0.8 -0.87 -1.43
P >0.05 >0.05 >0.05
Table (8): Correlation between Serum AFP, AFP-L3, AFP-L3/AFP ratio other parameters Among Patients of Group I
(HCC Group).
AFP AFPL3 Ratio
Child score R 0.23 0.36 0.15
P < 0.05 <0.05 >0.05
Tumor No. R 0.21 0.23 0.04
P < 0.05 < 0.05 >0.05
Tumor Size (cm2) : R 0.19 0.20 -0.14
P < 0.05 < 0.05 >0.05
Age (years): R -0.21 -0.09 0.22
P >0.05 >0.05 >0.05
AFP (ng/ml): (cut off 62) R - 0.8 -0.37
P - <0.01 <0.05
ALT (IU/L): (cut off 67) R -0.03 0.26 0.33
P >0.05 >0.05 <0.05
AST (IU/L): (cut off 87) R -0.29 -0.16 0.21
P >0.05 >0.05 >0.05
Albumin (g/ml): (cut off 3) R 0.12 0.26 0.12
P >0.05 >0.05 >0.05
Bilirubin (mg/dl): (cut off 2.9) R -0.06 -0.05 0.06
P >0.05 >0.05 >0.05
INR (cut off 1.6) R 0.2 0.14 -0.05
P >0.05 >0.05 >0.05
There was a significant correlation between
each of serum AFP, AFP-L3 and each of child
classification, tumor size and tumor number,
among patients of group I. There was a non-
significant correlation between age and each
of serum AFP, AFP-L3 and AFP-L3/AFP
ratio among group I. There was a significant
correlation between AFP and AFP-L3/AFP
ratio.
Table (9): Performance characteristics of AFP, AFP-L3and AFP-L3/AFP ratio in the present study as regard group I versus
II at cut-off 95th percentile specificity. Cut-off SP% SN% NPV% PPV% Efficacy%
AFP 110ng/Ml 95 50 45 96 64
AFP-L3 27ng/mL 95 70 58 97 76
AFP-L3/AFP
ratio 24% 95 61 52 97 71
By testing the performance at a cut-off of 95th
percentile specificity, it was found that AFP-
L3 had the best performance, sensitivity of
70%, NPV 52%, PPV 97% and efficacy of
76%.
Table (10): ROC and multi ROC analysis of AFP, AFP-L3and AFP-L3/AFP ratio in the present study
as regard group I versus II.
1- The best cut-off point for AFP as predictor
of HCC was 62 ng/mL (sensitivity 83%,
specificity 80%, PPV 91%, NPV 67% and
efficacy 82%). 2- The best cut off point for
AFP-L3 as predictor of HCC was 15 ng/mL
(sensitivity 83% specificity 85%, PPV 93%,
NPV 68% and efficacy 83%). 3- The best cut-
off point for AFP-L3/AFP ratio was 20 %
(sensitivity 70%, specificity 75%, PPV 87%,
NPV 52% and efficacy 71%). 4- Combination
of AFP and AFP-L3(subjects considered
having HCC when being positive for both
AFP and-or AFP-L3)revealed best cut-off of
AFP at 100 ng/mL and AFP-L3 at 4 ng/mL
giving sensitivity of 100%,specificity of
85%,PPV 94%,NPV100% and efficacy 96%.
The following ROC curves show also
sensitivity & specificity of AFP & AFP-L3 in
discrimination group I from group II.
Cut-off
SN
%
SP
%
NP
V%
PP
V%
Effica
cy%
AFP 62 ng/mL (Best cut-off) 83 80 67 91 82
AFP-L3 15 ng/mL (Best cut-off) 83 85 68 93 83
AFP-L3/AFP
ratio
20% (Best cut-off) 70 75 52 87 71
Combination of
AFP
And AFP-L3
AFP-L3
100 ng/mL for AFP and 4 ng/mL for AFP-L3 100 85 100 94 96
AUC : AFP = 0.748
AUC : AFPL3 = 0.808
Figure1: ROC curve analysis showing the diagnostic
performance of AFP for discriminating group I from
group II.
Figure 2: ROC curve analysis showing the diagnostic
performance of AFP-L3 for discriminating group I
from group II.
AUC : AFP-L3/AFP ratio = 0.768
AUC : AFP & AFP-L3 = 0.997
Figure-3: ROC curve analysis showing the diagnostic
performance of AFP-L3/AFP ratio for discriminating
group I from group II.
Figure-4:Multi-ROC curve analysis showing the
diagnostic performance of AFP-L3 and AFP for
discriminating group I from group II.
Receiver operator characteristics (ROC)
curves were constructed for serum AFP, AFP-
L3 and AFP-L3/AFP ratio as predictors of
HCC. Serum AFP-L3 had the largest area
under the curve (AUC) when compared to
serum AFP and AFP-L3/AFP ratio.
Discussion
The present study included forty-six patients
with HCC, twenty patients with benign
hepatic disorders such as chronic hepatitis and
liver cirrhosis, in addition to twenty apparent
healthy individuals serving as a normal
control group. The results of the present study
yielded that, the male to female ratio of HCC
patients was 4:1, which is an impact of
predominance in males, that is very close to
data proved by Kiyosawa et al(5). which
informed that male to female ratio is 3:1 as
regard HCC sex predominance.(5). Lui et al, (6). explained the cause of male predominance
is that 2-methoxyestradiol – an estrogen
metabolite – is effective in growth inhibition
of various tumor cells as well as in
angiogenesis inhibition. (6). Since estrogen is
metabolized in the liver, it is conceivable that
females have more estrogen metabolized in
their liver; consequently more 2-
methoxyestradiol produced which could
inhibit tumor growth in situ .The data concern
about sex were presented in table (2) & (3).
95.7% of the patients with HCC were positive
for HCV infection which is an impact of
increased prevalence of HCV infection among
the Egyptians in 14% of the general
population (7). HCV infected persons
represent 78.5% of all HCC patients in Egypt (8). Usage of glass syringes in the early
campaigns of schistosama treatment appeared
to be responsible for wide spread transmission
of HCV (9). Table (4), (5) and (6) show the
statistic details of prevalence of HCV
infection. ALT, AST and total bilirubin levels
were higher in HCC and chronic liver disease
patients than normal control individuals, as
they are indicators of impaired liver functions
and hepatocellular damage. That is close to
data supported by Suruki et al. (10). who stated
that liver enzymes are indicators of the
severity of hepatocellular damage which is
evident in HCC. Table (2), (4), (5) and (8)
yield that results. (10). Other value in this
study. It was found that the median value of
both serum AFP-L3 and AFP-L3/AFP ratio
are significantly higher in HCC patients group
when compared to chronic liver disease
patients and normal control individuals, that
is matching with the study by Davi et
al.(2001) revealing that AFP-L3 is produced
only from malignant liver cells. (11). In another
study, Li et al. (2001) studied the presence of
AFP-L3 in chronic liver disease patients
suspected to have HCC. They found that 57%
of AFP-L3 positive cases were diagnosed as
HCC in the following 6 months and 6 of them
were diagnosed to be single small HCC at the
early stage through ultrasonic diagnosis or
CT. (4). This proves that there is significant
implication for identification of benign or
malignant liver disease by detection of AFP-
L3. Results were presented in table (4), (5) &
(6). The median value for serum AFP- L3 and
the ratio was increasing from stage A to stage
C and was higher in stage C. Results were
presented in table 6,7,8 and 9. That is
matching with the data from Davi et al (11).
who stated that AFP-L3 increases with
advanced tumor stages and that the liver
cancer cells with expression of AFP- L3 have
tendency toward early vascular invasion and a
potential for rapid growth, so it may be used
as prognostic tool. (11). Also in study by Oka
et al(12). they studied 388 patients of HCC
during a year. They found that AFP-L3
positive patients were having advanced tumor
characteristics in the form of maximum
diameter, portal vein invasion and tumor
stage. (12). Another study, Tada et al(13).
studied the biological behavior of HCC
positive for AFP-L3 in a total of 111 HCC
patients who underwent hepatic resection.
Pathologic features of resected HCC
specimens were evaluated and classified
concerning growth pattern (expansive or
infiltrative growth), capsule formation,
capsule infiltration, septal formation, portal
vein invasion, hepatic vein invasion, bile duct
invasion, and intrahepatic metastasis. They
found that those features were significantly
higher in AFP-L3 positive tumors when
compared to AFP-L3 negative tumors (13).
This results confirmed by Yamashita et al (14).
who assayed the serum AFP-L3 in 55 patients
of HCC who underwent different methods of
treatment. They found that 50% converted to
positive and by further investigations these
patients were found to have HCC recurrence
and portal vein invasion. (14). This is also
supported by the data collected by Song et al.
(2002) who studied the serum AFP-L3 as
pretreatment assessment. They found that
those patients who were positive for AFP-L3
had a very poor response to therapy. (15).
Kumada et al. (1999) also stated that AFP-
L3/AFP ratio of 10% in patients having liver
cirrhosis is associated with 7 fold increased
risk for developing HCC in the next 21
months and could be detected 3 to 21 months
prior to standard imaging techniques. (16). A
study also by Kazohisha et al (16). who studied
the sera of 422 patients stated that AFP-
L3/AFP ratio with a cut-off value of 15%
could predict the occurrence of HCC in
cirrhotic liver four months before the imaging
techniques. (17). There was a positive
correlation between AFP, AFP-L3 and Child-
Pugh score which is also an impact of the
severity of liver damage. This is matching to
the data by Suruki et al (16). who stated that
hepatocellular damage predispose eventually
to HCC. (10) Table (2), (3), & (4) yield that
results. There was no correlation between
AFP, AFP-L3 and the ratio and the age of the
studied groups, which denotes that these
makers are not affected by the age of the
patients as also proved by Tada et al. (2005)
(13) , table (8) yield that result. Serum AFP
was significantly higher in HCC patients than
in patients with benign liver diseases and
control subjects. Assessment of the diagnostic
performance of AFP as a marker for
distinguishing HCC from benign liver
diseases and healthy candidates revealed that
at a cut-off 20 ng/mL (manufacturer cut-off)
the diagnostic sensitivity was 100%, the
specificity was 40%, PPV was 79%, NPV
was 100% and efficacy was 82%. Motawa et
al(18), examined the diagnostic performance of
AFP in the diagnosis of HCC. It gave 68.2%
sensitivity and 75% specificity but he used
19.8ng/mL as a cut-off for AFP but he used
different staging system and in the other side
we had larger tumor sizes (18). Table (10)
showed that. When the 95th percentile
specificity for the tested markers was used,
the sensitivities of all of them were low but
the AFP-L3 had the highest one, accordingly
ROC and multi ROC analysis were done for
them in table (9). AFP -L3 showed sensitivity
of 83%, specificity 85%, PPV 93% and NPV
68%, therefore, more specific than AFP. That
is close to study by Evi et al(19). in which
AFP- L3 showed sensitivity of 90% and 95%
specificity. A possible cause for their higher
performance is that they studied larger
number of cases (n=334) which included
larger tumor sizes. (19). In a study by Davi et
al(19) who assessed the diagnostic performance
of AFP-L3 in the diagnosis of HCC, the
sensitivity of AFP-L3 was found to be stage-
related in HCC. The overall sensitivity of
AFP-L3 for HCC was approximately 60%. In
small HCC (HCC <2 cm in diameter) AFP-L3
had a sensitivity of only 45%. The sensitivity
increased with increase in size of the HCC,
and reached 90% when HCC was 5 cm in
diameter or greater. (11). Shiraki et al(20).
examined the diagnostic performance of AFP-
L3 in HCC patients.AFP-L3 had a sensitivity
of 75% and specificity of 90% but he used
larger number of HCC patients (n=2000). (20)
Statistic analysis done for them in table (9) &
(10). AFP- L3/AFP ratio with 20% as a cut-
off value had a sensitivity of 70%, specificity
of 75%, PPV of 87% and NPV of 52%.This
was close to the study by Tanwandee et al. (21). who had a cut-off 15% yielding a
sensitivity of 82% specificity of 71%, PPV of
83% and NPV of 69% but he used different
staging system and had larger number of
HCC patients (n=61). (21). Apinya et al. (22).
studied 166 cases of HCC and 106 cases of
benign liver conditions. When a cut-off value
of 10% was used, it gave a sensitivity of 71%
and specificity of 63% and by using a cut-off
value of 35 %, it gave a sensitivity of 33%
and specificity of 100 %.(22). A study by
Sangiovanni et al (23). assessed the diagnostic
performance of AFP-L3/AFP ratio in 86 HCC
patients and 38 patients with other liver
conditions. By a cut-off value of 7%, it gave a
sensitivity of 60% and specificity of 80%. A
possible cause for the discrepancy in the cut-
offs in the above studies is the target of each
study in choosing the more specific or more
sensitive marker according to the need of
different countries. (23) Statistic analysis done
for them in table (10). When the diagnostic
performance of AFP and AFP-L3 was tested
in combination in table (10), they had a
sensitivity of 100% and specificity of 85 %,
so there is improvement in sensitivity. That is
contradictory to data by Leerapun et al (24).
who stated that determination of AFP-L3, in
combination with AFP, increases the
specificity of diagnosis of HCC in individuals
with HCC but he studied large number of
HCC patients (n=272). (24)
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Original Article
Study of Hepatic Neo-Angiogensis in Different Stages of Chronic Hepatitis C
Related Liver Diseases Mohamed Yousri Taher Rashed1, El Said Hassan Ibrahim1, Nahed Mohamed Baddour2, Amany Saleh Abd EL Aziz El
Yamany1, Mohamed Saed Said Shater1
Hepatobiliary Unit Alexandria University Internal Medicine department1, pathology department2, Faculty of Medicine,
University of Alexandria1,2 Egypt
ABSTRACT
Hepatitis C virus (HCV) infection causes chronic hepatitis(CHC), which gradually progresses to liver cirrhosis and
subsequently to hepatocellular carcinoma (HCC). Angiogenesis plays a major role in chronic inflammation and may
have prognostic value in disease progression. Aim of the study: to asses neo-angiogenesis of the liver in patients with
HCV related chronic hepatitis C (CHC), liver cirrhosis(LC) and hepatocellular carcinoma (HCC)by assessment of
CD34 positivity and PDGFr B expression in liver tissue. Subjects and Methods: Forty patients with chronic hepatitis
C virus infection and twenty patients with hepatocellular carcinoma (HCC) were subjected to history taking, clinical
examination, laboratory investigations to evaluate hepatitis C RNA , liver function tests, ultrasonographic and
Triphasic CT liver examinations. Liver biopsy was performed for histopathological evaluation of necroinflammatory
grading and Histopathological staging of fibrosis . Patients were divided into three groups according to stage of liver
fibrosis and the presence or absence of HCC: chronic hepatitis (CHC( (n=20), liver cirrhosis (LC) (n=20) and HCC
(n=20). Immunohistochemical staining for platelets derived growth factor (PDGFr B) and CD34 were done. Results:
showed increased positivity for CD34 and PDGFr B expression with increasing clinical stage of the liver disease, with
maximal positivity seen in HCC group. A statistically significant positive correlation between expression PDGFRB and
CD34, and severity and progress of the disease was reported especially with progression from LC to HCC. Conclusion
: Assessment of these parameters in chronic HCV (CHC) related liver disease correlate with severity and progression
of the disease .It may be helpful in choosing the best treatment strategy, and indicate that anti-angiogenic therapy may
be useful in preventing progression of the disease and avoid development of HCC.
Introduction
Chronic viral Hepatitis infections including the
hepatitis B virus (HBV) and hepatitis C Virus
(HCV) are major risk factors contributing to
HCC development(1). Whatever the etiologic
cause of chronic liver disease, liver injury
usually results in a form of excess scarring
termed liver cirrhosis where the liver
synthetic and metabolic functions are
compromised and there is also an increased
risk of developing liver cancer(2). With
progression of the scarring process, the
endothelial lining of the sinusoids undergoes
conversion to a non fenestrated cell leading to
an appearance which has been termed".
capillarization" of sinusoids (3). In liver,
resting endothelial cells rarely proliferate
under physiologic conditions. When the
process of "sinusoidal capillarization", occur
these endothelial cells form tight junctions
along with deposition of extracellular matrix
followed by a new vessel formation (4).According to Tosh and Strain (5), there are
two reasonably well defined types of stem
cells in the bone marrow. The hematopoietic
stem cells (HSC) and the mesenchymal stem
cells MSC . The HSC is the precursor for the
lymphoid and the myeloid cells of the blood
and they are quite well characterized and have
been isolated from humans as cells with a
CD34 +ve phenotype. According to Fausto (6)
oval cells can be induced to proliferate under
different pathological conditions. CD34 is a
cell surface, sialo-mucin-like glycoprotein
which is expressed on hematopoietic prog-
enitor cells, normal vascular endothelium and
fibroblasts (7).Poon et al. (8) used CD34 as an
endothelial cell marker and Amarapurkar et
al. (9) used it in evaluation of normal liver,
cirrhosis and HCC. CD34 has been widely
used for the assessment of sinusoidal like
neo-angiogenesis in HCC. Amongst, viral
infection, HCV has been demonstrated to
show more angiogenesis and has been
suggested to represent a risk factor for HCC
in patients with chronic HCV (10)It has been
reported as a potent angiogenic virus .
PDGFrB is a heparin-binding growth
factor(11-13) of disulfide bonded A, B, C, and
D polypeptides that assemble into the
homodimers PDGF-AA, -BB, -CC, and -
DD.(14) The A and B polypeptides also form
heterodimers, denoted PDGF-AB. PDGF
transduces cellular responses by binding to 2
related protein tyrosine kinase receptors,
PDGF receptor-α and -β (PDGFr-α and-β).
PDGF-BB binds to PDGFr- β and - α with
similar affinity, whereas PDGF-AA binds
only to PDGFr- α. (15)Binding of PDGF to its
receptors leads to activation of the receptor
tyrosine kinase and to subsequent initiation of
cytoplasmic signal transduction pathways, in
turn leading to the migration, proliferation,
and differentiation of PDGF-responsive cell
types. PDGF receptors (PDGFr) are present
on connective tissue cells such as fibroblasts
and smooth muscle cells, but they have also
been detected on other cell types. (16)
Aim of the study
Is to asses liver neo-angiogenesis in patients
with HCV related chronic hepatitis, liver
cirrhosis and hepatocellular carcinoma by
assessment of CD34 positivity and PDGFr B
expression in liver tissue.
Methods
Study design included non randomized non
blinded clinical trial. Setting. The study was
performed at Alexandria University Hospital,
Alexandria, Egypt. From December 2011 to
October 2013.
Patients and Methods
The study included 60 patients with CLD,
20with chronic hepatitis C (CHC) ,20 patients
with liver cirrhosis (LC) and 20 patients with
Hepatocellular carcinoma (HCC) . Patients
were selected from Alexandria University
Main Hospital, Alexandria, Egypt. Exclusion
criteria: Chronic Hepatitis B virus (HBV)
infection, alcohol consumption, obesity, con-
comitant schistosomiasis, bleeding diathesis,
hepatic decompensation patients were
excluded Also patients with chronic diseases
such as diabetes mellitus or connective tissue
diseases, malignancies, previous anti-viral or
systemic anti-cancer therapy were excluded .
An informed consent was obtained from all
patients before enrollment in the study.The
presence of HCV-RNA in patient's sera was
detected by real-time polymerase chain
reaction. All patients were subjected to
thorough clinical examination, liver function
tests, abdominal ultrasonography examination
and axial computed tomography (CT)
scanning. Percutaneous liver biopsy was
taken for histopathologic and immuno-
histochemical studies.The studied patients
were classified into three groups according to
the results of abdominal ultrasonography,
liver biopsy and alpha fetoprotein (AFP), and
CT scan examination .Liver biopsy was
subjected to light microscopic examination
for evaluating the histopathological and basic
classification of cases of CHC using Ishak
(modified Knodell score) scoring (17).
Immunohistochemistry for Detection of
tissue CD34 and PDGFR B antigens:
Paraffin blocks were cut at 5 micron thick
sections and stained with H and E stain for
histopathological examination, Masson
Trichrome stain for better evaluation of the
stage. Immunohistochemical staining for each
of PDGFr B polyclonal antibody (ready to use
antibody (Lab vision) (code no.RB-1627) and
CD34 polyclonal antibody (Lab Vision, clone
QB End/10). Sections were stained with the
universal polyclonal kit (Lab vision, UK)
(TP-015).Positive controls in the form of
infiltrating ductal carcinoma of the breast was
included in every run. Negative controls were
included by omission of the primary.
Immunohistochemical scoring of PDGFR
B: A combination of positive cell count and
staining intensity was used for scoring.
Positive cell count was scored based on the
average percentage of positive cells per 100
cells in 10 high-power fields, as follows:0–
10% score 0; 11–25%, score 1; 26–50% score
2 ; 51–75%, score 3; and >75% score 4.
Staining intensity was scored as follows:
negative, score 0; faint yellow, score 1;
yellow or deep yellow, score 2; brown or dark
brown, score 3. The final score was obtained
by multiplying the cell count and staining
intensity scores. A score of ≥ 3 was defined
as high expression group and a score of < 3
was defined as Low expression. Counting of
CD34 positivity: This was performed using
the point counting technique. Positive cells in
the lobules were counted. The inflammatory
cells in the portal tract were not considered.
At least 6 fields were counted. Presented data
represent the mean of the counted fields.
Statistical analysis : Data were analyzed
using statistical package SPSS version 20 .
Kruskal-Wallis, Mann-Whitney(U), Spear-
man’s rank correlation coefficient (r) Chi-
square (X2) and ANOVA (F) tests were done
at 5% level of significance (18). Results:
Patients were 37 males (66.3 %) and 24
females (33.7%), Table (1) shows comparison
between different studied groups regarding
gender, viral marker, and liver function tests,
alpha fetoprotein and ultrasound findings as
regard liver and spleen size. The highest
levels of AST and ALT were found among
CHC and LC patients. Figure (1,2) On the
other hand, the lowest levels of total protein
and albumin were detected among HCC
patients while the mean total serum bilirubin
levels and alkaline phosphatase (ALP) levels
showed significant increase in HCC group.
There was a significantly higher serum level
of AFP among HCC (p<0.000) and LC
(p<0.000) patients compared with CHC.Table
(2): shows Histopathological Findings of liver
biopsies. The degree of fibrosis (histological
“stage”) was significantly lower (p<0.002*)
in CHC group (2.65±0.49) comp-ared to HCC
and LC groups (3.75±.85, 3.5±1.64). Figures
(3). Eestimating CD34 positivity by
semiquantative methods Table 3 significant
positivity was not found in CHC group. CD34
in HCC cases was higher than CHC and LC
groups (P<0.000*). There was significant
difference in CD34 positivity between HCC
and LC liver (P<0.000*). Figure (4-6). The
results of PDGFr B expression are shown in
Table 4, 90% of CHC, LC showed positivity,
and 100% of HCC showed signinificant
positivity. Figure (8-13) PDGFr B expression
showed a significant increase (p<0.000*) in
LC and HCC groups compared to CHC.
Figure (7) Moreover, there was significant
correlations between PDGFr B expression,
CD34 positivity and AFP, serum albumin,
seum aminotransferases, total serum bilirubin,
Prothrombin time (PT), Alkaline phosphatase
(ALP) and Ishake score Table 5) and Figure
(14-17).
Table (1): General characteristics and liver function tests of the different studied groups
The studied parameter HCC
(No = 20)
CHC
(No = 20)
LC
(No = 20)
Test of
significance p value
Gender: male/female 15/5 12/8 10/10 >0.05
Hepatomegaly 0 5 0
Splenomegaly 19 0 10
Prothrombin Time % mean + SD 66.1±3.1 86.3±9.5 65.2±2.5 X2=38.0 0.000*
Albumin (gm/dl) 3.0±0.4 4.1±0.4 3.4±0.5 X2=33.3 0.000*
AST( IU/L) 65.5±33.6 42.5±17.2 94.4±63.0 X2=10.8 0.005*
ALT (IU/L) 55.5±15.8 59.5±25.6 89.8±34.0 X2=14.8 0.001*
Total bilirubin (mg/dl) 2.0±0.5 0.6±0.3 1.1±0.6 X2=35.9 0.000*
Alkaline phosphatase
(IU/L) 240.0±8.3 103.6±76.9 137.9±56.7 X2=35.7 0.000*
Serum AFP (ng/ml): 364.8±224.8 10.0±16.7 45.3±58.9 X2=35.2 0.000*
HCV.RNA(IU) 139000.0±
38134.1
1323792.9±
1623605.8
374420.5±
498192.6 X2=10.4 0.006*
X2: Kruskal-Wallis test for independent groups
* P < 0.05 (significant)
Table (2): Histopathological evaluating necroinflammatory activity and degree of fibrosis the three groups
HCC
(No = 20)
CHC
(No = 20)
LC
(No = 20)
Test of
significance p value
necroinflammatory activity
(histological “grade”).
Mean 3.75 2.65 3.50 12.3 0.002*
SD 0.85 0.49 1.64
degree of fibrosis (histological
“stage”)
Mean 4.25 1.35 4.60 45.4 0.000*
SD 0.44 0.88 0.94
X2: Kruskal-Wallis test for independent groups
* P < 0.05 (significant)
Table (3): CD34 expression in the three groups
HCC
(No = 20)
CHC
(No = 20)
LC
(No = 20)
Test of
significance p value
CD34
expression
Mean 0.96 0.00 0.47 54.7 0.000*
SD 0.04 0.00 0.12
X2: Kruskal-Wallis test for independent groups
* P < 0.05 (significant)
Table (4): PDGFr positive cases in each group
Variables
PDGFR BB
Number of positive cases for
PDGF
Percentage of positive area
Mean ± SD
CHC(n =20) 19/20 2.75±.97
LC (n=20) 19/20 5.70±2.56
HCC(n=20) 20/20 6.35±2.43
X2: Kruskal-Wallis test for independent groups
* P < 0.05 (significant)
Table (5): Correlation between CD 34, PDGFRB +ve with different parameters in total sample
Total sample
PDGFRB +ve CD 34
rs P rs P
PT -0.451* <0.001 -0.611* <0.001
S.Albumin -0.396* 0.002 -0.707* <0.001
ALP 0.475* <0.001 0.723* <0.001
AST 0.091 0.491 0.283* 0.028
ALT -0.018 0.891 -0.070 0.596
Total.bil 0.483* <0.001 0.765* <0.001
HCV.RNA -0.152 0.246 -0.399* 0.002
AFP 0.400* 0.002 0.718* <0.001
Histological grade 0.422* 0.001 0.411* 0.001
Histological stage 0.515* <0.001 0.740* <0.001
CD34 0.600* <0.001 1.000 0.0
X2: Kruskal-Wallis test for independent groups
* P < 0.05 (significant)
Figure (1): Comparison between the three studied groups according to AST
Figure (2): Comparison between the three studied groups according to ALT
0
10
20
30
40
50
60
70
80
90
100
CHC Cirrhosis HCC
Mea
n o
f A
ST
0
10
20
30
40
50
60
70
80
90
CHC Cirrhosis HCC
Mea
n o
f A
lT
Figure (3): Comparison between the three studied groups according to modified histological grading
Figure (4): Section of a case of chronic CHC showing total negativity for CD34.
(Streptavidin peroxidase technique, CD34 polyclonal antibody, DAB X100).
Figure (5): Section of a case of HCV positive cirrhosis showing patchy membranous positivity for CD34 .
(Streptavidin peroxidase technique, CD34 polyclonal antibody, DAB X100).
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
CHC Cirrhosis HCC
Mea
n o
f m
od
ifie
d H
AI
“gra
de”
Figure (6): Comparison between the three studied groups according to CD 34 positivity
Figure (7): Comparison between the three studied groups according to PDGFr B +ve positivity
Figure (8): Section of a case of chronic hepatitis C(CHC) showing cytoplasmic staining of the Kupffer cells (arrows)
for PDFrB (Streptavidin peroxidase technique, PDFr B polyclonal antibody, DAB X200).
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
CHC Cirrhosis HCC
Mea
n o
f C
D34
0
1
2
3
4
5
6
7
CHC Cirrhosis HCC
Mea
n o
f P
DG
FR
B +
ve
(A) (B)
Figure (9): Several hepatocytes also showed positive cytoplasmic staining in acinar zone 1 (a) as well as in the rest of the
acinus (b). (Streptavidin peroxidase technique, PDFr B polyclonal antibody, DAB, (a) X100, (b) x 200).
(A) (B)
Figure (10): Section of a case of chronic hepatitis C showing cytoplasmic positivity in the inflammatory cellular infiltrate in
the portal tracts (Streptavidin peroxidase technique, PDFr B polyclonal antibody, DAB, (a) X200, (b) x100).
(A) (B)
Figure (11): Section of a case of cirrhosis without HCC showing cytoplasmic positivity in the hepatocytes of acinar zone 3.
(Streptavidin peroxidase technique, PDFr B polyclonal antibody, DAB X200).
Figure (12): Section of a case of cirrhosis showing intense positivity in steatotic hepatocytes
(Streptavidin peroxidase technique, PDFr B polyclonal antibody, DAB X100).
(A) (B)
(C) (D)
(E)
Figure (13): Section of different cases of cirrhosis anf HCC (group 3) showing different grades of positivity for PDFR B.
(a) a few hepatocytes are positive with a weak intensity. (b) a larger number of hepatocytes show moderate positivity.
(c) a large number of hepatocytes are positive with a low intensity. (d) a large number of hepatocytes with a high intensity.
(e) totally negative result in the negative control.
(Streptavidin peroxidase technique, PDFr B polyclonal antibody, DAB, (a, c & d) X100, (b), x200).
Figure (14): Positive correlation between PDGFr B+ve
and modified histopathological grade
Figure (15): Positive correlation between CD34 and
PDGFr B+ve
Figure (16): Positive correlation between PDGFr B+ve
and histological stage
Figure (17): Positive correlation between CD34 and
histological stage
Discussion
Evidence of angiogenesis was reported to be
significantly more frequent in HCV positive
patients compared with HBV-positive patients
or controls. In addition, HCV-positive sera
and liver homogenates stimulated a higher
migration and proliferation of human
Endothlial cells (ECs) in vitro compared with
HBV-positive. These observations indicate
that angiogenesis is particularly linked to
HCV infection, suggesting a possible
contribution to HCV-related liver oncogenesis
The intensity of angiogenesis was positively
associated with the grade of inflammatory
activity in CHC. Evident CD34 expression,
which is a marker of neovascularization, was
found in the periportal area of lobules and
increased in parallel with the fibrosis stage. In
the case of septal fibrosis, new-formed blood
vessels were observed in the close neighbor-
hood of fibrous septa. The molecular
mechanisms involved in chronic viral
hepatitis-associated angiogenesis have not
been fully identified. Hepatic VEGF
expression was found to be significantly
higher in stages 3 and 4 compared with stages
1 and 2 of fibrosis and revealed a positive
correlation with the fibrosis stage .
Additionally, expression of PDGF in
macrophages and infiltrating inflammatory
cells and PDGF receptors in sinusoidal and
perisinusoidal cells in periportal areas is
increased during chronic viral hepatitis. These
0
1
2
3
4
5
6
7
8
9
0 2 4 6 8 10 12 14
HA
I gra
de
PDGFRB +ve
rs = 0.422*
p <0.001
0
2
4
6
8
10
12
14
0.0 0.2 0.4 0.6 0.8 1.0 1.2
PD
GF
RB
+ve
CD 34
Series1
Linear (Series1)rs = 0.600*
p <0.001
0
1
2
3
4
5
6
7
8
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
HA
I st
ag
e
PDGFRB +ve
Series1
Linear (Series1)
rs = 0.515*
p <0.001
0
1
2
3
4
5
6
7
0.0 0.2 0.4 0.6 0.8 1.0 1.2
HA
I S
tag
e
CD 34
rs = 0.740*
p <0.001
alterations facilitate the stabilization of
nascent blood vessels (19) Some studies
showed a significant development of hepatic
CD34-positive neovascularization in NASH,
whereas almost no development was observed
in simple steatosis. The degree of
angiogenesis was almost parallel to that of
liver fibrosis in NASH]. These results were
consistent with a previous finding that
angiogenesis increases stepwise during
hepatic fibrosis development in several
models of fibrosis, including the rodent
dietary NASH model (20,21,22). All analyzed
CHC patients with steatosis had a
significantly higher CD34 expression both in
portal tracts and fibrous septa and lobules
(19). In this study , we evaluated progression
of angiogenesis with the help of PDGFr-B
and CD 34 expression in CHC, HCV
cirrhosis, and HCV HCC patients. CD34 in
HCC cases was found higher than CHC and
LC groups (P < 0.000*). There was
significant difference in CD34 between HCC
and LC liver (P < 0.000*) indicating that
higher expression of CD34 can be an
indicatore for HCC development in CHC and
LC patients .In this study, while comparing
the stage of fibrosis, PDGFr- B expression
was significantly more in stage 3 and 4LC as
compared to stage 1 and 2 fibrosis.. PDGF-B
signaling through PDGFr is considered to be
most closely related to hepatic fibrosis. The
expressions of PDGF-B and PDGFr are
rapidly increased in both the experimental
hepatic fibrosis in rats and human fibrotic
liver.(23,24,35,25,26).Also in this study there were
significant correlation between PDGFr B
expression and CD34 positivity and serum
AFP, serum albumin, serum amino-
transferases, and total serum bilirubin,
Prothrobine (PT), Alkaline poshphatase
(ALP) and histological grade and stage
among all the studied groups. In vitro studies
have demonstrated that PDGF-B is the most
potent mitogenic factor for HSCs .(27,28)
reported that Platelet- derived growth factor
BB (PDGF-BB) and type β1 TGF (TGF β1)
are two key factors in fibrogenesis.The
relationship between PDGF signaling and
hepatic fibrosis has been evidenced by a
number of studies. Although all the four
PDGF isoforms might be involved in hepatic
fibrosis (25,22,26,27,22).In the present study
PDGFr-B expression was maximal in HCC
group as compared with LC and CHC groups
.PDGFr-B is involved in different stages of
liver disease progression through develop-
pment of liver fibrosis, via upregulation of
TGF-β receptors by PDGFr-B. Additionally,
overexpression of PDGFr-B also leads to an
increased expression of β-catenin as well as
vascular endothelial growth factor and platelet
endothelial cell adhesion molecule-1
(PECAM-1 / CD31), all these factors are
known to have established roles in hepatic
carcinogenesis. (29,30,31,32) Estimating CD34
expression, it was not markedly increased in
CHC group. CD34 expression in HCC cases
was significantly higher than in LC groups (P
< 0.05). Ohmori S; et al.(,33), stated that high
expression of CD34-positive sinusoidal
endothelial cells is a risk factor for HCC in
patients with HCV-associated chronic liver
diseases. Amarapukar et al. (9) suggested that
agiogenesis as assessed by CD34 expression
play an important role in carcinogenesis.Park
et al. (34) in their study found that there was a
gradual increase in CD34 expression from
cirrhotic nodules to dysplastic nodules to
HCC, with all the cases of HCC showing
diffuse strong CD34 positivity. Ma Jee et al. (35) concluded that CD34 is a useful marker
for distinguishing HCC from non cancerous
liver tissue; as CD34 was not present along
the sinusoidal wall in normal human liver,
was weakly present in the perinodules in few
cases of cirrhosis, while HCC showed a
diffuse capillarization with over expression.
Cui et al. (36) found enhanced CD34
expression of sinusoidal like vascular
endothelial cells in HCC. On the other hand
Kimura et al. (37) did not find any significant
difference in CD 34 among all clinical stages
and histological grades of HCC. Yang et al. (38), by Immunohistochemical staining, found
that CD34 was expressed in the vascular
endothelial cells in, paracarcinomatous tissue
. In the present study there was positive
correlation between CD34 expression and
PDGFr-B in LC and HCC groups CD34
expression was correlated positively and
significantly with degree of inflammation and
fibrosis in all studied patients. Conclusions:
The present study showed, that there is close
relationship between angiogenesis and
progression of HCV –related liver disease
from chronic infection to cirrhosis till HCC
development. PDGFr B and CD34 expression
together might be of great importance to
detect CLD progression especially to HCC.
High expression of PDGFr-β is an indicator
of poor prognosis (as it correlates with PT,
AFP and liver cirrhosis). Therapy against
PDGFr-B and antiangiogenic drugs for
chronic liver diseases might have dual
benefits of both suppressing fibrosis and
preventing carcinogenesis. However, anti-
angiogenic agents should be used with
caution and be carefully balanced in patients
with CLDs. Angiogenesis is a relevant
phenomenon in wound healing and excessive
blocking of angiogenesis is not desirable.
Also the angiogenic effect might be a
manifestation and consequence of the cause
of CLD.
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Original Article
Tenascin C: A New Promising Marker for Liver Inflammation and Fibrosis in
Hepatitis C Patients
Amal Sobhy El Sedfy1 and Marwa A. Madkour2 1Department of Pathology; 2Department of Clinical and Experimental Internal Medicine, Medical Research Institute,
University of Alexandria, Egypt.
ABSTRACT
Hepatitis C virus (HCV) infection represents a serious health problem in Egypt with high morbidity and mortality.
Fibrosis and cirrhosis represent a continuous wound-healing response to chronic liver injury due to HCV infection.
Tenascin C (TN-C) is an extracellular matrix (ECM) molecule highly expressed during tissue repair and in pathological
situations such as chronic inflammation and cancer. Although TN-C has been connected to liver inflammation and
fibrosis, however, it has not yet been elucidated whether TN-C promotes the fibrotic process. Alpha smooth muscle
actin (alpha SMA) has been established as a reliable marker of activated hepatic stellate cells (HSC). These are the main
cells involved in the production of excessive ECM seen in liver fibrosis. Aim of the Work: Our aim was to study the
serum concentration of TN-C as well as its immunohistochemical expression in the liver tissue of patients with chronic
hepatitis C (CHC), and to investigate their relevance to other clinical and laboratory aspects, as well as to the expression
of alpha SMA, in a trial to demonstrate the role and usefulness of TN-C as a marker of liver inflammation and fibrosis.
Patients and Methods: The present study was undertaken on 100 CHC patients and 5 normal healthy subjects as
control group. In addition to clinical and ultrasound assessment of all subjects, routine laboratory investigations were
performed including platelet count and liver profile. Serum level of TN-C high molecular weight (large) variants was
determined by ELISA. Liver core tissue biopsies were obtained and subjected to routine hematoxylin and eosin stain for
Ishak grading and staging, TN-C immunohistochemical staining using monoclonal antibodies and alpha SMA
immunohistochemical staining for assessment of HSC activity. Results: CHC patients with higher scores of
inflammatory activity and fibrosis stage of the liver had significantly higher serum levels of large TN-C variants
(p<0.01). Also, serum TN-C levels were significantly correlated with the severity of piecemeal necrosis (p<0.01), as
well as TN-C immunopositivity of liver tissue. CHC cases showed preferential accumulation of TN-C immunostain at
sites of necroinflammatory activity, particularly with piecemeal necrosis, with significant correlation between TN-C
expression and grade of hepatitis, as well as stage of fibrosis. HSC activity in hepatitis C cases (as determined by alpha
SMA immunostaining) demonstrated a significant correlation and co-localization with dense TN-C deposition,
indicating that activated HSCs are the most plausible cellular source of TN-C. Conclusion: serum concentration of TN-
C might be a useful marker for inflammatory activity (particularly piecemeal necrosis) and fibrosis in CHC. It reflects
well the liver tissue expression of TN-C, which is particularly upregulated in CHC, being specific to areas where tissue
injury and remodeling are in progress.
Introduction
Approximately 3% of the world population
suffers from chronic C Hepatitis (CHC), with
Egypt displaying the highest prevalence
worldwide.(1) Fibrosis and cirrhosis represent
a continuous wound-healing response to
chronic liver injury due to HCV infection
(from 20% to 30% of the cases), eventually
culminating in hepatic dysfunction.(2)
Furthermore, cirrhosis is a now considered a
preneoplastic condition that predisposes to
hepatocellular carcinoma (HCC) in one-third
of cirrhotic patients, with an estimated yearly
incidence of 3–5%.(3) So far, the best
treatment available is the antiviral
combination of pegylated interferon and
ribavirin, which eradicates HCV infection in
only 50% of patients with genotype 1, 80% of
patients with genotypes 2 or 3, and around
60% of patients with genotype 4 (the
predominant genotype in Egypt).(4,5) As a
consequence of the worldwide burden of
chronic hepatitis C infection and the
limitations of current antiviral treatment, new
approaches have assumed greater importance,
including antifibrotic therapies. Hence, the
management of fibrosis and cirrhosis due to
hepatitis C is expected to continue as a major
issue for hepatologists throughout the
world.(2). The understanding of the role of
hepatic stellate cells (HSCs) in liver fibrosis
is important as it is considered as a prognostic
indicator of progression of liver fibrosis as
well as a potential target for therapeutic
intervention to prevent the development of
cirrhosis. In the setting of chronic infection,
the immune system - attempting to eradicate
the virus – unintentionally promotes
hepatocyte damage and fibrosis through direct
cellular toxicity and release of inflammatory
cytokines. Under conditions of stress and
injury, HSCs become activated, acquire
myofibroblastic phenotype, and contribute to
excessive extracellular matrix (ECM)
deposition and fibrosis.(6-8). It has been
reported that histological activity index –
which is defined through liver biopsy – is a
good determinant to evaluate the prognosis in
patients with chronic hepatitis C.(9) However,
in spite of being the “gold standard”, liver
biopsy can be associated with sampling error,
interobserver variability, and potential
complications. Thus, there is a need for
simple and noninvasive means to assess
disease activity in patients with CHC.(10) So
far, several markers have been identified,
whose serum levels show relatively good
correlation with the stage of liver fibrosis,
while only few reported markers reflect
necroinflammatory activity of the disease.(11).
Tenascin C (TN-C) is the founding member
of a family of glycoproteins comprising also
Tenascin-X, -R and –W. Tenascins are found
in the ECM of many tissues. Their role is not
only to support the tissue structurally but also
to regulate the fate of the different cell types
populating the ECM.(12) Human TN-C is
composed of six subunits. A small-molecular-
weight variant of TN-C formed after splicing
exists constitutively in normal tissues,
whereas the large-molecular weight variants
are specifically expressed in tissues under
several pathological conditions.(13,14) TN-C
alters the adhesivity of cells to the ECM, and
modulates cell proliferation and migration.
TN-C also affects the adhesivity of immune
cells, such as lymphocytes and macrophages,
and plays a critical role in inflammation and
immune responses.(15) In case of liver
diseases, TN-C was proved to be a constituent
of the ECM of the space of Disse. It is
expressed and upregulated in several
pathological conditions, particularly in
fibrotic lesions in which HSC are possibly the
major cellular source of TN-C.(16) Although
TN-C has been connected to liver fibrosis,
however, it has not yet been elucidated
whether TN-C promotes the fibrotic process.
Alpha-smooth muscle actin (alpha SMA) is a
well-established reliable marker of HSC
activation, which precedes fibrous tissue
deposition in the setting of chronic hepatitis.
Activated HSCs lose lipid droplets stored in
their cytoplasm, proliferate and gain
abundance of microfilaments that consist
mainly of alpha SMA. Therefore, it is used in
identifying the earliest stages of hepatic
fibrosis and monitoring the efficacy of
therapy.(17). The aim of this work was to study
the serum concentration of TN-C as well as
its immunohistochemical expression in the
liver tissue of patients with CHC, and to
investigate their relevance to other clinical
and laboratory aspects, as well as to the
expression of alpha-smooth muscle actin, in a
trial to demonstrate the role and usefulness of
TN-C as a marker of liver inflammation and
fibrosis.
Subjects and Methods
The present study was undertaken on 100
cases of hepatitis C patients and five normal
healthy subjects as control group. Samples
were collected during a two-year period from
September 2010 and September 2012 from
the Hepatology Unit of Clinical and
Experimental Internal Medicine Department
in collaboration with Pathology and Chemical
Pathology Departments, Medical Research
Institute, Alexandria University, Egypt.
Biopsies and serum samples from patients
infected with hepatitis C virus were collected
as part of routine clinical evaluation prior to
interferon therapy, provided that they had
prothrombin activity ≥70%, INR ≤ 1.6,
platelet count ≥ 80,000/mm3 and no ascites or
peritoneal infections. HCV infection was
screened by third generation ELISA for HCV
antibodies and verified by polymerase chain
reaction (PCR). Normal liver tissue
specimens and serum samples were obtained
intraoperatively from volunteer subjects
undergoing elective hernia repair operations
who had normal liver function tests and
apparently normal liver appearance by
ultrasound examination. All patients and
control subjects were subjected to the
following (after obtaining their written
consent): 1-Clinical and ultrasound
assessment: Clinical focus was on the
presence or absence of symptoms and signs of
chronic liver disease. Abdominal ultrasound
was performed using a 3.5 mHz sector
transducer scanner to detect the presence or
absence of liver cirrhosis, portal hypertension,
ascites, splenomegaly and/or hepatocellular
carcinoma. 2- Routine laboratory investi-
gations: These included platelet count, serum
levels of transaminases, albumin, bilirubin,
prothrombin time and activity. 3- Serum level
of TN-C: It was determined using an ELISA
kit (Immuno-Biological Laboratories Co.
Ltd., Gunma, Japan). This kit can determine
TN-C high molecular weight (large) variants
including FN-III-C domain in human serum
by using two different kinds of specific
antibodies; a “coating antibody” [Anti-Human
Tenascin-C (19C4MS) mouse IgG
monoclonal antibody affinity purify specific
to human FN-III-C domain] and a “labeled
antibody” [horseradish peroxidase-conjugated
Anti-Human TN-C (4F10TT) mouse IgG
Fab’ affinity purify reacting to EGF-like
domain]. Serum samples were diluted 10-fold
and incubated in a 96-well ELISA plate
coated with 19C4MS. After washing HRP
conjugated 4F10TT Fab’ was added and
incubated. The range of this assay kit was
0.38–24 ng/ml.(18) . 4- Core liver biopsy:
Liver biopsy specimens were obtained from
patients percutaneously through a right
midaxillary approach after an overnight fast.
Biopsy site was confirmed by ultrasound
examination and color Doppler. Local
anesthesia at the site of puncture was applied
using 1-2% lignocaine and core liver biopsies
were obtained using gauge 16 automatic
needles. Only samples longer than 10 mm
with more than eight portal tracts were
included in this study. 5- Pathological
evaluation of biopsy specimens: Biopsies
were fixed at room temperature in 10%
neutral buffered formalin and processed for
routine paraffin embedding. Serial 5
micrometer thick paraffin sections were
subjected to routine hematoxylin and eosin
(H&E) staining as well as Masson’s
Trichrome staining to determine the grade of
activity and the stage of fibrosis according to
Ishak grading and staging systems.(19)
According to Ishak et al, the histological
grades of the necroinflammatory activity were
scored from 0-18 and designated as: None
(score: 0), minimal (score: 1-4), mild (score:
5-8), moderate (score: 9-12) and marked
(score: 13-18). The stages of fibrosis were
also scored from 0-6 according to Ishak et al,
where scores 5 and 6 were designated as
incomplete cirrhosis and probable or definite
cirrhosis, respectively.(20). 6- Immunohisto-
chemical evaluation of biopsy specimens:
Immunohistochemical (IHC) staining using
monoclonal mouse antibody against TN-C
(Labvision UK) (22) and monoclonal mouse
antibody against alpha SMA in (Labvision
UK) were performed.(23) All sections were
counterstained with hematoxylin and
examined using the light microscope. For the
positive control of each antibody, squamous
cell carcinoma was used for TN-C and
leiomyosarcoma for alpha SMA. Assessment
of TN-C immunoreactivity was performed
using a semi-quantitative method by
determining the percentage of positive cells in
(100x) magnification (Olympus BX50
microscope). The staining intensity was
denoted weaker or stronger than, or moderate
if equivalent to, that of the arterial wall,
which expressed Tenascin C constantly and
served as an internal positive control. TN-C
staining was graded as: 1 (weak), 2
(moderate), 3 (strong and patchy staining;
<50% of the positive area) and 4 (strong and
diffuse staining; >50% of the positive area).
Assessment of alpha SMA staining was
performed by counting the positive cells
within the parenchyma and developing septa
and their interfaces in 20 randomly selected
fields under a (20x) objective lens excluding
periductular and perivascular cells of portal
areas and perivascular cells of central veins.
The expression of alpha SMA was assessed in
spindle shaped cells. Quantification of the
staining was assessed in four categories:
SMA-negative (no cells stained for SMA),
SMA-weak (1-4 cells stained), SMA-
moderate (5-10 cells stained), SMA-strong
(>10 cells stained).(24). 7- Statistical analysis
of the data: Quantitative data were expressed
using range, mean and standard deviation
while qualitative data were expressed in
frequency and percent. Qualitative data were
analyzed using Chi-square, Fisher exact and
Monte Carlo tests. Quantitative data were
analyzed using student t- test and Mann
Whitney test. Correlations with the serum
levels of markers and sonographic
measurements were analyzed by Pearson’s
correlation, while the correlations with the
histological scoring systems were estimated
by Spearman’s rank correlation test. P value
was assumed to be significant at 0.05.
Results
I ) Clinical and sonographic data: The mean
age of studied cases was 44.25 ± 8 years;
ranging between 27 and 63 years. Among 100
hepatitis C patients; 82 were males and 18
were females, while among control subjects
there were 4 males and one female. Medical
history revealed that 15 out of 100 HCV
patients (15%) complained of easy
fatigability, while abdominal examination
only revealed enlarged liver in 5 patients
(5%). Abdominal ultrasound study revealed a
statistically significant difference between
patients and control group only in the
diameter of the right lobe of the liver (p
<0.01). Forty-seven percent of HCV patients
showed increased hepatic echogenicity
suggestive of fatty liver changes, while six
HCV patients (6%) showed coarse
echopattern and irregular border of the liver
suggestive of cirrhotic changes. All five
control subjects had normal sonographic
features of the liver, (Table 1).
Table 1: Results of ultrasound study in HCV patients and control subjects
Patient’s group
(n=100)
Control group
(n=5)
P
Right lobe of liver (cm) 14.98 ± 2.48 12.62 ± 1.04 <0.01*
Splenic diameter (cm) 10.4 0± 1.27 10.05±1.07 0.850
Portal vein diameter (mm) 11.50±0.71 11.40±0.51 0.110
Fatty changes n (%) 47 (47.0%) 0 (0.0%) <0.01*
Cirrhotic changes n (%) 6 (6.0%) 0 (0.0%) <0.01*
*Statistically significant (p<0.05), n: number. The values are expressed as mean ± standard deviation or number (percent).
II) Routine laboratory investigations: As
regards liver profile, there was a statistically
significant difference between the patient’s
group and control subjects only in serum
transaminase levels (p <0.01) as well as in
prothrombin time and activity (p <0.05),
while there was no statistically significant
difference in serum albumin, bilirubin or
alkaline phosphatase between the two groups.
The platelet count also showed a statistically
significant difference (p<0.01) between the
patient and control groups, (Table 2).
Table 2: Laboratory test results in patients and control subjects
Patients group
(n=100)
Control group
(n=5)
P
ALT (IU/L) 139.90 ± 57.33 26.60 ± 10.56 <0.01*
AST (IU/L) 78.60 ± 43.45 22.60 ± 9.33 <0.01*
Albumin (g/dl) 4.01 ± 0.40 4.10 ± 0.24 0.242
Bilirubin (mg/dl) 0.92 ± 0.10 0.77 ± 0.23 0.321
Alk-P (IU/L) 208 ± 74.27 179.00 ± 55.54 0.294
Prothrombin activity (%) 84.16 ± 11.57 90.40 ± 8.66 0.030*
Platelets (count x103/mm3) 169.75 ± 79.82 325.67 ± 49.32 <0.01*
* Statistically significant (p<0.05), ALT; alanine transaminase, AST: aspartate transaminase, Alk-P: alkaline phosphatase, n: number.
The values are expressed as mean ± standard deviation.
III) Serum level of TN-C: The mean value for
serum TN-C concentrations in hepatitis C
patients was 39.7 ± 14.2 ng/ml, whereas in the
control group it was 20.9 ± 4.3 ng/ml; being
significantly higher in CHC patients than in
control group (p<0.01). However, serum TN-
C levels showed no correlations with any of
the routine laboratory parameters, including
serum AST, ALT, albumin, bilirubin, alkaline
phosphatase, prothrombin activity or platelet
count. Similarly in the control group, serum
TN-C concentrations were not correlated with
any of the laboratory data, (Table 3). Also, no
significant correlation was found between
serum TN-C levels and any of the ultrasound
parameters, neither in CHC patients’ group,
nor in the control group.
Table 3: Correlation of serum Tenascin-C level with other laboratory parameters in CHC patients and control subjects
Patients group (n=100) Control group (n=5)
R P r p
ALT (IU/L) + 0.04 0.213 + 0.10 0.230
AST (IU/L) + 0.03 0.250 + 0.09 0.206
Albumin (g/dl) − 0.08 0.448 − 0.07 0.456
Bilirubin(mg/dl) + 0.13 0.152 + 0.12 0.134
Alk-P (IU/L) + 0.12 0.210 + 0.14 0.208
Prothrombin activity (%) − 0.11 0.185 − 0.05 0.200
Platelets (count x103/mm3) − 0.13 0.140 − 0.15 0.122
Statistically significant (p<0.05), ALT; alanine transaminase, AST: aspartate transaminase, Alk-P: alkaline phosphatase, n: number
IV) Histopathologic characteristics: The Ishak
grade of activity among hepatitis C patients
ranged between minimal to moderate, and the
grading scores ranged between 4 and 12 out
of 18. The mean Ishak grade was 7.57 ± 1.26,
and the mean Ishak stage was 2.97 ± 1.438,
(Table 4). The necroinflammatory activity
was determined by scoring of piecemeal, lytic
and confluent necrosis and portal tract
inflammation, as displayed in details in table
5. According to Ishak staging system eight
hepatitis C cases (8%) showed features of
cirrhosis, while 92 cases (92%) didn’t.
Different grades of steatosis were also found
among hepatitis C patients; 24% showed mild
steatosis, 20% showed moderate, 7% showed
marked and 50% showed no steatosis.
Increased bile duct proliferation was noticed
in 39% and was absent in 61% of cases.
Evidence of large cell change was present in
20% of hepatitis C cases, while none of the
cases showed evidence of small cell change,
(Figures 1-6).
Table 4: Ishak grades and stages in HCV cases
Score Frequency n (%)
Ishak Grade None (score 0) 0 (0.0%)
Minimal (score 1-4) 13 (13.0%)
Mild (score 5-8) 82 (82.0%)
Moderate (score 9-12) 5 (5.0%)
Marked (score 13-18) 0 (0.0%)
Total 100 (100%)
Ishak Stage 1 8 (8.0%)
2 44 (44.0%)
3 20 (20.0%)
4 14 (14.0%)
5 6 (6.0%)
6 8 (8.0%)
Total 100 (100.0%)
n: number of cases
Table 5: Detailed Ishak grading in studied cases
Frequency n (%)
Piecemeal necrosis
P1 6 (6.0%)
P2 36 (36.0%)
P3 46 (46.0%)
P4 12 (12.0%)
Confluent necrosis
Negative 100 (100%)
Lytic necrosis
L1 5 (5.0%)
L2 86 (86.0%)
L3 9 (9.0%)
Portal inflammation
PI-1 4 (4.0%)
PI-2 54 (54.0%)
PI-3 38 (38.0%)
PI-4 4 (4.0%)
n: number of cases
Figure 1: (H&E x 400). Interface hepatitis (piecemeal
necrosis, circle) and lymphocytic infiltrate eroding the
limiting plate (PN=2).
Figure 2: (H&E x 100). Fibrous expansion of the
portal tracts with porto-portal and porto-central
bridging (Ishak stage 4).
Figure 3: (H&E x 100). Occasional nodule formation
(circle) (Ishak stage 5).
Figure 4: (H&E x 100). Complete cirrhosis (Ishak
stage 6)
Figure 5: (H&E x 100). Steatosis in chronic hepatitis a
mixed micorvesicular and macrovesicular, mild and
patchy steatosis (circle) a pattern commonly seen in
HCV.
Figure 6: (H&E x 400).Foci of large call change
(arrow); note enlarged nuclei with abundant
cytoplasm but with preserved nucleo-cytoplasmic
ratio.
CHC patients showing higher Ishak scores of
inflammatory activity of the liver had
significantly higher serum levels of TN-C
(p<0.01), (Figure 7). Also, higher Ishak stages
of fibrosis tended to be associated with higher
serum TN-C levels with statistically
significant correlation (p=0.02), (Figure 8).
When analyzing the individual
necroinflammatory parameters of Ishak
grading system, there were no significant
correlations of the serum TN-C levels with
the degree of confluent necrosis, focal
necrosis, or portal inflammation. However,
serum TN-C levels were significantly
correlated with the severity of piecemeal
necrosis, which is an important parameter of
necroinflammatory activity (p<0.01), (Figure
9). On the other hand, no significant
correlation was found between Ishak grade or
stage and any of the other laboratory
parameters (p>0.05).
Figure 7: Correlation of serum Tenascin-C (TN-C) in
chronic hepatitis C patients with the Ishak histological
grade of activity
Figure 8: Correlation of serum Tenascin-C (TN-C) in
chronic hepatitis C patients with the Ishak histological
stage of fibrosis
Figure 9: Correlation of serum Tenascin-C (TN-C) in chronic hepatitis C patients with the degree of piecemeal necrosis
according to the Ishak histological grading system.
V) Immunohistochemical study: 1)
Tenascin C expression: All 5 biopsies from
control subjects were negative, while all 100
hepatitis C cases were positive for TN-C
expression, with different degrees of
positivity. Preferential accumulation of
immunostain was observed at connective
tissue-parenchymal interfaces and in areas of
piecemeal necrosis, (Table 6, figures 10-15).
Minimal Mild Moderate
Ishak grade
15.0
20.0
25.0
30.0
35.0
40.0
45.0
Serum
TN
-C
(ng/m
l)
1 2 3 4 5 6
Ishak stage of fibrosis
20.0
25.0
30.0
35.0
40.0
45.0
Seum
T
N-C
(ng/m
l)
P1 P2 P3 P4
Piecemeal necrosis
15.0
20.0
25.0
30.0
35.0
40.0
45.0
Serum
T
N-C
(ng/m
l)
Figure 10: IHC for TN-C (x400). Weak, discontinuous
TN-C staining.
Figure 11: IHC for TN-C (x100). Moderate TN-C
expression in a case of cirrhosis.
Figure 12: IHC for TN-C (X400). Moderate TN-C
expression along portal infiltration and piecemeal
necrosis.
Figure 13: IHC for TN-C (X400). Moderate TN-C
expression along foci of piecemeal and lytic necrosis.
Figure 14: IHC for TN-C (X400). Strong diffuse TN-C
expression.
Figure 15: IHC for TN-C (X400). Strong patchy TN-C
expression.
2) Alpha smooth muscle actin expression: Among the control subjects, alpha SMA
immunoreactivity showed variable results; it
was negative in two, weak in two and
moderate in 1 case. In the hepatitis C group it
showed positivity of variable degrees in all
cases. Both showed statistically significant
difference between patients group and control
group, (Table 6, figures 16-19).
Figure 16: IHC for alpha SMA (x400). Strong blood
vessel staining and weak expression within the portal
tracts highlighting activated HSC (arrows).
Figure 17: IHC for alpha SMA (x400). Strong blood
vessel staining (arrow) and moderate staining reaction
along porto-parenchymal interface (circle).
Figure 18: IHC for alpha SMA (x400). Strong blood
vessel staining (arrows) and strong expression within
the portal tracts and along the scar tissue.
Figure 19: IHC for alpha SMA (x100). Strong blood
vessel staining and strong expression within the portal
tracts and along the scar tissue in a case of cirrhosis
(arrows).
Table (6): Tenascin C and Alpha SMA distribution among studied cases:
Grades Patients group
n (%)
Control group
n (%)
Total
n (%)
Tenascin Negative 0 (0.0%) 5 (100%) 5 (4.8%)
Weak 20 (20.0%) 0 (0.0%) 20 (19.0%)
Moderate 48 (48.0%) 0 (0.0%) 48 (45.8%)
Strong-patchy 20 (20.0%) 0 (0.0%) 20 (19.0%)
Strong-diffuse 12 (12.0%) 0 (0.0%) 12 (11.4%)
Total 100 (100.0%) 5 (100%) 105 (100%)
X2 40.203
P 0.001*
Alpha- SMA Negative 0 (0.0%) 2 (40.0%) 2 (1.9%)
Weak 28 (28.0%) 2 (40.0%) 30 (28.6%)
Moderate 40 (40.0%) 1 (20%) 41 (39.0%)
Strong 32 (32.0%) 0 (0.0%) 32 (30.5%)
Total 100 (100.0%) 5 (100.0%) 105 (100.0%)
X2 31.241
P 0.001*
* Statistically significant (p< 0.05), X2: Qui-square, SMA: Smooth muscle actin, n: number
A statistically significant correlation of
Tenascin C expression was found with Ishak
grade of hepatitis (p=0.007), as well as with
Ishak stage of fibrosis (p=0.039) among
studied cases. Similarly, a significant
correlation of alpha SMA expression was
proved with Ishak grade of activity (p=0.043),
Ishak stage of fibrosis (p<0.01) as well as
Tenascin C expression (p=0.032). Also, a
significant correlation existed between the
serum levels of TN-C and both: TN-C
expression (p=0.022), as well as alpha smooth
muscle actin expression (0.013). No
statistically significant correlation of Ishak
grading or staging scores was proved neither
with the degree of liver steatosis nor with the
presence of large cell change, (Table 7).
Table (7): Correlation of Ishak grade and stage of chronic hepatitis C patients with various histological and
immunohistochemical findings
Ishak grade of activity Ishak stage of fibrosis
Tenascin C expression
X2 27.329 25.878
P 0.007* 0.039*
Alpha SMA expression
X2 0.615 86.430
P 0.043* 0.001*
Degree of steatosis
X2 5.639 20.02
P 0.131 0.171
Large cell change
X2 0.098 7.449
P 0.555 0.189
*Statistically significant (p< 0.05), SMA: Smooth muscle actin, X2: Qui-square
Discussion
Cirrhosis develops in approximately 10% to
15% of individuals with chronic HCV
infection.(25) The smaller percentage of cirrhosis
among CHC patients in our study (8%) may be
explained by the lack of eligibility in patients
with cirrhosis for core biopsy, due to their
inadequate bleeding and coagulation profiles.
About 50% of our cases showed different
grades of steatosis, which is in accordance
with a study by Ruggiero et al who stated in
a meta-analysis for patients data that steatosis
was present in (50.9%) out of the 3068 CHC
patients studied.(26). Our results showed no
significant correlation between Ishak grade of
activity or stage of fibrosis and any of the
routine laboratory parameters. The grade of
activity was assessed according to Ishak
grading system; one of the most widely
accepted systems for assessment of
necroinflammation in CHC. Confluent
necrosis (being usually a very rare finding in
CHC) was absent in all of our cases.
Therefore, in most of our patients the Ishak
score limit was 12 instead of 18, which may
be considered an “underscoring” of the actual
necroinflammation. Also, portal inflammation
(another parameter in the Ishak grading
system) may reflect immunological response
rather than necroinflammation. This may
explain the usual discrepancy between liver
enzyme elevation and necroinflammatory
grade as determined by Ishak score in
assessment of chronic hepatitis C biopsy
specimens, as was stated by Shiha et al.(27). In
clinical practice, the stage of fibrosis is more
important than the grade of activity (unless it
is severe), as it reflects the disease
progression and hence the prognosis. Also, it
determines which treatment strategy to
pursue; whether curative or conservative.(28)
The stage of fibrosis was assessed in our CHC
cases according to Ishak staging system with
a mean value of 2.97 ± 1.44. Ductular
proliferation was also noticed in 39% of
cases, which was probably due to the
compression exerted on bile ducts by the
fibrotic septa. As hepatitis C dramatically
increases the risk for hepatocellular
carcinoma, cellular atypia (previously referred
to as dysplasia) that may suggest early or
incipient neoplasia must be surveyed in
biopsy specimens. In our study none of the
cases showed small cell change, which is
considered to be a directly premalignant
finding. On the other hand, large cell change
was noticed in 12% of cases. In spite of
increasing evidence that large cell change
may actually be related to hepato-
carcinogenesis, nevertheless its significance is
still under debate, as it is thought by many to
be malignancy associated, rather than directly
premalignant.(29,30). Immunohistochemical
study in hepatitis C patients revealed that
sinusoidal immunoreactivity to TN-C was
increased compared to normal liver
specimens. It also showed a significant
correlation as well as co-localization of alpha
SMA positive cells with dense TN-C
deposition, indicating that myofibroblastic
cells (mostly activated HSCs) and
myofibroblasts are the most plausible cellular
source of TN-C. Our study also showed that
both TN-C and alpha SMA expression
increased as the stage of fibrosis increased.
This may be the result of continuous
activation of HSC and other myofibroblasts
during hepatitis and synthesis of large
amounts of extracellular matrix (ECM)
proteins, including TN-C. Most striking was
the preferential TN-C immunostain accumu-
lation at connective tissue-parenchymal
interfaces in areas of piecemeal necrosis.
Also, TN-C expression closely correlated
with scores of necroinflammatory activity, as
well as with fibrosis stage. Thus our results
indicate that TN-C deposition is particularly
upregulated in chronic hepatitis C and
correlates with progressive disease activity,
being specific to areas where tissue injury and
remodeling are in progress. These results are
in agreement with the findings of El-Karef et
al and Van Eyken et al.(31,32) who reported
that large TN-C variants were upregulated in
chronic hepatitis, especially at sites of
interface hepatitis and confluent
necrosis.(31,32). In humans, serum TN-C has
multiple variants: A small-molecular-weight
variant which exists constitutively in normal
tissues, whereas the high-molecular weight
(large) variants are specifically expressed in
tissues under several pathological
conditions.(13,14,18) In this study, the ELISA kit
used was chosen to determine serum TN-C
large variants (including FN-III-C domain).
Results revealed that the mean value for
serum TN-C concentrations was significantly
higher in CHC patients than in the control
group. However, it showed no correlations
with any of the routine laboratory parameters,
neither in CHC patients’ group, nor in the
control group. Thus, it appears that serum
large TN-C variant levels in CHC patients
were increased independently of other
laboratory data. On the other hand, a
significant correlation was found between the
serum levels of TN-C and TN-C immuno-
histochemical expression in liver tissue
among CHC patients. Moreover, CHC
patients with higher Ishak grades of
inflammatory activity and higher stages of
fibrosis of the liver had significantly higher
serum levels of TN-C. Also, serum TN-C
level was significantly correlated with the
severity of piecemeal necrosis, which is an
important parameter of necroinflammatory
activity in Ishak grading system. These
findings, when combined with the preferential
TN-C immunostain accumulation at areas of
piecemeal necrosis in liver tissue, strongly
suggests that the serum concentration of TN-
C might be a useful marker to indicate the
grade of inflammatory activity – particularly
piecemeal necrosis- in CHC. Similar to our
results, Tanaka et al .(33) showed in one study
that the serum concentration of large TN-C
variants significantly correlated with the
histological grade of necroinflammatory
activity. They also demonstrated in the same
study that positive immunolabeling of the FN-
III-C domain was associated with active
piecemeal necrosis, while negative labeling
was observed in the normal liver, concluding
that serum TN-C might hold promise of being
a clinically useful marker for evaluating
disease activity and the effectiveness of
treatment in patients with CHC. However,
unlike our results, they found no significant
correlation of the serum TN-C levels with the
stage of liver fibrosis in CHC.(33) Never-
theless, another study performed by
Yamauchi et al also demonstrated signify-
cantly higher serum levels of TN-C in patients
with chronic liver disease than in healthy
subjects. In their study – similar to our results
- they proved a positive correlation of serum
TN-C with the histological findings,
especially with the degree of fibrosis, as well
as with other serum markers of fibrosis
including type IV collagen, procollagen type
III peptide (PIIIP) and laminin.(34). However,
only a few reports have found some
correlation between rotine laboratory
parameters like serum transaminase levels and
the histological grade of disease activity.(35,36)
In our study, we did not observe any
correlation between both parameters. On the
other hand, we found that the serum
concentration of large TN-C variants might
indeed be a promising marker of
inflammatory activity as well as progress of
fibrosis in CHC. Further investigations on
TN-C and its role in the progression of liver
disease may be expected to provide new
information for the diagnosis and prevention
of liver fibrosis in the future.
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