Invasive and Noninvasive Monitoring of Hepatitis C Virusinduced Liver Fibrosis, Alternatives or Comp

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Invasive and Non-invasive Monitoring of Hepatitis
C Virus-induced Liver Fibrosis,
Alternatives or Complements? Znoiko Olga
O. (1, 2) Yuschuk Nikolai D. (1)
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INTRODUCTIONChronic hepatitis C is estimated to
affect 170 million persons worldwide, and is now
the single major reason for liver
transplantation. Natural history studies suggest
that 520 of persons develop cirrhosis during
the 20 years of HCV infection, while other
patients remain asymptomatic, without
significant liver disease for many decades, if
not for life 1, 2. Detection and
characterization of hepatic fibrosis by liver
histology and by non-invasive monitoring is a
primary instrument to assess chronic hepatitis C
natural history and prognosis. This topic review
will focus on the hepatic fibrosis diagnosis.
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However, in doing so liver function
mayultimately become impaired. With recurrent
bouts of inflammation, the liver's normal
architecture can be replaced by fibrous scar
tissue, ultimately resulting in the advanced
liver disease known as cirrhosis 1, 3.
Typically, injury is present for months to years
before significant scar accumulates, although the
time course may be accelerated in congenital
liver disease. Being a hallmark of hepatic
cirrhosis, the fibrosis worsening is probably the
best surrogate marker for the progression of
chronic liver disease.
5
Stellate cells undergo a process known as
activation, in response to any insult (Fig. (2)).
Upon activation, HSC is transformed from a
quiescent cell filled with retinoids to
anactivated cell characterized by a loss of
retinoids, expression of new receptors, cellular
proliferation, expression of new genes such as
a-smooth muscle actin, and synthesis of
extracellular matrix (ECM) proteins (Fig. (2))
Brenner
DA.The role of hepatitis C in hepatic fibrosis
http//www.postgrado.com/5htm).
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ECM is a dynamic regulator of cell function.
Early, subendothelial matrix accumulation leading
to "capillarization"of the subendothelial space
of Disse is a key event, and may be more
important than overall increases in matrix
content (Fig. 1 ) 7. Normally, this space
contains the components of a basement membrane.
Replacement of the normally low-density matrix of
basement membrane by high-density
interstitial matrix directly perturbs hepatocyte
function. Activated HSCs produce fibrogenic
environment through combination of ECM
overproduction, diminished activation of
metallproteinases responsible for degradation of
ECM proteins and inhibition of this degradation
by metalloproteinase tissue inhibitors.
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FIBROSIS STAGING AND GRADINGThe risk of
developing cirrhosis depends on the stage (degree
of fibrosis) and the grade (degree of
inflammation and necrosis) observed in the
initial liver biopsy (Figs. (3, 4) for more
images, see also http//janis7hepc.com/learning_
about_liver_fibrosis.htmPredicting). The
degrees of fibrosis, portal, then peripheral,
then bridging ending at cirrhosis) are
illustrated by Figure 3.
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It is widely excepted that the fibrosis intensity
grade correlates with the activity of
inflammatory changes in the liver (Fig. (4))
inflammation grades from I to IV as illustrated
at the web-site http//www.ikp.unibe.ch/lab2/hepC)
. This, however, is not absolutely true.
According tothe liver biopsy data, in 36 of
patients with chronic hepatitis C the severity of
hepatitis did not correspond to the grade of
fibrosis. If the recommendations to treat were
based on the degree of hepatitis intensity, 56
of patients would not undergo medical treatment
despite the severe fibrosis 9.
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FIBROSIS DEVELOPMENTFibrosis RateThe speed of
fibrosis development in the case of hepatitis C
is of top interest for the clinicians. The speed
of fibrosis development is determined as a ratio
of the difference in fibrosis stages expressed in
METAVIR and the interval between biopsies
expressed in years 9. This method may only be
deemed significant if one knows exactly
theinfection duration and if the speed of
fibrosis development is constant. However, the
rate of fibrosis development is not a constant
value.
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There are three excepted options of fibrosis
progress quick (10 and less years), average
(about 30 years) and slow (more than 50 years)
1, 9. For the untreated patients, the average
number of life years from the on-set of the
infection to the development of cirrhosis amounts
to 30, with 33 of the patients having the
expected time of cirrhosis development of less
than 20 years, while for 31 of patients the
expected time of cirrhosis development exceeds 50
or more years. Different patients progress to
cirrhosis at different rates. The overall rate of
fibrosis progression suggests that the average
patient would require 49 years to develop
cirrhosis.
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Factors Effecting Fibrosis DevelopmentLiver
fibrosis in chronic hepatitis C is related to
sex, age at infection, duration of infection,
and alcohol consumption. A link between age at
HCV infection and the presence of hepatic
fibrosis is supported by the strong and
consistent trend of increasing risk with age at
infection, and the independence of effect. This
relationship has been seen in several previous
cross-sectional studies (Fig. (5)) 17, 23,
24.Recent longitudinal studies had found very
low rates of progression to advanced liver
disease among young adults infected through
injecting drug use and contaminated anti-D
immunoglobulin injections 25-27 and among
children infected through blood transfusion 28.
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The progression of liver fibrosis seems to be
influenced by polymorphisms in the genes encoding
immunoregulatory proteins, proinflammatory
cytokines, and fibrogenic factors 29. These
genetic factors could explain the broad spectrum
of responses to HCV found in patients with
chronic liver diseases. For example, analysis of
patients with end stage liver cirrhosis and blood
donors demonstrated a reduced risk for
HCV-induced end stage liver disease in the
presence ofDRB111 and DQB103 30.
Large-scale, well-designed studies are required
to clarify the actual role of different factors
and genetic variants in liver fibrosis
development.
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ReversibilityDense cirrhosis, the end-stage
consequence of fibrosis, with nodule formation,
portal hypertension, and early liver failure is
generally considered irreversible. The exact
moment at which fibrosis becomes irreversible is
unknown, either in terms of a histologic marker
or a specific change in the matrix composition or
content 7 www.uptodate.com/patient_info/topic
pages/topics/Cirrhosi/9999.asp 13).Less
advanced lesions can show remarkable
reversibility when the underlying cause of the
liver injury is controlled.
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FIBROSIS DIAGNOSIS BY LIVER PUNCTURE BIOPSY AND
ITS RISKSLiver biopsy serves as a gold
standard for the diagnosis of fibrosis with
chronic hepatitis C being responsible for over
50 liver biopsy cases all over the world 19,
33. A morphological study of the punctate
determines the grade of hepatitis activity and
fibrosis stage, eliminates the alternative
diagnoses and helps to detect additional
pathologies, as well as to evaluate the
efficiency of antiviral therapy.
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However, the biopsy is an invasive technique
withpossible complications and even the lethal
outcomes. The number of lethal cases due to
biopsy varies from 0 to 3,3 out of 1000 9, 34,
35. The study by Gadranel et al. Contains data
analysis of 2,084 liver biopsies carried out in
89 medical centers in France during 1997 with 54
of cases due to HCV infection 33. A
percutaneous biopsy was carried in 91, and
transvenous, in 9 of cases. In 20 of
cases,moderate pains were registered after the
procedure, while 3 of patients had considerable
pains that required intravenous analgesia with
further hospitalization (in cases when the biopsy
was carried out in the outpatient setting).
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LIVER BIOPSY LIMITATIONSSome clinicians believe
that since liver biopsies sample only 1/50,000 of
the entire liver mass, sampling errors are bound
to occur. There is a probability of spotting
error when the biopsy needle hits the area of
tissue where thechanges are less or on the
contrary more intense than on the whole in the
liver. For example, liver biopsy samples taken
from the right and left hepatic lobes differed in
histological grading and staging in a large
proportion of hepatitis Cpatients 37.
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NON-INVASIVE BIOCHEMICAL SCALING OF FIBROSISThe
above-mentioned limitations of the liver biopsy
plus considerable biopsy costs substantiate the
necessity todevelop simple, inexpensive, and
reliable non-invasive tests to assess disease
severity in patients with chronic hepatitis C in
which peripheral blood is to be used 38-41.
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Three-Marker Biochemical Fibrosis IndexOne of
the biochemical approaches of non-invasivefibrosi
s monitoring makes use of three parameters the
number of thrombocytes, the prothrombin time and
the ALT/AST ratio with evaluation range from 0 to
11 points 45. The scale shows a specificity of
98 and a sensitivity of 46 45. The average
score for the patients having histologically
detected fibrosis of 0-2 amounts to 4.32.0,
while the score for the patients having stage 3
and 4 fibrosis is considerably higher (7.91.4
plt 0.0001). Thus, without recurring to liver
biopsy, one may suggest that patients with the
number of scores equal to 7 and more are more
likely to have fibrosis stage 3 to 4.
19
Thrombocytopenia is commonly seen in patients
with cirrhosis and in patients with acute liver
failure 50. Both splenomegaly and inadequate
thrombopoietin (TPO) production by the cirrhotic
liver are responsible for thrombocytopenia. In
addition, thrombocytopenia is frequently observed
in chronic hepatitis patients who received
interferon therapy, and may even lead to the
discontinuation of treatment. The serum TPO
response to interferon-induced thrombocytopenia
is interferon-dose dependent, and together with
the changesin platelet counts may serve as a
marker for the degree of liver fibrosis 51.
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Five-Marker Biochemical Fibrosis IndexThe newest
fibrosis index devised included a wide panel of
clinical and laboratory markers combined with age
and sex. The most informative markers were
alpha2 macroglobulin, alpha2 globulin (or
haptoglobin), gamma globulin, apolipoprotein A1,
gamma glutamyltranspeptidase (GGT), and total
bilirubin 52, 53. Five-markers index accurately
predicts significant fibrosis in patients with
HIV/HCV co-infection, and reduce the necessity
for liver biopsy by 55 while maintaining an
accuracy of 89 53.
21
IronIn CHC patients, hepatic iron concentration
correlates with liver fibrosis 21. In CHC
patients, hepatic iron could play a role in the
activation of hepatic stellate cells and in the
progression of fibrosis. In CHC patients with low
grading score (lt 3), hepatic iron concentration
correlates positively with the number of stellate
cells and proliferating hepatocytes (72 CHC
patients) 56. Ferritin serum level correlated
significantly with hepatic iron concentration (r
0.59, P lt 0.001), with grading (r 0.47, P lt
0.001) and staging (r 0.51, P lt 0.001)
scores for chronic hepatitis in the whole group
of patients 56.
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Enzymatic and Nonenzymatic AntioxidantsFree
radicals play a role in CHC liver
damage.Antioxidants (AO) (enzymatic and
nonenzymatic) scavenge free radicals and prevent
tissue injury. The levels of malondialdehyde
(MDA) and AO, namely retinol, alpha- and
gamma-tocopherol, lutein, beta-cryptoxanthin,
lycopene, andalpha- and beta-carotene levels,
were estimated by highpressure liquid
chromatography in the pretreatment serum and
liver biopsy specimens 58.
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Alpha-FetoproteinSerum levels of
alpha-fetoprotein (AFP) were analysed at the time
of diagnosis in 77 patients with CHC fibrosis and
HCC 59. AFP levels were elevated in 78 of
cases, but only 34 reached gt500 ng/ml, a value
considered to besignificant for the diagnosis of
HCC. AFP levels correlated with the stage of
fibrosis. The sensitivity of AFP serum levels as
a tumour marker is poor but might help to detect
atleast a minority of cases.
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Conflicting Biochemical Fibrosis MarkersThe
relationship between serum transaminase levels
and risk of liver disease progression remains
controversial.Although risk of disease
progression is considerably higher among people
with abnormal serum transaminase levels compared
with those with consistently normal levels
60.There is conflicting evidence on the
association between degree of ALT abnormality and
extent of histological fibrosis (see, for
example, Fig. (6)).
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A reasonable explanation for this discrepancy is
that ALT/AST levels vary during the course of
this disease, and the values found at the time of
a single liver biopsy may not accurately
reflect values that have occurred in previous
years.These findings indicate that regular
determination of aminotransferase values is a
helpful and reliable means of monitoring disease,
judging prognosis, and recommending therapy and
support the recommendation that patients with
normal aminotransferase levels and mild liver
histology can safely defer treatment 2.
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EXTRACELLULAR MATRIX TESTS OF FIBROSIS
SCALINGAnother route of non-invasive fibrosis
scaling that has recently received much attention
is the evaluation of serumlevels of
extracellular matrix break down products. The
rationale is that since there is extensive
deposition of fibrous tissue, serum levels of the
constituents of fibrous tissue will increase as a
result of remodeling and recurrent scarring.
27
The biochemical tests of fibrosis severity are
based on the detection of molecular junctions
that activate fibrosis, or participate in the
pathophysiology of the extracellular matrix
generation process. The most extensively studied
are hyaluronic acid (HA) metalloproteinases
(MMP) and inhibitors of metalloproteinases
(TIMP) type IV collagen (IV-C) and N-terminal
propeptide of type III procollagen(PIIIP) 21,
40, 68, 69.
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Hyaluronic Acid (HA)The particularly intense
discussion arises over thediagnostic
significance of the high content of HA. HA is a
glycosaminoglycan, polysaccharide with a heavy
molecular weight, widely present in extracellular
space. Some of the produced HA is supplied by the
lymphatic system to the venous blood and may be
identified in the blood by enzymeimmune
detection. In liver, HA is synthesized by
stellate cells and is considered to be a factor
contributing to fibrogenesis. The increase in HA
production may reflect the induction of stellate
cell proliferation and the synthesis of
extracellular matrix due to the inflammation.
29
According to Ueno et al., HA levels reflected the
degree of fibrosis in HCV-RNA positive
non-responders to antiviral therapy 73. Changes
in the levels of HA were compared among the
patients who showed a sustained normalization of
serum ALT levels with and without eradication of
serum HCV RNA (complete responders and
biochemical responders) and nonresponders.
Hyaluronate levels were significantly decreased
24 weeks after the end of the treatment, as
compared with those prior to the treatment 76.
In nonresponders, IFNalpha therapy was unable to
decrease the HA levels and did not improve the
histological outcome. It was reflected by
unchanged HA levels during the therapy and the
follow up 77.
30
Propeptide of Procollagen Type III (PIIIP) and
Type IV Collagen (IV-C)Collagens type III and V
and fibronectin accumulate in early, and
collagens type I, III, and IV, indulin, elastan
and laminin in late injury 80. Serum
pro-collagen III peptide levels (PIIIP) are
related to lobular necrosis in untreated patients
with chronic hepatitis C 71. Serum N-terminal
procollagen III peptide (PIIINP) displayed a
significant and persistent decrease in
sustainedresponders but not in non-responders
(14 sustainedresponders and 17 non-responders
81, 82, PIIIP normalized when fibrosis improved
(Plt 0.01).
31
CHC patients showed a mean level of serum type IV
collagen (IV-C) (133.6/-93.3 ng/ml)
significantly (P lt 0.01) higher than controls
(100.2/-10.5 ng/ml). A good relationship between
the degree of liver fibrosis and IV-C serum level
was found (r 0.68 P lt 0.005). In both
responders and non-responders IV-C levels
decreased during interferon therapy. During the
follow-up, in responders IV-C remained unchanged,
while in non-responders/relapsers it returned
rapidly to the pretreatment levels (139.1/-100.7
ng/ml). Thus, in CHC type IV collagen appears to
be a useful tool for evaluating fibrogenic
activity, and can be used to monitor the effects
of antiviral treatment 85.
32
Matrix MetalloproteinasesRecovery from fibrosis
involve remodelling and breakdown of multiple ECM
components with degradation of predominant
component collagen I. The matrix
metalloproteinases (MMP), a family of zink
dependent endoporteinases, have a capacity to
degrade these various ECM components. MMPs are
expressed particlularly by HSCs and Kupffer cells
88. Matrix metalloproteinases 1, 2, and 7
(MMP-1, -2, -7) and tissue inhibitor of
metalloproteinase-1 (TIMP-1) are closely involved
in the fibroproliferativeprocess in the liver
during chronic hepatitis C 88-91.
33
In another study, serum levels of MMP-1, MMP-2,
TIMP-1 and TIMP-2 before and after
interferon-alpha (IFNalpha) treatment
demonstrated a significant increase in the
MMP-1/TIMP-1 ratio in the responder group, while
in the non-responder group the MMP-1/TIMP-1 ratio
tended towards a decrease 93. Moreover, there
was not only a significant increase in TIMP-2
levels but also a tendency towards an increase in
TIMP-1 levels. The results of Ninomiya et al.
suggested that an elevated MMP-1/TIMP-1 ratio may
ameliorate liver fibrosis by interferon in CHC,
whereas elevated levels of TIMP-1 and TIMP-2
might impede improvement 93.
34
Cell Adhesion MoleculesLymphocyte adhesion to
endothelium, extravasation, and adhesion to
hepatocytes are mediated by adhesion molecules
and constitute important steps in the liver
inflammation dueto CHC. A number of publciations
has described increased levels of soluble
adhesion molecules in the serum of patients with
hepatitis C, and their correlation with cytokine
concentrations and liver inflammation and
fibrosis. Measurement ofsoluble adhesion
molecules may be a perspective way to follow
liver inflammation and fibrosis during CHC 94.
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CYTOKINES TRANSFORMING GROWTH FACTOR BETA
1Host genetic factors influencing fibrogenesis
may account for some of the variability in
progression of this disease. Progressive fibrosis
of other organs, particularly heart and kidney,
involve overproduction of the profibrogenic
cytokine transforming growth factor beta1
(TGF-beta1) that may be enhanced by angiotensin
II, the principal effector molecule of the
renin-angiotensin system. Powell et al. examined
the inheritance of polymorphisms in TGF-beta1,
interleukin 10 (IL-10), tumor necrosis factor
alpha (TNF-alpha), and genes of the
renin-angiotensin system in 128 CHC patients 96.
36
TGF-beta 1 is an important cytokine involved in
thepathobiology of tissue fibrosis through its
stimulation of the production of, and inhibition
of the degradation of, extracellular matrix
proteins. TGF-beta 1 impairment of extracellular
matrix breakdown is related to stimulation
oftissue inhibitor of metalloproteinases-1
(TIMP-1) gene. There is a close correlation
between TGF-beta serum levels and the rate of
fibrosis progression. Plasma TGF-beta 1 levels
are significantly elevated in patients with mild
and moderate chronic hepatitis, but not in those
with minimal chronic hepatitis, compared with the
levels in the controls.
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CONCLUSIONThere has been considerable effort to
identify serum markers as noninvasive measures of
hepatic fibrosis 67. However, noninvasive
markers have several limitations1. They
typically reflect the rate of matrix turnover,
not deposition, and thus tend to be more elevated
when there is high inflammatory activity. In
contrast, extensive matrix deposition can go
undetected if there is minimal inflammation.2.
None of the molecules are liver-specific, so that
concurrent sites of inflammation may contribute
to serum levels.
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ACKNOWLEDGEMENTSAuthors wish to thank Swedish
Institute for the financial support rendered
through the New Visby program. REFERENCES