Assessment of fibrosis in chronic liver diseases

March 8, 2011

http://onlinelibrary.wiley.com/doi/10.1111/j.1751-2980.2008.00356.x/full

Kun ZHOU, Lun Gen LU

Article first published online: 10 FEB 2009

DOI: 10.1111/j.1751-2980.2008.00356.x

Shanghai Branch, Chinese Society of Gastroenterology and Blackwell Publishing

Asia Pty Ltd

Issue

Journal of Digestive Diseases

Volume 10, Issue 1, pages 7â€“14, February 2009

The assessment of liver fibrosis provides useful information not only for

diagnosis but also for therapeutic decisions. Although liver biopsy is the

current gold standard for fibrosis assessment, it has some risks and

limitations, including intra-observer and inter-observer variation, sampling

error and variability. In recent years, many studies and great interest have

been dedicated to the development of non-invasive tests to substitute a liver

biopsy for fibrosis assessment and follow up. Advances in serological and

radiological tests such as serum marker panels, transient elastography and their

combinations can assess fibrosis accurately and reduce the need for a liver

biopsy. But at present, all have failed to completely replace a liver biopsy

because of their respective limitations and an imperfect gold standard used in

current researches. The searching for an ideal surrogate is still in progress.

INTRODUCTION

Chronic liver diseases (CLD) are very common. More than 400 million individuals

worldwide are chronically infected with hepatitis B virus (HBV), and more than

170 million are chronically infected with hepatitis C virus (HCV). Nonalcoholic

fatty liver disease also attracts more and more attention, paralleling the

increasing prevalence of obesity, diabetes mellitus and the metabolic syndrome,

and alcoholic liver disease (ALD) is prevalent in drinkers. Advanced fibrosis

and cirrhosis develop in about 20â€“40% of patients with CLD, some of whom

finally progress to end-stage liver disease or hepatocellular carcinoma. But the

progression may take years or decades. A CLD natural history is variable and its

long-term evolution differs in individual patients.

Previously, fibrosis was thought to be an irreversible process and received

little attention until the 1980s, when it was discovered that hepatic stellate

cells (HSC) played an important role during fibrosis. Following chronic liver

injury in CLD, HSC proliferate and activate under inflammatory milieu, acquiring

proinflammatory and fibrogenic properties. Activated HSC synthesize large

amounts of extracellular matrix (ECM) constituents. The accumulation of ECM

distorts the hepatic architecture by forming a fibrous scar, and the subsequent

development of nodules of regenerating hepatocytes defines cirrhosis. In the

1990s, it was demonstrated that liver fibrosis may be reversible when the cause

is treated. Since, researchers have been stimulated to develop therapies for

treating hepatic fibrosis. Although successful treatments, such as antiviral

therapies for HBV and HCV, seems to modify fibrosis and prevent progression to

cirrhosis and cancer, their response rates are low (especially in HCV genotype 1

patients) and with frequent side effects and high costs. Thus, assessing

fibrosis precisely is the main determinant in the management of CLD, to decide

when to begin a treatment and assess treatment response. Up to now we have

developed three main tools to assess fibrosis: liver biopsies, serological and

radiological tests. Unfortunately, they all have limitations and pitfalls. To

discuss their advantages and deficiencies will be helpful in scientific research

and clinical practice.

INVASIVE MEASUREMENTS

Liver biopsy

The liver biopsy has been considered the gold standard for confirming a clinical

diagnosis, for assessing the severity of necro-inflammation and fibrosis, for

identifying cofactors and comorbidities, and for monitoring the efficacy of

treatment ever since the first liver biopsy was performed by Ehrlich in

1883.1 The procedure is particularly useful for diagnosing the early stages of

fibrosis and identifying patients at high risk of progressing to fibrosis, but

it has also a number of limitations. Patient acceptance is somewhat low because

a biopsy is expensive, invasive and associated with some discomfort and

complications. Pain appears in about one-quarter of patients, and other

complications include bleeding, biliary peritonitis, pneumothorax and a

mortality rate of about 0.01%.2 A sampling error of at least 24% is reported,

usually because of specimen fragmentation or inadequate length. Colloredo et al.

concluded that an optimum specimen should be at least 20 mm in length with 11

complete portal tracts.3 Even with adequate-sized biopsies, the interpretation

might be unreliable, because the distribution of necro-inflammation and fibrosis

is not homogeneous and a liver biopsy samples only 0.00002 of the mass of the

liver.

Several semi-quantitative scoring systems have been proposed to describe and

quantify necro-inflammation, steatosis and fibrosis in the liver, particularly

for chronic viral hepatitis. These include the Knodell histological activity

index (HAI) first proposed in 1981, then modified to the Scheuer system, the

METAVIR system and the Ishak modified HAI.4 However, all these scoring systems

could only provide qualitative descriptors to stage fibrosis, and the staging of

certain histopathological changes differ in different systems (Table 1). This

could cause considerable intra-observer and inter-observer variation and

difficulty in comparison.

Table 1. Scoring systems for staging fibrosis Pathologic features Knodell

Scheuer METAVIR Ishak

No fibrosis 0 0 0 0

Enlargement of some portal tracts 1 1 1 1

Enlargement of most portal tracts 1 1 1 2

Periportal septa 1 2 1 2

Occasional portalâ€“ portal septa 3 2 2 3

Numerous septa (portalâ€“portal and/ or portalâ€“central) 3 3 3 4

Occasional nodules 4 4 4 5

Definite cirrhosis 4 4 4 6

Using computerized digital image analysis, the amount of fibrosis in liver

biopsy specimens can be evaluated by a quantitative score. Though this is

thought to be less reliable in determining early stage fibrosis, recent advances

such as a higher resolution digital camera can improve discrimination between

the varying stages of liver fibrosis, including mild fibrosis. It may be a more

precise method than semi-quantitative histological stages for monitoring

fibrosis progression or regression during clinical therapeutic trials.5

Considering the irregular shape of specimens, fractal and spectral dimension

analysis can also be used to improve accuracy.6

The detection of genes correlated with fibrosis from biopsy samples has renewed

interest in liver biopsies. Changes in liver gene expression can indicate

fibrosis progression precisely at an early stage.7 Genetic studies have

identified possible genetic polymorphisms that influence the progression of

liver fibrosis.8 The identification of panels of key genes correlating with

differences in the progression of CLD could lead to establishing excellent

prognostic/diagnostic tools.

Hepatic venous pressure gradient (HVPG)

HVPG, as an expression of intrahepatic resistance, does not exceed 5 mmHg in the

absence of significant fibrotic evolution. The measurement of HVPG is a

validated, safe and highly reproducible technique. It may be considered a

dynamic marker of disease progression in patients with HCV and an end point in

antiviral therapy, irrespective of the antiviral response.9 However, the

technique is invasive and expensive; it requires technical expertise and has a

low patient acceptance.

SEROLOGICAL TESTS

The limitations of liver biopsy led to the search for non-invasive tests to

assess liver fibrosis. Afdhal and Nunes et al.10 suggest the following criteria

for an ideal marker of liver fibrosis: it should be liver specific; it should

not be influenced by alterations in liver, renal, or reticulo-endothelial

function; it should measure one or more of the processes related to fibrosis

(the stage of fibrosis, the activity of matrix deposition, or the activity of

matrix removal) and should be easy to perform.

Direct serum markers

The key step in the pathophysiology of liver fibrosis is a balance between ECM

deposition and removal. The accumulation of ECM results from both increased

synthesis and decreased degradation. The principal ECM constituents are

synthesized by activated HSC, while they are broken down by a family of enzymes

known as matrix metalloproteinases (MMP). Many studies have been dedicated to

find serum ECM markers for fibrosis assessment: (i) collagens: N-terminal

peptide of type pro-collagen (PIIINP), type IV collagen 7s domain(IV-7S); (ii)

proteoglycans: hyaluronic acid; (iii) glycoproteins: laminin, human cartilage

glycoprotein 39; (iv) collagenases and their inhibitors: MMP, tissue inhibitor

of metalloproteinases; and (v) cytokines: transforming growth factor Î²,

platelet-derived growth factor (PDGF), tumor necrosis factor Î².

The clinical applications of such markers appear innovative and they are useful

for assessing the speed of liver fibrogenesis and estimating the response to

antiviral therapies or anti-fibrotic drugs. But most of them are insensitive in

milder fibrosis, and it must be stressed that these markers reflect fibrogenesis

and fibrolysis more than fibrosis itself. In other words, there may be a highly

active fibrotic process in the liver, although fibrotic tissue has not yet been

developed, or there may be heavy fibrosis in the liver but fibrotic activity is

temporarily discontinued.

Serum marker panels

Since present direct markers cannot satisfy yet the clinical need of measuring

the fibrosis, an alternative approach turn out to be combining a number of serum

markers to generate algorithms capable of evaluating fibrosis. A large number of

panels have been studied by groups worldwide11â€“32 (Table 2).

Table 2. Studies of serum markers panels for assessment of liver fibrosis

These panels are mainly based on two kinds of markers, direct and indirect.

Direct markers are those directly linked to the modifications in ECM metabolism,

such as hyaluronic acid and PIIINP. Indirect markers include a broad range of

blood tests which have no direct link with liver fibrosis. They reflect liver

dysfunction or other phenomena caused by fibrosis rather than fibrosis per se.

Generally speaking, indexes including direct markers, such as the Fibrometer,

may perform with greater accuracy, but indexes composed by only indirect markers

are effective as well, and are usually more useful because they are based on

routine blood tests that are easy to be performed in a general laboratory.

The diagnostic value of the models was assessed by calculating the area under

the receiver operating characteristic curves (AUROC). Most studies reported an

AUROC > 0.80 in differentiating significant fibrosis (fibrosis spread out the

portal tract with septa) from no/mild fibrosis (no fibrosis or portal fibrosis

without septa). Improved performance with a higher AUROC value was shown in

differentiating between no cirrhosis and cirrhosis. But it must be underlined

that the AUROC values in Table 2 each came from differently designed studies and

are not suitable for making a comparison. Some well designed validation studies

were done in the last 2 years, which may give us more reliable results.33,34

There are still some limitations of these marker panels to be considered. First,

the design of every study differed in population characteristics, patient

selection, significant fibrosis prevalence, blood test inclusion, biochemical

measurement and liver histological assessment, which resulted in various panels

with different markers and parameters. The agreement among these indexes is poor

and a validation study is needed to choose a proper panel and cut-off value for

clinical use. Second, none of the studies controlled for the degree of

necro-inflammatory activity, most of the panels include markers likely to

reflect or be affected by inflammation in the liver, which is much more mobile

than fibrosis stage. Third, the formulas can easily fail because many markers

included will be influenced by extrahepatic diseases or conditions such as

inflammation, hemolysis, cholestasis, hypercholesterolaemia and renal failure.

Finally, few of the studies include treated patients. It is not clear whether

these indexes are suitable for assessing treatment response. However, a few

studies by Poynard et al. suggested that FibroTest (BioLiveScale, Angers,

France) could also be used as surrogate markers of the histological impact of

treatments in patients infected by HCV and HBV.35

These indexes, in their current form, are not able to give us the exact stage of

fibrosis in most studies. Their main value is to reduce the need for a liver

biopsy by distinguishing significant fibrosis from no/mild fibrosis, and showing

the presence of cirrhosis. It does not seem appropriate to completely replace

liver biopsy with serum marker panels at the present time, but it can be

anticipated that these indexes will become very useful in the clinical

management of CLD by offering an attractive alternative to liver biopsy, as they

are non-invasive, convenient, and inexpensive, and may allow the dynamic

assessment of fibrosis. Validation in larger cohorts of patients with different

CLD is needed before an index is proposed for extensive clinical use.

Proteomics and glycomics

Over the last 2 years it was reported that the use of proteomic patterns in

serum to distinguish individual stages of fibrosis could achieve perfect

diagnostic sensitivity and specificity. Using a proteome-based fingerprinting

model generated by surface-enhanced laser desorption/ionization time-of-flight

(SELDI-TOF) ProteinChip (Ciphergen Biosystems, California, USA) arrays, Poon et

al.36 achieved an AUROC of 0.93 in identifying significant fibrosis. Another

proteomic index combining eight peaks established by Morra et al.37 could

diagnosis advanced fibrosis with an AUROC of 0.88, significantly greater than

the FibroTest AUROC of 0.81. Besides, The SELDI-TOF ProteinChip technology is

useful for the early detection and prediction of HCC in patients with chronic

HCV infection. Similar technologies have also been used to generate profiles of

serum N-glycan profile for identifying liver fibrosis.38 Further studies

identifying the altered peaks in these models to understand their origins may

help to find new biomarks for fibrosis, or even improve our understanding in the

mechanism of liver fibrosis.

RADIOLOGICAL TESTS

Since significant structural changes are present only in advanced CLD, routine

examinations by ultrasound (US), computed tomography and magnetic resonance

imaging (MRI) could produce specific findings, but with very limited

sensitivity. Thus, persistent efforts have been made to search for technological

developments.

Perfusion examinations

MR and Doppler US techniques are being studied to find sensitive perfusion

changes in the progression of fibrosis. For example, the circulatory changes

will result in a decrease of hepatic vein transit time (HVTT), which can be

measured by microbubble-enhanced US. Using HVTT measurements, Lim et al.

achieved 100% sensitivity and 80% specificity for diagnosis of cirrhosis, and

95% sensitivity and 86% specificity for the differentiation of mild hepatitis

from more severe liver disease.39 Recent studies of hepatic microcirculation

discovered that there is a progressive arterialization of the hepatic parenchyma

during the progression of fibrosis, especially in the peripheral areas of the

liver. The â€˜centralâ€“peripheral phenomenonâ€™ belonging to the transient

hepatic arterial differences (THAD)40 can be detected in patients with advanced

fibrosis or cirrhosis by computed tomography, which is a reliable qualitative

measurement of liver perfusion changes. However, these techniques are not strong

enough to distinguish precisely between different stages of fibrosis.

Liver stiffness measurement

Recently, an important technological advance in fibrosis assessment was made by

the FibroScan (Echosens, Paris, France), a new medical device based on

one-dimensional transient elastography, which assesses fibrosis through liver

stiffness measurement (LSM). A special probe generates an elastic shear wave

propagating through the liver tissue: the harder the tissue, the faster the

shear wave propagates. Transient elastography could accurately predict different

stages of fibrosis or cirrhosis (AUROC: 0.79 for F â‰¥ 2, 0.91 for F â‰¥ 3, and

0.97 for F = 4. by the METAVIR scoring system).41

The major advantage of transient elastography compared with serum markers and

marker panels is that it measures the liver directly and there is no

interference from extrahepatic diseases or conditions. Furthermore, the test is

standardized and completely non-invasive. Though assessing earlier fibrosis is

the common shortcoming of various non-invasive tests, Colletta et al.42 reported

that the agreement between transient elastography and a liver biopsy was much

better than a FibroTest in normal transaminase HCV carriers with early stages of

fibrosis.

Compared to a liver biopsy, transient elastography is painless, and rapid: it

has no risk of complications and is, therefore, very well accepted. Transient

elastography measures a volume of liver stiffness which is 100 times bigger than

the biopsy specimen. The high reproducibility (the intra-observer and

inter-observer agreement intra-class correlation coefficient was 0.9843) and

acceptance of transient elastography makes it an attractive alternative to

biopsy for individual follow-up.

There are also some physical limitations of transient elastography. The signal

penetrates only 25â€“65 mm, making obesity (particularly the fatness of the

chest wall) the most important cause of failure. But new technological

developments may overcome this limitation. Additional limitations include a

narrow intercostal space and ascites. However, the presence of ascites generally

indicates cirrhosis by itself. Steatosis could have been expected to soften the

liver because of the fat. Though current studies did not report that steatosis

and necro-inflammation influence LSM,41 Fraquelli et al. found that transient

elastography reproducibility is significantly reduced in patients with

steatosis, an increased body mass index and lower degrees of hepatic fibrosis.43

So, larger cohorts with more severe grades of steatosis need to be studied

before we make a conclusion.

The main reason that transient elastography cannot totally replace a liver

biopsy is that it is only a means to stage disease. It is unable to diagnose

liver disease by distinguishing between subtle diagnostic differences. Nor can

transient elastography identify cofactors and comorbidities or grade

necro-inflammation and steatosis. But it represents a totally different approach

to assessing fibrosis and therefore could be combined with other non-invasive

modalities to better assess liver fibrosis. The combined use of transient

elastography and the FibroTest to evaluate liver fibrosis could avoid a biopsy

procedure in most patients with chronic hepatitis C.44 Recently, Foucher et al.

suggested that transient elastography might also be useful for predicting

clinical complications of end-stage liver diseases such as esophageal varices

and hepatocellular carcinomas,45 indicating the potential usefulness of

transient elastography in the management of cirrhosis patients.

Real-time elastography

Real-time elastography is another ultrasound technique developed by Hitachi

Medical Systems that can reveal the physical property of tissue using

conventional ultrasound probes during a routine sonography examination. In the

first study assessing real-time elastography for the detection of liver

fibrosis,46 the AUROC was 0.75 for the diagnosis of significant fibrosis. Much

higher diagnostic accuracy (AUROC = 0.93) was obtained by a mathematic

combination of the elasticity score and two routine laboratory values (platelet

count and Î³-glutamyltransferase), which provided a superior way to combine

serological and radiological tests together.

Magnetic resonance elastography

Magnetic resonance elastography (MRE) is a technique using a modified

phase-contrast MRI sequence to image propagating shear waves in tissue. The

technique has been previously applied to quantitatively assess the viscoelastic

properties of the breast, brain and muscle in humans. Several recent studies

showed that MRE is also a feasible method for assessing the stage of liver

fibrosis.47 MRE has several potential advantages compared with ultrasound

transient elastography. It can be performed in obese patients. It can assess

larger volumes and provide full three-dimensional information about the

viscoelastic parameters of tissues. With MR techniques a comprehensive

examination of the liver can be performed, including MRE, contrast-enhanced MRI

to detect hepatocellular carcinomas and perfusion MRI to assess liver function.

However, direct comparison of the two methods is still not available. More

studies are needed to define the sensitivity and specificity of this new

technique.

Double contrast material-enhanced MRI

The conspicuity of gadolinium-enhanced lesions is increased in the setting of a

decreased signal intensity from an uninvolved liver parenchyma following a

superparamagnetic iron oxide injection. This MRI technique has been used to

improve detection of focal hepatic lesions and hepatocellular carcinomas.

Recently, Aguirre et al.48 examined 101 CLD patients who underwent

double-enhanced MR imaging to detect hyperintense reticulations, which are

postulated to represent septal fibrosis. They achieved an accuracy of greater

than 90% for the diagnosis of fibrosis compared with histopathological analysis.

Diffusion weighted magnetic resonance imaging

Diffusion weighted magnetic resonance imaging (DWMRI) has been widely used in

brain imaging for the evaluation of acute ischemic stroke. With the advent of

the echo-planar MRI technique, it became possible to be applied in the abdomen

for the characterization of focal hepatic lesions. Recently, using DWMRI to

measure the apparent diffusion coefficient (ADC) of water, a parameter that is

dependent on the tissue structure, has been introduced in the assessment of

liver fibrosis. The ADC value is lower in livers with heavier fibrosis because

of the restriction of water diffusion in fibrotic tissue. Lewin et al. assessed

the performance of DWMRI in 54 patients with chronic HCV infection with

reference to several other non-invasive methods.49 In discriminating significant

fibrosis, the area under the curve (AUC) values were 0.79 for DWMRI, 0.87 for

transient elastography, 0.68 for the FibroTest, 0.81 for aspartate

aminotransferase to platelet ration index, 0.72 for the Forns index and 0.77 for

hyaluronate. DWMRI performed better in discriminating between patients staged

F3â€“F4, when the AUC value increased to 0.92, the same as in transient

elastography. But besides fibrosis, it seems that ADC values might also reflect

the intensity of inflammation, necrosis and steatosis. However, DWMRI still

benefits from the intrinsic advantages of MRI. Several other MR techniques have

also been introduced in the area of fibrosis assessment, such as ultra-short

echo time MRI50 and magnetic resonance spectroscopy,51 but the research data are

still insufficient.

CONCLUSION

The increase of potentially effective management for CLD such as antiviral and

antifibrotic therapies has led to an urgent need for a rapid, safe and

repeatable tool to assess fibrosis of CLD and to follow up progression or

regression of fibrosis during treatment. A liver biopsy has been the gold

standard for the assessment of hepatic fibrosis, but the invasive procedure has

considerable limitations and fails to satisfy current needs. Many non-invasive

methods have been proposed with the aim of substituting for a liver biopsy. The

numerous advances in serological and radiological techniques and their

combinations have allowed clinicians to satisfactorily identify patients without

resorting to a liver biopsy. But each has some deficiencies and a liver biopsy

will still have an important role to play. Applying new techniques for the

detection of fibrosis may potentially circumvent the pitfalls and deficiencies

of existing surrogates mentioned above. However, further studies are needed to

develop or validate non-invasive tests that can accurately reflect the full

spectrum of hepatic fibrosis in CLD. But an incorrigible defect in our studies

will be the questionable gold standard we have to use. Biopsy failure is more

common than diagnostic failure of markers.52 Mathematical modeling suggests

that, assuming either 80% or 90% diagnostic accuracy of a liver biopsy,

non-invasive tests cannot achieve an AUROC better than 0.9 and are likely to

perform between 0.75 and 0.9, exactly where they are today. We may find a better

surrogate for a liver biopsy, but how we can prove it will be a challenge. A

laparoscopic biopsy can decrease sampling error and increase the reliability of

a histopathological assessment. Using automated image analysis to assess texture

features and the shape representation of the fibrosis structural expansion can

turn the current semiquantitative methods of liver fibrosis assessment into real

quantitative methods with significant reduction in variability and

subjectivity.53 Validating non-invasive tests against not only histological

stage scores but also digital image analysis and clinical outcomes may also be a

better choice.

FINANCIAL SUPPORT

This study was supported by the Prominent National Projects of Science &

Technology (No: 2008ZX10203), the National High Technology Research and

Development Program of China (863 Program, No: 2006AA02A411), Science and

Technology Commission of Shanghai Municipality (No: 064119519), and Shanghai

Leading Academic Discipline Project (No: Y0205).

REFERENCES <CUT>

March 8, 2011