| |
What
does it all mean? (Interpreting Liver Function Tests)
| Special
Considerations in Interpreting Liver Function Tests.
Author/s: David E. Johnston
Issue: April 15, 1999
A number of pitfalls can be encountered in the interpretation of
common blood liver function tests. These tests can be normal in patients
with chronic hepatitis or cirrhosis. The normal range for
aminotransferase levels is slightly higher in males, nonwhites and obese
persons. Severe alcoholic hepatitis is sometimes confused with
cholecystitis or cholangitis. Conversely, patients who present soon
after passing common bile duct stones can be misdiagnosed with acute
hepatitis because aminotransferase levels often rise immediately, but
alkaline phosphatase and g-glutamyltransferase levels do not become
elevated for several days. Asymptomatic patients with isolated, mild
elevation of either the unconjugated bilirubin or the g-glutamyltransferase
value usually do not have liver disease and generally do not require
extensive evaluation. Overall hepatic function can be assessed by
applying the values for albumin, bilirubin and prothrombin time in the
modified Child-Turcotte grading system.
The commonly used liver function tests (LFTs) primarily assess liver
injury rather than hepatic function. Indeed, these blood tests may
reflect problems arising outside the liver, such as hemolysis (elevated
bilirubin level) or bone disease (elevated alkaline phosphatase [AP]
level).
Abnormal LFTs often, but not always, indicate that something is wrong
with the liver, and they can provide clues to the nature of the problem.
However, normal LFTs do not always mean that the liver is normal.
Patients with cirrhosis and bleeding esophageal varices can have normal
LFTs. Of the routine LFTs, only serum albumin, bilirubin and prothrombin
time (PT) provide useful information on how well the liver is
functioning.
The general subject of LFTs1,2 and the differential diagnosis of
abnormal LFTs in asymptomatic patients3-5 have been well reviewed. This
article discusses some common pitfalls in the interpretation of LFTs.
Hints for interpreting these tests are presented in Table 1.
Markers of Hepatocellular Injury
The most commonly used markers of hepatocyte injury are aspartate
aminotransferase (AST, formerly serum glutamic-oxaloacetic transaminase
[SGOT]) and alanine aminotransferase (ALT, formerly serum glutamate-pyruvate
transaminase [SGPT]). While ALT is cytosolic, AST has both cytosolic and
mitochondrial forms.
Hepatocyte necrosis in acute hepatitis, toxic injury or ischemic
injury results in the leakage of enzymes into the circulation. However,
in chronic liver diseases such as hepatitis C and cirrhosis, the serum
ALT level correlates only moderately well with liver inflammation. In
hepatitis C, liver cell death occurs by apoptosis (programmed cell
death) as well as by necrosis. Hepatocytes dying by apoptosis presumably
synthesize less AST and ALT as they wither away. This probably explains
why at least one third of patients infected with hepatitis C virus have
persistently normal serum ALT levels despite the presence of
inflammation on liver biopsy.6,7 Patients with cirrhosis often have
normal or only slightly elevated serum AST and ALT levels. Thus, AST and
ALT lack some sensitivity in detecting chronic liver injury. Of course,
AST and ALT levels tend to be higher in cirrhotic patients with
continuing inflammation or necrosis than in those without continuing
liver injury.
As markers of hepatocellular injury, AST and ALT also lack some
specificity because they are found in skeletal muscle. Levels of these
aminotransferases can rise to several times normal after severe muscular
exertion or other muscle injury, as in polymyositis,8 or in the presence
of hypothyroidism, which can cause mild muscle injury and the release of
aminotransferases. In fact, AST and ALT were once used in the diagnosis
of myocardial infarction.
Slight AST or ALT elevations (within 1.5 times the upper limits of
normal) do not
necessarily indicate liver disease. Part of this ambiguity has to do
with the fact that unlike the values in many other biochemical tests,
serum AST and ALT levels do not follow a normal bell-shaped distribution
in the population.9 Instead, AST and ALT values have a skewed
distribution characterized by a long "tail" at the high end of the scale
(Figure 1).5 For example, the mean values for ALT are very similar from
one population to another, but the degree to which the distribution is
skewed varies by gender and ethnicity. The ALT distributions in males
and nonwhites (i.e., blacks and Hispanics) tend to have a larger tail at
the high end, so that more values fall above the upper limits of normal
set for the average population.10,11
AST and ALT values are higher in obese patients, probably because
these persons commonly have fatty livers.12 ALT levels have been noted
to decline with weight loss.13 Depending on the physician's point of
view, the upper limits of normal for AST and ALT levels could be set
higher for more obese persons.
Rare individuals have chronically elevated AST levels because of a
defect in clearance of the enzyme from the circulation.14 For both AST
and ALT, the average values and upper limits of normal in patients
undergoing renal dialysis are about one half of those found in the
general population.15 Mild elevations of ALT or AST in asymptomatic
patients can be evaluated efficiently by considering alcohol abuse,
hepatitis B, hepatitis C and several other possible diagnoses (Table
2).5
Various liver diseases are associated with typical ranges of AST and
ALT levels (Figure 2). ALT levels often rise to several thousand units
per liter in patients with acute viral hepatitis. The highest ALT
levels-often more than 10,000 U per L-are usually found in patients with
acute toxic injury subsequent to, for example, acetaminophen overdose or
acute ischemic insult to the liver. AST and ALT levels usually fall
rapidly after an acute insult.
Lactate dehydrogenase (LDH) is less specific than AST and ALT as a
marker of hepatocyte injury. However, it is worth noting that LDH is
disproportionately elevated after an ischemic liver injury.16
It is especially important to remember that in patients with acute
alcoholic hepatitis, the serum AST level is almost never greater than
500 U per L and the serum ALT value is almost never greater than 300 U
per L. The reasons for these limits on AST and ALT elevations are not
well understood. In typical viral or toxic liver injury, the serum ALT
level rises more than the AST value, reflecting the relative amounts of
these enzymes in hepatocytes. However, in alcoholic hepatitis, the ratio
of AST to ALT is greater than 1 in 90 percent of patients and is usually
greater than 2.17 The higher the AST-to-ALT ratio, the greater the
likelihood that alcohol is contributing to the abnormal LFTs. In the
absence of alcohol intake, an increased AST-to-ALT ratio is often found
in patients with cirrhosis.
The elevated AST-to-ALT ratio in alcoholic liver disease results in
part from the depletion of vitamin B6 (pyridoxine) in chronic
alcoholics.18 ALT and AST both use pyridoxine as a coenzyme, but the
synthesis of ALT is more strongly inhibited by pyridoxine deficiency
than is the synthesis of AST. Alcohol also causes mitochondrial injury,
which releases the mitochondrial isoenzyme of AST.
Patients with alcoholic hepatitis can present with jaundice,
abdominal pain, fever and a minimally elevated AST value, thereby
leading to a misdiagnosis of cholecystitis. This is a potentially fatal
mistake given the high surgical mortality rate in patients with
alcoholic hepatitis.19
Markers of Cholestasis
Cholestasis (lack of bile flow) results from the blockage of bile
ducts or from a disease that impairs bile formation in the liver itself.
AP and g- glutamyltransferase (GGT) levels typically rise to several
times the normal level after several days of bile duct obstruction or
intrahepatic cholestasis. The highest liver AP elevations-often greater
than 1,000 U per L, or more than six times the normal value-are found in
diffuse infiltrative diseases of the liver such as infiltrating tumors
and fungal infections.
Diagnostic confusion can occur when a patient presents within a few
hours after acute bile duct obstruction from a gallstone. In this
situation, AST and ALT levels often reach 500 U per L or more in the
first hours and then decline, whereas AP and GGT levels can take several
days to rise.
Both AP and GGT levels are elevated in about 90 percent of patients
with cholestasis.20 The elevation of GGT alone, with no other LFT
abnormalities, often results from enzyme induction by alcohol or
aromatic medications in the absence of liver disease. The GGT level is
often elevated in persons who take three or more alcoholic drinks (45 g
of ethanol or more) per day.21 Thus, GGT is a useful marker for
immoderate alcohol intake. Phenobarbital, phenytoin (Dilantin) and other
aromatic drugs typically cause GGT elevations of about twice normal. A
mildly elevated GGT level is a typical finding in patients taking
anticonvulsants and by itself does not necessarily indicate liver
disease.22,23
Serum AP originates mostly from liver and bone, which produce
slightly different forms of the enzyme. The serum AP level rises during
the third trimester of pregnancy because of a form of the enzyme
produced in the placenta. When serum AP originates from bone, clues to
bone disease are often present, such as recent fracture, bone pain or
Paget's disease of the bone (often found in the elderly). Like the GGT
value, the AP level can become mildly elevated in patients who are
taking phenytoin.22,23
If the origin of an elevated serum AP level is in doubt, the
isoenzymes of AP can be separated by electrophoresis. However, this
process is expensive and usually unnecessary because an elevated liver
AP value is usually accompanied by an elevated GGT level, an elevated
5[acute accent]-nucleotidase level and other LFT abnormalities.
In one study,24 isolated AP elevations were evaluated in an
unselected group of patients at a Veterans Affairs hospital. Most mild
AP elevations (less than 1.5 times normal) resolved within six months,
and almost all greater elevations had an evident cause that was found on
routine clinical evaluation.
Persistently elevated liver AP values in asymptomatic patients,
especially women, can be caused by primary biliary cirrhosis, which is a
chronic inflammatory disorder of the small bile ducts. Serum
antimitochondrial antibody is positive in almost all of these patients.
Indicators of How Well the Liver Functions
Bilirubin
Bilirubin results from the enzymatic breakdown of heme. Unconjugated
bilirubin is conjugated with glucuronic acid in hepatocytes to increase
its water solubility and is then rapidly transported into bile. The
serum conjugated bilirubin level does not become elevated until the
liver has lost at least one half of its excretory capacity. Thus, a
patient could have obstruction of either the left or right hepatic duct
without a rise in the bilirubin level.
Because the secretion of conjugated bilirubin into bile is very rapid
in comparison with the conjugation step, healthy persons have almost no
detectable conjugated bilirubin in their blood. Liver disease mainly
impairs the secretion of conjugated bilirubin into bile. As a result,
conjugated bilirubin is rapidly filtered into the urine, where it can be
detected by a dipstick test. The finding of bilirubin in urine is a
particularly sensitive indicator of the presence of an increased serum
conjugated bilirubin level.
In many healthy persons, the serum unconjugated bilirubin is mildly
elevated to a concentration of 2 to 3 mg per dL (34 to 51 [micro
sign]mol per L) or slightly higher, especially after a 24-hour fast. If
this is the only LFT abnormality and the conjugated bilirubin level and
complete blood count are normal, the diagnosis is usually assumed to be
Gilbert syndrome, and no further evaluation is required. Gilbert
syndrome was recently shown to be related to a variety of partial
defects in uridine diphosphate-glucuronosyl transferase, the enzyme that
conjugates bilirubin.25
Mild hemolysis, such as that caused by hereditary spherocytosis and
other disorders, can also result in elevated unconjugated bilirubin
values, but hemolysis is not usually present if the hematocrit and blood
smear are normal. The presence of hemolysis can be confirmed by testing
other markers, such as haptoglobin, or by measuring the reticulocyte
count.
Severe defects in bilirubin transport and conjugation can lead to
markedly elevated unconjugated bilirubin levels, which can cause serious
neurologic damage (kernicterus) in infants. However, no serious form of
liver disease in adults causes elevation of unconjugated bilirubin
levels in the blood without also causing elevation of conjugated
bilirubin values.
When a patient has prolonged, severe biliary obstruction followed by
the restoration of bile flow, the serum bilirubin level often declines
rapidly for several days and then slowly returns to normal over a period
of weeks. The slow phase of bilirubin clearance results from the
presence of delta-bilirubin, a form of bilirubin chemically attached to
serum albumin.26 Because albumin has a half-life of three weeks, delta-bilirubin
clears much more slowly than bilirubin-glucuronide. Clinical
laboratories can measure delta-bilirubin concentrations, but such
measurements are usually unnecessary if the physician is aware of the
delta-bilirubin phenomenon.
Albumin
Although the serum albumin level can serve as an index of liver
synthetic capacity, several factors make albumin concentrations
difficult to interpret.27 The liver can synthesize albumin at twice the
healthy basal rate and thus partially compensate for decreased synthetic
capacity or increased albumin losses. Albumin has a plasma half-life of
three weeks; therefore, serum albumin concentrations change slowly in
response to alterations in synthesis. Furthermore, because two thirds of
the amount of body albumin is located in the extravascular,
extracellular space, changes in distribution can alter the serum
concentration.
In practice, patients with low serum albumin concentrations and no
other LFT abnormalities are likely to have a nonhepatic cause for low
albumin, such as proteinuria or an acute or chronic inflammatory state.
Albumin synthesis is immediately and severely depressed in inflammatory
states such as burns, trauma and sepsis, and it is commonly depressed in
patients with active rheumatic disorders or severe end-stage
malnutrition. In addition, normal albumin values are lower in pregnancy.
Prothrombin time
The liver synthesizes blood clotting factors II, V, VII, IX and X.
The prothrombin time (PT) does not become abnormal until more than 80
percent of liver synthetic capacity is lost. This makes PT a relatively
insensitive marker of liver dysfunction. However, abnormal PT
prolongation may be a sign of serious liver dysfunction. Because factor
VII has a short half-life of only about six hours, it is sensitive to
rapid changes in liver synthetic function. Thus, PT is very useful for
following liver function in patients with acute liver failure.
An elevated PT can result from a vitamin K deficiency. This
deficiency usually occurs in patients with chronic cholestasis or fat
malabsorption from disease of the pancreas or small bowel. A trial of
vitamin K injections (e.g., 5 mg per day administered subcutaneously for
three days) is the most practical way to exclude vitamin K deficiency in
such patients. The PT should improve within a few days.
Blood ammonia
Measurement of the blood ammonia concentration is not always useful
in patients with known or suspected hepatic encephalopathy. Ammonia
contributes to hepatic encephalopathy; however, ammonia concentrations
are much higher in the brain than in the blood and therefore do not
correlate well.28 Furthermore, ammonia is not the only waste product
responsible for encephalopathy. Thus, blood ammonia concentrations show
only a mediocre correlation with the level of mental status in patients
with liver disease. It is not unusual for the blood ammonia
concentration to be normal in a patient who is in a coma from hepatic
encephalopathy.
Blood ammonia levels are best measured in arterial blood because
venous concentrations can be elevated as a result of muscle metabolism
of amino acids. Blood ammonia concentrations are most useful in
evaluating patients with stupor or coma of unknown origin. It is not
necessary to evaluate blood ammonia levels routinely in patients with
known chronic liver disease who are responding to therapy as expected.
Grading Liver Function by Child-Turcotte Class
In communicating among themselves, many physicians use the Child-Turcotte
class as modified by Pugh, often termed the "Child class," to convey
information about overall liver function and prognosis (Table 3).29 This
grading system can be used to predict overall life expectancy and
surgical mortality in patients with cirrhosis and other liver
diseases.30
For elective general abdominal surgery, perioperative mortality is in
the neighborhood of several percent for patients who fall into the Child
class A, 10 to 20 percent for those in class B and approximately 50
percent for those in class C.31 These percentages must be balanced by
prognostic considerations when transplantation becomes an option. The
presence of cirrhosis by itself is not an indication for liver
transplantation, and transplantation is rarely performed in patients who
fall into Child class A. For example, the 10-year survival rate is as
high as 80 percent in patients with hepatitis C and cirrhosis who have
Child class A liver function and no variceal bleeding.32 However, once
patients with any type of liver disease fall into the Child-Turcotte
class B or class C category, survival is significantly reduced and
transplantation should be considered.
REFERENCES
1.Kaplan MM. Laboratory tests. In: Schiff L, Schiff ER, eds. Diseases
of the liver. 7th ed. Philadelphia: Lippincott, 1993:108-44.
2.Kamath PS. Clinical approach to the patient with abnormal liver
function test results. Mayo Clin Proc 1996;71:1089-94.
|
Noninvasive Markers of Fibrosis for Longitudinal Assessment of Fibrosis in
Chronic Liver Disease: Are They Ready for Prime Time? EDITORIAL
The American
Journal of Gastroenterology
September 2005
Paul J. Thuluvath, M.D., F.R.C.P.1, and Karen L. Krok, M.D.1
1Department of Medicine, The Johns Hopkins University School of Medicine,
Baltimore, Maryland
….. the current published evidence suggests that FibroTest is not
yet ready for prime time either to diagnose the severity of fibrosis or
for longitudinal assessment of fibrosis since 15-20% are likely to be
misdiagnosed by these markers….
The study published that this Editorial refers to follows the
Editorial below.
SUMMARY
Over the past decade, there has been a renewed enthusiasm to develop
noninvasive serum markers or tests to assess the presence and severity of
fibrosis in chronic liver disease. Although a single marker or test has
lacked the necessary accuracy to predict fibrosis, different combinations
of these markers or tests have shown encouraging results. However,
interlaboratory variability and inconsistent results with liver diseases
of varying etiologies have made it difficult to assess the reliability of
these markers in clinical practice. In this issue of the Journal, Poynard
and colleagues describe the "histological" response to lamivudine in
patients with chronic HBV over a 24-month period using surrogate serum
biomarkers (FibroTest-ActiTest) without corroborating histological data.
Investigators found improvement in fibrosis and inflammation in 85% and
91%, respectively, despite the emergence of YMDD mutation in 41.5% of
patients. The higher improvement rates reported in this study should be
interpreted with caution for a number of reasons including the absence of
data on virological response rates, corroboratory histological data, and
data on the validity of FibroTest to evaluate fibrosis in a longitudinal
manner. Although FibroTest has been studied extensively by the authors of
the current study, to date there are only few independent studies. In
addition to significant interlaboratory variations, these studies have
shown that significant fibrosis could be missed, or conversely significant
fibrosis diagnosed in the absence of minimal or no fibrosis in about
15-20% of patients. We may be approaching a time when serum biomarkers may
become an integral part of the assessment of patients with chronic liver
disease, but published evidence suggests that these markers are not yet
ready for prime time.
ARTICLE TEXT
The assessment of the presence and severity of liver fibrosis is of
paramount importance in determining treatment strategies, response to
treatment, prognosis, and the potential risk for complications in patients
with chronic liver disease. Liver biopsy, the gold standard for assessing
the severity of necroinflammatory activity and fibrosis, is invasive, and
even in expert hands, it is associated with rare but serious complications
including bleeding, pneumothorax, and perforation of colon or gallbladder
(1, 2). It is also not practical or cost-effective to perform serial
biopsies within a short time interval to assess treatment-related
response. In addition to the sampling error that is inherently associated
with percutaneous liver biopsies, there is an intra- and interobserver
variability that may range from 15% to 33% in determining the fibrosis
staging (3-5). Nevertheless, liver biopsy has remained the "gold standard"
to assess the severity of inflammation and fibrosis in patients with
chronic liver disease.
In the past decade, many investigators have proposed noninvasive tests to
replace liver biopsy using either a single biochemical marker or a
combination of tests. An ideal noninvasive marker for the evaluation of
liver fibrosis should accurately predict the presence or absence of
significant fibrosis (high sensitivity, specificity, positive, and
negative predictive value [NPV]). In addition, it should be readily
available and reproducible with low interlaboratory variability and with
applicability in liver disease of various etiologies. Although liver
biopsy does not fulfill many of these criteria, it has remained the "gold
standard" mainly because of the absence of better alternatives. Recently,
there has been a renewed interest in noninvasive markers of fibrosis
because of evolving novel therapies for hepatitis C and B (6-9). While
some investigators have focused on a combination of laboratory tests such
as reversal of aspartate aminotransferase (AST)/alanine aminotransferase
(ALT) ratio, or AST/platelet ratio index (APRI), others have been
searching for more novel markers of fibrosis and inflammation (8-12).
Despite the common pathway for fibrosis, an imbalance between synthesis,
and degradation of extra cellular matrix by activated stellate cells,
there are currently no liver-specific markers that could accurately
reflect liver fibrosis in liver disease of different etiologies. Many
serum markers of matrix degradation products have been studied previously,
either as a single marker or in combination, including N-terminal
propeptide of type III collagen (PIIINP), collagen type-IV, hyaluronic
acid, prolyl hydroxylase, laminin, matrix metalloproteinase-1 (MMP-1), and
tissue inhibitors of metalloproteinase-1 (TIMP-1) and metalloproteinase-2
(TIMP-2). Many of these tests are not widely available, and when
critically evaluated, they have shown disease-specific variations and
inconsistencies suggesting that they are nonspecific. Many of these
markers are nonspecifically elevated in the presence of inflammation or
expressed in others tissues including skin, blood vessels, bone, and
kidney. Other markers that have been studied include alpha2-macroglobulin,
haptoglobins, and apolipoprotein A1. Increased serum levels of
alpha2-macroglobulin, a protease inhibitor expressed by activated stellate
cells, may relate to increased fibrosis. Haptoglobin levels may be
decreased in liver fibrosis because of its association with profibrotic
cytokine TGF-beta1, and apolipoprotein A1 levels may be decreased because
of its binding affinity with extracellular matrix. Other tests that merits
further independent corroboration include YKL-40 (a growth factor for
fibroblasts that is expressed in the human liver), transient elastography,
13C-caffeine breath test, and DNA sequenced-based serum protein glycomics
(10-13). The recent trend has been to use a combination of these tests to
improve accuracy of predicting fibrosis in a consistent and reproducible
manner (6-9).
The prospective multicenter study reported in this issue of American
Journal of Gastroenterology is a longitudinal assessment of FibroTest and
ActiTest (FT-AT) in 283 patients with chronic hepatitis B being treated
with lamivudine (14). The biochemical tests were done in a central
laboratory, but liver biopsy was done only at baseline. They found a
statistically significant decrease in both fibrosis and inflammation as
measured by FT-AT at 24 months irrespective of the presence of YMDD
mutation. Interestingly, there was an immediate improvement at 6 month
with a plateau between 6-12 months and then further improvement at 24
months. Lamivudine had previously been shown to significantly improve the
histological features both for necroinflammatory activity and fibrosis. In
the current study, 85% showed improvement in fibrosis and 91% showed
improvement in inflammation at 24 months. It is reassuring to note the
continued improvement in both fibrosis and inflammatory scores despite the
emergence of YMDD mutation in 41.5% of patients. The higher improvement
rates reported in this study should be interpreted with caution for a
number of reasons including the absence of data on virological response
rates, no corroboratory histological data, and a paucity of previously
published data on the validity of FibroTest to evaluate fibrosis in a
longitudinal manner.
The same investigators have published many previous articles on the
validity of this proprietary test score (FibroTest) that uses five
biochemical components including alpha2-macroglobulin, haptoglobin,
apolipoprotein A1, gamma-glutamyl transpeptidase (GGT), and total
bilirubin. ActiTest includes the same components plus serum ALT levels.
The score is computed (using a complex formula) by entering patient's age
and sex (older age and male sex were shown to be associated with more
fibrosis in previous studies) along with the five components into a
proprietary program (available for a fee) available at a website (http://www.biopredictive.com)
and this composite value is used to determine the presence or absence of
significant fibrosis. Poynard and colleagues have previously reported that
the FibroTest score has a NPV (absence of fibrosis) of 100% and positive
predictive value (presence of significant fibrosis) of 91% in patients
with appropriate cut-off scores (6). This would be a remarkable
development if it could be verified independently in a consistent fashion
in liver disease of varying etiology. However, when the same investigators
compared the test scores using laboratory results from eight centers with
the reference laboratory, the interlaboratory agreement for fibrosis stage
using this test score was very poor with kappa (kappa statistics) ranging
from 0.32 to 0.94 with a mean 0.6 (15). In the current study, authors do
not comment on the reproducibility of the tests in their reference
laboratory over an unspecified period of time. Only a few studies have
independently assessed FibroTest outside the current study group (12, 16).
In an independent study, Rossi et al. found that 21% of patients who were
predicted to have significant fibrosis by the FibroTest scores had only
minimal fibrosis by histology and conversely, 18% of patients who were not
supposed to have fibrosis by the test score had significant fibrosis (16).
When FibroTest was studied in patients with chronic HBV in a previous
study by Poynard's group, a FibroTest scores <= 0.20 had a NPV of 92% to
exclude F2-F4 fibrosis, but the specificity was only 52% (17). The
discrepancy in the published results could be explained by the inadequate
length or sampling error of liver biopsy specimens, and inconsistencies in
the measurement of biochemical markers (5, 15, 16, 18). Nevertheless, the
current published evidence suggests that FibroTest is not yet ready for
prime time either to diagnose the severity of fibrosis or for longitudinal
assessment of fibrosis since 15-20% are likely to be misdiagnosed by these
markers (15, 16, 18, 19).
Readers may find it disturbing to note that the first author of the study
has a capital interest in Biopredictive, the company that markets FT-AT at
their website for a fee, and another author is an employee of
Biopredictive. Appropriately, most publications on the validity of the
FT-AT have come from the same authors who have a research interest to
develop noninvasive markers of fibrosis. The authors, rightfully so, have
declared their conflict of interest. Another disturbing aspect of this
article is that the authors are withholding important information on
virological response rates perhaps for a later publication. This important
piece of information is critical to interpret the results of the study.
REFERENCES
1. James CH, Lindor KD. Outcome of patients admitted with
complications after outpatient liver biopsies. Ann Intern Med 1993;118:
96-8.
2. McGill DB, Rakela J, Zinsmeister AR, et al.. A 21-year
experience with major hemorrhage after percutaneous liver biopsy.
Gastroenterology 1990;99: 1396-400.
3. Westin J, Lagging LM, Wejstal R, et al.. Interobserver study of
liver histopathology using the Ishak score in patient with chronic
hepatitis C virus infection. Liver 1999;19: 183-7.
4. Bedossa P, Dargere D, Paradis V. Sampling variability of liver
fibrosis in chronic hepatitis C. Hepatology 2003;38: 1449-57.
5. Regev A, Berhho M, Jeffers LJ, et al.. Sampling error and
intraobserver variation in liver biopsy in patients with chronic HCV
infection. Am J Gastroenterol 2002;97: 2614-8.
6. Imbert-Bismut F, Ratziu V, Pieroni L, et al.. Biochemical
markers of liver fibrosis in patients with hepatitis C virus infection.
Lancet 2001;357: 1069-75.
7. Forns X, Ampurdanes S, Llovet JM, et al.. Identification of
chronic hepatitis C patients without hepatic fibrosis by simple predictive
model. Hepatology 2002;36: 986-92.
8. Wai CT, Greenson JK, Fontana RJ, et al.. A simple noninvasive
index can predict both significant fibrosis and cirrhosis in patients with
chronic hepatitis C. Hepatology 2003;38: 518-26.
9. Rosenberg WM, Voelker M, Thiel R, et al.. Serum markers detect
the presence of liver fibrosis: A cohort study. Gastroenterology 2004;127:
1704-13.
10. Saitou Y, Shiraki K, Yamanaka Y, et al.. Noninvasive
estimation of liver fibrosis and response to interferon therapy by a serum
fibrinogenesis marker, YKL-40, in patients with HCV-associated liver
disease. World J Gastroenterol 2005;11: 476-81.
11. Callewaert N, Van Vlierberghe H, Van Hecke A, et al.
Noninvasive diagnosis of liver cirrhosis using DNA sequencer-based total
serum protein glycomics. Nat Med 2004;10: 429-34.
12. Caster L, Vergniol J, Foucher J, et al.. Prospective
comparison of transient elastography, Fibrotest, APRI, and liver biopsy
for the assessment of fibrosis in chronic hepatitis C. Gastroenterology
2005;l128: 343-50.
13. Park GJ-H, Katelaris PH, Jones DB, et al.. Validity of
C-caffeine breath test as a non-invasive, quantitative test of liver
function. Hepatology 2003;38: 1227-36.
14. Poynard T, Zoulim F, Ratziu V, et al.. Longitudinal assessment
of histology surrogate markers (Fibrotest-Actitest) during lamivudine
therapy in patients with chronic hepatitis B infection. Am J Gastroenterol
2005;100: 1970-80.
15. Halfon P, Imbert-Bismut F, Messous D, et al.. A prospective
assessment of inter-laboratory variability markers of fibrosis (FibroTest)
and activity (ActiTest) in patients with chronic liver disease. Comp
Histol 2002;1: 3.
16. Rossi E, Adams L, Prins A, et al.. Validation of Fibro Test
biochemical markers score in assessing liver fibrosis in hepatitis C
patients. Clin Chem 2003;49: 450-4.
17. Myers RP, Tainturier MH, Ratziu V, et al.. Prediction of liver
histological lesions with biochemical markers in patients with chronic
hepatitis B. J Hepatol 2003;39: 222-30.
18. Rosenthall-Allieri MA, Peritore ML, Tran A, et al.. Analytical
variability of the Fibrotest proteins. Clin Biochem 2005;38: 473-8.
19. Bissell DM. Assessing fibrosis without a liver biopsy: Are we
there yet? Gastroenterology 2004;127: 1847-9.
Longitudinal
Assessment of Histology Surrogate Markers (FibroTest-ActiTest) During
Lamivudine Therapy in Patients with Chronic Hepatitis B Infection
The American
Journal of Gastroenterology
Volume 100 Issue 9 Page 1970 - September 2005
Thierry Poynard, M.D., Ph.D.1, Fabien Zoulim, M.D., Ph.D.2, Vlad Ratziu,
M.D., Ph.D.1, Françoise Degos, M.D., Ph.D.3, Francoise Imbert-Bismut,
Ph.D.4, Paul Deny, M.D.5, Paul Landais, M.D.6, Abdelkader El Hasnaoui,
M.D.7, Alain Slama, M.D.7, Patrick Blin, Ph.D.8, Vincent Thibault, M.D.9,
Parviz Parvaz, M.D.10, Mona Munteanu, M.D.11, and Christian Trepo, M.D.2
ABSTRACT
OBJECTIVES: The noninvasive serum markers, FibroTest-ActiTest
(FT-AT), are an alternative to liver biopsy in patients with chronic
hepatitis C and B. The aim was to use these markers in a prospective study
of patients treated with lamivudine in order to assess the impact of
treatment, as well as the factors associated with fibrosis progression.
METHODS: Two hundred and ninety-eight patients were included in a
prospective longitudinal study in 50 hospitals across France. FT-AT were
measured at baseline, and then after 6, 12, and 24 months of lamivudine
100-mg treatment. Epidemiological, clinical, and virologic characteristics
were analyzed by univariate and multivariate analysis.
RESULTS: Two hundred and eighty-three patients were included for
analysis. The accuracy of FT-AT versus biopsy was validated with the area
under the ROC curve, 0.77 (SE = 0.03) for bridging fibrosis and 0.75 (SE =
0.06) for severe activity (A3). At baseline, bridging fibrosis (METAVIR
stages F2-F3-F4) was highly associated (p< 0.001) in multivariate analysis
with male gender and age and marginally associated with anti-HBe presence
(p= 0.05) and non-Asian ethnic origin (p= 0.046). Lamivudine treatment had
a very significant impact overall. FT decreased significantly from 0.51 at
baseline to 0.37 at 24 months (p< 0.001), and 85% of patients had
improvement at 24 months. AT also decreased significantly from 0.56 to
0.13 (p< 0.0001), and 91% of patients had improvement at 24 months. A
three-phase kinetics was observed for both fibrosis and activity; there
was a marked improvement during the first 6 months, followed by a plateau
between 6 and 12 months, and another improvement between 12 and 24 months.
The occurrence of a YMDD variant does not entirely explain these
three-phase variations. The first phase impact on fibrosis rates was
higher in Asian patients (p= 0.01) and in patients younger than 40 yr (p<
0.001).
CONCLUSIONS: In patients with chronic hepatitis B, a 24-month course of
lamivudine treatment leads to a significant decrease in necroinflammatory
grades and fibrosis stages as assessed by noninvasive markers, with the
occurrence of a three-phase kinetics. FT-AT should be useful in the
noninvasive follow-up of lamivudine treatment.
AUTHOR DISCUSSION
In this large multicenter cohort study of patients with chronic
hepatitis B starting lamivudine therapy, we used noninvasive markers to
estimate the histological impact of the first 24 months of treatment.
The use of biomarkers has many advantages and few disadvantages. The main
advantage is the simplicity of the estimation procedure. Such simplicity
would be unachievable using the classical estimate of four liver biopsies
in 24 months. The detailed study by Dienstag et al. was possible only on a
small sample of 63 patients who had already been included in a randomized
trial, and used only three estimates (35). Another advantage of biomarkers
is the reduced variability related to time differences. There is much more
time variability for biopsy estimates than for biomarker estimates. In
previous studies, which described 3 yr of treatment between the baseline
biopsy and the end of follow-up biopsy, the exact interval was often
actually 24 wk longer, which, therefore, included time without treatment
(20, 35). This time variability is probably more significant for
necroinflammatory activity than for fibrosis, as the variation in grades
is generally more rapid than the variation in stages.
One disadvantage of biomarkers is that they are indirect markers of
histological features and have 20-30% of discordance with biopsy. However,
prospective studies in chronic hepatitis C have demonstrated that many of
these discordances were in fact due to sampling error from biopsies that
were too small (17, 20, 25). The diagnostic values of FT-AT observed in
the present multicenter study were similar (AUROC = 0.74-0.77) to those
observed in patients contaminated with HCV (10-26) and in the first
validation study in patients contaminated with HBV (27).
The present study with biomarkers confirms previous findings made with
biopsy on a greater number of patients but also presents new findings. In
patients treated with lamivudine for 2 yr, there was an improvement of
histological features both for necroinflammatory activity and fibrosis.
Compared with Dienstag et al.'s study (35) and using a definition of
improvement as a decrease in 0.01 units for FT or AT, we observed a higher
improvement rate, probably because biomarkers are more sensitive than
biopsy. If results were expressed in worsening percentages, the overall
results for activity were similar: 11% for Dienstag at 3 yr versus 9% at 2
yr in the present study (35). The results were also similar for fibrosis
impact. In patients with baseline bridging fibrosis, Dienstag et al.
observed a 30% improvement rate (12 out of 19 patients with a drop of HAI
score from 3 to 0 or 1) (35). Here, we observed a 94% FT improvement rate,
including 31 patients (32%) with a drop of FT values of 0.30, which is
equivalent to a 2 histological stage improvement. In cirrhotic patients,
significant fibrosis regression was observed in 32%, confirming the
reversibility of cirrhosis. This confirms previous studies using liver
biopsy in a smaller group of HBV patients (35), and FT in HCV treated
patients, without the risk of biopsy sampling error (20).
It was also confirmed that there had been an overall clear and significant
histological benefit in patients with YMDD-variant HBV at 2 yr, without
any case of dramatic worsening. However, the histological benefit was
reduced after prolonged YMDD-variant HBV. We observed that the occurrence
of a YMDD-variant significantly increased the mean AT compared to patients
without a YMDD-variant; this occurred as early as the 6 month follow-up
and during 2 yr of treatment. This early difference in AT at 6 months was
not expected, as only 8% of patients already had a detectable YMDD-variant
at this date. AT is, therefore, a sensitive marker which could be useful
for the management of patients treated by lamivudine. FT values were not
significantly higher in patients with a YMDD-variant, but results were
close to those observed in Dienstag et al.'s study (35).
The new findings from this study were first, the kinetics of the
histological impact of lamivudine. There was, both for fibrosis and
activity estimates, a first phase of rapid improvement in the first 6
months of treatment, followed by a second phase with a plateau between 6
and 12 months and a third phase with another improvement between 12 and 24
months.
The first phase was particularly marked in patients with baseline bridging
fibrosis who had two significant independent favorable prognostic factors:
younger age and Asian ethnicity. This observation suggests that long-term
management (duration of treatment and follow-up) of patients with HBV
could be different in patients according to age and ethnic origin.
Contrary to expectations, the plateau observed in the second phase could
not be entirely explained by the occurrence of a YMDD-variant, as the
plateau was also observed in patients without a YMDD-variant. The only
differences were a small increase of FT-AT between 6 and 12 months in
patients with a YMDD-variant, which was not observed in patients without a
YMDD-variant. We have no clear explanation for this plateau in patients
without a YMDD-variant. The presence of an occult variant could be one
explanation, as could be an immune response.
Weaknesses of the present study were the lack of intermediate estimates
and a longer follow-up. We have assessed biomarkers at four different
times only, baseline, 6, 12 and 24 months. We speculate that there is
perhaps a plateau between 6 and 12 months in the antifibrotic efficacy of
lamivudine. Other scenario are, however, possible such as rapid early
improvement, followed by more steady improvement in inflammation and,
particularly, fibrosis scores. The three phase pattern should be validated
in another study to eliminate an artefact. However, these findings suggest
that lamivudine treatment should not be stopped too early in patients with
high risk factors such as non-Asian patients or those older than 40 yr.
Another original finding is the difference in the lamivudine impact
according to baseline liver injury. In patients with baseline bridging
fibrosis, the impact of treatment was already significant at 6 months, a
point that encourages starting treatment very early in these patients. In
patients without bridging fibrosis at baseline, the effect on fibrosis was
much slower, with a significant decrease observed only at 24 months. For
necroinflammatory activity, the impact of treatment was also very rapid
for patients with moderate or severe activity at baseline. In patients
without activity or with minimal activity, the impact was much slower,
with a significant decrease only observed at 24 months. This observation
is interesting, as it does not support the classical view of the
nonefficacy of treatment in patients with low ALT, which are considered to
lack a strong spontaneous immune response. Although delayed, an
improvement of necroinflammatory features was observed. However, the
risk-benefit of treatment in patients without significant activity at
baseline should be discussed, as the benefit in terms of necroinflammatory
features was only observed in patients without a YMDD-variant.
Factors Associated with Baseline Fibrosis
This study has demonstrated for the first time in a large number of
subjects that sophisticated viral characteristics, such as HBV genotype or
precore mutations, were marginally associated with bridging fibrosis
compared with gender and age. Most of the significance observed in
univariate analysis was no longer seen when adjusted for age and sex. It
is, therefore, mandatory that studies assessing the prognostic value of
molecular characteristics take into account age and gender. The present
study was not perfect, as it is very difficult to assess the duration of
contamination in our population with mixed ethnic origin. One advantage
was that in contrast with the Phase III trials, which imposed rigorous
inclusion and exclusion criteria, our study recruited a consecutive sample
of patients with hepatitis B infections fulfilling broad inclusion
criteria, thus representative of patients presenting at specialist
hepatology departments in France.
Symptoms Observed During Treatment
In terms of clinical outcome, all patients responded to lamivudine
treatment with a progressive decrease in symptoms of liver disease and ALT
levels during the study period, HBe-seroconversion, and reduction of HBV
DNA viral load detected by PCR. This desirable therapeutic response was
observed in both patients with and without HBV polymerase mutations. There
were no differences in the rate of disappearance of symptoms or in the
incidence of new symptoms according to the presence or absence of severe
activity or bridging necrosis estimated by biopsy or FT-AT (data not
shown).
ALT flares accompanied by hepatic decompensation (i.e., jaundice, liver
failure) have been observed at the time of lamivudine resistance and viral
breakthrough (43). These cases were mainly described in patients with
severe liver disease at baseline and/or with a precore mutant infection.
In our cohort study, no cases of acute exacerbation and liver disease
decompensation were observed, at least during the study period.
In conclusion, our cohort study provides new information on the
histological impact of lamivudine in patients with chronic hepatitis B.
This began with a rapid improvement in patients with baseline bridging
fibrosis during the first 6 months, particularly in Asian and young
patients, followed by a plateau between the 6th and 12th month, and
another phase of improvement during the second year of treatment. The
emergence of a YMDD-variant was associated with higher necroinflammatory
activity at 2 yr. The use of FT-AT every 6 months could be a considerable
help for making treatment decisions in patients treated with lamivudine.
Future protocols assessing the histological impact of other drugs, alone
or in combination, should make use of these noninvasive markers.
INTRODUCTION
Chronic hepatitis B virus (HBV) infection affects 350 million
individuals globally. Approximately 15-40% may develop serious
complications, including end-stage liver disease and hepatocellular
carcinoma (1). Patients with significant hepatic inflammation and fibrosis
are at the highest risk of these complications (2). Prior to considering
antiviral treatment, current guidelines recommend liver biopsy (3). This
procedure provides important information regarding the severity of
necroinflammatory activity and fibrosis, features potentially useful for
predicting treatment response and prognosis. Unfortunately, liver biopsy
is invasive (4), costly, and limited by sampling error and poor intra- and
interobserver concordance (5-7).
Considering these limitations and patient reluctance to undergo liver
biopsy, noninvasive predictors of histology have been studied in the last
4 yr, particularly in patients with chronic hepatitis C (8). Aspartate
(AST) and alanine amino transferase (ALT) are widely used for assessing
hepatitis activity, but the ideal cut-offs are unclear (9). We developed a
panel of biochemical markers, FibroTest-ActiTest (FT-AT), which have
repeatedly demonstrated high predictive values for fibrosis and
necroinflammatory histological activity in patients with chronic hepatitis
C (10-26). FT-AT was also validated in patients with chronic hepatitis B
in a single study (27).
The introduction of lamivudine in 1998 represented a significant advance
in the treatment of chronic hepatitis B infections (28). This drug is a
nucleoside analog that prevents replication of the HBV by inhibiting the
viral RNA-dependent DNA polymerase enzyme. Lamivudine has been shown to
stimulate viral clearance and improve clinical status in several
randomized clinical trials and to provide sustained benefit over periods
up to 4 yr (1, 28, 29). The efficacy of lamivudine in the treatment of
hepatitis B is, however, compromised by the development of viral
resistance (30). This is due to the selection of HBV mutants containing
mutations in the YMDD motif of the hepatitis B polymerase. These viral
strains are present as minor species in the pretreatment viral
quasi-species and thus become the dominant species due to selection
pressure during drug treatment. However, these polymerase mutants have
reduced viability due to impaired catalytic activity and are replaced by
the wild-type virus if lamivudine treatment is stopped (31).
The histological consequences of YMDD mutations are not well established.
Only a few prospective studies have been undertaken using liver biopsy to
determine the progression of fibrosis and activity in patients treated
with lamivudine mostly at 1 year (28, 32-34). Two studies, including one
study on 63 patients (35), have investigated treatment longer than 1 year,
being the histological consequences of HBV mutants resistant to lamivudine
by repeated estimates of histological features during 3 yr of treatment
(35, 36).
The aim of this study was to use noninvasive markers, instead of biopsy,
in a prospective study of patients treated with lamivudine in order to
assess the dynamic impact of treatment on fibrosis and necroinflammatory
histological activity while taking risk factors, particularly the
occurrence of YMDD mutations, into account.
METHODS
Subject Selection
This prospective longitudinal study was carried out in 50 hospital
hepato-gastroenterology or internal medicine departments across France.
Patients were to be included in the study if they were over the age of 18
yr and presented with active chronic hepatitis B infection that the
investigator decided to treat with lamivudine. Exclusion criteria included
previous exposure to lamivudine, dialysis, HIV infection,
immunosuppressant chemotherapy, or organ graft recipients. In addition,
lamivudine was only provided if the conditions set out in the official
prescribing information for the drug were fulfilled, notably with respect
to pregnancy.
Study Design
Following initiation of treatment with lamivudine, patients were
followed up for a total of 24 months. A blood sample was taken for
virologic characterization and for biochemical markers at baseline, 6, 12,
and 24 months.
At the baseline visit, data were recorded on the sociodemographic
characteristics of the patient, alcohol consumption, risk factors for
hepatitis B infections (intravenous drug use, at-risk sexual practices,
travel to countries where hepatitis B is endemic, familial or perinatal
contamination), other viral infections including hepatitis C and D,
duration of hepatitis B infection, symptom presentation, and results of
previous serological tests of liver biopsies and previous treatments for
hepatitis B.
Estimates of Liver Injury
We used the previously validated FT-AT (Biopredictive, Paris, France;
FibroSURE LabCorp, Burlington, NC) (10-26). FT-AT is a noninvasive blood
test that combines the quantitative results of six serum biochemical
markers (alpha2-macroglobulin, haptoglobin, gamma glutamyl transpeptidase
(GGT), total bilirubin, apolipoprotein A1, and ALT) with patients' age and
gender in a patented artificial intelligence algorithm (USPTO 6631330) in
order to generate a measure of fibrosis and necroinflammatory activity in
the liver. FT-AT is a continuous linear biochemical assessment of fibrosis
stage and necroinflammatory activity grade. It provides a numerical
quantitative estimate of liver fibrosis, ranging from 0.00 to 1.00,
corresponding to the well-established METAVIR scoring system (37) of
stages F0-F4 and of grades A0-A3. The same conversions that had been
established in HCV patients were used. Corresponding stages and grades
were calculated from median scores and 95% confidence intervals (CIs) were
observed in 1,270 HCV patients and 300 healthy blood donors (22, 24, 25).
Among the 300 controls, the median FT value (±SE) was 0.08 ± 0.004 (95%,
0.23) and the median AT value was 0.07 ± 0.004 (95%, 0.26). Among the 1270
HCV-infected patients, the FT conversion was 0.000-0.2100 for F0;
0.2101-0.2700 for F0-F1; 0.2701-0.3100 for F1; 0.3101-0.4800 for F1-F2;
0.4801-0.5800 for F2; 0.5801-0.7200 for F3; 0.7201-0.7400 for F3-F4; and
0.7401-1.00 for F4. The AT conversion was 0.00-0.1700 for A0;
0.1701-0.2900 for A0-A1; 0.2901-0.3600 for A1; 0.3601-0.5200 for A1-A2;
0.5201-0.6000 for A2; 0.6001-0.6200 for A2-A3; and 0.6201-1.00 for A3.
Samples stored at -80°C were centralized in the reference laboratory at
Pitié Salpêtrière Hospital. GGT, ALT, and total bilirubin were measured by
Hitachi 917 Analyzer and Roche Diagnostics reagents (both Mannheim,
Germany). alpha2-Macroglobulin, apolipoprotein A1, and haptoglobin were
measured using a Modular analyzer (BNII, Dade Behring; Marburg, Germany).
All coefficients of variation assays were lower than 6%.
Liver biopsies were fixed, paraffin-embedded, and stained with at least
hematoxylin-eosin-safran, and Masson's trichrome or picrosirius red for
collagen. At each center pathologists analyzed in a blinded fashion the
biopsies using the METAVIR classification (37). Fibrosis was staged from
F0 to F4: F0, no fibrosis; F1, portal fibrosis without septa; F2, portal
fibrosis with few septa; F3, numerous septa without cirrhosis; and F4,
cirrhosis. Necroinflammatory activity was graded from A0 to A3: A0, no
activity; A1, mild activity; A2, moderate activity; and A3, severe
activity.
Virology
Blood samples taken at each study visit were subjected to virological
analysis in a single center (INSERM 271, Lyon) without knowledge of the
clinical data. Blood samples were analyzed for viral genome centrally in a
blinded fashion with respect to the clinical data. Viral DNA was extracted
from serum using the QIAmp DNA Blood Mini Kit (QIAGEN, Courtaboeuf,
France). Baseline HBV DNA was quantified using the Versant branched DNA
kit version 3.0 (Bayer, France) with a lower limit of detection of 357 IU/ml
(1 IU/ml represents 5.6 copies/ml). The HBV polymerase gene was sequenced
following amplification by polymerase chain reaction (PCR) using
appropriate oligonucleotide primers. The lower limit of detection of the
PCR assay was 70 IU/ml (data not shown). The sequencing reaction was
performed using labeled nested primers (CY5.5-POL3M and CY5.0-P4M),
CY5/CY5.5-Dye Primer Kit, Long-Read TOWER sequencer, and Opengene System
software (Visible Genetics, Evry, France). INNO-LiPA genotyping and
Precore mutant detection kits were used, and the blot reactions were
performed using AUTOLiPA kits on nitrocellulose strips.
Statistical Analysis
We performed statistical analyses on all patients with at least one
sample and in patients with all four samples (sensitivity analysis).
Categorical variables were compared with the chi2 test or Fisher's exact
test (in cases with less than five theoretical subjects per cell),
quantitative variables with the Mann-Whitney test, and ANOVA for repeated
measures used Bonferroni and Tukey-Kramer multiple comparison tests (38).
Multivariate analysis used logistic regression analysis. Diagnostic value
was assessed by the area under the receiver operating characteristics
curve (AUROC). A sensitivity analysis was performed according to the
duration between biopsy and baseline serum, using the median as a cutoff.
Three methods were used in the statistical comparison of liver injury
dynamics: the difference between FT-AT values, the percentage of patients
with FT-AT improvement, and the fibrosis rate per year as previously used
for HCV modeling (39-41). A sensitivity analysis included date of birth to
calculate the fibrosis progression before treatment in order to exclude
bias due to inaccurate date of infection (42). As we had already observed
a strong interaction between baseline fibrosis and treatment impact in a
previous analysis of treatment impact in patients with chronic hepatitis
C, we stratified the analysis according to the baseline fibrosis stage
(bridging vs non bridging fibrosis) (20). All data were centrally analyzed
using BMDP statistical software (38).
RESULTS
Patients Included
Between October 1999 and March 2001, 298 subjects were included.
Fifteen (5.1%) were excluded from the analysis due to the absence of
follow-up data in 11 and protocol violation in 4. In all, 274 subjects
(96.8% of the analyzed population of 283 subjects and 91.9% of the
included population) completed the 24-month course of treatment. A total
of 916 FT were assessed in the analyzed population; 258 were done at
baseline, 229 at 6 months, 227 at 12 months, and 202 at 24 months (Fig.
1). In the remaining patients (no different from included patients, data
not shown), serum was not available for FT assessments.
At the time of inclusion, 55.1% of the population was treatment naive; the
rest had received interferon alpha predominantly. Only 20 patients had
received previous nucleoside analog antiviral drugs, principally
vidarabine (12 patients) and/or famciclovir (10 patients).
At the time when biochemical markers were obtained, liver biopsy results
were available in 214 subjects, at a median of 5.2 months before inclusion
in the study. At the time of biopsy, 66.7% of patients had bridging
fibrosis (F2-F3-F4), including 18.7% cirrhosis F4. At the time of
inclusion, as estimated by biochemical markers, 57.8% of patients had
bridging fibrosis, including 25.2% cirrhosis (Table 1).
Hepatitis B virus DNA was quantified at baseline in all patients. At
inclusion, basic core promoter (BCP) and precore mutants were identified
in the majority of subjects (81.0%). There was a slight preponderance of
promoter mutations compared to stop codon mutations, with both mutations
being observed in 33.6% of subjects. A total of 119 patients (42%) were
HBeAg-positive and 164 (58%) were HBeAg-negative. Genotype D was the most
frequent genotype, followed by genotype A. Genotypes F and G were very
rare.
Validation of Biochemical Markers
Using the previous biopsy as "a gold standard," the AUROC for the
diagnosis of bridging fibrosis (METAVIR stages F2-F3-F4) was 0.77 (SE =
0.03); for many septa (F3) or cirrhosis (F4), 0.74 (SE = 0.03); for the
diagnosis of moderate or severe activity (METAVIR grades A2, A3), 0.62 (SE
= 0.04); and for severe activity (A3), 0.75 (SE = 0.06). When AUROCs were
compared according to the duration between biopsy and biomarkers
measurements (less vs more than 4 months), there was no significant
difference for FT (for F2-F3-F4): 0.76 (0.05) versus 0.79 (0.05) (p= 0.53)
and a significance for AT (for A2A3): 0.69 (0.06) versus 0.52 (0.06) p=
0.04.
Six patients were at high risk of having false positive results for
baseline FT due to possible hemolysis (haptoglobin <0.11 g/l); 1 patient
was at risk of having a false negative result due to acute inflammation
(A2M 6.22 g/l, haptoglobin 1.95 g/l).
Factors Associated with Fibrosis at Baseline
In univariate analysis, bridging fibrosis (METAVIR stages F2-F3-F4)
was associated with age, male gender, BMI, alcohol consumption, baseline
activity grade, anti-HBe presence in the serum, and genotypes A, D, E
(Table 2).
In multivariate analysis, baseline bridging fibrosis was highly associated
(p< 0.001) with male gender (odds ratio (OR) = 0.14, 95% CI 0.06-0.32) and
age (OR = 0.93, CI = 0.91-0.96), and marginally associated with anti-HBe
presence (OR = 0.53, CI = 0.28-1.00; p= 0.05) and non-Asian ethnic origin
(OR = 2.31, CI = 1.01-5.30; p= 0.046).
Evolution of Fibrosis and Histological Activity During Treatment
The values of FT-AT before and during treatment are shown in Table 3.
Lamivudine treatment had a very significant impact overall. FT decreased
significantly from 0.51 at baseline to 0.37 at 24 months (p< 0.001), and
85% of patients had an improvement at 24 months. AT also decreased
significantly from 0.56 to 0.13 (p< 0.0001), and 91% of patients had an
improvement at 24 months. The mean fibrosis progression rate was 0.15 FT
units per year (0.02; n = 254) before treatment, -0.12 (0.02; n = 217)
during the first 6 months, 0.04 (0.02; n = 187) between 6 and 12 months,
and -0.09 (0.02; n = 166) between 12 and 24 months (the progression rates
before treatment and between 6 and 12 months were significantly different
vs all) (Table 4). A total of 44 patients had at baseline a cirrhosis
estimated by FT and a second FT assessment at 24 months; 95% (42/44) had
an improvement of FT at 24 months, including a significant regression of
fibrosis greater than 0.30 in 32% (14/44).
The impact of treatment on fibrosis was greater during the first 6 months,
with a plateau between 6 and 12 months and a greater impact between 12 and
24 months. The impact of treatment on necroinflammatory activity was
marked at 6 months (87% improved), with a plateau between 6 and 12 months
and another impact between 12 and 24 months.
Factors Associated with Lamivudine Impact
The impact was greater in patients with bridging fibrosis at baseline.
The significant impact of treatment was observed both in patients with and
without an occurrence of YMDD mutation.
The impact on AT was lower at 12 and 24 months in patients with YMDD
mutation than in patients without (Table 3).
During the course of the study, YMDD mutations were observed in 114
patients. This corresponds to an incidence rate of 41.5% (95% CI limits
35.6% and 47.3%). The time course for the emergence of mutations was
linear, with 17 (6%) occurring in the first 6 months, 47 (17%) between 6
and 12 months, and 51 (18%) between 12 and 24 months. The FT changes were
from 0.41 ± 0.07 to 0.33 ± 0.07 (NS) in the group of mutation occurrence
in the first 6 months; from 0.59 ± 0.04 to 0.43 ± 0.05 (p< 0.0001) in the
group of mutation occurrence between 6 and 12 months; from 0.46 ± 0.04 to
0.36 ± 0.04 (p< 0.0001) in the group of mutation occurrence after 12
months. In 44 patients with baseline cirrhosis, the only 2 patients with
worsening FTs were those with YMDD-variant HBV (2/20); there was a
significant FT reduction in 25% of these patients (5/20).
The AT changes were from 0.53 ± 0.10 to 0.10 ± 0.03 (p= 0.04) in the group
of mutation occurrence in the first 6 months; from 0.59 ± 0.04 to 0.20 ±
0.03 (p< 0.0001) in the group of mutation occurrence between 6 and 12
months; from 0.55 ± 0.04 to 0.19 ± 0.04 (p< 0.0001) in the group of
mutation occurrence after 12 months.
The fibrosis progression rates during the three phases according to the
baseline fibrosis and YMDD mutation are shown in Table 4. In the group of
patients with baseline bridging fibrosis, very significant differences in
the kinetics of the first 12 months of treatment were observed in Asian
patients, who had a greater decrease in fibrosis rate during the first
month compared with non-Asian patients (p= 0.01) (Fig. 2A), and patients
younger than 40 yr (p< 0.001) (Fig. 2B), both still significant in
multivariate analysis (p= 0.03 and p= 0.04, respectively). Kinetics were
not different according to gender or anti-HBe status (data not shown).
Sensitivity Analyses
The same results were obtained when the population of patients with
all completed FT or AT at the four follow-up dates was used (Table 3 panel
B). When patients were stratified according to stage or grade as estimated
by liver biopsy instead of FT-AT, the results were similar (data not
shown). When baseline fibrosis progression rates were calculated according
to the date of birth instead of the date of contamination, results were
similar (data not shown).
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Prometheus
Laboratories Introduces Non-Invasive Test for the Detection of Liver
Fibrosis
Prometheus Laboratories Introduces FIBROSpect(SM) II
Tuesday January 20, 2004 5:16 pm ET
- A New Improved Non-Invasive Test for
the Detection of Liver Fibrosis -
SAN DIEGO, Calif., Jan. 20 /PRNewswire/ -- Prometheus Laboratories Inc., a
specialty pharmaceutical company, announced today the introduction of
FIBROSpect II, a unique non-invasive diagnostic panel to aid in the
detection of liver fibrosis in patients with chronic hepatitis C. This
simple blood test helps physicians differentiate the presence of no/mild
from significant liver fibrosis without the pain, anxiety and risk
associated with liver biopsy.
(Logo:
http://www.newscom.com/cgi-bin/prnh/20030416/PROMETHEUSLOGO )
"In chronic hepatitis C infection, liver biopsy has been the favored
approach to evaluate the extent of liver fibrosis and help guide treatment
decisions; however, it is expensive, associated with possible
complications, and limited by sampling error and observer variability,"
stated Dr. F. Fred Poordad, Associate Director, Hepatology and Liver
Transplant at Cedars Sinai Medical Center. "Non-invasive methods to aid in
assessing liver disease severity, such as FIBROSpect II, provide
additional diagnostic options."
FIBROSpect II, an enhanced version of the original FIBROSpect test,
eliminates indeterminate test results that were occasionally reported with
the first generation test, and improves the ability to accurately
differentiate patients with and without significant liver fibrosis.
FIBROSpect II is based on three extracellular matrix remodeling proteins
utilizing a new algorithm and unique index, thereby providing physicians
with even more clinically useful information.
According to the most recent estimates by the Centers for Disease
Control and Prevention, 3.9 million people are currently infected with
hepatitis C in the U.S., with 2.7 million being chronically infected.
Worldwide, over 170 million are infected. Chronic hepatitis C varies in
its cause and outcome. At one end of the spectrum are patients who have no
sign of liver disease and for whom the overall prognosis may be good. At
the other end of the spectrum are patients with chronic hepatitis C and
advanced fibrosis that may ultimately develop end-stage liver disease. The
major consequence of liver disease is the progression to fibrosis and
cirrhosis, which can lead to liver cancer or the need for a liver
transplant. Therefore, early and accurate diagnoses and staging are
critical for proper patient management.
Prometheus Laboratories Inc. is a specialty pharmaceutical company
committed to developing new ways to help physicians individualize patient
care. The Company focuses on the treatment, diagnosis and detection of
gastrointestinal, autoimmune and inflammatory diseases and disorders. The
Company's strategy includes the marketing and delivery of pharmaceutical
products complemented by its proprietary, high-value diagnostic testing
services. By integrating these therapeutic, diagnostic and treatment
monitoring services, Prometheus addresses the full continuum of care,
thereby providing physicians with a comprehensive solution to treat
chronic diseases. Prometheus' corporate offices are located in San Diego,
California. Additional information about Prometheus Laboratories Inc. can
be found at
www.prometheuslabs.com .
LabCorp® Announces
U.S. Launch Of Exclusive Liver Fibrosis Assay HCV Fibrosure™
Noninvasive Blood Test Provides Alternative to Liver Biopsy for Assessing
Status of Hepatitis C Patients
Burlington, NC, March 17, 2004 - Laboratory Corporation of America®
Holdings (LabCorp®) (NYSE: LH) today announced the availability of HCV
FIBROSURE™, a noninvasive blood test for assessing liver status in hepatitis
C virus (HCV) patients. Developed by leading hepatologists at the
Pitie-Salpetriere Hospital and BioPredictive in France, HCV FIBROSURE™ is
only available in the United States through LabCorp.
HCV FIBROSURE™ provides an easily accessible alternative to liver biopsy,
which physicians use to assess liver fibrosis and necroinflammatory activity
in HCV patients. While liver biopsy has long been considered the gold
standard to monitor the status of HCV and determine therapy options, it is
an invasive procedure that carries a risk of serious complications. HCV
FIBROSURE™ uses a combination of six serum biochemical markers plus age and
gender in a patented algorithm to determine the degree of liver fibrosis and
the level of ongoing necroinflammatory activity. The test, which has been
clinically available in Europe for the past two years, has been shown in
several studies to enable quantitative, reproducible assessment of
fibrogenic and necrotic activity in the liver of HCV patients.
"The launch of this important new test once again validates LabCorp's
strategy of creating a world-class national laboratory with the best and
broadest array of diagnostic testing services," said Myla P. Lai-Goldman,
M.D., executive vice president, chief scientific officer and medical
director at LabCorp. "Our focus on bringing forth innovative new
technologies and tests, coupled with our scientific expertise and national
scope, helps us broadly deliver vital new tools like BioPredictive's liver
fibrosis assay to U.S. physicians managing HCV patients."
BioPredictive is currently researching clinical use of this test for
other disease populations, including hepatitis B, HIV-HCV, and alcoholic and
non-alcoholic steato hepatitis (NASH). "We anticipate that HCV FIBROSURE™
will prove to be just the first in a family of innovative, noninvasive
diagnostic testing products aimed at hepatitis and non-hepatitis-related
conditions," said Dr. Thierry Poynard, a world-renowned hepatologist, head
of Hepato-Gastrotroenterology department in Pitie-Salpetriere Hospital in
Paris, and researcher and founder of BioPredictive. "We look forward to
continuing our relationship with LabCorp and building upon their expertise
in the world of hepatitis testing for future products."
HCV FIBROSURE™ is recommended for use to assess liver status following a
diagnosis of HCV, as a baseline determination of liver status before
initiating HCV therapy, as post-treatment assessment of liver status six
months after therapy completion, and for noninvasive assessment of liver
status in patients at risk of complications from a liver biopsy. The blood
sample for HCV FIBROSURE™ can be collected in minutes and results can be
returned to the physician within days. The test uses six biochemical markers
that are routine and considered standard of care in the United States.
About BioPredictive
Founded at Paris University in 2002, BioPredictive is focused on the
study, design and development of medically important biological tests. At
the center of the company's scientific efforts is the desire to improve
disease management by replacing invasive strategies with noninvasive
alternatives. BioPredictive has developed two noninvasive tests for chronic
liver disease - FibroTest and ActiTest. FibroTest is a biochemical marker of
liver fibrosis and ActiTest is a biochemical marker of inflammation and
necrosis of the liver. BioPredictive licenses the FibroTest and ActiTest
technology from Assistance Public-HTMpitaux de Paris (AP-HP). The company
performs more than 2,000 tests per month, and services 150 private and 12
public hospital laboratories in France, Switzerland, Portugal, Morocco and
Mexico. To learn more about BioPredictive, visit the company Web site at:
www.BioPredictive.com.
FibroTest and ActiTest are available in the U.S. exclusively through
Laboratory Corporation of America® Holdings (LabCorp) under the name HCV
FIBROSURE™.
About LabCorp
Laboratory Corporation of America® Holdings is a pioneer in
commercializing new diagnostic technologies and the first in its industry to
embrace genomic testing. With annual revenues of $2.9 billion in 2003,
approximately 23,000 employees nationwide, and more than 220,000 clients,
LabCorp offers over 4,400 clinical assays ranging from blood analyses to HIV
and genomic testing. LabCorp combines its expertise in innovative clinical
testing technology with its Centers of Excellence: The Center for Molecular
Biology and Pathology, in Research Triangle Park, NC; National Genetics
Institute, Inc. in Los Angeles, CA; ViroMed Laboratories, Inc. based in
Minneapolis, MN; The Center for Esoteric Testing in Burlington, NC; and
DIANON Systems, Inc. based in Stratford, CT. LabCorp clients include
physicians, government agencies, managed care organizations, hospitals,
clinical labs, and pharmaceutical companies. To learn more about our growing
organization, visit our Web site at: www.LabCorp.com.
Each of the above forward-looking statements is subject to change based
on various important factors, including without limitation, competitive
actions in the marketplace and adverse actions of governmental and other
third-party payors. Actual results could differ materially from those
suggested by these forward-looking statements. Further information on
potential factors that could affect LabCorp's financial results is included
in the Company's Form 10-K for the year ended December 31, 2003, and
subsequent SEC filings.
For questions or comments about this site, please contact the LabCorp
Webmaster.
Privacy Statement
Blood Tests Provide Alternative to Liver Biopsy
for Assessing Status of Liver Disease in HCV Patients
Oneida TheraDiagnostics
Ltd has launched two non-invasive blood tests for assessing the extent of
liver disease in patients infected with
hepatitis C virus (HCV).
Developed by hepatologists at the Pitie-Salpetriere Hospital and
BioPredictive in France, the two tests are called FibroTest and ActiTest.
The new tests provide
easily accessible alternatives to
liver biopsy, which is currently used to assess liver fibrosis
and necroinflammatory activity in these patients. The tests do not replace
the use of liver biopsy, however, which may be the appropriate diagnostic in
many cases. Whether to use these new blood tests instead of a liver biopsy
should be discussed between each individual patient and his/her primary care
physician or liver specialist.
Although liver biopsy has
long been considered the gold standard for monitoring the status of HCV and
to determine therapy options, it is an invasive procedure that carries some
risk of serious complications. The new assays use a combination of six serum
biochemical markers, plus age and gender data, in a patented algorithm to
determine the degree of liver fibrosis and the level of ongoing
necroinflammatory activity.
The tests have been
available in parts of Europe for the past two years and have recently been
launched in the USA. Each test has been clinically validated in HCV patients
to enable quantitative, reproducible assessment of fibrogenic and necrotic
activity in the livers of HCV patients.
Oneida TheraDiagnostics
Ltd. is a recently established company based in the UK offering molecular
diagnostic services for clinical studies with particular emphasis on viral
disease.
“Our intention is to
establish a world-class portfolio of molecular diagnostics that will
facilitate individual patient management and clinical trial studies
performed in the UK and Ireland. The tests launched today are very well
validated and address one of the major challenges in patients with chronic
viral hepatitis. They will be the cornerstone of our viral hepatitis
diagnostic service,” said Berwyn Clarke, CEO at Oneida.
FibroTest and
ActiTest may be recommended by physicians for use in assessing
liver status following a diagnosis of HCV. This provides a baseline
determination of liver status before the initiation of
HCV therapy
and a post-treatment assessment of liver status six months after the
completion of therapy.
The tests can also be
applied for the non-invasive assessment of liver status in patients at risk
of complications from a liver biopsy. The blood sample for the tests can be
collected in minutes and results normally returned to the doctor within
days.
10/11/04
Source
Oneida TheraDiagnostics.
www.oneidathd.co.uk
“Validated blood tests
provide new alternative to liver biopsy for assessing status of liver
disease in hepatitis C patients.” Press Release. October 8, 2004.
Index to
Hepatitis C News Articles by Topic [ A -- Z ]
Internet Conference Report
39th
EASL -
April 14 - 18, 2004, Berlin
Germany
Biochemical Markers (Fibrotest)
Reliably Predict Extensive Fibrosis and Cirrhosis in Non Alcoholic Fatty
Liver Disease (NAFLD)
The aim of the
current study was to assess the predictive value of Fibrotest (FT) a
biochemical marker of
fibrosis for the diagnosis of fibrosis
stage in patients with non-alcoholic fatty liver disease (NAFLD).
The study participants
consisted of 89 patients with steatosis on ultrasound, abnormal
transaminases and no other causes of liver disease. Fibrosis was staged: F0=
none; F1=mild (perisinusoidal or portal); F2=moderate (portal+perisinusoidal
and/or bridging); F3F4 =severe (extensive bridging fibrosis or cirrhosis).
300 prospectively included blood donors (BD) were used as controls.
Results
Severe fibrosis was
present in 11%, moderate or severe in 45%. The mean FT (se) value was 0.09
(0.01) in BD; 0.19 (0.04) in F0 (n=18); 0.21 (0.03) in F1 (n=31); 0.33
(0.03) in F2 (n=30); 0.62 (0.05) in F3F4 (n=10) (p<0.05 between F3F4 and
F0,F1,F2, and between F2 and F0, F1 and all stages vs BD).
For the diagnosis of
severe fibrosis, AUROC= 0.94 (0.05); accuracy rate= 90%, kappa= 0.51 (0.11);
Spearman correlation= 0.50 (p<0.001). An FT cutoff of 0.60 had a 95% PPV
for severe fibrosis (Sp 97%, Se 60%). An FT cutoff of 0.30 had a 100% NPV
for severe fibrosis (Sp=73%, Se 100%). Sensitivity analyses found an
improvement of F2F3F4 AUROC for a biopsy size longer than 15mm as compared
to a size smaller than 15 mm, 0.84 (0.07) vs. 0.63 (0.20) (p=0.04).
The authors conclude,
“Fibrotest, reliably predicts extensive fibrosis and cirrhosis in NAFLD.
This should improve the identification of the population at risk of
significant liver injury and reduce the number of unnecessary liver
biopsies.”
04/30/04
Reference
V Ratziu and others. DIAGNOSTIC VALUE OF BIOCHEMICAL MARKERS (FIBROTEST)
FOR THE PREDICTION OF LIVER FIBROSIS IN PATIENTS WITH NON-ALCOHOLIC FATTY
LIVER DISEASE (NAFLD). Abstract 597. 39th EASL. April 14-18,
2004. Berlin, Germany.
www.hivandhepatitis.com
Internet
Conference Report
39th EASL -
April 14 - 18, 2004, Berlin Germany
A New,
Sensitive, Real Time PCR-based Assay for Quantification of HCV RNA from
Roche Diagnostics
Management of therapy of patients with
hepatitis C is based on qualitative and quantitative measurement of HCV-RNA
by different assays. A new real-time RT-PCR based assay (COBAS TaqMan, Roche
Diagnostics, Pleasanton, USA) was designed for highly sensitive
quantification of HCV-RNA thereby covering the range of qualitative and
quantitative assays in one test.
COBAS TaqMan a single tube, single enzyme,
real-time RT-PCR-based assay was evaluated on the basis of reference panels
(AcroMetrix, clinical), and clinical samples (n=100) for testing of
sensitivity, specificity, linearity, intra-, inter-assay variability (6-18
repeats) and comparability with COBAS Amplicor Monitor 2.0.
HCV-RNA extraction was performed with the
high-pure-system (HPS, Roche Diagnostics).
The lower detection limit of the COBAS TaqMan
was 100% and 72% at 10 and 5 IU/mL, respectively. The specificity was 99%
with equal amplification of HCV genotypes 1-6.
The mean intra- and inter-assay variability
within the range of 7 Mill. to 1000 IU/mL was between a SD of 0.076 and
0.096 log compared to a SD of 0.107 and 0.254 log by the COBAS Amplicor
assay.
The linearity tests showed a regression
coefficient of 0.99 between 7 Mill. and 30 IU/ml.
Comparison of HCV RNA quantification by COBAS
TaqMan and COBAS Amplicor showed a high concordance with a correlation
coefficient of 0.95.
In conclusion the authors state, “The new
COBAS TaqMan assay is a highly sensitive, precise, and linear assay for HCV
RNA quantification and has the potential to be used instead of previous
qualitative and quantitative measurements before, during and after antiviral
therapy.”
Saarland University Clinic, Homburg.
04/30/04
Reference
C Sarrazin and
others. EVALUATION OF A NEW, SENSITIVE, REAL TIME PCR BASED ASSAY FOR
QUANTIFICATION OF HCV RNA. Abstract 511. 39th EASL. April 14-18,
2004. Berlin, Germany.
www.hivandhepatitis.com
Quest Diagnostics Announces Availability of HEPTIMAX
Ultra-Sensitive Quantitative Hepatitis C Virus Test
TETERBORO, N.J., July 23 /PRNewswire/ -- Quest Diagnostics Incorporated
(NYSE: DGX - news),
T he
nation's leading provider of gene-based medical testing, information and
services, announced the availability of a new ultra-sensitive viral load
test for hepatitis C virus (HCV) that is approximately 10 times more
sensitive than any other commercially available test. The test detects the
level of hepatitis C virus based on an innovative application of
transcription mediated amplification (TMA) technology to HCV testing. Quest
Diagnostics developed the test and is the first laboratory in the world to
offer it.
The new offering, called the HEPTIMAX(TM) viral load test, is capable of
detecting minute quantities of hepatitis C virus down to as few as 5
International Units (IUs) per milliliter (ml). The ability to detect minute
quantities of virus is useful to physicians in monitoring the effectiveness
of various treatments for hepatitis C in patients. The HEPTIMAX(TM) viral
load test not only delivers the maximum sensitivity but also offers the
maximum range (5 IUs/ml to 8.3 million IUs/ml).
This proprietary test is ordered by physicians to monitor and confirm
hepatitis C viral infection and to demonstrate post-treatment resolution of
the infection. Because the HEPTIMAX(TM) viral load test combines the new TMA
technology with traditional quantitative viral load testing using branched
DNA technology, it simplifies patient management by allowing physicians to
order just one test to cover the complete range of possible viral load
values from 5 IUs to 8.3 million IUs per ml.
``The improved sensitivity of the HEPTIMAX(TM) viral load test is
particularly relevant to physicians with the advent of new forms of
hepatitis C therapeutics,'' said Jorge Leon, Ph.D., Vice President for
Applied Genomics at Quest Diagnostics. ``New combination therapies utilizing
sustained-action forms of interferon have shown better treatment responses
in clinical trials that translate to lower levels of virus in patients.''
``Preliminary studies comparing the HEPTIMAX(TM) viral load test to other
technologies and laboratories confirm it has superior sensitivity,'' said
Dr. Peter Heseltine, Medical Director of Infectious Diseases for Quest
Diagnostics. ``We are planning additional studies with other outside
investigators to confirm and publish our findings.''
A study published in the October 2000 issue of the journal ``Hepatology''
utilizing a hepatitis C qualitative version |
