U.S. patent application number 13/257456 was filed with the patent office on 2012-01-12 for non-invasive method for assessing liver fibrosis progression.
This patent application is currently assigned to CENTRE HOSPITALIER UNIVERSITAIRE D'ANGERS. Invention is credited to Paul Cales.
Application Number | 20120010824 13/257456 |
Document ID | / |
Family ID | 42227782 |
Filed Date | 2012-01-12 |
United States Patent
Application |
20120010824 |
Kind Code |
A1 |
Cales; Paul |
January 12, 2012 |
Non-Invasive Method for Assessing Liver Fibrosis Progression
Abstract
The present invention relates to a non-invasive method for
assessing liver fibrosis progression in an individual, said method
comprising the steps of calculating the ratio of fibrosis level to
cause duration and to a non-invasive method for assessing liver
fibrosis progression in an individual, said method comprising the
steps of measuring, at two different times t.sub.1 and t.sub.2, the
fibrosis levels FL (t.sub.1) and FL (t.sub.2) and calculating the
ratio FL (t.sub.2)-FL (t.sub.1) to (t.sub.2-t.sub.1) and to a
non-invasive method for assessing if an individual is a slow,
medium or fast fibroser.
Inventors: |
Cales; Paul; (Avrille,
FR) |
Assignee: |
CENTRE HOSPITALIER UNIVERSITAIRE
D'ANGERS
Angers
FR
UNIVERSITE D'ANGERS
Angers
FR
|
Family ID: |
42227782 |
Appl. No.: |
13/257456 |
Filed: |
March 18, 2010 |
PCT Filed: |
March 18, 2010 |
PCT NO: |
PCT/EP2010/053548 |
371 Date: |
September 19, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61161474 |
Mar 19, 2009 |
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13257456 |
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Current U.S.
Class: |
702/21 |
Current CPC
Class: |
G01N 33/50 20130101;
G01N 2800/52 20130101; G01N 2333/78 20130101; A61K 31/00 20130101;
G01N 33/6893 20130101; G16H 50/20 20180101; G16B 5/00 20190201;
G01N 33/576 20130101; G01N 2800/60 20130101; A61K 31/4178 20130101;
A61K 38/21 20130101; G01N 2400/40 20130101; G01N 2800/085 20130101;
G01N 2800/7052 20130101; G01N 2333/4713 20130101; G16H 50/30
20180101; G01N 2333/495 20130101; G01N 33/57438 20130101 |
Class at
Publication: |
702/21 |
International
Class: |
G06F 19/10 20110101
G06F019/10 |
Claims
1.-14. (canceled)
15. A non-invasive method for assessing liver fibrosis progression
in an individual comprising: measuring a fibrosis level in a
patient; and calculating a ratio of fibrosis level to cause
duration.
16. The method of claim 15, further comprising: measuring at two
different times t1 and t2 fibrosis levels FL(t1) and FL(t2); and
calculating a ratio FL(t.sub.2)-FL(t.sub.1) to
(t.sub.2-t.sub.1).
17. The method of claim 15, further comprising: a) measuring in a
sample of the individual at least one variable or score further
defined as: biological variables further defined as .alpha.-2
macroglobulin (.alpha.2M), Hyaluronic acid (HA), Apolipoprotein A1
(ApoA1), Type III procollagen N-terminal propeptide (P3P),
.gamma.-glutamyltranspeptidase (GGT), Bilirubin, .beta.-globulin,
.gamma.-globulin (GLB), Platelets (PLT), Prothrombin time (PT),
Prothrombin index (PI), Aspartate aminotransferase (AST), Alanine
aminotransferase (ALT), Urea, Sodium (NA), Glycemia, Triglycerides,
Albumin (ALB), Alkaline phosphatase (ALP), Human cartilage
glycoprotein 39 (YKL-40), Tissue inhibitor of matrix
metalloproteinase 1 (TIMP-1), Matrix metalloproteinase 2 (MMP-2),
Ferritin, TGF.beta.1, Laminin, .beta..gamma.-block, Haptoglobin,
C-Reactive protein (CRP), and/or cholesterol, complex biological
variable; clinical variables further defined as age at first
contact, age, cause duration, firm liver, Splenomegaly, Ascites,
collateral circulation, cause of CLD, and/or oesophageal varices
(EV grade); score further defined as Metavir F stage, Area of
fibrosis (AOF), fractal dimension, Fibrosis score, PGA score, PGAA
score, Hepascore, Aspartate-aminotransferase to platelet ratio
index (APRI), and/or European Liver Fibrosis (ELF), and/or
combinations thereof: and b) combining the selected variables in a
mathematical function, further defined as a multiple linear
regression function, a non-linear regression function, or simple
mathematic function.
18. The method of claim 17, further comprising measuring in a
sample of the individual at least two variables or scores.
19. The method of claim 18, further comprising measuring in a
sample of the individual at least three variables or scores.
20. The method of claim 17, wherein AST/ALT is measured.
21. The method of claim 17, wherein the mathematical function is an
arithmetic operation.
22. The method of claim 21, wherein the arithmetic operation is
division.
23. The method of claim 17, wherein liver fibrosis progression is
assessed by measuring Metavir F progression established by:
measuring in any combination: at least one biological variable
further defined as Type III procollagen N-terminal propeptide
(P3P), Hyaluronic acid (HA), Prothrombin index (PI),
.gamma.-glutamyl transpeptidase (GGT) and/or .beta..gamma.-block;
at least one complex biological variable further defined as
AST/ALT; at least one clinical variable further defined as age at
first contact and/or cause duration; at least one score further
defined as a fibrosis score, AOF, and/or fractal dimension; and/or
combining the selected variables in the mathematical function.
24. The method of claim 17, wherein the liver fibrosis progression
is assessed by measuring the area of fibrosis (AOF) progression
established by: measuring in any combination: at least one
biological variable further defined as .alpha.-2 macroglobulin
(.alpha.2M), Hyaluronic acid (HA), .beta.-globulin, Prothrombin
index (PI) and/or .beta..gamma.-block; the complex biological
variable AST/ALT; at least one clinical variable further defined as
age at first contact, age, cause duration, sex, firm liver,
Splenomegaly, Ascites, Collateral circulation and/or cause of CLD;
and/or at least one score further defined as Metavir F stage, area
of fibrosis (AOF), FibroMeter.TM., PGA score and/or PGAA score; and
combining the selected variables in the mathematical function.
25. The method of claim 17, wherein the variables comprise in any
combination: the biological variable .beta.-globulins; the complex
biological variable AST/ALT; the clinical variable cause duration;
and/or the score Area of fibrosis (AOF) or Metavir F stage.
26. The method of claim 17, wherein the variables comprise in any
combination: at least one biological variables chosen among
.beta.-globulins or Prothrombin index (PI); the complex biological
variable AST/ALT; and/or at least one clinical variable chosen
among age at first contact, cause duration, or firm liver.
27. The method of claim 17, wherein the variables comprise in any
combination: at least one biological variable defined as
.beta.-globulins and/or .alpha.-2 macroglobulin (.alpha.2M); the
complex biological variable AST/ALT; and/or at least one clinical
variable further defined as age at first contact or cause
duration.
28. A method for assessing an individual comprising performing at
least once the method of claim 15.
29. The method of claim 28, further defined as a method of
assessing if an individual is a fast fibroser, using binary
logistic regression, and a fast fibroser is identified as having an
increased AOF progression, younger inclusion age and older start
age or alternatively cause duration by stepwise binary logistic
regression.
30. The method of claim 28, further defined as a method of
assessing if an individual is a slow, medium or fast fibroser using
discriminant analyses and the individual is ranked as a slow,
medium or fast fibroser with reference to a ranking of patients
determined by statistical analysis.
31. The method of claim 28, further defined as a method of
assessing if an individual is at risk of suffering or is suffering
from a condition further defined as chronic liver disease, a
hepatitis viral infection, a hepatoxicity, a liver cancer, a non
alcoholic fatty liver disease (NAFLD), an autoimmune disease, a
metabolic liver disease and/or a disease with secondary involvement
of the liver.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of hepatology and
in particular to a non-invasive method for assessing the liver
fibrosis progression, especially in alcohol or viral or metabolic
chronic liver disease.
BACKGROUND OF THE INVENTION
[0002] Liver fibrosis refers to the accumulation of fibrous scar
tissue in the liver. In order to diagnose liver fibrosis, various
techniques can be used. One of these techniques is the liver needle
biopsy (LNB), leading to a classification based on observation of
lesions in the liver, particularly in the hepatic lobe. Indeed, one
of the most commonly used classifications is the Metavir
classification, which classifies liver fibrosis into five stages
from F0 to F4. According to the Metavir classification, an
F.gtoreq.2 stage means that fibrosis is clinically significant,
whereas a F4 stage corresponds to the ultimate stage, namely
cirrhosis.
##STR00001##
[0003] Other techniques, such as the measurement of the presence or
the severity of fibrosis in an individual through a Fibrosis score
(such as for example Fibrometer.TM.), an area of fibrosis (AOF)
score as well as quantitative image analysis can also be used alone
or in combination with LNB or Metavir classification, in order to
determine with more accuracy the extent of liver fibrosis in an
individual.
[0004] However, if detecting the presence or the severity of liver
fibrosis is of high importance, it is observed that progression
rate of the fibrosis differs from an individual to another. Thus,
the assessment of liver fibrosis progression would be a very
important and useful tool in clinical practice for both prognostic
and therapeutic reasons.
[0005] First, considering that liver fibrosis progression depends
on various genetic and host factors, it may indeed be useful to
determine ahead of time whether it is reasonable to expect that
liver fibrosis will progress towards cirrhosis during the patient's
lifetime and if it does, at what rate will this progression
occur.
[0006] Second, assessing the progression rate of liver fibrosis can
also be useful in order to help physicians decide whether or not to
treat a patient or in order to help them monitor patients who are
already following a treatment regimen. Until now, physicians relied
mostly on fibrosis staging (ex. Metavir stage .gtoreq.F2) in order
to justify an antiviral treatment for chronic viral hepatitis.
However, it would be very useful to know early on, such as for
example but not limited to patients showing a stage F0 or F1,
whether his liver fibrosis will evolve rapidly or not into
clinically significant fibrosis or cirrhosis, in order for the
physicians to anticipate the treatment.
[0007] Several documents have disclosed techniques developed in
order to assess liver fibrosis progression. WO 03/064687 discloses
a method for assessing a patient's risk of development and
progression of liver cirrhosis, said method comprising the step of
determining the patient's genotype or phenotype for a coagulation
factor. WO 2006/003654 discloses methods and kits for determining
the predisposition of an individual affected by chronic hepatitis C
infection to develop a fast progression rate of liver fibrosis.
This method essentially consists in determining the presence or
absence, in the CYP2D6 locus of the individual, of at least one
fast progression liver fibrosis associated genotype. EP 1887362A1
discloses a hepatic disease-evaluating method comprising a step of
calculating an index indicating the degree of hepatic fibrosis from
amino acid concentration data. Although, the aforementioned methods
can assess liver fibrosis progression, they require sophisticated
biological analysis which is not easily available in clinical
practice.
[0008] Consequently, there is still a need for a low cost and
easily available method which can evaluate the progression of
fibrosis, said method being non-invasive, non-traumatizing,
accurate and reliable, as well as simple to use.
DESCRIPTION OF THE INVENTION
[0009] For the purpose of the present invention,
[0010] "Score" is a combination of markers (or variables) aimed at
predicting a clinical event or a lesion such as fibrosis degree.
Usually, and especially when using the binary logistic regression,
the score ranges from 0 (0% risk) to 1 (100% risk), i.e. the
probability of the diagnostic target. When the score relies on
multiple linear regression, the score produces a result in the same
units as the diagnostic target. In the present invention, the main
scores are derived from multiple linear regression and measure a
progression rate of fibrosis, i.e. expressed as a fibrosis unit per
time unit.
[0011] "Progression" means the evolution of the fibrosis level over
time.
[0012] "Regularly" means at regular intervals, such as for example,
every 10-day, every month, or every year, etc.
[0013] "Sample" means a biological fluid of an individual, such as
for example blood, serum, plasma, urine or saliva of an
individual.
[0014] "Non-invasive" means that no tissue is taken from the body
of an individual (blood is not considered as a tissue).
[0015] "Individual" means a woman, a man or an animal, young or
old, healthy or susceptible of being affected or clearly affected
by a hepatic pathology, such as a liver fibrosis of viral origin,
of alcohol origin, a chronic liver steatosis or by any other
pathology.
[0016] "Cause" means the risk factor that induces the lesions and
the ensuing pathology.
[0017] "Cause duration" is the time between the age when the cause
started ("start age") and the age at inclusion when fibrosis level
was measured ("inclusion age").
[0018] "Fibrosis level" is reflected by a Fibrosis Score, AOF or
fractal dimension, preferably fibrosis level is a fibrosis score or
an AOF score or a fractal dimension score.
[0019] "Fibrometer" may refer to a fibrosis score or to a AOF
score.
[0020] The present invention proposes a solution to the technical
issue of assessing the progression rate of fibrosis in all and any
condition or disease involving fibrosis. This invention results in
a very accurate diagnosis of fibrosis progression and in the
ability of distinguishing slow, medium and fast fibrosers.
In a preferred embodiment, the condition or disease is alcohol or
viral chronic liver disease (CLD). According to another embodiment,
in order to assess the progression rate of fibrosis, the
progression rate of area of fibrosis (AOF) is assessed.
[0021] According to a first embodiment of the invention, the liver
fibrosis progression is assessed by calculating the ratio fibrosis
level/cause duration. According to a preferred embodiment, fibrosis
level is measured by a non-invasive method. Advantageously, the
fibrosis level is a fibrosis score, preferably Fibrometer.TM., AOF
score or fractal dimension score.
[0022] According to a second embodiment of the invention, the liver
fibrosis progression is assessed by measuring, at two different
intervals t.sub.1 and t.sub.2, the fibrosis levels FL(t.sub.1) and
FL(t.sub.2) and calculating the ratio FL(t.sub.2)-FL(t.sub.1) to
(t.sub.2-t.sub.1).
[0023] According to the invention, "t.sub.1": is the time at which
a first measure is performed in an individual and a first fibrosis
level FL(t.sub.1) is determined;
[0024] "t.sub.2": is the time at which a second measure is
performed in the same individual and a second fibrosis level
FL(t.sub.2) is determined;
[0025] "t.sub.2-t.sub.1" is a period of time of at least 10 days;
in an embodiment, t.sub.2-t.sub.1 is a period of 1 to 6 months; in
another embodiment, t.sub.2-t.sub.1 is a period of 1 year.
[0026] Advantageously, the fibrosis level is a fibrosis score, AOF
score or fractal dimension score.
[0027] According to this invention, "Fibrosis Score" is a score
obtained by measuring in a sample of an individual and combining in
a logistic or linear regression function at least three, preferably
6 to 8, markers selected in the group consisting of .alpha.-2
macroglobulin (A2M), hyaluronic acid (HA or hyaluronate),
apoliprotein A1 (ApoA1), N-terminal propeptide of type III
procollagen (P3P), gamma-glutamyltranspeptidase (GGT), bilirubin,
gamma-globulins (GLB), platelet count (PLT), prothrombin index
(PI), aspartate amino-transferase (AST), alanine amino-transferase
(ALT), urea, sodium (NA), glycemia (GLY), triglycerides (TG),
albumin (ALB), alkaline phosphatases (ALP), human cartilage
glycoprotein 39 (YKL-40), tissue inhibitor of matrix
metalloproteinase 1 (TIMP-1), matrix metalloproteinase 2 (MMP-2),
ferritin, weight, age and sex.
[0028] Preferably, the Fibrosis Score is measured by combining the
levels of at least three markers selected from the group consisting
of glycemia (GLY), aspartate aminotransferase (AST), alanine
amino-transferase (ALT), ferritin, hyaluronic acid (HA),
triglycerides (TG), prothrombin index (PI) gamma-globulins (GLB),
platelet count (PLT), weight, age and sex.
[0029] More preferably, the Fibrosis score is established by
combining in a binary linear regression function, the levels of
four to eight markers, preferably selected from the group
consisting of Alpha2 macroglobulin (A2M), hyaluronic acid or
hyaluronate (AH), Prothrombin index (PI) Platelets (PLQ), ASAT,
Urea, GGT, Age and Sex. According to a preferred embodiment, the
Fibrosis score is a Fibrometer.TM. or Fibrotest.TM. or
Fibrospect.TM. or Hepascore.
[0030] According to a specific embodiment, the markers of the score
may be selected depending on the fact that the liver condition is
of viral or alcoholic origin.
[0031] "Area Of Fibrosis" may be determined by image analysis, or
by a non invasive method wherein a score is obtained by measuring
in a sample of said patient and then combining in a logistic or
linear regression function, preferably in a multiple linear
regression function, at least two, preferably 3, more preferably 6
to 8 variables selected from the group consisting of .alpha.-2
macroglobulin (A2M), hyaluronic acid (HA or hyaluronate),
apoliprotein A1 (ApoA1), procollagen Type III-N-terminal propeptide
(P3P), gamma-glutamyltranspeptidase (GGT), bilirubin,
gamma-globulins (GLB), platelet count (PLT), prothrombin index
(PI), aspartate amino-transferase (AST), alanine amino-transferase
(ALT), urea, sodium (NA), glycemia, triglycerides, albumin (ALB),
alkaline phosphatases (ALP), human cartilage glycoprotein 39
(YKL-40), tissue inhibitor of matrix metalloproteinase 1 (TIMP-1),
matrix metalloproteinase 2 (MMP-2), ferritin, age, weight, body
mass index.
[0032] "Fractal dimension" reflects the liver architecture and may
be obtained by image analysis or by a non invasive method wherein a
score is obtained by measuring in a sample of an individual and
combining in a logistic or linear regression function (preferably a
multiple linear regression function) at least three, preferably 4
markers selected in the group comprising or consisting of .alpha.-2
macroglobulin (A2M), albumine (ALB), Prothrombin index (PI),
hyaluronic acide (HA ou hyaluronate), alanine amino-transferase
(ALAT), aspartate amino-transferase (ASAT) and age.
[0033] According to a preferred embodiment of the invention, the
fibrosis level is selected from the scores set forth in the table
below:
TABLE-US-00001 FibroMeter Virus Alcohol NAFLD Fibrosis Score Area
Score Area Score Area Age x x x Sex x Body weight x alpha2
macroglobulin x x x x Hyaluronate x x x x Prothrombin index x x x X
Platelet x x x x X AST x x X Urea x x GGT x x Bilirubin x ALT x X
Ferritin x Glycemia x x
[0034] This invention also relates to a non-invasive method for
assessing whether or not an individual is a fast fibroser,
including measuring the liver progression of said individual by
using a non-invasive method here above described, preferably by
calculating FL/cause duration and/or FL(t2)-FL
(t.sub.1)/t.sub.2-t.sub.1, wherein FL preferably is a fibrosis
score, an AOF score or a fractal dimension score. According to the
invention, the fast fibroser is identified with reference to
statistical data as having an increased AOF, younger inclusion age
and older start age (or cause duration replacing the two previous
variables) by stepwise binary logistic regression. According the
Applicant experiments, the diagnostic accuracy seems to be of
100.0% by stepwise binary logistic regression.
[0035] This invention also relates to a non-invasive method for
assessing if an individual is a slow, medium or fast fibroser using
discriminant analyses with reference to a population of fibrosers,
ranked from their fibrosis progression rate in three categories,
i.e. slow, medium and fast fibrosers: first, a method of assessing
the fibrosis progression, preferably by AOF progression, as
described above, is implemented, and the individual is ranked in
slow, medium, fast fibrosers categories determined by statistical
analysis. In the Example 2 below, cut-offs were 0.58 and 1.36%/yr
distinguishing slow (52.5%), medium (34.5%) and fast (12.9%)
fibrosers where AOF progression was: 0.42.+-.0.10, 0.81.+-.0.21 and
2.43.+-.0.81%/yr (p<10.sup.-3), respectively. Fibrosers,
preferably defined by AOF progression, are in agreement with
Fibrosis progression: 0.09.+-.0.06, 0.15.+-.0.06 and 0.43.+-.0.18
MU/yr (p<10.sup.-3), respectively slow, medium and fast
fibrosers. According to a preferred embodiment, the non-invasive
method here above described is preferably FL/cause duration or
FL(t2)-FL(t1)/t2-t1, wherein FL preferably is AOF score.
[0036] According to a fourth embodiment of the invention, liver
fibrosis progression is assessed by a score.
[0037] According to a first object, the invention relates to a
non-invasive method for assessing liver fibrosis progression in an
individual, said method comprising the steps of:
a) measuring in a sample of said individual, at least one,
preferably at least two, more preferably at least three, even more
preferably six to eight variables selected from the group
consisting of [0038] biological variables chosen among .alpha.-2
macroglobulin (.alpha.2M), Hyaluronic acid (HA), Apolipoprotein A1
(ApoA1), Type III procollagen N-terminal propeptide (P3P),
.gamma.-glutamyltranspeptidase (GGT), Bilirubin, .beta.-globulin,
.gamma.-globulin (GLB), Platelets (PLT), Prothrombin time (PT),
Prothrombin index (PI), Aspartate aminotransferase (AST), Alanine
aminotransferase (ALT), Urea, Sodium (NA), Glycemia, Triglycerides,
Albumin (ALB), Alkaline phosphatase (ALP), Human cartilage
glycoprotein 39 (YKL-40), Tissue inhibitor of matrix
metalloproteinase 1 (TIMP-1), Matrix metalloproteinase 2 (MMP-2),
Ferritin, TGF.beta.1, Laminin, .beta..gamma.-block, Haptoglobin,
C-Reactive protein (CRP) or Cholesterol, preferably chosen among
.alpha.-2 macroglobulin (.alpha.2M), Hyaluronic acid (HA), Type III
procollagen N-terminal propeptide (P3P),
.gamma.-glutamyltranspeptidase (GGT), .beta.-globulin, Platelets
(PLT), Prothrombin time (PT), Prothrombin index (PI), Aspartate
aminotransferase (AST), Alanine aminotransferase (ALT), Glycemia,
Triglycerides, Tissue inhibitor of matrix metalloproteinase 1
(TIMP-1) or .beta..gamma.-block, more preferably chosen among
.alpha.-2 macroglobulin (.alpha.2M), Hyaluronic acid (HA), Type III
procollagen N-terminal propeptide (P3P),
.gamma.-glutamyltranspeptidase (GGT), .beta.-globulin, Prothrombin
index (PI) or .beta..gamma.-block, [0039] complex biological
variables such as for example AST/ALT, [0040] clinical variables
chosen among Age at 1st contact "start age", Age, Cause duration,
Sex, Firm liver, Splenomegaly, Ascites, Collateral circulation,
Cause of CLD or Oesophageal varices (EV grade), preferably chosen
among Age at 1st contact, Age, Cause duration, Sex or Cause of CLD,
[0041] scores chosen among Metavir F stage, Area of fibrosis (AOF),
Fibrosis score (such as for example FibroMeter.TM., Fibrotest.TM.,
Fibrospect.TM., Fibroscan.TM., preferably FibroMeter.TM.), PGA
score, PGAA score, Hepascore, Aspartate-aminotransferase to
platelet ratio index (APRI) or European Liver Fibrosis (ELF), and
[0042] any combination thereof, b) combining the selected variables
in a mathematical function selected from the group consisting of
multiple linear regression function, a non-linear regression
function, or simple mathematic function such as arithmetic
operation, for example division.
[0043] According to a preferred embodiment, the method includes
combining at least two biological variables or at least two scores,
and at least one clinical variable selected from cause duration,
especially Chronic Liver Disease duration and age at first contact
with cause (also named "start age").
[0044] Preferably, the at least one clinical variable is cause
duration. Alternatively, the at least one clinical variable is age
at first contact with cause ("start age"). Preferably, the method
includes two clinical variables. According to a preferred
embodiment, the two clinical variables are cause duration and start
age.
[0045] Advantageously, the at least one score is selected from the
group consisting of Area of Fibrosis (AOF) and/or the Fibrosis
Score and/or Fractal dimension.
[0046] According to a first embodiment of the invention, the liver
fibrosis progression is assessed by measuring Metavir F
progression, said Metavir F progression being established by
measuring the following: [0047] biological variables chosen among
Type III procollagen N-terminal propeptide (P3P), Hyaluronic acid
(HA), Prothrombin index (PI), .gamma.-glutamyl transpeptidase (GGT)
or .beta..gamma.-block, [0048] the complex biological variable
AST/ALT, [0049] clinical variables chosen among Age at 1st contact,
Cause duration, [0050] scores chosen among Metavir F stage, Area of
fibrosis (AOF), PGA score, PGAA score or FibroMeter.TM., and [0051]
any combination thereof, and combining the selected variables in a
mathematical function selected from the group consisting of
multiple linear regression function, a non-linear regression
function, or simple mathematic function such as arithmetic
operation, for example division.
[0052] In this embodiment, preferably, the variable sex is not
selected.
[0053] In this embodiment, according to a first object, the
variables are: [0054] the biological variable Prothrombin index
(PI), [0055] the complex biological variable AST/ALT, [0056] the
clinical variable Cause duration, [0057] the score Metavir F stage,
and
[0058] any combination thereof.
[0059] In this embodiment, according to a second object, the
variables are: [0060] the complex biological variable AST/ALT,
[0061] clinical variables chosen among Cause duration or Age at 1st
contact, [0062] the score FibroMeter.TM., and [0063] any
combination thereof.
[0064] In this embodiment, according to a third object, the
variables are: [0065] the complex biological variable AST/ALT,
[0066] clinical variables chosen among Cause duration, and [0067]
any combination thereof.
[0068] According to a second embodiment of the invention, the liver
fibrosis progression is assessed by measuring the area of fibrosis
(AOF) progression, said AOF progression being established by
measuring the following: [0069] biological variables chosen among
.alpha.-2 macroglobulin (.alpha.2M), Hyaluronic acid (HA),
.beta.-globulin, Prothrombin index (PI) or .beta..gamma.-block,
[0070] the complex biological variable AST/ALT, [0071] clinical
variables chosen among Age at 1st contact, Age, Cause duration,
Sex, Firm liver, Splenomegaly, Ascites, Collateral circulation or
Cause of CLD, [0072] scores chosen among Metavir F stage, Area of
fibrosis (AOF), FibroMeter.TM., PGA score or PGAA score, and [0073]
any combination thereof, and [0074] combining the selected
variables in a mathematical function selected from the group
consisting of multiple linear regression function, a non-linear
regression function, or simple mathematic function such as
arithmetic operation, for example division.
[0075] In this second embodiment, according to a first object, the
variables are: [0076] the biological variable .beta.-globulins,
[0077] the complex biological variable AST/ALT, [0078] the clinical
variable Cause duration, [0079] the score Area of fibrosis (AOF),
and [0080] any combination thereof.
[0081] In this second embodiment, according to a second object, the
variables are: [0082] the biological variable .beta.-globulins,
[0083] the complex biological variable AST/ALT, [0084] the clinical
variable Cause duration, [0085] the score Metavir F stage, and
[0086] any combination thereof.
[0087] In this second embodiment, according to a third object, the
variables are: [0088] biological variables chosen among
.beta.-globulins or Prothrombin index (PI), [0089] the complex
biological variable AST/ALT, [0090] clinical variables chosen among
Cause duration or Firm liver, and [0091] any combination
thereof.
[0092] In this second embodiment, according to a fourth object, the
variables are: [0093] biological variables chosen among
.beta.-globulins or Prothrombin index (PI), [0094] the complex
biological variable AST/ALT, [0095] clinical variables chosen among
Age at 1.sup.st contact, Cause duration or Firm liver, and [0096]
any combination thereof.
[0097] In this second embodiment, according to a fourth object, the
variables are: [0098] biological variables chosen among
.beta.-globulins or .alpha.-2 macroglobulin (.alpha.2M), [0099] the
complex biological variable AST/ALT, [0100] clinical variables
chosen among Age at 1.sup.st contact or Cause duration, and [0101]
any combination thereof.
[0102] According to a particular embodiment, the non-invasive
method of the invention includes at least two fibrosis scores,
measured at regular intervals, such as for example, every 10-day,
every month, or every year.
[0103] According to the invention, the individual may be at risk of
suffering or is suffering from a condition selected from the group
consisting of a chronic liver disease, a hepatitis viral infection,
an hepatoxicity, a liver cancer, a non alcoholic fatty liver
disease (NAFLD), an autoimmune disease, a metabolic liver disease
and a disease with secondary involvement of the liver.
[0104] Hepatitis viral infection may be caused by a virus selected
from the group consisting of hepatitis C virus, hepatitis B virus
and hepatitis D virus. Hepatoxicity may be alcohol induced
hepatoxicity and/or drug-induced hepatoxicity (i.e. any xenobiotic
like alcohol or drug). According to the invention, autoimmune
disease is selected from the group consisting of autoimmune
hepatitis (AIH), primary biliary cirrhosis (PBC) and primary
sclerosing cholangitis (PSC). Metabolic liver disease may be
selected from the group consisting of Hemochromatosis, Wilson's
disease and alpha 1 anti trypsin. Secondary involvement of the
liver may be celiac disease or amyloidosis.
[0105] Other objects, advantages and features of the present
invention will become more apparent upon reading of the following
non restrictive description of preferred embodiments thereof, given
by way of examples with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
[0106] FIGS. 1-7 are to be read with regard to Example 1.
[0107] FIG. 1 is a graph showing the correlation of progression
rates between Metavir F and area of fibrosis (rs=0.77,
r.sub.p=0.90, p<10.sup.-4) as a function of Metavir fibrosis (F)
stage. r.sub.s is the coefficient of correlation of Spearman;
r.sub.p is the coefficient of correlation of Pearson.
[0108] FIG. 2 is a graph showing the progression rate of fibrosis
as a function of Metavir F stage. The progression rate of Metavir F
(F) or area of fibrosis (AOF) is correlated to Metavir F stages
(r.sub.s=0.58, p<10.sup.-4, r.sub.s=0.49, p<10.sup.-4,
respectively) and significantly different as a function of Metavir
F grade (ANOVA: p<10.sup.-4, p=0.001, respectively).
[0109] FIG. 3 is a graph showing the fibrosis progression rates for
Metavir F (3A) and AOF (3B) in alcoholic and viral chronic liver
disease (CLD). Transition lines are drawn only to show the
differences between patient groups.
[0110] FIG. 4 is a graph showing the AOF as a function of cause
duration according to CLD cause (alcoholic in black and viral in
grey) and to Metavir F stage.
[0111] FIG. 5 is the AOF progression rate as a function of cause
duration according to Metavir fibrosis (F) stage. The curve has an
inverse shape (1/x) by definition.
[0112] FIG. 6 is the relationship between fibrosis progression
rate, Metavir fibrosis stage (6A) and AOF (6B) and age at 1st
contact. Lines are provided by polynomial regression. The axis of
AOF progression was truncated at 3.
[0113] FIG. 7 is the effects of antifibrotic treatment on area of
fibrosis and Metavir F stage. Box plots indicate median, quartiles
and extremes.
FIGS. 8-16 are to be read with regard to Example 2.
[0114] FIG. 8 is a graph showing the correlation between Metavir
fibrosis (F) stage and area of fibrosis (AOF) progression in
populations 1 (panel a) and 2 (panel b) of Example2 Lines depict
linear regression.
[0115] FIG. 9 is a graph showing relationship between Metavir
fibrosis (F) stage (left panels) or area of fibrosis (AOF)
progression (right panels), during cause duration, as a function of
Metavir fibrosis (F) stage at inclusion age in populations 1 (top
panels) and 2 (bottom panels).
[0116] FIG. 10 is a graph showing the correlation between Metavir
fibrosis (F) stage (left panels) or area of fibrosis (AOF)
progression (right panels) and respective predicted progression in
populations 1 (top panels, alcoholic CLD only) and 2 (bottom
panels, viral CLD).
[0117] FIG. 11 shows the relationship between Metavir fibrosis (F)
stage (left panels) or area of fibrosis (AOF) (right panels) and
cause duration in populations 1 (top panels) and 2 (bottom panels).
Curves depict Lowess regression.
[0118] FIG. 12 shows the Relationship between Metavir fibrosis (F)
stage (left panels) or area of fibrosis (AOF) (right panels)
progression and start age in populations 1 (top panels) and 2
(bottom panels). Curves depict Lowess regression.
[0119] FIG. 13 shows the relationship between Metavir fibrosis (F)
stage (left panels) or area of fibrosis (AOF) (right panels) and
start age in populations 1 (top panels) and 2 (bottom panels).
Curves depict Lowess regression.
[0120] FIG. 14 shows the relationship between Metavir fibrosis (F)
stage progression (left panels) or area of fibrosis progression
(medium panels) or area of fibrosis (right panels) and inclusion
age in populations 1 (top panels) and 2 (bottom panels). Curves
depict Lowess regression.
[0121] FIG. 15 shows the relationship between fibrosis
characteristics and cause duration showing different fibrosers as a
function of fibrosis progression in population 2. Curves depict
Lowess regression.
[0122] FIG. 16 shows the impact of special patient subgroups on
curves of Metavir fibrosis (F) stage as a function of different
times in population 2. The impact was determined according to the
method shown in FIG. 11a.
EXAMPLE 1
Methods
1. Patients
Populations
[0123] All 201 patients included in this study were admitted to the
hepatogastroenterology unit of the University hospital in Angers,
France. A 1.sup.st population of 185 patients (all of which had
been subjected to one liver biopsy) was selected according to the
availability of an estimation of the contact date (or exposure) to
the risk factor (or cause) of CLD. The difference between inclusion
date and contact date is herein called "duration of cause". A
2.sup.nd population of 16 patients (all of which had been subjected
to two liver biopsies) was selected.
Population 1
[0124] The 185 patients included in this population were admitted
for alcoholic liver disease, or for chronic viral hepatitis B or C.
Patients were included who had drunk at least 50 g of alcohol per
day for the past five years or were positive for serum hepatitis B
surface antigen or C antibodies. None of the patient had clinical,
biological, echographic or histological evidence of other causes of
chronic liver disease (Wilson's disease, hemochromatosis,
.alpha.1-antitrypsin deficiency, biliary disease, auto-immune
hepatitis, hepatocellular carcinoma). Blood samples were taken at
entry and a transcostal (suction needle) or transjugular (cutting
needle) liver biopsy was performed within one week.
[0125] These patients might have had liver decompensation and
different CLD causes. In fact, the duration of cause was recorded
in only 179 patients but in the 6 other patients with Metavir F
stage 0, the rate of Metavir F progression could be fixed at 0 by
definition. However, the area of fibrosis could be measured in only
153 patients due to specimen fragmentation in 26 patients whereas
the progression rate could not be fixed in the 6 patients with
Metavir F stage 0 since baseline area of fibrosis is not null. The
date of 1.sup.st exposure was estimated according to the recording
of 1.sup.st blood transfusion or drug abuse in viral CLD and the
1.sup.st date of chronic excessive alcohol intake in alcoholic CLD.
This population allowed calculating an estimated progression rate
of fibrosis. In addition, explanatory variables of progression were
recorded a posteriori.
Population 2
[0126] These 16 patients had two liver biopsies, different CLD
causes and 10 underwent putative antifibrotic treatment like
interferon and sartan between both biopsies. This population
allowed measuring an observed progression rate of fibrosis. In
addition, explanatory variables of progression were recorded a
priori, thus being true predictive factors.
2. Clinical Evaluation
[0127] A full clinical examination was performed by a senior
physician. The recorded variables were: age, age at 1.sup.st
contact to the cause of liver disease (available only for alcoholic
patients and in C hepatitis attributed to blood transfusion and
drug abuse), sex, size, body weight (before an eventual
paracenthesis), mean alcohol consumption (g/d) before eventual
withdrawal, duration of alcohol abuse, alcohol withdrawal, duration
of alcohol withdrawal, known duration of liver disease (since the
first clinical or biochemical abnormality suggestive of CLD),
Child-Pugh score and other clinical abnormalities. Population 1
underwent also an upper gastro-intestinal endoscopy to evaluate
signs of portal hypertension and liver Doppler-ultrasonography.
3. Blood Tests
[0128] Analyses of blood samples provided the following
measurements: hemoglobin, mean corpuscular volume, lymphocyte
count, platelet count, cholesterol, urea, creatinine, sodium (NA),
bilirubin, .gamma.-glutamyltranspeptidase (GGT), alkaline
phosphatases (ALP), aspartate aminotransferase (AST) and alanine
aminotransferase (ALT), albumin (ALB), .alpha.1 and
.alpha.2-globulins, .beta.-globulins, .gamma.-globulins,
.beta..gamma.-block, prothrombin index (PI), apolipoprotein A1
(ApoA1). Some of them are indirect blood markers of fibrosis
(1).
[0129] The direct blood markers of fibrosis used in this study were
the following: .alpha.-2-macroglobulin (A.sub.2M), the N-terminal
peptide of type III procollagen (P3P), hyaluronic acid (HA), TGF
.beta.1, and laminin. The following blood tests were calculated:
AST/ALT ratio, PGA score (2), PGAA score (3), APRI (4), different
FibroMeters (5), and Hepascore (6). Sera were kept at -80.degree.
C. for a maximum of 48 months for assay.
4. Liver Histological Assessment
Microscopic Analysis
[0130] Biopsy specimens were fixed in a formalin-alcohol-acetic
solution and embedded in paraffin; 5 .mu.m thick sections were
stained with haematoxylin-eosin-saffron and 0.1% picrosirius red
solution. Fibrosis was staged by two independent pathologists
according to the Metavir staging (7). The Metavir staging is also
well adapted to the semi-quantitative evaluation of fibrosis in
alcoholic CLD since porto-septal fibrosis is more frequent and
developed than centrolobular fibrosis (8). Observers were blinded
for patient characteristics. When the pathologists did not agree,
the specimens were re-examined under a double-headed microscope to
analyse discrepancies and reach a consensus. All specimens were
also evaluated according to the following grades: Metavir activity
(7), steatosis and centrolobular fibrosis (CLF) as previously
described (9).
Image Analysis
[0131] AOF was measured on the same sections as the microscopic
analysis using a Leica Quantimet Q570 image processor as previously
described (9). Fractal dimension of fibrosis was also measured in
population 2 (10).
5. Observers
[0132] Overall there were 2 pathologists with 1 senior expert and 1
junior expert working in academic hospital. Image analysis was
performed by the junior expert pathologist experienced in this
technique.
6. Statistical Analysis
[0133] Quantitative variables were expressed as mean.+-.SD, unless
otherwise specified. The Pearson's rank correlation coefficient
(r.sub.p) was used for correlations between continuous variables or
Spearman correlation coefficient (r.sub.s) when necessary. To
assess independent predictors, multiple linear regression for
quantitative dependent variables and binary logistic regression for
qualitative dependent variables were used with forward stepwise
addition of variables. The predictive performance of each model is
expressed by the adjusted R.sup.2 coefficient (.sub.aR.sup.2) and
by the diagnostic accuracy, i.e. true positives and negatives,
respectively. A .alpha. risk <5% for a two-sided test was
considered statistically significant. The statistical software used
was SPSS version 11.5.1 (SPSS Inc., Chicago, Ill., USA).
7. Example of Mathematical Function
[0134] The estimation of the progression rate (PR) is provided by
multiple linear regression according to the following formula:
PR=a.sub.0+a.sub.1x.sub.1+a.sub.2x.sub.2+ . . . , where a.sub.x is
the coefficient of marker or variable x.sub.x and a.sub.0 is a
constant.
[0135] An example of formula for the PR of area of fibrosis is the
predictive model including AST/ALT, cause duration, firm liver,
.beta.-globulins, and FibroMeter.TM. where the coefficients are the
followings:
[0136] Constant: -0.0978158087539 with limits of confidence
interval at 95%: 0.8363614252041 & -1.035103236918,
[0137] AST/ALT: 0.5412244415007 with limits of confidence interval
at 95%: 2.07804027617.e-006 & 0.3283727153579,
[0138] Cause duration: -0.07623687627859 with limits of confidence
interval at 95%: 5.016575306101.e-011 & -0.09671608407235,
[0139] Firm liver: 0.7172332316927 with limits of confidence
interval at 95%: 0.006563850544752 & 0.2047931685256,
[0140] .beta.-globulins: 0.1594071294621 with limits of confidence
interval at 95%: 0.001915414369681 & 0.06022006972876,
[0141] FibroMeter.TM.: 1.15299980586 with limits of confidence
interval at 95%: 0.002487655344947 & 0.4161078148282.
Results
1. General Characteristics
[0142] The general characteristics of different populations are
presented in table 1.
TABLE-US-00002 TABLE 1 Main characteristics of populations
Population 1 2 n 185 16 Age (y) 48.5 .+-. 12.3 44.5 .+-. 10.4 Sex
(% M) 67.6 62.5 Cause (% virus) 26.5 75.0 Metavir F (%): 0 9.7 18.8
1 18.9 31.3 2 15.1 25.0 3 8.1 6.2 4 48.1 18.8 Complication (%) 21.6
12.5
2. Main Characteristics of Fibrosis Progression
[0143] There were calculated in population 1. The rate of
progression, expressed in Metavir unit (MU) per year, ranged from 0
to 2.0 MU/yr for Metavir F (mean: 0.22.+-.0.29, median: 0.13) and
from 0.1 to 17.2%/yr for the area of fibrosis (mean: 1.8.+-.2.6,
median: 1.0).
[0144] Both fibrosis progression rates were highly correlated (FIG.
1). The progression rate of fibrosis increased as a function of
fibrosis F stage (FIG. 2). We then tested the other factors linked
to the progression of fibrosis.
3. Predictive Factors of Fibrosis Progression
Metavir F Progression
[0145] The most marked correlations of Metavir F progression were
observed with Metavir F stage (r=0.33, p<10.sup.-4), the area of
fibrosis (r=0.28, p<10.sup.-4), age at 1.sup.st contact
(r=0.46), cause duration (r=-0.48, p<10.sup.-4), P3P (r=0.26,
p<10.sup.-4), HA (r=0.27, p<10.sup.-4), PI (r=-0.22,
p<10.sup.-4), GGT (r=0.32, p<10.sup.-4), AST/ALT (r=0.38,
p<10.sup.-4), FibroMeter.TM. (r=0.27, p<10.sup.-4), PGA score
(r=0.27, p<10.sup.-4) and PGAA score (r=0.28, p<10.sup.-4).
The only significant links with qualitative variables were observed
with .beta..gamma.block (p=0.03) and sex (p=0.001).
[0146] With linear regression, the independent predictors of the
Metavir F progression were: AST/ALT, cause duration, Metavir F
stage and PI (.sub.aR.sup.2=0.605). CLD cause had no independent
role (p=0.63). If Metavir F stage was removed, there was no
pathological variable in the predictive model: cause duration,
AST/ALT, age at 1.sup.st contact, and FibroMeter.TM.
(.sub.aR.sup.2=0.488). It should be noted that "age at 1.sup.st
contact"+"cause duration"=age, however if the two former were
removed, the latter was not selected, while .sub.aR.sup.2 decreased
to 0.195 with AST/ALT and sex.
Area of Fibrosis Progression
[0147] The most marked correlations of the area of fibrosis
progression were observed with Metavir F stage (r=0.32,
p<10.sup.-4), the area of fibrosis (r=0.41, p<10.sup.-4), age
at 1.sup.st contact (r=0.43), cause duration (r=-0.43,
p<10.sup.-4), HA (r=0.34, p<10.sup.-4), PI (r=-0.24,
p<10.sup.-4), .beta.-globulins (r=0.32, p<10.sup.-4), AST/ALT
(r=0.51, p<10.sup.-4), FibroMeter.TM. (r=0.29, p<10.sup.-4),
PGA score (r=0.29, p<10.sup.-4) and PGAA score (r=0.30,
p<10.sup.-4). Several significant links with qualitative
variables were observed: .beta..gamma.-block (p=0.004), sex
(p=0.004), firm liver (p=0.04), splenomegaly (p=0.02), ascites
(p=0.001), EV grade (p=0.04), collateral circulation (p=0.001) and
the cause of CLD (p=0.03).
[0148] With linear regression, the independent predictors of the
area of fibrosis progression were: AST/ALT, cause duration, area of
fibrosis, and .beta.-globulins (.sub.aR.sup.2=0.716). It should be
noted that steatosis had a borderline signification (p=0.057) but
not activity (p=0.53) and CLD cause (p=0.39). If the area of
fibrosis was removed, the Metavir F stage took its place in the
model (.sub.aR.sup.2=0.689) and if Metavir F stage was removed,
i.e. without any pathological variables, the predictive model
included AST/ALT, cause duration, firm liver, .beta.-globulins, and
PI (.sub.aR.sup.2=0.643). If "cause duration" was removed, "age at
1.sup.st contact" took its place in the model (.sub.aR.sup.2=0.643)
and if "age at 1.sup.st contact" stage was removed, the model
included objective variables: AST/ALT, age, .beta.-globulins and
A2M with .sub.aR.sup.2=0.509.
4. Kinetics of Fibrosis Progression
Estimated Progression (Population 1)
[0149] FIG. 3 shows a progressive but irregular increase in
fibrosis rate as a function of Metavir F stage. As expected, the
progression rate of metavir F stage was more linked to F stage than
did the area of fibrosis as also reflected by correlation
coefficients (r.sub.s=0.58 and 0.49, respectively, p<10.sup.-4).
FIG. 3 shows a rather stable progression rate of area of fibrosis
from F stage 0 to 3 and a dramatic increase in patients with
cirrhosis whereas the increase was progressive through all F stages
for progression rate of Metavir F stage.
[0150] The correlation between the area of fibrosis and cause
duration was weak (r.sub.p=0.32, p<10.sup.-4). In fact, FIG. 4
shows that the area of fibrosis as a function of cause duration
markedly varied among patients, so patients might develop cirrhosis
within a short period and others after a prolonged period. However,
all patients with the fastest rate, as expected, and those with the
longest follow-up, as less expected, had cirrhosis. A short cause
duration was surprising in cirrhosis, however this was mainly
observed in alcoholic CLD. Moreover, patient age was significantly
lower when cause duration was <15 yr: 45.5.+-.8.9 vs
55.0.+-.10.2 yr for 15 yr (p=0.002) in alcoholic CLD whereas the
figures were similar in viral CLD: 54.4.+-.14.4 vs 56.6.+-.15.2 yr
(p=0.81), respectively. This figure also does not suggest
particular groups of patients according to progression rate.
[0151] The graph of AOF progression plotted against cause duration
(FIG. 5) clearly shows that individual patients had different
patterns of progression rate of area of fibrosis within each F
stage. In fact, previous multivariate analyses indicated that
"cause duration" or "age at 1.sup.st contact" was the main clinical
independent predictor of Metavir F or area of fibrosis progression.
FIG. 6 shows that the F progression dramatically increased by 40
years in viral and alcoholic CLD. However, the AOF progression
displayed a linear increase over age in alcoholic CLD whereas there
was a plateau followed by a linear increase by 40 years in viral
CLD.
Observed Progression (Population 2)
[0152] The mean interval between biopsies (follow-up duration) was
4.1.+-.2.6 years in the whole group and 4.8.+-.2.5 in the 6
patients without treatment compared to 3.6.+-.2.6 (p=0.38) in the
10 patients with anti-fibrotic treatment between the 2 liver
biopsies. The yearly rate of progression in untreated patients was
for Metavir F: mean: 0.17.+-.0.27, median: 0.09 MU and for the area
of fibrosis: mean: 1.3.+-.3.4, median: 1.2%. These values were not
significantly different than those estimated (p=0.66 for F and
p=0.72 for AOF).
[0153] AOF was far more sensitive than Metavir F stage to detect
effects of anti-fibrotic treatment: percent changes in AOF: p=0.03,
progression rate of AOF: p=0.09; percent changes in F stage:
p=0.85, progression rate of F stage: p=0.71 (by Mann-Whitney test,
FIG. 10) or proportion of F stage increase: p=0.61 (by McNemar
.chi..sup.2 test).
EXAMPLE 2
[0154] Fibrosis progression was calculated as the ratio fibrosis
level/cause duration, with fibrosis level indicating stage or
amount AOF. So, this is a mean value as a function of time. As the
main aim was to precisely describe fibrosis progression, through
the amount of fibrosis reflected by the AOF, we used LB as
reference for fibrosis level determination and we chose for the non
invasive diagnosis a blood test that can both evaluate fibrosis
staging and AOF (14). For time recording, we used two descriptors
of fibrosis progression: the progression rate and the progression
course. The progression rate is a mean as a function of cause
duration, cause duration being the time between the age when the
cause started ("start age") and the age at inclusion when fibrosis
level was measured ("inclusion age"). Progression course is the
trend as a function of time (increase, stability, decrease). Thus,
according to the methods used for fibrosis determination (LB or
non-invasive test) and duration recording
(retrospective/transversal or prospective/longitudinal), we
distinguished 4 methods to calculate fibrosis progression. Their
characteristics, advantages and limits are detailed in table 2.
Because the availability of these methods has markedly evolved as a
function of time, we had to indirectly compare them by collecting
different populations in our database.
Patients
[0155] Population Aims (table 3)
[0156] 5 populations including 1456 patients were used. All
patients included in this study were admitted to the
Hepatogastroenterology unit of the University hospital in Angers,
France, except in population 3 that is described elsewhere
(15).
[0157] Populations 1 and 2 were selected according to the
availability of estimation of the age when the cause started
("start age"). The period between start age and age at inclusion
when fibrosis level was measured ("inclusion age"), was called
"cause duration". Population 1 provided comparison between
alcoholic and viral CLD. Population 2 with viral CLD had a
sufficient high number of patients to validate the previous viral
subpopulation and to allow subgroup analysis. Population 3 was a
large population with viral CLD providing a validation of inclusion
age effect. Population 4 allowed validating in patients with 2 LB
the previous progression estimated with 1 LB. Finally, population 5
was used to validate the progression calculated with two blood
tests.
Population Characteristics (Table 4)
[0158] Population 1--It included 185 patients with alcoholic CLD or
chronic hepatitis B or C between 1994 and 1996. This population is
detailed elsewhere (16). The date of 1.sup.st cause exposure was
estimated according to the 1.sup.st date of chronic excessive
alcohol intake for alcoholic CLD and the recording of 1.sup.st
blood transfusion or drug abuse for viral CLD. These patients might
have liver decompensation. In fact, the cause start was recorded in
only 179 patients but in 6 other patients with Metavir F stage 0,
the rate of Metavir F progression could be fixed at 0 by
definition. However, the AOF could be measured in only 153 patients
due to specimen fragmentation in 26 patients whereas the
progression could not be fixed in the 6 patients with Metavir F
stage 0 since baseline AOF is not null.
[0159] Population 2--It included 157 patients with chronic
hepatitis C between 1997 and 2002 detailed elsewhere (14). Mean
inclusion age was 43.4.+-.12.4 yr and 59.4% of patients were
male.
[0160] Population 3--It included 1056 patients with chronic
hepatitis C, LB recruited in 9 French centres between 1997 and 2007
detailed elsewhere (15). Mean age was 45.4.+-.12.5 yr at inclusion
and 59.6% of patients were male.
[0161] Population 4--It included 16 patients with various causes of
CLD having two LB between 1997 and 2002 and different CLD
causes.
[0162] Population 5--It included 42 patients with chronic hepatitis
C between 2004 and 2008. The blood tests were yearly measured for
2.4.+-.0.5 yr.
Clinical Evaluation and Blood Tests
[0163] A full clinical examination was performed by a senior
physician. The main clinical variables recorded were: inclusion
age, start age, sex and CLD cause. Other variables are described
elsewhere (14-16). Analyses of blood samples provided the usual
variables as well as direct blood markers of fibrosis to calculate
blood fibrosis tests. Thus, blood tests were calculated to estimate
either fibrosis stage or AOF (14).
Liver Histological Assessment (Populations 1, 2 and 4)
[0164] Microscopic analysis--Biopsy specimens were fixed in a
formalin-alcohol-acetic solution and embedded in paraffin; 5 .mu.m
thick sections were stained with haematoxylin-eosin-saffron and
0.1% picrosirius red solution. Fibrosis was staged by two
independent pathologists, blinded for patient characteristics,
according to the Metavir staging (6). The Metavir staging is also
well adapted to the semi-quantitative evaluation of fibrosis in
alcoholic CLD (17). In case of discrepancy, the specimens were
re-examined under a double-headed microscope to reach a
consensus.
[0165] Image analysis--AOF was measured on the same sections as the
microscopic analysis using either a Leica Quantimet Q570 image
processor as previously described from 1996 to 2006 (10) or an
Aperio digital slide scanner (Scanscope.RTM. CS System, Aperio
Technologies, Vista Calif. 92081, USA) image processor providing
high quality images of 30,000.times.30,000 pixels and a resolution
of 0.5 .mu.m/pixel (magnification .times.20) since 2007. A binary
image (white and black) was obtained via an automatic thresholding
technique using an algorithm developed in our laboratory.
[0166] Observers--Overall there were 2 pathologists with 1 senior
expert and 1 junior expert working in academic hospital. Image
analysis was performed by the junior expert pathologist experienced
in this technique (17) or by an engineer for the fully automated
system.
Statistical Analysis
[0167] Quantitative variables were expressed as mean.+-.SD, unless
otherwise specified. The Pearson's rank correlation coefficient
(r.sub.p) was used for correlations between continuous variables or
the Spearman correlation coefficient (r.sub.s) when necessary. The
Lowess regression by weighted least squares was used to determine
the average trend of relationships between variables, mainly the
progression course (18). The line rupture observed in these curves
were checked by cut-offs determined according to maximum Youden
index and diagnostic accuracy (data not shown). The curve shape was
evaluated by corresponding test, e.g. quadratic trend test. To
assess independent predictors, multiple linear regression for
quantitative dependent variables, binary logistic regression for
qualitative dependent variables and discriminant analysis for
ordered variables were used with forward stepwise addition of
variables. The prediction of each model is expressed by the
adjusted R.sup.2 coefficient (.sub.aR.sup.2) and/or by the
diagnostic accuracy, i.e. true positives and negatives,
respectively. An .alpha. risk <5% for a two-sided test was
considered statistically significant. The statistical software used
was SPSS version 11.5.1 (SPSS Inc., Chicago, Ill., USA).
Results
General Characteristics
[0168] The general characteristics of core populations 1 and 2 are
presented in table 4. In population 1, variables at baseline
(inclusion) were significantly different between alcoholic and
viral causes, except for start age. Baseline variables were not
significantly different between viral populations 1 and 2. It
should be noted that the start age was similar between populations
whereas the inclusion age was significantly older in alcoholic CLD
which was responsible to a longer cause exposure.
Overall Description of Fibrosis Progression
Retrospective Measurement
[0169] Population 1--The progression, expressed in Metavir unit
(MU) per year, ranged from 0 to 2.0 MU/yr for Metavir F (mean:
0.22.+-.0.29, median: 0.13) and from 0.1 to 17.2%/yr for the AOF
(mean: 1.8.+-.2.6, median: 1.0). Both fibrosis progressions were
highly correlated (r.sub.p=0.90, p<10.sup.-4, FIG. 8a). The
fibrosis progression increased as a function of fibrosis Fstage
(FIGS. 9a and 9b). The AOF progression was significantly faster in
alcoholic CLD than in viral CLD but not that of Metavir F (table
4).
[0170] Population 2--The rate of progression, expressed in Metavir
unit (MU) per year, ranged from 0 to 0.8 MU/yr for Metavir F (mean:
0.16.+-.0.14, median: 0.11) and from 0.2 to 4.5%/yr for the AOF
(mean: 0.8.+-.0.7, median: 0.6). AOF and F progressions were also
well correlated r.sub.p: 0.795 (p<10.sup.-3) (FIG. 8b). The
fibrosis progressions were significantly different according to F
stage (ANOVA, p<10.sup.-3) (FIGS. 9c and 9d). By Bonferroni post
hoc comparison, the progressions were significantly different
between each F stage for F progression (except between F2 and F3)
but only in F4 vs F1 and F3 for AOF progression.
[0171] Comparison as a function of sex (table 4)--In alcoholic
patients, F or AOF at inclusion were not significantly different
between females and males, but cause duration was significantly
shorter in females than in males. Consequently, the F or AOF
progression was significantly faster in females than in males in
alcoholic CLD. F or AOF at inclusion in population 2 were
significantly higher in males than in females, but cause duration
was not significantly different between males and females.
Consequently, and conversely to alcoholic CLD, the F or AOF
progression was significantly faster in males than in females in
viral CLD (significant in more numerous population 2).
[0172] Comparison as a function of cause (table 5)--F and AOF
progressions were dramatically and significantly increased in
alcoholic CLD compared to viral CLD only in females.
[0173] Comparison between viral populations--The AOF progression
were significantly higher in population 1 than in population (table
4); this can be due to difference in AOF technique since AOF was
significantly different or in populations since the F progression
tended to be different.
Prospective Measurement
[0174] Population 4--The mean interval between biopsies (follow-up
duration) was 4.1.+-.2.6 years. The yearly rate of progression was
for Metavir F: mean: 0.17.+-.0.27, median: 0.09 MU and for the area
of fibrosis: mean: 1.3.+-.3.4, median: 1.2%. These values were not
significantly different than those estimated in population 1
(p=0.481 for F and p=0.567 for AOF).
Course of Fibrosis Progression
[0175] We described the average trends in course of fibrosis
progression, as reflected by the plots of Lowess regression,
according to three variables linked to times: cause duration, age
at start cause and age at inclusion which is the sum of the two
formers. Age at start cause was correlated with cause duration in
population 1 (r.sub.p=-0.449, p<10.sup.-4), due to alcoholic
CLD, but not in population 2 (r.sub.p=-0.084 p=0.319). Particular
trends in extremes of plots have to be cautiously interpreted since
this could be due to a decreased robustness linked to fewer
patients.
[0176] Cause duration--In population 1, the cause duration was
weakly correlated with fibrosis level: F stage: r.sub.s=0.357,
p<10.sup.-3 (FIG. 11a), AOF: r.sub.s=0.316, p<10.sup.-3 (FIG.
11b). In population 2, the cause duration was weakly correlated
with F stage (r.sub.s: 0.241, p=0.004) (FIG. 11c) or AOF (r.sub.s:
0.201, p=0.018) with the same course in males and females (FIGS.
11c and 11d). All these figures show an unexpected decrease in the
first 15 years and thereafter a progressive increase.
[0177] Start age--FIG. 12a shows that the F progression
dramatically increased by 30-40 years of start age in alcoholic
(.apprxeq.40 years) and viral (.apprxeq.30 years) CLD (population
1). The latter figure was confirmed in population 2 especially in
men (FIG. 12c). This resulted in a progressive increase in F stage
with start age in viral CLD (FIG. 13c) but this was not observed in
alcoholic CLD (FIG. 13a) or in young patients with viral CLD
(explanation below). However, the AOF progression displayed an
almost linear increase over start age in alcoholic CLD whereas
there was a plateau followed by a linear increase by .apprxeq.40
years of start age in viral CLD (population 1) (FIG. 12b). This was
confirmed in population 2 especially in men (FIG. 12d). Globally,
the AOF was relatively stable a function of start age in population
1 (FIG. 13b) and 2 (FIG. 13d). However, there were some
peculiarities: a slow decrease in the first 20 years in males with
viral CLD in F stages (FIG. 13c) or AOF (FIG. 13d) as well as a
decrease by 40 yrs of start age in females (FIG. 13d).
[0178] Inclusion age--Considering F progression, in alcoholic CLD
there was a stable progression until 50 yr (FIG. 14a) then a
decrease whereas in viral CLD after a initial decrease below 35 yr,
especially in men, there was thereafter an increase (FIGS. 14a and
14d). Considering F level, the increase was linear with age in
alcoholic CLD and occurred by 40-50 yr in viral CLD (FIG. 15a).
Populations 2 and 3 stated that this increase occurred by age 40 yr
in males and 50 yr in females in viral CLD (FIGS. 15b and 15c).
There was an initial F decline in viral CLD (FIG. 15a), especially
in men (FIG. 15b) which was not confirmed in population 3 (FIG.
15c) but there were less young patients in this latter population
(as reflected by an older age: p=0.06).
[0179] AOF progression did not depend on the inclusion age in
alcoholic CLD (FIG. 14b) whereas there was a late increase in viral
CLD (FIGS. 14b and 14e). Consequently, the AOF level linearly
increased with age in alcoholic CLD (FIG. 14c) whereas this
occurred by age 50 yr in viral CLD (FIG. 14f).
[0180] Sex--We state here the particular relationship between sexes
and CLD cause since sex effect has been already mentioned in viral
CLD. Whereas there was a global parallelism between males and
females in viral CLD, females in alcoholic CLD had two
particularities: a slowdown between 30-50 yr and a late increase in
fibrosis progression and level by 50 yr of start age (data not
shown). The same differences were observed for inclusion age at the
difference that the slowdown was observed later between 45-50 yr,
as expected.
Times to Cirrhosis
[0181] In population 1, times to cirrhosis was 24.7.+-.13.3 yr in
alcoholic CLD vs 22.1.+-.15.9 yr in viral CLD (p=0.495) and
28.0.+-.12.5 yr in males vs 16.1.+-.11.4 yr (p=0.001) in females in
alcoholic CLD. In (viral) population 2, it was 17.0.+-.8.0 yr in
males vs 24.0.+-.10.0 yr (p=0.017) in females.
Non Invasive Evaluation
[0182] observed FibroMeter.TM. progression [(FibroMeter.TM.
t2-FibroMeter.TM. t1)/(t2-t1)] was 0.049.+-.0.058/yr in population
5 whereas the estimated FibroMeter.TM. progression (FibroMeter.TM.
t2/cause duration) was 0.038.+-.0.033/yr in population 2
(p=0.217).
Identifying Categories of Fibrosers
[0183] In population 2, it was possible to distinguish three
categories of fibrosers as a function of AOF progression (FIG. 15b)
rather on F progression (FIG. 15a). The cut-offs were 0.58 and
1.36%/yr distinguishing slow (52.5%), medium (34.5%) and fast
(12.9%) fibrosers where AOF progression was: 0.42.+-.0.10,
0.81.+-.0.21 and 2.43.+-.0.81%/yr (p<10.sup.-3), respectively.
Fibrosers, defined by AOF progression, were in agreement with F
progression: 0.09.+-.0.06, 0.15.+-.0.06 and 0.43.+-.0.18 MU/yr
(p<10.sup.-3), respectively slow, medium and fast fibrosers
(FIG. 15c). The start age increased with fibroser degree:
25.2.+-.10.5, 28.7.+-.10.8 and 33.0.+-.13.6 yr, respectively
(p<10.sup.-3). The proportion of males increased with fibroser
degree: 53.4%, 66.7% and 77.8%, respectively (p=0.034). By stepwise
discriminant analysis, fibrosers were predicted by Metavir F, AOF,
F progression and cause duration (diagnostic accuracy: 91.4%). The
fast fibrosers were predicted by increased AOF, younger inclusion
age and older start age with diagnostic accuracy: 100.0% by
stepwise binary logistic regression.
TABLE-US-00003 TABLE 2 Fibrosis evaluation Fibrosis evaluation
Fibrosis progression Method Technique Calculation.sup.a Description
Advantages Limits Single 1 biopsy FL/cause Transversal Availability
+ Linearity biopsy duration (retrospective) Start measurement
measurement estimation Repeated 2 (FLt2 - FLt1)/ Longitudinal
Precision Variability biopsy biopsies (t2 - t1) (prospective)
Reference Unavailability measurement measurement Short duration
Single non 1 test.sup.b FL/cause Transversal Availability Linearity
invasive duration (retrospective) ++ Start test estimation
estimation estimation Repeated 2 tests (FLt2 - FLt1)/ Longitudinal
Precision non (t2 - t1) (prospective) Repeatability invasive
estimation test estimation .sup.aFL is the fibrosis level and t is
the corresponding date .sup.bNon-invasive (blood test in the
present study)
TABLE-US-00004 TABLE 3 Main characteristics of different
populations used in this study. Pa- Popu- tients Fibrosis Area of
Duration Fibrosis lation Cause (n) evaluation fibrosis.sup.a Time
progression 1 Alcohol 185 1 LB, 1 Yes Cause Retrospective virus
blood duration.sup.b measurement + test estimation 2 Virus 157 1
LB, 1 Yes Cause Retrospective blood duration.sup.b measurement +
test estimation 3 Virus 1056 1 LB, 1 No No Retrospective blood
measurement + test estimation.sup.c 4 Miscell 16 2 LB Yes Follow-
Prospective aneous up measurement 5 Virus 42 0 LB, 2 No Follow-
Prospective blood up estimation tests .sup.aOn LB, .sup.bCause
duration = time between age at inclusion when liver fibrosis level
was measured and age at the start of the liver disease;
.sup.cLimited to the plot fibrosis level vs age.
TABLE-US-00005 TABLE 4 Clinical characteristics of populations 1
and 2. Population 1 Population 2 Cause Alcohol Virus p.sup.a Both
Virus p.sup.b N 136 49 -- 185 157 -- Age at inclusion 49.9 .+-.
11.2 44.2 .+-. 14.6 0.02 48.5 .+-. 12.3 43.4 .+-. 12.4 0.793 (yr)
Age at cause start 28.8 .+-. 9.5 28.2 .+-. 13.5 0.779 28.8 .+-.
10.8 27.4 .+-. 11.2 0.707 (yr) Cause duration 21.3 .+-. 13.2 15.8
.+-. 10.7 0.006 19.8 .+-. 12.9 16.5 .+-. 7.3 0.604 (yr) Sex (% M)
72.8 53.1 0.011 67.6 59.4 0.550 Cause (% virus) -- -- -- 26.5 100
-- Metavir F (%): 0.002 -- 0 9.6 10.2 9.7 10.3 0.998 1 14.0 32.7
18.9 33.5 0.886 2 13.2 20.4 15.1 25.8 0.419 3 6.6 12.2 8.1 11.0
0.303 4 56.6 24.5 48.1 19.4 0.414 Area of fibrosis 23.5 .+-. 14.7
13.6 .+-. 11.7 p < 10.sup.-3 20.7 .+-. 14.6 10.7 .+-. 6.5 0.005
(%) Complication (%) 29.4 0 p < 10.sup.-3 21.6 0 -- Progression
rate: Metavir F (MU/yr) 0.23 .+-. 0.32 0.19 .+-. 0.21 0.424 0.22
.+-. 0.29 0.16 .+-. 0.14 0.120 Area of fibrosis 2.0 .+-. 2.9 1.3
.+-. 1.4 0.027 1.8 .+-. 2.6 0.8 .+-. 0.7 0.017 (%/yr) .sup.aalcohol
vs virus; .sup.bvs viral population 1 NA: not available
TABLE-US-00006 TABLE 5 Fibrosis: data at inclusion and course as a
function of sex in populations 1 and 2. MALES FEMALES P.sup.A
POPULATION 1 AGE AT CAUSE START (YR) ALCOHOL 26.9 .+-. 8.1 34.1
.+-. 11.0 0.001 VIRUS 27.5 .+-. 15.0 29.0 .+-. 11.7 0.337 P 0.354
0.160 -- BOTH 27.0 .+-. 9.9 32.2 .+-. 11.5 0.001 AGE AT INCLUSION
(YR) ALCOHOL 50.6 .+-. 12.0 48.0 .+-. 8.4 0.358 VIRUS 42.2 .+-.
15.1 46.7 .+-. 13.6 0.400 P 0.001 0.680 -- BOTH 48.8 .+-. 13.1 47.5
.+-. 10.7 0.623 CAUSE DURATION (YR) ALCOHOL 23.9 .+-. 13.1 14.2
.+-. 11.0 <10.sup.-3 VIRUS 14.6 .+-. 9.3 17.2 .+-. 12.2 0.626 P
0.001 0.287 -- BOTH 22.0 .+-. 12.9 15.3 .+-. 11.4 0.001 METAVIR F
SCORE ALCOHOL 2.8 .+-. 1.5 2.9 .+-. 1.4 0.724 VIRUS 2.1 .+-. 1.3
2.1 .+-. 1.4 0.984 P 0.012 0.024 -- BOTH 2.7 .+-. 1.5 2.6 .+-. 1.5
0.737 F PROGRESSION (MU/YR) ALCOHOL 0.17 .+-. 0.23 0.41 .+-. 0.43
<10.sup.-3 VIRUS 0.20 .+-. 0.21 0.18 .+-. 0.22 0.609 P.sup.A
0.685 0.019 -- BOTH 0.17 .+-. 0.23 0.32 .+-. 0.38 0.011 AREA OF
FIBROSIS (%) ALCOHOL 22.9 .+-. 14.7 25.0 .+-. 14.8 0.483 VIRUS 14.3
.+-. 11.9 12.2 .+-. 11.4 0.199 P 0.014 0.001 -- BOTH 20.8 .+-. 14.5
20.2 .+-. 14.9 0.636 AOF PROGRESSION (%/YR) ALCOHOL 1.4 .+-. 1.8
3.5 .+-. 4.2 0.001 VIRUS 1.4 .+-. 1.4 1.1 .+-. 1.4 0.146 P 0.762
0.001 -- BOTH 1.4 .+-. 1.7 2.7 .+-. 3.6 0.106 POPULATION 2 AGE AT
CAUSE START (YR) 26.1 .+-. 10.9 29.4 .+-. 11.4 0.021 AGE AT
INCLUSION (YR) 41.8 .+-. 11.8 47.1 .+-. 13.1 0.015 CAUSE DURATION
(YR) 15.7 .+-. 6.8 17.7 .+-. 8.0 0.195 METAVIR F SCORE 2.3 .+-. 1.2
1.9 .+-. 1.2 0.030 F PROGRESSION (MU/YR) 0.18 .+-. 0.14 0.13 .+-.
0.13 0.004 AREA OF FIBROSIS (%) 11.4 .+-. 6.9 9.6 .+-. 5.8 0.018
AOF PROGRESSION (%/YR) 0.91 .+-. 0.74 0.67 .+-. 0.67 0.004
COMPARISON VIRAL POPULATIONS 1 AND 2 (P): AGE AT CAUSE START (YR)
0.665 0.888 -- AGE AT INCLUSION (YR) 0.903 0.903 -- CAUSE DURATION
(YR) 0.516 0.830 -- METAVIR F SCORE 0.473 0.549 -- F PROGRESSION
0.659 0.311 -- AREA OF FIBROSIS (%) 0.274 0.301 -- AOF PROGRESSION
0.095 0.213 -- .sup.aMann Whitney test
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[0221] Although the present invention has been described
hereinabove by way of preferred embodiments thereof, it can be
modified, without departing from the spirit and nature of the
subject invention as defined in the appended claims.
* * * * *