NON-INVASIVE METHOD FOR ASSESSING LIVER FIBROSIS PROGRESSION

Abstract

A method for implementing an adapted patient care for an individual suffering from liver fibrosis after assessing liver fibrosis progression in the individual, and thus determining whether the individual is a slow, medium or fast fibroser. Also, a method for treating an individual suffering from liver fibrosis and identified as a fast fibroser, which includes the steps of identifying the individual as a fast fibroser by assessing fibrosis progression and treating the individual by administering without delay at least one therapeutic agent for treating liver fibrosis, or for treating the underlying cause responsible for liver fibrosis, or both.

Claims

1. A method for treating an individual suffering from liver fibrosis comprising: assessing the fibrosis progression in said individual to determine whether said individual is a slow fibroser, a medium fibroser or a fast fibroser, by measuring at least three markers in an individual, wherein said markers are measured in a blood sample obtained from the individual and are selected from the group consisting of α-2 macroglobulin, hyaluronic acid, gamma-glutamyl transpeptidase, bilirubin, platelet count, prothrombin index, aspartate amino-transferase, alanine amino-transferase, urea, glycemia, and ferritin; and/or said markers are clinical markers selected from weight, age and sex; and combining said at least four measured markers in a logistic or linear regression function, thereby determining a fibrosis level in the individual; calculating a ratio of said fibrosis level to cause duration, thereby obtaining a value-useful for assessing the liver fibrosis progression in the individual; and implementing an adapted patient care depending on whether the subject is a slow fibroser, a medium fibroser or a fast fibroser.

2. The method of claim 1, wherein fibrosis progression is assessed by measuring at two different times t1 and t2 the fibrosis levels FL(t1) and FL(t2); and calculating a ratio FL(t2)−FL(t1) to cause duration, wherein cause duration is defined as (t2−t1).

3. The method according to claim 1, wherein the individual is determined to be a slow fibroser and wherein the adapted patient care consists in monitoring said individual by assessing the fibrosis level at regular intervals.

4. The method according to claim 1, wherein the individual is determined to be a medium fibroser and wherein the adapted patient care consists in monitoring said individual by assessing the fibrosis level at regular intervals and delaying treatment until the individual is determined to suffer from clinically significant liver fibrosis.

5. The method according to claim 1, wherein the individual is determined to be a fast fibroser and wherein the adapted patient care consists in administering without delay at least one therapeutic agent or starting a complication screening program for applying early prophylactic or curative treatment.

6. The method according to claim 5, wherein the at least one therapeutic agent is an antifibrotic agent selected from the group consisting of simtuzumab, GR-MD-02, stem cell transplantation (in particular MSC transplantation), Phyllanthus urinaria, Fuzheng Huayu, S-adenosyl-L-methionine, S-nitrosol-N-acetylcystein, silymarin, phosphatidylcholine, N-acetylcysteine, resveratrol, vitamin E, losartan, telmisartan, naltrexone, RF260330, sorafenib, gleevec, nilotinib, INT747, FG-3019, oltipraz, pirfenidone, halofuginone, polaorezin, gliotoxin, sulfasalazine, rimonabant and combinations thereof.

7. The method according to claim 5, wherein the at least one therapeutic agent is for treating the underlying cause responsible for liver fibrosis, and/or ameliorating or alleviating the symptoms associated with the underlying cause responsible for liver fibrosis, including liver fibrosis.

8. The method according to claim 6, wherein the underlying cause responsible for liver fibrosis is a viral infection and the at least one therapeutic agent is selected from the group consisting of interferon, peginterferon 2b (pegylated IFNalpha-2b), infliximab, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, daclatasvir, elbasvir, grazoprevir, velpatasvir, lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir, clevudine, ANA380, zadaxin, CMX 157, ARB-1467, ARB-1740, ALN-HBV, BB-HB-331, Lunar-HBV, ARO-HBV, Myrcludex B, GLS4, NVR 3-778, AIC 649, JNJ56136379, ABI-H0731, AB-423, REP 2139, REP 2165, GSK3228836, GSK33389404, RNaseH Inhibitor, GS 4774, INO-1800, HB-110, TG1050, HepTcell, TomegaVax HBV, RG7795, SB9200, EYP001, CPI 431-32 and combinations thereof.

9. The method according to claim 6, wherein the underlying cause responsible for liver fibrosis is excessive alcohol consumption and the at least one therapeutic agent is selected from the group consisting of topiramate, disulfiram, naltrexone, acamprosate and baclofen.

10. The method according to claim 6, wherein the underlying cause responsible for liver fibrosis is a non-alcoholic fatty liver disease (NAFLD) and the at least one therapeutic agent is selected from the group consisting of orlistat, metformin, pioglitazone, atorvastatin, ezetimine, vitamin E, sylimarine, pentoxyfylline, ARBs, EPL, EPA-E, multistrain biotic (L. rhamnosus, L. bulgaricus), simtuzumab, obeticholic acid, elafribanor (GFT505), DUR-928, GR-MD, 02, aramchol, RG-125, cenicriviroc CVC and combinations thereof.

11. A method for treating an individual suffering from liver fibrosis comprising: assessing the fibrosis progression in said individual to determine whether said individual is a slow fibroser, a medium fibroser or a fast fibroser, by measuring in a sample of the individual at least one variable or score further defined as: biological variables further defined as ≢-2 macroglobulin (α2M), Hyaluronic acid (HA), Apolipoprotein A1 (ApoA1), Type III procollagen N-terminal propeptide (P3P), γ-glutamyl transpeptidase (GGT), bilirubin, β-globulin, γ-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, TGFβ1, Laminin, βγ-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 esophageal 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 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; and implementing an adapted patient care depending on whether the subject is a slow fibroser, a medium fibroser or a fast fibroser.

12. The method according to claim 11, wherein the individual is determined to be a slow fibroser and wherein the adapted patient care consists in monitoring said individual by assessing the fibrosis level at regular intervals.

13. The method according to claim 11, wherein the individual is determined to be a medium fibroser and wherein the adapted patient care consists in monitoring said individual by assessing the fibrosis level at regular intervals and delaying treatment until the individual is determined to suffer from clinically significant liver fibrosis.

14. The method according to claim 11, wherein the individual is determined to be a fast fibroser and wherein the adapted patient care consists in administering without delay at least one therapeutic agent or starting a complication screening program for applying early prophylactic or curative treatment.

15. The method according to claim 14, wherein the at least one therapeutic agent is an antifibrotic agent selected from the group consisting of simtuzumab, GR-MD-02, stem cell transplantation (in particular MSC transplantation), Phyllanthus urinaria, Fuzheng Huayu, S-adenosyl-L-methionine, S-nitrosol-N-acetylcystein, silymarin, phosphatidylcholine, N-acetylcysteine, resveratrol, vitamin E, losartan, telmisartan, naltrexone, RF260330, sorafenib, gleevec, nilotinib, INT747, FG-3019, oltipraz, pirfenidone, halofuginone, polaorezin, gliotoxin, sulfasalazine, rimonabant and combinations thereof.

16. The method according to claim 14, wherein the at least one therapeutic agent is for treating the underlying cause responsible for liver fibrosis, and/or ameliorating or alleviating the symptoms associated with the underlying cause responsible for liver fibrosis, including liver fibrosis.

17. The method according to claim 16, wherein the underlying cause responsible for liver fibrosis is a viral infection and the at least one therapeutic agent is selected from the group consisting of interferon, peginterferon 2b (pegylated IFNalpha-2b), infliximab, ribavirin, boceprevir, telaprevir, simeprevir, sofosbuvir, daclatasvir, elbasvir, grazoprevir, velpatasvir, lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir, clevudine, ANA380, zadaxin, CMX 157, ARB-1467, ARB-1740, ALN-HBV, BB-HB-331, Lunar-HBV, ARO-HBV, Myrcludex B, GLS4, NVR 3-778, AIC 649, JNJ56136379, ABI-H0731, AB-423, REP 2139, REP 2165, GSK3228836, GSK33389404, RNaseH Inhibitor, GS 4774, INO-1800, HB-110, TG1050, HepTcell, TomegaVax HBV, RG7795, SB9200, EYP001, CPI 431-32 and combinations thereof.

18. The method according to claim 16, wherein the underlying cause responsible for liver fibrosis is excessive alcohol consumption and the at least one therapeutic agent is selected from the group consisting of topiramate, disulfiram, naltrexone, acamprosate and baclofen.

19. The method according to claim 16, wherein the underlying cause responsible for liver fibrosis is a non-alcoholic fatty liver disease (NAFLD) and the at least one therapeutic agent is selected from the group consisting of orlistat, metformin, pioglitazone, atorvastatin, ezetimine, vitamin E, sylimarine, pentoxyfylline, ARBs, EPL, EPA-E, multistrain biotic (L. rhamnosus, L. bulgaricus), simtuzumab, obeticholic acid, elafribanor (GFT505), DUR-928, GR-MD, 02, aramchol, RG-125, cenicriviroc CVC and combinations thereof.

20. A method for treating an individual suffering from liver fibrosis and identified as a fast fibroser, said method comprising: identifying an individual suffering from liver fibrosis as a fast fibroser by assessing fibrosis progression in the individual, measuring at least three markers in an individual, wherein said markers are measured in a blood sample obtained from the individual and are selected from the group consisting of α-2 macroglobulin, hyaluronic acid, gamma-glutamyl transpeptidase, bilirubin, platelet count, prothrombin index, aspartate amino-transferase, alanine amino-transferase, urea, glycemia, and ferritin; and/or said markers are clinical markers selected from weight, age and sex; and combining said at least four measured markers in a logistic or linear regression function, thereby determining a fibrosis level in the individual; calculating a ratio of said fibrosis level to cause duration, thereby obtaining a value-useful for assessing the liver fibrosis progression in the individual; and treating the individual suffering from liver fibrosis identified as a fast fibroser administering without delay at least one therapeutic agent, wherein said therapeutic agent is for treating liver fibrosis, or for treating the underlying cause responsible for liver fibrosis, or both.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0207] FIGS. 1-7 are to be read with regard to Example 1.

[0208] 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.

[0209] 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).

[0210] FIGS. 3A and 3B are a combination of graphs 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.

[0211] 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.

[0212] FIG. 5 is the AOF progression rate as a function of cause duration according to Metavir fibrosis (F) stage.

[0213] The curve has an inverse shape (1/x) by definition.

[0214] FIGS. 6A and 6B are 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.

[0215] FIG. 7 is the effects of antifibrotic treatment on area of fibrosis and Metavir F stage. Box plots indicate median, quartiles and extremes.

[0216] FIGS. 8A-16 are to be read with regard to Example 2.

[0217] FIGS. 8A and 8B are a combination of graphs showing the correlation between Metavir fibrosis (F) stage and area of fibrosis (AOF) progression in populations 1 (8A) and 2 (8B) of Example 2. Lines depict linear regression.

[0218] FIGS. 9A-9D are a combination of graphs showing relationship between Metavir fibrosis (F) stage (9A and 9C) or area of fibrosis (AOF) (9B and 9D) progression, during cause duration, as a function of Metavir fibrosis (F) stage at inclusion age in populations 1 (9A and 9B) and 2 (9C and 9D).

[0219] FIGS. 10A-10D are a combination of graphs showing the correlation between Metavir fibrosis (F) stage (10A and 10C) or area of fibrosis (AOF) (10B and 10D) progression and respective predicted progression in populations 1 (10A and 10B, alcoholic CLD only) and 2 (10C and 10D, viral CLD).

[0220] FIGS. 11A-11D show the relationship between Metavir fibrosis (F) stage (11A and 11C) or area of fibrosis (AOF) (11B and 11D) and cause duration in populations 1 (11A and 11B) and 2 (11C and 11D). Curves depict Lowess regression.

[0221] FIGS. 12A-12D show the relationship between Metavir fibrosis (F) stage (12A and 12C) or area of fibrosis (AOF) (12B and 12D) progression and start age in populations 1 (12A and 12B) and 2 (12C and 12D). Curves depict Lowess regression.

[0222] FIGS. 13A-13D show the relationship between Metavir fibrosis (F) stage (13A and 13C) or area of fibrosis (AOF) (13B and 13D) and start age in populations 1 (13A and 13B) and 2 (13C and 13D). Curves depict Lowess regression.

[0223] FIGS. 14A-14E show the relationship between Metavir fibrosis (F) stage progression (14A and 14D) or area of fibrosis progression (14B and 14E) or area of fibrosis (14C and 14F) and inclusion age in populations 1 (14A, 14B and 14C) and 2 (14D, 14E and 14F). Curves depict Lowess regression.

[0224] FIGS. 15A-15D show the relationship between fibrosis characteristics, i.e., Metavir fibrosis (F) stage (15A), area of fibrosis (15B and 15D), and Metavir fibrosis (F) stage progression (15C), and cause duration (15A, 15B and 15D) or area of fibrosis progression (15C) showing different fibrosers as a function of fibrosis progression in population 2. Curves depict Lowess regression.

[0225] 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.

EXAMPLES

Example 1

Methods

1. Patients

Populations

[0226] 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

[0227] 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, α1-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.

[0228] 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 1st 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

[0229] 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

[0230] 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 paracentesis), 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

[0231] Analyses of blood samples provided the following measurements: hemoglobin, mean corpuscular volume, lymphocyte count, platelet count, cholesterol, urea, creatinine, sodium (NA), bilirubin, γ-glutamyl transpeptidase (GGT), alkaline phosphatases (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), albumin (ALB), α1 and α2-globulins, β-globulins, γ-globulins, βγ-block, prothrombin index (PI), apolipoprotein A1 (ApoA1). Some of them are indirect blood markers of fibrosis (1).

[0232] The direct blood markers of fibrosis used in this study were the following: α-2 macroglobulin (α2M), the N-terminal peptide of type III procollagen (P3P), hyaluronic acid (HA), TGF β1, 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° C. for a maximum of 48 months for assay.

4. Liver Histological Assessment

Microscopic Analysis

[0233] Biopsy specimens were fixed in a formalin-alcohol-acetic solution and embedded in paraffin; 5 um 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

[0234] 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

[0235] 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

[0236] 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 α 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

[0237] 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.

[0238] An example of formula for the PR of area of fibrosis is the predictive model including AST/ALT, cause duration, firm liver, β-globulins, and FibroMeter™ where the coefficients are the followings: [0239] Constant: −0.0978158087539 with limits of confidence interval at 95%:0.8363614252041 & -1.035103236918, [0240] AST/ALT: 0.5412244415007 with limits of confidence interval at 95%: 2.07804027617.e-006 & 0.3283727153579, [0241] Cause duration: −0.07623687627859 with limits of confidence interval at 95%: 5.016575306101.e-011 & −0.09671608407235, [0242] Firm liver: 0.7172332316927 with limits of confidence interval at 95%: 0.006563850544752 & 0.2047931685256, [0243] β-globulins: 0.1594071294621 with limits of confidence interval at 95%: 0.001915414369681 & 0.06022006972876, [0244] FibroMeter™: 1.15299980586 with limits of confidence interval at 95%: 0.002487655344947 & 0.4161078148282.

Results

1. General Characteristics

[0245] 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

[0246] 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).

[0247] 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

[0248] 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™ (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 βγ-block (p=0.03) and sex (p=0.001).

[0249] 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™ (.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

[0250] 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), β-globulins (r=0.32, p<10.sup.−4), AST/ALT (r=0.51, p<10.sup.−4), FibroMeter™ (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: βγ-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).

[0251] With linear regression, the independent predictors of the area of fibrosis progression were: AST/ALT, cause duration, area of fibrosis, and β-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, β-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, β-globulins and A2M with .sub.aR.sup.2=0.509.

4. Kinetics of Fibrosis Progression

Estimated Progression (Population 1)

[0252] 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.

[0253] 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.

[0254] 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)

[0255] 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).

[0256] 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, FIGS. 10A-10D) or proportion of F stage increase: p=0.61 (by McNemar χ.sup.2 test).

Example 2

[0257] 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

Population Aims (Table 3)

[0258] 5 populations including 1456 patients were used. All patients included in this study were admitted to the Hepato-gastroenterology unit of the University hospital in Angers, France, except in population 3 that is described elsewhere (15).

[0259] 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)

[0260] 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.

[0261] 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.

[0262] Population 3—It included 1056 patients with chronic hepatitis C, LB recruited in 9 French centers between 1997 and 2007 detailed elsewhere (15). Mean age was 45.4±12.5 yr at inclusion and 59.6% of patients were male.

[0263] Population 4—It included 16 patients with various causes of CLD having two LB between 1997 and 2002 and different CLD causes.

[0264] 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

[0265] 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)

[0266] Microscopic analysis—Biopsy specimens were fixed in a formalin-alcohol-acetic solution and embedded in paraffin; 5 μm thick sections were stained with hematoxylin-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.

[0267] 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® CS System, Aperio Technologies, Vista Calif. 92081, USA) image processor providing high quality images of 30,000×30,000 pixels and a resolution of 0.5 μm/pixel (magnification ×20) since 2007. A binary image (white and black) was obtained via an automatic thresholding technique using an algorithm developed in our laboratory.

[0268] 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

[0269] 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 a 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

[0270] 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

[0271] 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 F stage (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).

[0272] 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.

[0273] 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).

[0274] 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.

[0275] Comparison between viral populations—The AOF progression were significantly higher in population 1 than in population 2 (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

[0276] 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

[0277] 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.

[0278] Cause duration—In population 1, the cause duration was weakly correlated with fibrosis level: F stage: r.sub.2=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.

[0279] Start age—FIG. 12a shows that the F progression dramatically increased by 30-40 years of start age in alcoholic (≈40 years) and viral (≈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 ≈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 (FIGS. 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).

[0280] 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 an 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).

[0281] 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 (FIGS. 14f).

[0282] 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

[0283] 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

[0284] observed FibroMeter™ progression [(FibroMeter™ t2−FibroMeter™ t1)/(t2 −t1)] was 0.049±0.058/yr in population 5 whereas the estimated FibroMeter™ progression (FibroMeter™ t2/cause duration) was 0.038±0.033 /yr in population 2 (p=0.217).

Identifying Categories of Fibrosers

[0285] 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 estimation measurement measurement Repeated 2 biopsies (FLt2-FLt1)/ Longitudinal Precision Variability biopsy (t2-t1) (prospective) Reference Unavailability measurement measurement Short duration Single non- 1 test.sup.b FL/cause Transversal Availability++ Linearity invasive test duration (retrospective) Start estimation estimation estimation Repeated 2 tests (FLt2-FLt1)/ Longitudinal Precision non-invasive (t2-t1) (prospective) Repeatability test estimation 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. Patients Fibrosis Area of Duration Population Cause (n) evaluation fibrosis.sup.a Time Fibrosis progression 1 Alcohol  185 1 LB, 1 Yes Cause Retrospective virus blood test duration.sup.b measurement + estimation 2 Virus  157 1 LB, 1 Yes Cause Retrospective blood test duration.sup.b measurement + estimation 3 Virus 1056 1 LB, 1 No No Retrospective blood test measurement + estimation.sup.c 4 Miscella-   16 2 LB Yes Follow-up Prospective neous measurement 5 Virus   42 0 LB, 2 No Follow-up Prospective estimation blood 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 Population 1 2 Cause Alcohol Virus p.sup.a Both Virus p.sup.b N 136 49 — 185 157 — Age at 49.9 ± 44.2 ± 0.02 48.5 ± 43.4 ± 0.793 inclusion 11.2 14.6 12.3 12.4 (yr) Age at 28.8 ± 28.2 ± 0.779 28.8 ± 27.4 ± 0.707 cause 9.5 13.5 10.8 11.2 start (yr) Cause 21.3 ± 15.8 ± 0.006 19.8 ± 16.5 ± 0.604 duration 13.2 10.7 12.9 7.3 (yr) Sex (% M) 72.8 53.1 0.011 67.6 59.4 0.550 Cause — — — 26.5 100 — (% virus) 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 23.5 ± 13.6 ± <10.sup.−3 20.7 ± 10.7 ± 0.005 fibrosis 14.7 11.7 14.6 6.5 (%) Complication 29.4 0 <10.sup.−3 21.6 0 — (%) Progression rate: Metavir F 0.23 ± 0.19 ± 0.424 0.22 ± 0.16 ± 0.120 (MU/yr) 0.32 0.21 0.29 014 Area of 2.0 ± 1.3 ± 0.027 1.8 ± 0.8 ± 0.017 fibrosis 2.9 1.4 2.6 0.7 (%/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.A Mann Whitney test

REFERENCES

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[0321] 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.