Investigator’s Brochure - Laboratorio · PDF fileInvestigator’s Brochure ... FibroSURE™ LabCorp and BioPredictive Inc have entered into an exclusive agreement to offer ... Maddrey

Version 1 – January 2007

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FibroMAX:

FibroTest, SteatoTest, NashTest, ActiTest and AshTest

Investigator’s Brochure

Contents Index of abbreviations and definitions 1. Introduction ...................................................................................................................................................... 5 2. Limitations of liver biopsy for the diagnostic of steatosis, fibrosis and NASH .......................................... 5 3. Scientific rationale of FibroMax tests ............................................................................................................. 6 4. Administrative data on FibroMax, Patent and Marketing ........................................................................... 8

4.1. The most recent recommendations of the French Health Authority for the use of FibroTest ................. 9 5. Pre-analytic recommendations for FibroMax................................................................................................ 9 6. FibroMax pack biomarkers: FibroTest, ActiTest, SteatoTest, NashTest and AshTest ........................... 10

6.1. FibroTest-ActiTest-FibroSURE overview................................................................................................. 10 6.1.1. Standardisation of observed AUROC according to the prevalence of individual fibrosis stages ..... 10 6.1.2. FibroTest-FibroSURE Meta-analysis: fully validation in the four most frequent liver diseases .... 12 6.1.3. ActiTest diagnostic value in chronic viral hepatitis ........................................................................... 17

6.2. SteatoTest - universal serum marker of steatosis (fatty liver) .................................................................. 19 6.2.1. Introduction ......................................................................................................................................... 19 6.2.2. Discovery, internal validation, comparisons with ultrasonography, GGT and ALT ........................ 19 6.2.3. SteatoTest external validation in CYTOL Study Group NAFLD patients ........................................ 23 6.2.4. Steatosis improvement by SteatoTest after HCV treatment ............................................................... 23

6.3. NashTest – serum marker of non-alcoholic steatohepatitis (NASH) ..................................................... 24 6.3.1. Introduction ......................................................................................................................................... 24 6.3.2. Discovery and external validation (CYTOL Study Group NAFLD patients).................................... 24


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6.4. AshTest - marker of gravity of the alcoholic steatohepatitis (ASH) ........................................................ 28 6.4.1. Introduction ......................................................................................................................................... 28 6.4.2. Discovery and validation ..................................................................................................................... 28

7. Screening strategies with non-invasive biomarkers: FibroMAX (FibroTest, SteatoTest, NashTest) ..... 32 7.1. Efficacy of screening of hyperlipidemics with non-invasive biomarkers ................................................ 32 7.2. Efficacy of screening of diabetics with non-invasive biomarkers............................................................ 35

8. Conclusion ....................................................................................................................................................... 37 9. FibroMAX pack: how and when to use non-invasive biomarkers, recommended algorithms................ 38

9.1. Best management of the patients with metabolic risk factors (overweight, dyslipidemia, diabetes). FibroMAX pack: FibroTest, SteatoTest and NashTest ........................................................................... 38

9.2. Best management of the patients with excessive alcohol intake. FibroMAX pack: FibroTest, SteatoTest and AshTest .............................................................................. 39

9.3. Best management of the patients with chronic viral hepatitis FibroMAX pack: FibroTest, SteatoTest and ActiTest ............................................................................. 40

10. References...................................................................................................................................................... 41


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Index of abbreviations and definitions A0, A1, A2, A3: METAVIR scoring system for grading activity: A0=no liver activity, A1=minimal

activity, A2=moderate activity, A3= severe activity A2M: alpha2-macroglobulin AdAUROC: adjusted Area Under the ROC curve [AdAUROC=0.582+0.105 x (DANA)] AFEF: Association Française pour l’Etude du Foie ALD: alcoholic liver disease ALT, ALAT: alanine aminotransferase AP-HP: Assistance Publique Hôpitaux de Paris, Public Assistance Hospitals of Paris APO A1: Apolipoprotein A1 APRI: AST to platelet ratio index ASH: alcoholic steatohepatitis AST, ASAT: aspartate aminotransferase AT: ActiTest AUROC: Area Under the ROC curve BD: blood donors BIL or BILI: total bilirubin BMI: body mass index [weight (kg)/height²(cm)] CHC: chronic hepatitis C CNRS: Centre National de la Recherche Scientifique CT : total cholesterol DANA: difference between the mean fibrosis stages in the advanced fibrosis and non

advanced fibrosis groups EALT: Elevated ALT ESSEC: Ecole Supérieure des Sciences Economiques et Commerciales F0, F1, F2, F3, F4: METAVIR scoring system: F0=no liver fibrosis, F1=portal fibrosis, F2=portal

fibrosis with few septas, F3=portal fibrosis with many septas, F4=cirrhosis FibroSURE™ LabCorp and BioPredictive Inc have entered into an exclusive agreement to offer

FibroSURE™ combining both FibroTest (FT) and ActiTest (AT) as a single test option. Published literature refers to the tests as FibroTest-ActiTest. FibroSURE™ is a trademarked LabCorp name.

FT: FibroTest GGT: gamma-glutamyl-transpeptidase Gilbert’s syndrome: congenital partial enzymatic deficiency of glucuronyl-transferase with increased

fluctuating non-conjugated bilirubin level with occasional asymptomatic jaundice. HAPTO: haptoglobin HBV: hepatitis B virus, refered in text at chronic hepatitis B HCV: hepatitis C virus HCV-RNA: hepatitis C viral load HDL, HDL-C: high density lipoprotein, HDL-cholesterol HIV: human immunodeficiency virus


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HOMA-IR: homeostasis model assessment of insulin resistance HR, HRFP, HRFN: high risk false positive, high risk false negative IGF: Insulin Growth Factor IGFBP-1: Insulin-like Growth Factor Binding Protein-1 INSERM : Institut National de la Recherche et de la Santé Médicale Knodell score: histological scoring system, (ranging from 0 to 22), assessing the piecemeal necrosis

(0-10), intralobular necrosis (0-4), portal inflammation (0-4) and fibrosis (0-4) Maddrey-DF: Maddrey –discriminant function METAVIR: histological scoring system assessing the activity (necro-inflammatory lesions) in 4

grades (A0 to A3) and liver fibrosis in 5 stages (F0 to F4) NAFLD: Non-Alcoholic Fatty Liver Disease NAS: new histological score for the diagnostic of non-alcoholic steatohepatitis

(Kleiner&al.2005) NASH: non-alcoholic steatohepatitis NPV: negative predictive value ObAUROC: observed Area Under the ROC curve OR: Odds Ratio P: probability level, significant difference is considered as p<0.05 PEG-IFN (alpha2b): pegylated alpha2b-interferon PMN: polymorphonuclear neutrophils PNALT: persistently normal ALT (alanine amino transferases) PPV: positive predictive value R: correlation coefficient ROC: receiver operating characteristic curve SAA: serum amyloid A SAA HDL: complexe between serum amyloid A and HDL S0, S1, S2, S3-S4: S0=No steatosis, S1=mild steatosis (1-5%), S2=moderate steatosis (6-32%),

S3S4=marked to severe steatosis (>33%) Se: sensitivity se, SE: standard error Sp: specificity ST: SteatoTest SVR: sustained viral responder TB: total bilirubin TG: serum triglycerides USPTO: United States Patent and Trademark Office


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1. Introduction

FibroMax is the combination of up to five non-invasive liver tests: FibroTest, ActiTest, SteatoTest, NashTest and AshTest. These markers use a blood sample and therefore could be used in the first line before treatment decision; liver biopsy could be used as a secondary alternative only if necessary.

FibroTest and ActiTest are non-invasive alternatives to liver biopsy first developed in patients with hepatitis C virus (HCV) and thereafter in patients with hepatitis B virus chronic infection (HBV) and alcoholic (ALD) and non alcoholic (NAFLD) steatosis.

The FibroTest assesses the degree of fibrosis (scarring) in HCV, HBV, ALD and NAFLD patients. 1-6 The ActiTest the degree of activity (necrosis and inflammation) in HCV and HBV patients.1 Both tests are detailed in a separate brochure, which last version is available on the web site www.biopredictive.com.

Recently Biopredictive has developed three new non invasive tests: one (SteatoTest)7 for the diagnosis of liver steatosis in HCV, HBV, ALD and NAFLD patients, one for steato-hepatitis in ALD (AshTest)8 and one for NAFLD (NashTest)9. New panels, SteatoTest, NashTest and AshTest were developed combining the same 6 components of the FibroTest-ActiTest (alpha2-macroglobulin, haptoglobin, apolipoprotein A1, GGT, total bilirubin and ALT) adjusted for age, gender and BMI, plus serum fasting glucose, triglycerides, cholesterol and AST.

Data regarding liver histology is considered essential for basing treatment decisions upon and for the follow-up of patients suffering of the most frequent chronic liver diseases: HCV, HBV, ALD and NAFLD.

Until the development of FibroTest-ActiTest, liver histology could only be assessed correctly by liver biopsy, usually done via the intercostal route. However, the numerous limitations of liver biopsy does not make it a simple procedure nor suitable on a large scale. What is more, the high sampling variability in chronic liver diseases makes biopsy an indirect marker of reality, the true “gold-standard” being the liver in its entirety.

The release of these healthcare products on the market will simplify the medical management and supervision in patients worldwide.

2. Limitations of liver biopsy for the diagnostic of steatosis, fibrosis and NASH

Several studies on liver biopsy in chronic hepatitis C showed that this procedure is not completely adapted10,11 to the management of chronic hepatitis C. Liver biopsy is costly,12 can be experienced by patients as an aggressive procedure, prone to complications (30%) including death (0.3%),13 has considerable variability in staging fibrosis and grading activity mainly due to the sampling error.14-17 The advantages and disadvantages of the biomarkers compared to liver biopsy are debated in detail in the FibroTest Brochure available on the web site www.biopredictive.com).

The same is true for the other chronic liver diseases, which are even more frequent than chronic hepatitis C: chronic hepatitis B, 350 millions, alcoholic and non alcoholic steatosis with more than 1 billion of people concerned worldwide.

Sample variability for the diagnostic of steatosis, fibrosis, and steatohepatitis in NAFLD patients

A recent study18 in a NAFLD cohort with two liver samples collected by Ratziu et al. assessed the sampling error of liver biopsy and its impact on the diagnosis and staging of NASH and its individually histological features (steatosis, ballooning and acidophilic bodies).


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Ratziu et al. reported a discordance of one stage or more of fibrosis in 41%. 6/17 (35%) patients with bridging fibrosis on one sample had only mild or no fibrosis on the other and could have been under staged with only one sample biopsy.

The same study reported that a significant (more than 20%) difference in the magnitude of steatosis was noted in 18% of patients. Discordance rate between paired biopsies was 33% for the semiquantitative assessment of hepatocyte ballooning. A higher level of discordance was noted for acidophlic bodies (27%). Among the paired biopsies in which perisinusoidal fibrosis was detected, the staging was different between the two samples in 49%. Interface hepatitis and Mallory bodies was absent in most cases and discordances were low 4% and 16% respectively. For a composite diagnosis of NASH comprising ballooning and perisinusoidal fibrosis, the diagnosis would have been missed in 36%. This study suggests that not only diagnostic but also staging was significantly altered by sampling variability of liver biopsy in NAFLD/NASH patients. Liver biopsy is not an adequate screening tool for NAFLD patients

The number of patients at risk for NAFLD is high enough that liver biopsy is not apractical and efficient tool for identifying those at risk of advanced fibrosis. Indeed an estimated 15 to 20% of theWestern European population has steatosis19 while more than half of Americans are overweight or obese.

The inability of liver biopsy to meet several challenges in mass screening made the development of non-invasive, readily available and easy to perform serum markers an essential step for more effective management of patients with frequent liver pathologies. 3. Scientific rationale of FibroMax tests

Scientific rationale of FibroTest-ActiTest is detailed in the FibroTest-Fibrochure available on the web site www.biopredictive.com.

SteatoTest, NashTest and AshTest are combining the same six components of the FibroTest-

ActiTest (alpha2-macroglobulin, haptoglobin, apolipoprotein A1, GGT, total bilirubin and ALT) adjusted for age, gender and BMI, plus serum fasting glucose, triglycerides, cholesterol and AST. The chosen markers could be easily automated, and provide simple and robust quantification. A prospective first phase study was done which allowed for the identification and the optimal combination of markers. A second phase was carried out for the first validation. As for the FibroTest-ActiTest, in addition to quality control procedures, additional algorithms were also developed testing outliers (99% percentiles) for the risk of errors. Each of the components provides a rationale for, and adds complementary value to, the estimation of individual histological features predicted of each test.

Alpha2-macroglobulin (A2M) is a protein synthesized by the liver (hepatocytes, stellate cells, granulomas). Haptoglobin is a protein synthesized by the liver. Apolipoprotein A1 (ApoA1) is a protein synthesized by the liver, which transports cholesterol. Bilirubin is a pigment resulting from the degradation of erythrocytes (hemoglobin), which is normally taken up from the blood by the liver and excreted in bile. Gamma glutamyl transpeptidase (GGT), transaminases ALT and AST are enzymes synthesized by hepatocytes. Glucose (a carbohydrate), and total cholesterol and triglycerides (lipids) are serum composants which serum levels are dependent of food intake and metabolism.


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SteatoTest rationale GGT, ALT, triglycerides, cholesterol, glucose and BMI had been previously associated with steatosis

of different origins20,21,22. ApoA1 is highly associated with HDL-cholesterol 21and a negative association was also expected with steatosis when fibrosis or inflammation occured. The advantage of combining biomarkers of steatosis and those more specific for fibrosis such as A2M, haptoglobin and bilirubin is to adjust the predictive values according to the associated stage of fibrosis.

NashTest rationale

The most significant components of NashTest were the metabolic factors (mostly weight, triglycerides and glucose), as previously observed23-27, but also A2M and apoA1. These proteins have been proven to be associated with fibrosis28 but also with steatosis7, steato-hepatitis6,29 and insulin resistance pathways6. A2M is a protease inhibitor but also has multiple functions as a binding, carrier and targeting protein30. Other studies have observed an increase of A2M in diabetic patients31. Insulin is covalently bound to A2M32 in plasma and A2M is a binding protein of Insulin-like Growth Factor Binding Protein-1 (IGFBP-1) which modifies the IGFBP-1/IGF interaction33. Therefore A2M can be directly involved both in the hepatic mechanisms of insulin resistance and fibrogenesis.

Comparisons with biomarkers of alcoholic steato hepatitis (ASH)8 are important as ASH and NASH share many physio-pathological mechanisms and histological features. The same associations were observed for proteins in univariate analysis with a decrease in ApoA1, haptoglobin and an increase of A2M in NASH. However the decrease of apolipoprotein A1 in NASH was much lower than in patients with ASH.

AshTest rationale

AST, GGT and bilirubin, are increasing with ASH and were previously associated with ASH7,34-37. The most striking observation was the dramatic reduction of APOA1 in the presence of PMN infiltrate.38-39 The association of APOA1 with ASH independent of fibrosis had been indirectly suggested 5,40. During the acute phase response circulating HDL are charged in serum amyloid A (SAA) and depleted in APOA141,42. When APOA1 is liberated from HDL, it interacts with inflammatory cells such as PMN and decreases neutrophil degranulation and superoxyde production43. APOA1 facilitates the adhesive responses of PMN to lipopolysaccharide44. Other possible mechanisms and cascades include the down regulation of PMN by APOA145. In the absence of APOA1, SAAHDL is not formed and free SAA increases46 which can also activate PMN47. Finally activated PMN are a source of myeloperoxydase and hydrogen peroxide, which can destroy the integrity of APOA148-51.


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4. Administrative data on FibroMax, Patent and Marketing Professor Thierry Poynard and his team have worked for over 15 years on biochemical markers of liver fibrosis [Public Assistance Hospitals of Paris (AP-HP), University of Paris 6, CNRS UMR 8149 and University of Paris 5]. An international patent, (USPTO 6,631,330) which was registered by the AP-HP, protects the discovery of these biochemical markers of fibrosis (FibroTest), activity (ActiTest), steatosis (SteatoTest) and steatotohepatitis (NashTest and AshTest).

In 2002, and in agreement with the Innovation and Research Act (1999) pertaining to public research, the task of exploiting this patent was assigned to Biopredictive, a biotech start-up company. This assignment was made after examination by the scientific board of Paris-Biotech [which includes an incubator from the University of Paris 5, the City of Paris, INSERM (Institut National de la Santé et de la Recherche Médicale), l’Ecole Centrale and ESSEC (Ecole Supérieure des Sciences Economiques et Commerciales)]. The operating license was then transferred from the Public Assistance AP-HP to the limited public company, Biopredictive. In 2002, this project was a winner of the ANVAR (Agence Nationale de Valorisation de la Recherche) Award of the “Fourth National Competition of Aid for the Creation of Innovative Technological Enterprises” in the “Creation and Development” category. This competition, organized by the French Ministry of Research, designated the winners of the award after jury deliberation on the commercial and financial aspects of the project. In September 2002, the first health product, FibroTest-ActiTest, was released on the French market and in April 2006 the recently developed SteatoTest, NashTest and AshTest were released. This company provides a service to public or private laboratories. Using ten registered biologic tests, an algorithm-system expert takes into account the age, gender and BMI of the patient, and generates quantitative estimators of fibrosis stage (FibroTest), necroinflammatory activity (ActiTest), steatosis (SteatoTest) and alcoholic steatohepatitis (AshTest) and a semi quantitative estimator of non alcoholic steatohepatitis (NashTest). This is done via secured and anonymous Internet connection. Security algorithms are also provided with the results, in order to detect the risk of false positive/false negative results due of an extremely high or extremely low value of one of the ten parameters.

There has been widespread acknowledgement abroad of FibroTest and ActiTest through presentations at scientific meetings. These tests are currently marketed in France, Morocco (where they are performed by 30 laboratories), in Mexico, Portugal, Switzerland, Belgium, Germany, Italy, United Kingdom, Brazil, Egypt, Turkey, Canada and USA, Argentina, Austria, Australia, United Emirates, Spain, Georgia, Lebanon, Romania, Ukrain. In France, more than 500 private laboratories currently perform these tests and 37 public hospitals have signed a contract with Biopredictive for their use. FibroMax is performed in the same laboratories as FibroTest-ActiTest.

In USA, LabCorp and BioPredictive Inc have entered into an exclusive agreement to offer FibroSURE™, NASH-FibroSURE™ and ASH-FibroSURE™. Published literature that includes data on FibroSURE refers to the tests as FibroTest and ActiTest, NASH-FibroSURE refers to the tests as FibroTest, SteatoTest and NashTest and ASH-FibroSURE refers to the tests as FibroTest, SteatoTest and AshTest. FibroSURE™, NASH-FibroSURE™ and ASH-FibroSURE™ are trademarked LabCorp names that combines FibroTest with ActiTest, SteatoTest, NashTest and AshTest according to pathology, as a single test option. In Europe, FibroMax refers to the results of all five tests regrouped on the same results sheet.

FI-BROCHURE is the official investigator document of FibroTest-ActiTest, launched in March 2004 with the latest version available on the web site www.biopredictive.com .


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4.1. The most recent recommendations of the French Health Authority for the use of FibroTest The French Health Authority (La Haute Autorité de Santé) recommended the use of the non-invasive serum marker of fibrosis FibroTest as a first line estimate of liver injury to reduce the need for liver biopsy in chronic hepatitis C management. These official recommendations issued from several years of studies were oral communicated at the occasion of the French Liver Meeting (AFEF) in October 2006 and will be soon published.52 5. Pre-analytic recommendations for FibroMax Fasting versus non-fasting

For FibroTest-ActiTest the intra-individual variation of biochemical markers was low, and it was shown that measurement of FibroTest, ActiTest and their components are not significantly modified by meal intake.

However, the other FibroMax tests in particular SteatoTest and NashTest are including composants that need be dosed fasting (glucose, triglycerides and cholesterol) because of high variation of their serum level with the meal.53 Conditions of transport, pre-treatment, serum storage temperature

The measurements of the six parameters of FibroTest-ActiTest are made preferably on fresh serum (or plasma) or that which has been stored between +2°C and +8°C for a maximum of four days in an unlit area (for the protection of bilirubin). Centrifugation of the vial for the fasting glucose measurement should be done rapidly after been collected on a special fluorated vacutainer in order to empeach glucose metabolism by red cells enzyms. For deferred measurements, the serum should be quickly frozen to -80°C. After thawing, it should be centrifuged for 10 minutes at 15,000g.

One study recommended that to avoid the risk of false negative or false positive results for non-invasive liver markers, validation studies need to be done on fresh sera and if retrospective analyses are needed, sera storage at -80°C was recommended. 54


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6. FibroMax pack biomarkers: FibroTest, ActiTest, SteatoTest, NashTest and AshTest 6.1. FibroTest-ActiTest-FibroSURE overview 6.1.1. Standardisation of observed AUROC according to the prevalence of individual fibrosis stages Prevalence of fibrosis stages is a major factor of variability when assessing the area under the ROC curves (AUROC) in the diagnostic evaluation of liver fibrosis markers.55

Poynard, T, Halfon P, Castera L, Munteanu M, Imbert Bismut F, Ratziu V, Bourliere M, de Ledinghen V, FibroPaca study group. Gastroenterol Clin Biol. 2006; 30: 1039 (Abstract)

The area under the ROC curve (AUROC) is widely used as an estimate of the diagnostic value for liver fibrosis markers. AUROC combines sensitivity (Se) and specificity (Sp) of a quantitative marker. Se is usually assessed in patients with advanced fibrosis (METAVIR stages F2, F3, F4) and Sp in no advanced fibrosis (METAVIR stages F0, F1).

The AUROC’s variability due to a change in the prevalence of each fibrosis stage inside advanced fibrosis or non-advanced fibrosis groups is unknown.

The aim was to assess the relationships between the prevalence of each stage of fibrosis and the

AUROC and to construct an index for standardizing comparisons of AUROCs between studies. An integrated database of 1,312 HCV patients with contemporaneous FibroTest and biopsy was

constructed. The FibrpTest AUROC for the diagnostic of advanced fibrosis was computed in 19 populations with different stage prevalence.

An index (DANA) was constructed = difference between the mean of advanced fibrosis stages and the mean of non advanced fibrosis stages, range 0 to 4 fibrosis METAVIR units. An overview database of 19 published FibroTest studies was constructed to validate the association between DANA and AUROC.

According to stage prevalence, FibroTest AUROC varies (P<0.001) from 0.67 to 0.98 and from 0.65 to 0.89, DANA from 1.00 to 4.00 and 1.47 to 3.05, in the integrated database and in the overview, respectively. There was a close correlation between the FibroTest AUROC and DANA: R=0.89, both among the integrated database (R=0.95 P<0.0001) and the overview (R=0.69 P=0.001) (Figure 1). No other factors including biopsy size, fragments, author, and alcohol consumption had a significant association with FibroTest AUROC.

The formula for standardizing FibroTest AUROCs estimated from different stage prevalence was AUROC=0.582+0.105 x (DANA) with FibroTest AUROC=0.85 for a standard DANA of 2.5 (each stage prevalence =0.20).

The prevalence of each fibrosis stage must be taken into account when fibrosis markers are

evaluated using AUROCs for the diagnosis of advanced fibrosis. The formula for standardizing AUROCs estimated from different stage prevalence was

AUROC=0.582+0.105x (DANA) with FibroTest AUROC=0.85 for a standard DANA of 2.5 (each stage prevalence =0.20).


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Figure 1. Association between DANA and AUROC in the 19 studies included in the integrated database. Yellow points represent the 20 possible combinations between two different stages of fibrosis and the red points the DANA the representation of each publish study. For a study including a population with very different stages of fibrosis (only F0 and F4) the DANA=4 and the observed AUROC will be artificially increased (0.98); contrarily to a study including a population with adjacent stages of fibrosis (only F1 and F2) for which observed AUROC will be lower (0.67). For a standardized DANA =2.5 representing a population with equal prevalences of fibrosis (20% of each stage F0, F1, F2, F3, and F4) the standardized AUROC obtained is only 0.85.

0.85

0.00

1.00

2.00

3.00

4.00

0.5 0.6 0.7 0.8 0.9 1.0

F1234

NoF0

NoF1

NoF2

NoF3

NoF4

NoF02

NoF03

NoF04

NoF12

NoF13

NoF14

NoF23

NoF24

NoF34

NoF023

NoF034

NoF134

NoF123

Callewaert

Myers

Rossi

Poynard1

Wilson

Cales2

Poynard2

Cales1

Halfon

Varaud1

Varaud2

Imbert1

Poynard3

Imbert2Sebastiani1

Sebastiani2

Castera

Sene

AUROC for Advanced vs Non Advanced Fibrosis

A

d

v

a

n

c

e

d

m

i

n

u

s

N

o

n

A

d

v

a

n

c

e

d

F

i

b

r

o

s

i

s

M

e

a

n

s

F1 vs F2 DANA=1 AUROC=0.67

Standardized Point :

DANA=2.5

AUROC=0.85

F4 vs F0 DANA=4 AUROC=0.98

DANA=2.5


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6.1.2. FibroTest-FibroSURE Meta-analysis: fully validation in the four most frequent liver diseases

Complete information about FibroTest-ActiTest internal and independent validations, analytical standardisation and comparisons with other markers is published on FibroTest FIBROCHURE latest version available in open access on the web site www.biopredictive.com .

Meta-analysis of FibroTest (FT) for the diagnosis of fibrosis in the four more frequent liver diseases. Poynard T, Halfon P, Castera L, Ratziu V, Imbert-Bismut F, Naveau S, Thabut D, Lebrec D, Zoulim F, Munteanu M, Bourlière M, de Ledinghen V.2 Gastroenterol Clin Biol. 2006; 30: 1057 (Abstract) Diagnostic value of FibroTest -ActiTest among published studies (meta-analysis2 and integrated database)

For 24 groups of patients detailed in 19 publications,3-6,28,56-69 it was possible to assess the prevalence of significant fibrosis (F2F3F4), and AUROC values as well as the Se and Sp for different FibroTest cut-offs (Table 1) (exept for 4 studies for which AUROC, Se or Sp were not reported).

A new integrated database was constructed with the studies where individual data were available cumulating a total of 3,282 patients (2,431 HCV, 322 HBV, 267 NAFLD and 262 ALD) being 74% of the literature. Comparison of FibroTest diagnostic value between different liver diseases

Meta-analysis was performed twice, once according to the absolute value of the observed AUROCs (ObAUROC) and once according to the AUROCs adjusted for the spectrum of fibrosis stages (AdAUROC).55 (Figure 2, Panel A and B). When two publication were excluded because in one of them patients were already included in other study [Thabut et al.] and in the other AUROC [Colletta et al.] was not reported, the mean observed AUROCs for the diagnosis of significant fibrosis (F2F3F4) was 0.79 (95% CI, 0.77-0.82) and the mean adjusted AUROC was 0.84 (95% CI, 0.82-0.86); all observed AUROC ranged from 0.71 (for 0.39 prevalence of F2F3F4) to 0.87 (for 0.45 prevalence of F2F3F4). The heterogeneity of observed AUROC between studies disappears after adjustement of AUROCs (Cochran Q=26 p=0.19). Figure 2. Meta-analysis of the FibroTest ObAUROCs (Panel A) and AdAUROCs (Panel B) in published studies. Ob and AdAUROCs were all higher for FibroTest than the random 0.50 value (P<0.001). There was no significant difference between the different liver diseases for Ob and AdAUROCs. The red line is the best AUROC it can be obtained (0.90) with 20mm biopsy vs the whole liver [Bedossa 2003]. Panel A. Panel B.

0.00 0.10 0.20 0.30 0.40 0.50 0.60

AUROCs Difference vs 0.50

Mean Difference

A

u

t

h

o

r

HBVPoynard4MyersAve

HCVVaraudSebastiani1Poynard2RossiWilsonPoynard1Poynard3HalfonSebastiani2SeneImbert1CasteraMyersCales2Imbert2Ave

MixedCales1CallewaertAve

NAFLD ALDRatziu2NaveauRatziu1Ave

Total

Disease

Combined

HBV

HCV

Mixed

NAFLD ALD

0.00 0.10 0.20 0.30 0.40 0.50 0.60

AUROCs Difference vs 0.50

Mean Difference

Author

HBVMyers2Poynard4Ave

HCVSebastiani1VaraudSeneRossiWilsonPoynard2Poynard3Poynard1HalfonSebastiani2Imbert1Myers1Cales2Imbert2CasteraAve

MixedCales1CallewaertAve

NAFLD ALDRatziu2NaveauRatziu1Ave

Total

Ajusted AUROCs

CombinedHBVHCVMixedNAFLD ALD


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Table 1. Summary of ObAUROC (se) of FibroTest for the staging of hepatic fibrosis in patients with chronic liver disease (hepatitis C, B, ALD and NAFLD) from the published studies.

First author No Pts

Methodology Stage Prevalence

Observed AUROC (SE)

DANA1 Cut-off Sensitivity Specificity

HCV Imbert-1, 2001

189 Prospective Single center Training cohort

F2F3F4 0.38

0.84 0.03

2.03 0.10 0.30 0.60 0.80

0.97 0.79 0.51 0.29

0.24 0.65 0.94 0.95

Imbert-2, 2001

134 Prospective Single center Validation cohort

F2F3F4 0.45

0.87 0.03

2.16 0.10 0.30 0.60 0.80

1.00 0.87 0.70 0.38

0.22 0.59 0.95 0.97

Poynard-1, 2001

165 Retrospective Randomized trial Multicenter

F3F4 Knodell 0.32

0.74 0.03

1.42 0.10 0.30 0.60 0.80

0.96 0.81 0.50 0.13

0.24 0.65 0.92 0.98

Poynard-2, 2003

352 Retrospective Randomized trial Multicenter Before treatment

F2F3F4 0.39

0.73 0.03

1.71 0.10 0.30 0.60 0.80

0.97 0.86 0.50 0.20

0.08 0.45 0.79 0.95

Poynard-3, 2003

352 Retrospective Randomized trial Multicenter After treatment

F2F3F4 0.32

0.77 0.03

1.73 0.10 0.30 0.60 0.80

0.98 0.85 0.46 0.16

0.15 0.39 0.81 0.97

Rossi, 2003 125 Prospective Multicenter Non-validated analyzers

F2F3F4 0.38

0.74 0.05

1.95 0.10 0.30 0.60 0.80

0.92 0.75 0.42 0.22

0.29 0.61 0.94 0.96

Myers-1, 2003

130 Retrospective Single center HCV-HIV Co-infection

F2F3F4 0.45

0.86 0.04

2.15 0.10 0.30 0.60 0.80

0.98 0.90 0.66 0.34

0.17 0.60 0.92 0.96

Castéra, 2005

183 Prospective Single center

F2F3F4 0.38

0.85 0.03

1.95 0.48 NA NA

Calès-2, 2005


F2F3F4 0.48

0.86 0.04

2.09 0.48 NA NA

Colletta, 2005

40 Prospective, Multicenter, PNALT

F2F3F4 0.35

NA 1.47 NA NA NA

Varaut-1, 2005

50 Retrospective, Single center Dialysis patients

F2F3F4 0.42

0.532 0.04

1.61 NA NA NA

Varaut-2, 2005

60 Retrospective, Single center Kidney recipients

F2F3F4 0.48

0.71 0.04

1.84 0.20 0.60

0.90 0.28

0.56 0.88

Halfon, 2006

504 Prospective, Multicenter

F2F3F4 0.45

0.79 0.02

1.87 0.10 0.30 0.36 0.60 0.80

0.97 0.77 0.73 0.44 0.20

0.27 0.65 0.72 0.91 0.98

Sebastiani-1, 2006

65 Prospective, PNALT, Training set

F2F3F4 0.39

0.71 0.04

2.21 0.48 0.58 0.91

Sebastiani-2, 2006

125 Prospective, Elevated ALT Training set

F2F3F4 0.71

0.81 0.03

1.83 0.48 0.65 0.81


14

First author No

Pts Methodology Stage

Prevalence AUROCs DANA3 Cut-off Sensitivity Specificity

Wilson, 2006

115 Retrospective Multicenter 30%HIV

F2F3F4 0.38

0.74 0.05

1.87 NA NA NA

Sene, 2006

258 Prospective Single center Cryoglobulinemia vasculitis

F2F3F4 0.47

0.83 0.04

3.05 0.37 0.85 0.85

HBV Myers-2, 2003

209 Prospective (42) and Retrospective (167)

F2F3F4 0.29

0.78 0.04

2.31 0.20 0.40 0.60 0.80 0.90

0.89 0.54 0.34 0.18 0.08

0.52 0.80 0.93 0.99 1.00

Poynard-4, 2005

214 Prospective

F2F3F4 0.45

0.77 0.03

2.17 NA NA NA

ALD Naveau, 2005

221 Prospective F2F3F4 0.64

0.84 0.03

2.34 0.30 0.70

0.84 0.55

0.66 0.93

NAFLD Ratziu-1, 2006


0.86 0.03

2.30 0.30 0.70

0.83 0.18

0.78 0.98

Ratziu-2, 2006


0.75 0.04

2.04 0.30 0.70

0.71 0.13

0.74 0.98

Mixed Callewaert, 2004

106 Prospective HCVand alcohol

F4 0.29

0.89 0.04

2.08 NA NA NA

Calès-1, 2005

478 Retrospective Single center Mixed causes Training cohort

F2F3F4 0.59

0.82 0.03

2.73 NA NA NA

*Details for stages given out of 119 and not out of 115: ** half of Knodell F3 has been counted as METAVIR F2

In the four most common liver diseases there was a very significant difference for the diagnostic value (AUROC) of FibroTest versus random diagnosis (0.50) in each study using either meta-analysis or pooled data in the integrated database; the only significant difference of AUROC between differents etiologies was a higher ObAUROC before adjustment in ALD that in HCV (p=0.001); this difference was no longer significant after adjustment of AUROCs to the prevalence of each stage of fibrosis. (Table 2) Table 2. Characteristics of the new integrated data-base and the FibroTest diagnostic value in the four most common liver diseases Disease No

Age yrs

Biopsy Length mm

F0 (%)

F1 (%)

F2 (%)

F3 (%)

F4 (%)

ObAUROC 95% CI

DANA AdAUROC 95% CI

HCV 2,431 47 17 204 (8.4)

1123 (46.2)

531 (21.8)

298 (12.3)

275 (11.3)

0.77 0.75-0.79

1.92 0.83 0.81-0.85

HBV 322 42 17 86 (26.7)

94 (29.2)

61 (18.9)

38 (11.8)

43 (11.4)

0.81 0.76-0.86

2.30 0.83 0.78-0.88

ALD 262 48 14 18 (6.9)

67 (25.6)

50 (19.1)

23 (8.8)

104 (39.7)

0.85 0.80-0.89*

2.52 0.85 0.80-0.89

NAFLD

267 51 19 102 (38.2)

94 (35.2)

35 (13.1)

23 (8.6)

13 (4.9)

0.81 0.74-0.86

2.21 0.84 0.77-0.89

All 3,282 47 17 410 (12.5)

1378 (42.0)

677 (20.6)

382 (11.6)

435 (13.3)

0.79 0.77-0.80

2.07 0.84 0.82-0.86

*P=0.001 between HCV and ALD for ObAUROC, Non Significant for AdAUROC


15

Sensitivity analysis Prospective studies, inventors’ studies and fragmented biopsies studies were associated with higher

observed ObAUROC, but not with higher adjusted AdAUROCs (Table 3, Panel A). There was no difference between FibroTest Ob AUROCs or AdAUROCs according to elevated or nonelevated baseline ALT (Table 3 Panel A and B).

Table 3. Sensitivity analyses of FibroTest diagnostic values according to published studies (Panel A) or patient characteristics (Panel B). Panel A.

Characteristic (number of studies)

Observed AUROC Adjusted AUROCs

All (23) 0.79 0.77-0.82 0.84 0.82-0.86 Design Prospective (15) Retrospective (7)

0.82 0.79-0.84*

0.75 0.72-0.78

0.85 0.83-0.88 0.83 0.80-0.85

Authors Independent (12) Mixed (5) Inventor (6)

0.79 0.75-0.83 0.76 0.73-0.80

0.83 0.80-0.87**

0.84 0.80-0.87 0.83 0.80-0.86 0.85 0.81-0.89

Guidelines/Fresh No (8) Yes (15)

0.76 0.72-0.79 0.81 0.78-0.83

0.83 0.80-0.86 0.85 0.82-0.88

Length biopsy <18 (8) >=18 (10)

0.81 0.77-0.84 0.80 0.77-0.83

0.86 0.82-0.90 0.84 0.81-0.87

Interval serum-biopsy >30 days (7) <30 (14)

0.77 0.71-0.83 0.80 0.78-0.33

0.81 0.77-0.85 0.85 0.83-0.87

Co-morbidity No (18) Yes (5)

0.79 0.76-0.81 0.80 0.74-0.86

0.85 0.82-0.87 0.81 0.78-0.88

Normal baseline ALT Yes (1) No (22)

0.71 0.60-0.82 0.80 0.77-0.82

0.74 0.63-0.85 0.84 0.83-0.86

P=0.01 **P=0.04 Panel B. Characteristic (number of patients)

Observed AUROC DANA Adjusted AUROC

Length biopsy <25mm (2,446) >=25mm (492)

0.80 0.78-0.82 0.77 0.72-0.81

2.10 1.91

0.84 0.82-0.86 0.83 0.78-0.87

More than 2 fragments Yes (575) No (606)

0.86 0.82-0.89* 0.78 0.73-0.81

2.45 2.05

0.87 0.83-0.90 0.83 0.78-0.86

Normal baseline ALT No (1,833) Yes (493)

0.80 0.78-0.82 0.79 0.74-0.84

2.16 2.39

0.84 0.82-0.86 0.81 0.76-0.86

*P=0.002 not significant for adjusted AUROCs


16

Diagnostic value of FibroTest for intermediate and adjacent stages in HCV carriers on the first integrated data-base1

The AUROCs between different stages’ combinations are given in the Table 4. Between two contiguous stages (one stage difference) the AUROCs were not significantly different and ranged from 0.63 to 0.71. Between patients with a 2 stages difference, the AUROCs were not significantly different and ranged from 0.75 to 0.86. Between patients with a 3 stages difference, the AUROCs were not significantly different and ranged from 0.87 to 0.95. Between patients with a 4 or 5 stage differences (blood donors versus F3 or F4, and F0 versus F4), the AUROCs were not significantly different and ranged from 0.95 to 0.99. 1 Table 4. Summary of the diagnostic value of FibroTest for the diagnosis of all stages combinations of hepatic fibrosis (AUROC) in HCV carriers (first integrated published data-base). Between two contiguous stages (one stage difference), the AUROCs are given in bold. Between patients with a 2 stages difference, the AUROCs are given in italics. Between patients with 3 stages difference, the AUROCs are given in bold and italics. Between patients with 4 or 5 stages differences (blood donors versus F3 or F4, and F0 versus F4), the AUROCs are given underlined. Significant differences were observed between AUROCs when there was a difference of two or more stages. F0 F1 F2 F3 F4 BD F0 F0F1 F1F2 F2F3F4 F3F4

Blood Donor BD n=300 0.71 0.86 0.95 0.99 0.99 - 0.84 0.88 0.97 0.99 F0 n=95 -

F1 n=688 0.66 - F2 n=253 0.82 0.69 - F3 n=111 0.92 0.80 0.63 - F4 n=123 0.95 0.87 0.75 0.65 - BD F0 0.71 0.81 0.92 0.98 0.98 - F0F1 - - 0.71 0.82 0.88 - - - F1F2 0.71 - 0.69 0.81 0.82 0.84 - - - F2F3 0.85 0.76 - - 0.72 0.92 0.80 - - F3F4 0.94 0.81 0.81 - - 0.98 0.89 0.80 - F2F3F4 0.83 0.78 - - - 0.95 0.78 - - - BD F0F1 - - 0.77 0.87 0.91 - - - 0.83 0.89

Comparison of FibroTest diagnostic values between adjacent stages in the four chronic liver diseases Overall AUROC for the diagnosis of intermediate stages F2 vs. F1 (0.66, n=2,055) was not different

compared to the extreme stages: F3 vs. F4 (0.69, n=817) or F1 vs. F0 (0.62, n=1788). No AUROCs difference was observed between adjacent stages for all four chronic liver disease. (Table 5)

Table 5. FibroTest diagnostic values between adjacent stages in the four chronic liver diseases.

AUROCs (95%CI) Number of patients Etiology F1 vs F0 F2 vs F1 F3 vs F2 F4 vs F3

ALL 0.62 0.59-0.65 N=1,788 0.66 0.63-0.68 N=2,055 0.67 0.64-0.70 N=1,059 0.69 0.65-0.72 N=817 HCV 0.64 0.60-0.68 N= 1,327 0.66 0.63-0.69 N=1,654 0.66 0.62-0.69 N=829 0.66 0.61-0.70 N=573 HBV 0.64 0.56-0.72 N=180 0.63 0.53-0.71 N=155 0.78 0.67-0.86 N=99 0.54 0.40-0.65 N=81* ALD 0.47 0.32-0.60 N=85 0.65 0.53-0.74 N=117 0.66 0.50-0.77 N=73 0.82 0.69-0.90 N=127*

NAFLD 0.53 0.45-0.61 N=196 0.69 0.57-0.78 N=129 0.69 0.52-0.80 N=58 0.71 0.45-0.86 N=36 * P=0.001 between HBV and ALD


17

6.1.3. ActiTest diagnostic value in chronic viral hepatitis

Diagnostic value of ActiTest for intermediate and adjacent grades in HCV carriers-first integrated database The AUROCs between different grades’ combinations in the first integrated database (only HCV

carriers) are given in the Table 6. Between two contiguous grades (one grade difference) the AUROCs were not significantly different and ranged from 0.60 to 0.70. Between patients with 2 stages difference the AUROCs were not significantly different and ranged from 0.75 to 0.86. Between patients with a 3 stages difference, the AUROCs were not significantly different and ranged from 0.87 to 0.95. Between patients with a 4 stages difference (blood donors versus F3 and F0 versus F4) the AUROCs were not significantly different and ranged from 0.95 to 0.99.

Table 6. Summary of the diagnostic value of ActiTest for the differential diagnosis of all grades of histologic activity (AUROC) in HCV carriers (first integrated published data-base). Between two contiguous grades (one grade difference) the AUROCs are given in bold. Between patients with a 2 grades difference, the AUROCs are given in italics. Between patients with 3 grades difference, the AUROC are given in bold and italics. Between patients with a 3 or 4 grades difference (blood donors versus A2 or A3, and A0 versus A3) the AUROCs are given underlined. Significant differences were observed between AUROCs when there was difference of two or more grades. A0 A1 A2 A3 BD A0 A0A1 A1A2 A2A3

Blood Donor BD n=300 0.67 0.84 0.96 0.99 - 0.79 0.89 0.97 A0 n=185 - A1 n=443 0.69 - A2 n=370 0.87 0.70 - A3 n=272 0.93 0.79 0.60 -

A0A1 - - 0.70 0.83 - A1A2 0.77 - - 0.70 0.85 - A2A3 0.89 0.74 - - 0.94 0.78 -

A0A1A2 - - - 0.75 - - - - BD A0A1 - - 0.82 0.88 - - - 0.84

ActiTest diagnostic value

For four groups of patients detailed in two publications28,60 it was possible to assess the prevalence of significant activity, and the ActiTest AUROC values as well as the sensitivity and specificity for 4 different ActiTest cut-offs (Table 7). For the diagnosis of significant histological activity by the METAVIR scoring system, the AUROCs ranged from 0.75 to 0.86. There was a very significant difference versus random diagnosis, in each study, using either meta-analysis (Mean Difference in AUROC=0.29, random effect model Chi square=556 P<0.001) or pooled data in the integrated database. For the cut-off of 0.36, the ActiTest negative predictive value for excluding significant activity (prevalence 0.41) was 85%.


18

Table 7. Summary of the diagnostic value of ActiTest for the diagnostic of necrotico-inflammatory hepatic activity (AUROC) First Author Number Methodology Marker Grade

Prevalence AUROC SE

Cut off

Sensitivity Specificity

Imbert-Bismut, 2001


ActiTest A2A3 0.33

0.79 0.03

0.10 0.30 0.60 0.80

0.99 0.91 0.70 0.49

0.07 0.42 0.75 0.88

Imbert-Bismut, 2001

134 Prospective Single center Validation cohort

ActiTest A2A3 0.28

0.75 0.03

0.10 0.30 0.60 0.80

1.00 0.94 0.67 0.42

0.07 0.33 0.65 0.87

Poynard, 2003 352 Retrospective Randomized trial Multicenter Before treatment

ActiTest A2A3 0.83

0.75 0.03

0.10 0.30 0.60 0.80

1.00 0.90 0.49 0.20

0.00 0.38 0.87 0.99

Poynard, 2003 352 Retrospective Randomized trial Multicenter After treatment

ActiTest A2A3 0.39

0.86 0.02

0.10 0.30 0.60 0.80

0.91 0.75 0.38 0.14

0.59 0.83 0.98 0.996


19

6.2. SteatoTest - universal serum marker of steatosis (fatty liver) 6.2.1. Introduction

Steatosis or fatty liver is defined as an excessive accumulation of triglycerides in hepatocytes.70 The prevalence of steatosis is very high, and is associated with several factors such as alcohol, diabetes, overweight, hyperlipidemia, insulin resistance, hepatitis C genotype 3, abetalipoproteinemia and administration of some drugs.20,70-72

Fatty liver disease involves not only simple steatosis, but also other histological lesions and often fibrosis with possible progression to cirrhosis, hepatocellular cancer and liver-related death. In patients with alcoholic liver disease (ALD), chronic hepatitis C, and possibly in those with hepatitis B, the presence of steatosis is also associated with fibrosis progression. 70,72-74

Therefore the diagnosis of liver steatosis is mandatory to better manage all at risk patients.

6.2.2. Discovery, internal and external validations, comparisons with ultrasonography, GGT and ALT The diagnostic value of biomarkers (SteatoTest) for the prediction of liver steatosis.7 Poynard T, Ratziu V, Naveau S, Thabut D, Charlotte F, Messous D, Capron D, Abella A, Massard J, Ngo Y, Munteanu M, Mercadier A, Manns M, Albrecht J. Comp Hepatol. 2005. 23; 4: 10.

For the diagnosis of steatosis, there is no standard recommendation. The usual recommendation is to measure GGT and ALT and, in addition, to perform liver biopsy for grading and staging. It is impossible to perform liver biopsy in such a number of patients. 20,70,72,75

The objective of the current study was to create a new panel of biomarkers known as SteatoTest

with sufficient predictive values for the diagnosis of steatosis due to alcohol, non-alcoholic fatty liver disease (NAFLD) and chronic hepatitis C and B.

A secondary aim was to compare the diagnostic value of SteatoTest was compared to ultrasonography and serum GGT and ALT considered as the standard biochemical markers.

Independently, the SteatoTest diagnostic value was validated on an independent cohort from.

A total of 2,272 subjects were analyzed, being 884 subjects included in the biomarker validation study with a distribution as follows: 310 patients in the training group (various etiologies: NAFLD, ALD, HCV, HBV); 171 in the validation group 1 (HCV detectable at baseline); 201 in the validation group 2 (HCV undetectable at 24 weeks follow-up); 62 in the validation group 3 (ALD); and 140 in the control group (blood donors, fasting and not-fasting volunteers).

The advantage of validation group 2 was the inclusion of a group of patients having liver biopsies that are clinically and biologically close to a "normal" population, as these patients are sustained virologic responders (SVR) and had quasi-normal liver function tests. Characteristics of the patients

The 1,388 non-included patients were not significantly different from the 884 patients integrated in the validation group. Patients included in the four groups were similar in age with a predominance of male subjects. The prevalence of steatosis greater than 5% (grades 2 to 4) varied from 11% in hepatitis C virus (HCV) cured patients to 94% in patients with ALD.


20

Training group results: construction of the test In the training group (n=310) the most significant components associated with the presence of grade

2–4 steatosis in univariate analysis were body mass index (BMI), age, ALT, AST, GGT, glucose, and triglycerides. The logistic regression defining the SteatoTest included 12 components – ALT, α2-macroglobulin (A2M), apolipoprotein A-I (ApoA1), haptoglobin (HAPTO), total bilirubin (TB), GGT, cholesterol (CT), triglycerides (TG), glucose, age, gender and BMI and the most significant components were BMI (P = 0.0002), GGT (P = 0.002), ApoA1 (P = 0.01), A2M (P = 0.02), ALT (P = 0.03) and TG (P = 0.04).

In the validation groups, similar differences were observed, most significantly for BMI, GGT, ALT and triglycerides.

Conversion between the SteatoTest results and the corresponding steatosis histological grades

SteatoTest is a continuous linear quantitative assessment of steatosis grade. SteatoTest provides a numerical quantitative estimate of liver steatosis ranging from 0.00 to 1.00, corresponding to a steatosis scoring system of grades S0 to S4.

Among the 140 controls, the median SteatoTest value (± SE) was 0.08 ± 0.004 (95th percentile, 0.23). Among the 744 patients with liver biopsy, the SteatoTest conversion was linear. (Table 8 and Table 9) Table 8. Median SteatoTest values according to steatosis grade at liver biopsy in patients and according to control category (fasting, non-fasting, blood donors)

Subjects Group/Category Median SteatoTest

Fasting 0.13 Non-fasting 0.18

Controls

Blood donors 0.14 No steatosis 0.26 Grade 1 steatosis 0.43 Grade 2 steatosis 0.62 Grade 3 steatosis 0.70

Patients (biopsy) Grade 4 steatosis 0.75

Table 9. Conversion between the SteatoTest results and the corresponding steatosis

Diagnostic value of SteatoTest compared to GGT and ALT in the training group

SteatoTest had high AUROCs (SE) in the training and validations groups 1, 2 and 3: 0.79 (0.03); 0.80 (0.04); 0.86 (0.03); and 0.72 (0.05), respectively. The AUROCs of SteatoTest were always significantly higher than that of GGT and ALT (for the training and validation 1 groups). For the diagnosis of grade 2–4 steatosis, the sensitivity of SteatoTest at the 0.30 cut-off was 0.91, 0.98, 1.00 and 0.85 and the specificity at the 0.70 cut-off was 0.89, 0.83, 0.92, and 1.00, for the training and validation groups, respectively.

SteatoTest Predicted histological grade

Quantitative estimation

0.0000 – 0.3000 S0 No steatosis 0.3001 – 0.3800 S0-S1 0.3801 – 0.4800 S1

Mild steatosis (1-5%)

0.4801 – 0.5700 S1-S2 0.5701 – 0.6700 S2

Moderate steatosis (6-32%)

0.6701 – 0.6900 S2-S3 Marked steatosis (33- 66%) 0.6901 – 1.0000 S3-S4 Severe steatosis (>66%)


21

Results of the integrated database (all groups polled together), comparisons with ALT and GGT Integrated database (n=884) combined the training group, the three validation groups and the control group. There was a very significant overall correlation between SteatoTest and the steatosis grades from controls to S3. For SteatoTest, there was a significant difference between all histological grades for all pairwise differences between means (P < 0.05). Contrarily, for both GGT and ALT, there was no significant difference between S0 and S1. Moreover for ALT, there was no significant difference between S0 and S2, S1 and S2, and S2 and S3, either. For SteatoTest, a cut-off of 0.30 had 90% sensitivity and a cut-off of 0.70 had 88% specificity permitting to achieve useful predictive values for steatosis grade 2–4, 93% negative predictive value (NPV) and 63% positive predictive value (PPV) for a steatosis prevalence of 30%. The 90% specificity was obtained for a 0.72 cut-off with a corresponding 63% PPV. The overall percentage of patients classified with at least 90% sensitivity or 90% specificity was 59% (363+156/884). SteatoTest versus Ultrasonography for steatosis diagnosis Ultrasonography has been preformed together with SteatoTest and biopsy in 304 patients. Concordance between steatosis diagnosed, at ultrasonography and at biopsy, was lower (kappa coefficient = 0.32 ± 0.05) than the concordance with SteatoTest (at 0.50 cut-off, kappa = 0.44 ± 0.06; P = 0.02), as well as lower AUROC 0.65 ± 0.03 for ultrasonography versus 0.78 ± 0.03 for ST (P = 0.001). SteatoTest versus classical serum markers GGT and ALT for steatosis diagnosis Among the 744 patients with biopsy, for the diagnosis of steatosis S3S4, the SteatoTest AUROC was 0.79 (0.02), significantly higher than GGT 0.74 (0.02) (P = 0.03), and ALT 0.71 (0.02) (P = 0.007). The 90% sensitivity was obtained for a 0.32 cut-off; the 90% specificity was obtained for a 0.81 cut-off. (Table 10) SteatoTest could distinguish better than GGT and ALT between differents stages of fibrosis. (Figure 3, Panels A, B and C, respectively) Table 10. AUROCs of SteatoTest, GGT and ALT for the diagnosis of steatosis greater than 5%. Diagnostic panel

Training Group N=310

Validation group 1 (HCV before treatment) N=171

Validation group 2 (HCV sustained responders) N=201

Validation group 3 (Alcoholic liver disease) N=171

All groups

SteatoTest 0.79 (0.03)* 0.80 (0.04)£ 0.86 (0.03)$ 0.72 (0.05)** 0.80 (0.02)££ GGT 0.66 (0.03) 0.67 (0.05) 0.74 (0.05) 0.50 (0.09) 0.66 (0.02) ALT 0.58 (0.03) 0.62 (0.05) 0.79 (0.04) 0.66 (0.07) 0.61 (0.02) * – Higher than GGT (P < 0.0001) and ALT (P < 0.0001); £ – Higher than GGT (P = 0.007) and ALT (P < 0.0001); $ – Higher than GGT (P = 0.02); ** – Higher than GGT (P = 0.002); ££ Higher than GGT (P < 0.0001) and ALT (P < 0.0001). Sensitivity analyses: better diagnostic value with good quality biopsy A total of 670 (78%) patients had a biopsy sample length smaller than 20 mm. The AUROC of SteatoTest was slightly smaller in those patients (0.76, 95% CI 0.71–0.79) than in the 161 (15%) patients with greater biopsy sample (0.82, 95% CI 0.74–0.88; P = 0.10). There it was not found any significant differences in SteatoTest AUROCs according to ethnicity.


22

These results highlight the utility of a new panel of biochemical markers (SteatoTest) for the

prediction of steatosis of different origins (chronic viral hepatitis C and B, and alcoholic and non-alcoholic fatty liver diseases).

SteatoTest prediction for steatosis was better than the prediction provided by previous usual markers GGT, ALT and ultrasonography, as demonstrated by the significantly increase of AUROCs. Figure 3. Relationship between SteatoTest (Panel A), GGT (Panel B) and ALT (Panel C) and the grades of liver steatosis in the training group. A four grades scoring system was used to assess histological steatosis: S0 –no steatosis; S1- 1 to 5%, mild; S2- 6 to 32% moderate; S3-S4 -33 to 100%, marked or severe.

Panel A Panel B Panel C


23

6.2.3. SteatoTest external validation in CYTOL Study Group NAFLD patients SteatoTest was validated in a fourth independent group of NAFLD patients from the multicenter prospective CYTOL study cohort. In this independent group of patients the diagnostic value by AUROC (SE) for Steatosis more than 5% was 0.78 (SE=0.04). (Figure 4)

Figure 4. SteatoTest diagnostic value for 126 NAFLD patients of CYTOL prospective study group

6.2.4. Steatosis improvement by SteatoTest after HCV treatment7

The presence of steatosis at liver biopsy is often associated with chronic HCV infection and it was observed that steatosis diminishes in sustained viral responders (SVR).

The validation study of SteatoTest included 171 HCV patients before treatment. Among them, 75 patients with HCV were investigated twice (once before and then two years after treatment) permitted to assess the SteatoTest in HCV patients before and in HCV SVR after 2 years of follow-up. (Figure 5) Results of this analysis were not published in the validation study.

Mean SteatoTest improved significantly compared to baseline from 0.60 to 0.41 (p=0.001) after

2 years of follow-up in SVR patients. Figure 5. SteatoTest improvement in 75 HCV treated patients with Paired Biopsies (p=0.001)

0.4

0.6

0.8

Steatosis Improvement

Base l i n e 2 year


24

6.3. NashTest – serum marker of non-alcoholic steatohepatitis (NASH) 6.3.1. Introduction

Non-alcoholic steatohepatitis (NASH) is a histological pattern of the non-alcoholic fatty liver disease (NAFLD) which manifest as an excessive accumulation of hepatic fat in the absence of alcohol consumption. NAFLD is an increasingly recognized cause of liver-related morbidity and mortality.76-78 Although the majority of patients do not develop complications, 28% may develop serious liver sequelae, including end-stage liver disease and hepatocellular carcinoma. Those at highest risk include patients with significant hepatic necro-inflammation and fibrosis. 76-81

Liver biopsy, therefore, has been recommended for confirming its diagnosis and for providing prognostic information.82 The diagnosis of advanced fibrosis or NASH could be very important for motivating patients to make diet or lifestyle modifications, for the intensive treatment of complications of the metabolic syndrome or for providing weight in favor of anti-obesity surgery. FibroTest6 and SteatoTest7 has demonstrated high predictive values for advanced fibrosis and steatosis, respectively, in patients with NAFLD.

Therefore, early detection of NASH along with advanced fibrosis is the first step reducing future cirrhosis-related death.

6.3.2. Discovery and external validation (CYTOL Study Group NAFLD patients) Diagnostic value of biochemical markers (NashTest) for the prediction of non-alcoholo steato hepatitis in patients with non-alcoholic fatty liver disease.9 Poynard T, Ratziu V, Charlotte F, Messous D, Munteanu M, Imbert-Bismut F, Massard J, Bonyhay L, Tahiri M, Thabut D, Cadranel JF, Le Bail B, de Ledinghen V, LIDO Study Group , CYTOL Study Group. BMC Gastroenterol. 2006; 6: 34.

An estimated 15 to 20% of the Western European population has steatosis,19 while more than half of Americans are overweight or obese. Because liver biopsy is impossible to perform in such large cohorts, authors have tried to identify simple non-invasive markers of liver injury in patients with NAFLD.

The aim of this study was to develop and validate the diagnostic utility of a new panel of biomarkers of non alcoholo steato-hepatitis (NASH), the NashTest in patients with non-alcoholic fatty liver disease (NAFLD). Study population Study population included a training group, an independent validation group and a control group of 383 prospectively included blood donors and healthy volunteers. The inclusion criteria were: suspected NAFLD having steatosis at liver biopsy. Exclusion criteria: no histological steatosis, daily alcohol consumption >50 g/d for males and >30 g/d for females during the preceding year, concomitant liver diseases, HIV antibodies, immunosuppression, and an interval >3 months between serum sampling and liver biopsy.

Training group: 238 patients were hospitalized for suspected NAFLD; 160 patients were included and 78 patients were excluded.


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Validation group: between February 2002 and August 2004 166 patients with chronic abnormal ALT or GGT and steatosis at biopsy were considered for inclusion, all coming from a prospective multicenter study (CYTOL study) with the same inclusion/exclusion criteria as the training group. 97 patients were included and 69 patients were excluded. These patients were more representative of less specialized centers. Serum markers histological scoring

All the biochemical components had been prospectively assessed on fresh serum. Fibrosis was staged as follows: stage 0 = no fibrosis; stage 1 = perisinusoidal or periportal fibrosis;

stage 2 = perisinusoidal and portal/periportal fibrosis; stage 3 = bridging fibrosis; stage 4 = cirrhosis. Steatosis was scored S0 to S3: S0=no steatosis or <5%, S1=5%-33%, S2= 33%-66%, S3 >66%.

Two definitions were used for histological diagnosis of NASH. 1) A histological scoring system of NASH recently published by Kleiner et al was used for the construction of NashTest: steatohepatitis was defined as a NASH score (NAS) of 5 to 8, defined as the sum of the scores for steatosis (0-3), lobular inflammation (0-3), and ballooning (0-2); NAS of 0- 2 was considered not diagnostic of NASH; scores of 5-8 NASH.; scores 3-4 borderline (probable) NASH. 2) A second diagnostic of NASH was made by the pathologist independently of the NAS score. Construction of NashTest

The new panel, NashTest, was constructed in the training group combining age, gender, the 6 components of the FibroTest-ActiTest-HCV-FibroSURE (Biopredictive, Paris, France, patented artificial intelligence algorithm USPTO 6,631,330) plus weight, height, AST, serum glucose, triglycerides, cholesterol and SteatoTest.

When compared to patients with no or borderline NASH, those with NASH were older, had higher A2M, higher ALT and AST, higher glucose, higher triglycerides, higher FibroTest and SteatoTest scores. In multivariate analysis the most discriminator parameters were female gender and glucose. Characteristic of the patients Patients included in the training group were found to be older, and to have more diabetes and arterial hypertension, more elevated weight and less NASH than those of validation group and the biopsy size was longer with more portal tracts. Diagnostic value of NashTest According to Kleiner et al. diagnosis of NAS there was no difference between the AUROCs in the training and validation groups, respectively, for No NASH [AUROC = 0.77 (95%CI 0.68-0.84) versus 0.83 (95%CI 0.67-0.90; P=0.34)]; for Borderline NASH [AUROC = 0.69 (95%CI 0.60-0.77) versus 0.69 (95%CI 0.57-0.78; P=0.98)]; or for NASH [AUROC = 0.79 (95%CI 0.69-0.86) versus 0.79 (95%CI 0.67-0.78; P=0.87)]. (Figure 6) Table 11. Sensitivity, specificity and predictive values of NashTest for the diagnosis of NASH. NashTest diagnosis

Histological Diagnosis

Training Group n=160 Validation Group n=97 All patients n=257 Se Sp PPV NPV Se Sp PPV NPV Se Sp PPV NPV

NASH 11/28 39%

122/132 92%

11/21 52%

122/139 88%

10/35 29%

61/62 98%

10/11 91%

61/86 71%

21/63 33%

183/194 94%

21/32 66%

183/225 81%

Prevalence=0.18 Prevalence=0.36 Prevalence=0.25

Borderline or NASH

96/103 93%

20/57 35%

96/133 72%

20/27 74%

44/56 79%

29/41 71%

44/56 79%

29/41 71%

140/159 88%

49/58 50%

140/189 74%

49/68 72%

Prevalence=0.64 Prevalence=0.58 Prevalence=0.62


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Diagnostic value of NashTest for the diagnostic of NASH determined by the pathologist independent of NAS The AUROC for the diagnostic of NASH determined by the pathologist independent of NAS in the

overall population was 0.78 (95% CI 0.71-0.83) without significant difference between the raining and validation groups. Concordance between NashTest and histological diagnosis

Concordances of NashTest with biopsy results for predicting borderline NASH and NASH in the training (n=160), validation (n=97) groups and in all groups pooled together (n=257) were kappa (se) 0.28±0.05, 0.36±0.06 and 0.33±0.04, respectively.

Among 64/383 (17%) controls without biopsy and with more than 30% steatosis no one had NASH. Sensitivity analyses Sensitivity analyses of NashTest for the diagnosis of Nash vs No NASH or borderline and for the diagnosis of no NASH vs borderline NASH and NASH showed no significant differences in AUROCs, according to group, ALT values, alcohol consumption, exclusion of high risk of biomarkers failures, and quality of biopsy.

NashTest AUROCs for the diagnosis of NASH versus borderline or no NASH were unchanged in patients with ALT values lower than 50 IU/L versus ≥50UI/L which represents 50% of patients (0.78 vs 0.73, p NS); therefore NashTest could be used to diagnose NASH even in patients that are not eligible for liver biopsy. Analysis of discordance

A total of 11 (4%) clinically significant discordances (two stages or more) were been observed, nine attributable to NashTest failure (eight false negatives and one false positive) and two to biopsy (false negatives).

In the training group, there was a clinically significant (two stages or more) discordance in three patients, all with NT predicting NASH and biopsy showing no NASH. One of these cases had a good quality biopsy (30 mm non fragmented) but with piecemeal necrosis and no cause of NashTest false positive; two cases had a poor quality biopsy (15mm and five fragments, 22 mm and two fragments) with piece-meal necrosis at biopsy and no cause of NashTest false positive.

In the validation group there was clinically significant (two stages) discordance in eight patients (8%): all were predicted to have no NASH with NashTest and NASH at biopsy. None of these eight patients had a good quality biopsy. There was a very low triglyceride level (0.1 and 0.3 mmol/L) in two patients suggesting two instances of NashTest’s false negatives.

A new panel of biochemical markers (NashTest) is validated as a semi quantitative estimate with 3 classes: “No NASH”, “Borederline NASH”, “NASH”. In this study 4% of patients had discordances, 3.5% attributable to NashTest and 0.5% to biopsy.

These results highlight the utility of NashTest for patients with suspected NAFLD that will permit, along with previously validated FibroTest and SteatoTest, to reduce the use of liver biopsy, to identify the patients at risk and to reassure those without fibrosis or NASH.


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Figure 6. ROC curves of the NashTest for the diagnosis of NASH (Panel A) and No Nash (Panel B) in Training and in Validation Groups.

Panel A Panel B


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6.4. AshTest - marker of gravity of the alcoholic steatohepatitis (ASH) 6.4.1. Introduction

Chronic alcoholic liver disease (ALD) affects millions of individuals worldwide and is a major cause

of liver transplantation and death. 15–40% of ALD patients may develop end-stage liver disease, digestive hemorrhage and hepatocellular carcinoma. Those at the highest risk include patients with cirrhosis and alcoholic steato-hepatitis (ASH).72, 83-87

ASH is a necrotizing inflammatory lesion that in its severe form - Maddrey discriminant function (DF) >32 - is associated with high mortality88 despite corticosteroid treatment.89-90 Accessibility of transjugular liver biopsy permitting the diagnosis of ASH is low in many centers, dramatically reducing the proportion of patients treated with corticosteroids.91

6.4.2. Discovery and validation The diagnostic value of biomarkers (AshTest) for the prediction of alcoholic steato hepatitis in patients with chronic alcoholic liver disease8 Thabut D, Naveau S, Charlotte F, Massard J, Ratziu V, Imbert-Bismut F, Cazals-Hatem D, Abella A, Messous D, Beuzen F, Munteanu M, Taieb J, Moreau R, Lebrec D, Poynard T. J Hepatol. 2006; 44: 1175-85

The objectives of the current study were to evaluate the diagnostic utility of a new panel (AshTest) for the detection of alcoholic steato-hepatitis (ASH) in patients with chronic alcoholic liver disease (ALD).

Inclusion criteria were self-reported daily alcohol consumption > 50 g of pure ethanol during the

preceding year. Non-inclusion criteria were concomitant liver diseases, HIV antibodies and immunosuppression, non-available serum or biopsy or more than 1 month apart between both.

A total of 720 subjects were analyzed and were included: 70 patients in the training group, 62 in validation group 1 - called severe patients-, 93 in validation group 2-non-severe patients-, and 299 apparently healthy blood donors in the control group.

The main histological end point was the presence of ASH, defined by the presence of both polymorphonuclear neutrophil infiltrate (PMN) and hepatocellular necrosis. The secondary end points included ASH features: necrosis, PMN, Mallory bodies and ballooning, and a scoring system of ASH combining the detailed alcoholic features. Each feature was scored from 0 to 2 with a total score ranging from 0 to 8 with a four-grade scoring system: 0, no ASH; 1–2, mild; 3–4, moderate; 5–8, severe ASH. Fibrosis staging was adapted from the METAVIR: F0, no fibrosis; F1, fibrosis without septa; F2, few septa; F3, numerous septa, F4 cirrhosis.

The prevalence of histological diagnosis of ASH was different in the three groups, whatever its definition: the association of PMN and necrosis was more frequent (60%) in the training group vs the validation groups (19% vs 24%); and there was an absence of any ASH features in 13%, 48% and 30% of the groups, respectively. The prevalence of cirrhosis was similar in the training group (81%) and validation group 1 (90%) but lower in validation group 2 (25%).


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Construction of the test A new panel (AshTest), was constructed combining differently the six components of the FibroTest– ActiTest (patented artificial intelligence algorithm USPTO 6,631,330), with the serum AST activity and new algorithms adjusted by age and gender. The combination of the components was totally different from that of FibroTest–ActiTest.

AshTest provides a numerical quantitative estimate of liver ASH ranging from 0.00 to 1.00, corresponding to an ASH scoring system of grades ASH0–ASH1 to ASH3. (Table 12)

Table 12. Panel A. Conversion between the AshTest results and the corresponding histological grade. Panel B. Median AshTest values in blood donors and in patients according ASH grade at liver biopsy. Panel A. Panel B.

Diagnosis of alcoholic steatohepatitis (ASH)

The most significant components associated with the presence of ASH were BILI, GGT, and APOA1. In logistic regression analysis, the most significant component was APOA1, with an odds ratio=8.9, P=0.007. There was a dramatic decrease of serum apoA1 according to PMN infiltrate: 1.29 (0.04) g/L in 133 patients without PMN, 0.68 (0.09) in 57 patients with mild and 0.51 (0.07) in patients with severe PMN infiltrate (P<0.0001 between no vs either mild or severe PMN infiltrate). Diagnostic value of AshTest

For the main endpoint, ASH diagnosed as the presence of both necrosis and PMN infiltrate, AshTest had higher AUROCs in the training and validations groups 1 and 2, respectivement: [0.90, 0.88, 0.89] than Maddrey-DF [0.75, 0.64, 0.83] and the AST/ALT ratio [0.80, 0.79, 0.76] (all P<0.05 except between AshTest and AST/ALT in validation group 2-not severe patients).

The AUROC (se) of AshTest [0.89 (0.05)] in the integrated database (n=225) was higher than the AUROCs of each component and of Maddrey-DF and AST/ALT for each group (Table 13 and Figure 8)

An AshTest score of 0.50 had similar excellent diagnostic values in the training and validation groups: 88%, 92% and 59% sensitivity; and 79%, 72% and 94% specificity, respectively, higher than Maddrey-DF and the AST/ALT ratio.

There was a very significant overall correlation between AshTest and the ASH grades in all patients from controls to severe ASH. For AshTest there was a significant difference between all grades except between ASH grade 0 and 1. For Maddrey-DF and the AST/ALT ratio there was no significant difference between ASH grades 0 and 1 and between ASH grades 2 and 3. (Figure 7, Panels A, B and C, respectively)

AshTest Predicted histological grade

0.0000 – 0.1700 ASH0 – ASH1 0.1701 – 0.5535 ASH1 – ASH2 0.5536 – 0.7799 ASH2 0.7800 – 1.0000 ASH3

Category Median AshTest Value

Blood donors 0.005 No ASH 0.05 ASH grade 1 0.05 ASH grade 2 0.64 ASH grade 3 0.84


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Table 13. AUROCs of AshTest, AST - ALT ratio and Maddrey DF for the diagnosis of ASH (defined as presence of necrosis and PMN infiltrate) and its different components.

Diagnostic panel Alcoholic

hepatitis Hepatocellular necrosis

Polymorphonuclear infiltrate

Mallory bodies

Ballooning

Training group AshTest 0.90 0.68 0.87 0.75 0.78 AST/ALT 0.80 0.61 0.81 0.66 0.70 Maddrey 0.75 0.66 0.74 0.54 0.70

Validation group 1 AshTest 0.88 0.79 0.82 0.80 0.72 AST/ALT 0.79 0.63 0.65 0.77 0.69 Maddrey 0.64 0.63 0.67 0.61 0.64

Validation group 2 AshTest 0.89 0.64 0.87 0.90 0.74 AST/ALT 0.76 0.64 0.77 0.80 0.67 Maddrey 0.83 0.63 0.81 0.76 0.74

All groups AshTest 0.89 0.66 0.87 0.85 0.76 AST/ALT 0.78 0.60 0.76 0.76 0.69 Maddrey 0.78 0.60 0.78 0.72 0.71

Sensibility analysis according to the length of biopsy

When the AUROCs of AshTest, Maddrey-DF and AST/ALT ratio were compared according to the length of biopsy, there were always slightly better in patients with biopsies of 15 mm or more meanwhile NS, For biopsies higher and less than 15 mm leght, respectively AUROCs for AshTest were always significantly higher vs Maddrey-DF (P=0.005 and p=0.0004) and vs the AST/ALT ratio (P=0.01 and p=0.001).

Analysis of discordance

In the training and the two validation groups there were 39/225 (17%) cases of discordance between the diagnosis of ASH predicted by AshTest and that predicted by liver biopsy. Failure attributable to biopsy (false negative) was suspected in 15 cases (7%). Nine cases (3%) were indeterminate as small and fragmented biopsies but no sign of ASH. The 16 remaining cases (7%) were classified as false negatives of AshTest.

This study suggests that AshTest diagnostic value by AUROCs is significant better than Maddrey-DF at a cut-off of 32, which should not be used to make therapeutic decisions. Similarly, AshTest have better sensitivity and specificity in comparison with AST/ALT ratio for the diagnostic of severe alcoholic steatohepatitis.

AshTest is focused on the diagnosis of advanced ASH (with ASH defined as necrosis and PMN) in order to replace the transvenous liver biopsy with biomarkers in severe patients who could benefit from early treatment with corticosteroids.


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Figure 7. Box plots of AshTest (Panel A), Maddrey (Panel B) and AST/ALT (Panel B) ratio according to severity of alcoholic hepatitis (n=225). Panel A Panel B Panel C

Figure 8. AUROC curves of AshTest (solid black line), Maddrey (grey line) and AST/ALT ratio (dotted line) for the diagnosis of alcoholic hepatitis (n=225).


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7. Screening strategies with non-invasive biomarkers: FibroMAX (FibroTest, SteatoTest, NashTest) 7.1. Efficacy of screening of hyperlipidemics with non-invasive biomarkers Screening for liver disease using noninvasive biomarkers (FibroTest-SteatoTest-NashTest -FIBROSURE™) in patients with hyperlipidemia.92 Ratziu V, Giral P, Munteanu M, Messous D, Mercadier A, Bernard M, Morra R, Imbert-Bismut F, Bruckert E, Poynard T. Aliment Pharmacol Ther. 2007; 25: 207-218

Mortality related to complications of cirrhosis is increasing in patients with insulin-resistance factors.78 Hyperlipidemic patients could have increased serum levels of GGT or ALT and frequently have multiple risk factors of insulin resistance.93 Liver biopsy could not be performed in such a great number of patients and has the disadvantage of the sampling error, which has been well demonstrated for hepatitis C and also recently for non-alcoholic fatty liver disease (NAFLD).18 Two studies demonstrated that biomarkers had less sampling errors than small and fragmented liver biopsies for clinical endpoints.94,95

Non-invasive screening methods should be developed for hyperlipidemics to identify patients with advanced liver fibrosis (F2F3F4) or with steatohepatitis (NASH). Sensitivity analysis performed on studies which validated biomarkers (NashTest and SteatoTest) with liver biopsy, found identical diagnostic value of NashTest and SteatoTest according to the presence or absence of diabetes and hypertriglyceridemia.7,9

The main aim was to use non-invasive surrogate biomarker FibroTest, fully validated in several

pathologies, and newly developped biomarkers, SteatoTest and NashTest, previously validated in non-alcoholic steatosis (NAFLD), to assess the prevalence of advanced liver disease in a large population of hyperlipidemic patients free of other liver pathology.

A consecutive cohort of hyperlipidemics (HCV, HBV negative, hyperlipidemia secondary to renal

disease or hypothyroidism excluded) was prospectively followed in a lipid center and the sera (stored at –80° C) were retrospectively analyzed; a control group of blood donors (BD) was prospectively included (diabetics BD were excluded).

Advanced fibrosis, predicted when FibroTest was greater than 0.48, was defined as many septa, numerous septa, or cirrhosis. Advanced steatosis (more than 5%) was predicted when SteatoTest was greater than 0.57. The diagnosis of NASH was predicted in the three categories according to Kleiner et al: NASH (NAS 5-8), Borderline NASH (NAS 3-4), No NASH (NAS of 0-2).

A total of 2,933 subjects were pre-included; 54 hyperlipidemics and 34 BD were then excluded as high risk profile, 30 hyperlipidemics and 78 BD for missing data, leaving a total of 2,834 included subjects (51% female, median age 49 years, 1,909 hyperlipidemics and 925 BD). Among hyperlipidemics included, 83% had cholesterol >200mg/dl, 59% HDL-C <1.03mmol/L in males and <1.29mmol/L in females, 40% TG >=1.70mmol/L, 36% BMI >27, central obesity (by waist>102cm in male and >88cm female), 54% arterial hypertension or treatment, and 15% had fasting glucose >6.1mmol or diabetes treatment. GGT or ALT were elevated more than 50 IU/L in only 24% hyperlipidemics. Prevalence of liver fibrosis predicted by FibroTest, factors of advanced fibrosis

Presumed advanced fibrosis (F2F3F4) was predicted by FibroTest in 53/1909 (2.8%) hyperlipidemics, this included 31 F2, 14 F3, 3 F3F4 and 5 F4 versus none (0%) among the 925 BD (P<0.0001). Among diabetic hyperlipidemics patients the prevalence of F2F3F4 was higher (6.1%), with a maximum in patients with HOMA over 3.0 (9.5%).


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Clinical characteristics significantly associated with presumed fibrosis at FibroTest in univariate analysis were age, male gender, waist circumference (p<0.001). Drugs and alcohol consumption were not associated with fibrosis. In multivariate logistic regression taking in account metabolic factors not included in FibroTest algorithm, triglycerides increase and HDL-C decrease were risk factors of fibrosis. The prevalence of presumed advanced fibrosis increased linearly according to the number of metabolic syndrome factors (P<0.0001), reaching 9.4% in hyperlipidemic patients with 5 factors. Prevalence of steatosis predicted by SteatoTest, risk factors of severe steatosis

Advanced steatosis in 569/1893 hyperlipidemic patients (30%) versus 8/164 (5%) BD (P<0.0001); The prevalence of advanced steatosis was higher for type 2 diabetic hyperlipidemics (66%). The prevalence of presumed steatosis increased according to the number of metabolic syndrome factors, reaching 96% in hyperlipidemics with 5 factors, p<0.0001. Prevalence of steato-hepatitis predicted by NashTest, risk factors for NASH The highest prevalence of presumed NASH was observed among type 2 diabetic patients (23.6%), in patient with hypertriglyceridemia or on fibrate treatment (16%) and in patients with central obesity (15%). NASH prevalence was higher in hyperlipidemics with metabolic syndrome versus hyperlipidemics without (18.4% vs 1.8%, p<0.0001) and the prevalence increased lineary (p<0.0001) with the number of metabolic factors (0% with no factor to 50.9% in hyperlipidemics with 5 factors). Prevalence of liver injury according to the number of metabolic factors

The prevalence of presumed advanced fibrosis increased linearly according to the number of metabolic syndrome factors (P<0.0001), reaching 9.4% in hyperlipidemic patients with 5 factors.

The prevalence of presumed advanced steatosis increased according to the number of metabolic syndrome factors, reaching 96% in hyperlipidemics with 5 factors, p<0.0001.

NASH prevalence was higher in hyperlipidemics with metabolic syndrome versus hyperlipidemics without (18.4% vs 1.8%, p<0.0001) and the prevalence increased lineary (p<0.0001) with the number of metabolic factors (0% with no factor to 50.9% in hyperlipidemics with 5 factors). (Figure 9) Sensitivity analysis Fifty percent of patients were treated with lipid lowering agents (50% had statins) and this didn’t have an impact on presumed steatosis and NASH.

The use of biomarkers allows the estimation of the prevalence of advanced fibrosis, steatosis and steatohepatitis and their risk factors in a large sample of hyperlipidemic patients. The estimated prevalence.

The estimated prevalence of liver injury was 30% for advanced steatosis and 7% for NASH; hyperlipidemics had 3% advanced fibrosis compared to 0% in 925 blood donors. There was a clear increase of risk when two metabolic syndrome factors or more where present. The highest prevalence has been observed for hyperlipidemic diabetic patients: advanced steatosis 66%, NASH 24% and advanced fibrosis 6%.

The use of these biomarkers should help the clinician to reassure patients with only steatosis and to better manage patients with severe injuries such as advanced fibrosis and NASH.


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Figure 9. Prevalence (%) of liver injury (steatosis, NASH and fibrosis) according to the number of metabolic risk factors.

0

10

20

30

40

50

60

70

80

90

100

Steatosis NASH Fibrosis

0

1

2

3

4

5


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7.2. Efficacy of screening of diabetics with non-invasive biomarkers Efficacy of screening for advanced fibrosis (AF) in diabetic patients (DB) using non-invasive biomarkers: FibroTest -FibroMAX and Fibroscan. 96 Jacqueminet S, Morra R, Munteanu M, Devers L, Messous D, Bernard M, Jeanne S, Sachon C, Heurtier A, Bonnefont-Rousselot D, Mercadier A, Imbert Bismut F, Ratziu V, Grimaldi A, Poynard T. J Hepatol. 2006; 44 : S260 (Abstract) Diabetics are at risk of non-alcoholic fatty liver disease (NAFLD) leeding to cirrhosis and liver cancer. Due to high prevalence of diabetes liver biopsy cannot be performed to identify advanced fibrosis.

The primary aim was to identify advanced fibrosis (many septa F2, numerous septa F3 and cirrhosis F4) using non-invasive fibrosis biomarkers, FibroTest-FibroSURE in type 2 diabetics and concomitantly to estimate the prevalence of severe steatosis (more than 30%) and non-alcoholic steatohepatitis (NASH) using previously validated non-invasive biomarkers, SteatoTest and NashTest respectively.

The secondary aim was to confirm the clinical interest of the screening by FibroTest by reinvestigation of suspected advanced fibrosis with FibroScan (FSc).

Consecutive diabetics seen in a diabetes unit, were prospectively included, as well as a prospective control group of blood donors (BD). Security algorithms permitted to exclude high-risk (HR) profiles of serum biomarkers. FibroMax was performed blinded to all characteristics. Advanced fibrosis, predicted when FibroTest was greater than 0.48, was defined as many septa, numerous septa, or cirrhosis. Advanced steatosis (more than 5%) was predicted when ST was greater than 0.57. The diagnosis of NASH was predicted in the three categories according to Kleiner et al: NASH (NAS 5-8), Borderline NASH (NAS 3-4), No NASH (NAS 0-2).

Diabetics predicted advanced fibrosis (AF) at FibroTest were re-investigated by a hepatologist with FSc, APRI, and if necessary, ultrasonography, endoscopy or biopsy. Diabetics with previous suspected advanced fibrosis (pAF) were analyzed only for FibroTest validation purpose and diabetics without pAF as a true “naïve” population (nP). From 2,401 subjects pre-included, 252 were excluded (75 HR and 177 reinvestigated), 1,997 were included: 1,161 diabetics and 988 BD. 54% diabetics were male, mean age 54yrs, 68% diabetics type 2; 81% had fasting glucose ≥6.1 mmol/L, 68% had glycohaemoglobin ≥7.5%, 39% cholesterol ≥2.00 g/L, 27% triglycerides≥1.5g/L, 53% BMI ≥27, 50% arterial hypertension, 47% no alcohol consumption, 40% <50g/d, and 13% >50 g/d. Blood donors were younger (36years), had lower BMI (p<0.0001) and less blood donors had none alcohol consumption. Prevalence of hepatic lesions predicted by biomarkers In 1,161 diabetics, FibroTest predicted 94 (8.1%) advanced fibrosis: 28 F4, 26 F3 and 40 F2, versus 0 in BD (P<0.0001); 25 diabetics of the 94 had previous suspected advanced fibrosis (pAF) and 69 were a true “naïve” diabetic population (nP). FibroTest predicted 69 advanced fibrosis among 1136 nP diabetics (6.1%). Among 94 F2F3F4, 49 diabetics (12 pAF and 37 nP) from 94 AF have been reinvestigated at the time of this writing:

-among 25 diabetics known as pAF 12 were reinvestigated and all were confirmed as AF by Fibroscan (Fibroscan range 10.2-75kPa) that is 100% PPV in per protocol and 48% (12/25) in intention to detect analysis.


36

-among 69 nP, 37 were reinvestigated and AF was confirmed in 25 (range FS 6.9-34.3 kPa, 8 biopsies and 2 endoscopies), yielding a PPV of FibroTest = 67.6% (25/37) in per protocol and 36.2% (25/69) in intention to detect analysis For the 25 nP diabetics reinvestigated and with FibroTest results confirmed, the repartition of fibrosis

was as follows: 10 F2, 8 F3 and 7 cirrhosis (2 with confirmed and one suspected hepatocellular carcinoma, 1 moderate varices). The cause of advanced fibrosis was NAFLD alone in 15/25 (NASH in 7 and borderline NASH in 6) and NAFLD + HCV in 3, NAFLD + alcohol in 4 and NAFLD + metastasis, PBC and HBV, one in each respectively.

In multivariate analysis two factors were strongly associated (p<0.0001) with AF: type 2 diabetes (88%) and age more than 45 years (97%). In this high risk group for advanced fibrosis, the prevalence of predicted AF = 9.1% (62/679) vs 1.5% (7/457) in the non-high risk group (p<0.0001) and 0% in blood donors. The positive predictive value of FibroTest for confirmed AF in the high risk group = 25/35 (71.4%) in per protocol and 25/62 (40.3%) in intention to detect analysis. Steatosis and NASH predicted by SteatoTest and NashTest 388/912 (42.5%) diabetics had steatosis predicted by SteatoTest versus 11/222 (5%) BD (p<0.0001). In type 2 diabetics with advanced fibrosis versus no or minimal fibrosis, steatosis was predicted by SteatoTest in 74% versus 59%, respectively (p=0.03). 63/778 (8%) diabetics had NASH detected by NashTest versus no BD (p<0.0001). In type 2 diabetics with advanced fibrosis versus no or minimal fibrosis, NASH was predicted by NashTest in 22% versus 9%, respectively (p=0.005).

A screening strategy using FibroTest detected advanced fibrosis (F2F3F4) in 6.1% of diabetics followed in a tertiary center and not suspected previously of liver disease. 36% of these had already been confirmed including 7 asymptomatic cirrhosis. The prevalence of predicted NASH in diabetics estimated by NashTest is 8% vs 0 blood donors (p<0.0001).

This study strongly suggests that non-invasive biomarkers could be very useful for the screening of advanced fibrosis and NASH in type 2 diabetics older than 45 years to prevent liver mortality. (Figure 10)


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8. Conclusion

More than 170,000,000 people worldwide are chronically infected with the hepatitis C virus (HCV), more than 350,000,000 with the hepatitis B virus (HBV) and more than one billion are at risk of alcoholic and non alcoholic steatosis. These four diseases are responsible for more than 500, 000 annual cases of liver cancer and similar numbers of digestive hemorrhage and ascites episodes. Major breakthroughs have been achieved in recent years in for the specific diagnosis and treatment of these diseases.

Unfortunately, even in developed countries, mortality due to chronic liver diseases is increasing due to a lack of detection and treatment.

FibroMax’s tests (FibroTest, SteatoTest, ActiTest, NashTest and AshTest) are simple and non-

invasive alternatives to liver biopsy. The liver biopsy, though considered the gold standard, does not have the expected advantages of

a first line procedure to assess the severity of chronic liver diseases or acute hepatitis in excessive drinkers. The main disadvantages of liver biopsy are sampling errors, cost and severe adverse events.

FibroMax will be useful in decreasing the human, social and economic costs of the management of chronic liver diseases.

The early use of these biomarkers will allow for faster and simpler access to the healthcare system for patients before the occurrence of end stage liver diseases.


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9. FibroMAX pack: how and when to use non-invasive biomarkers, recommended algorithms 9.1. Best management of the patients with metabolic risk factors (overweight, dyslipidemia, diabetes). FibroMAX pack: FibroTest, SteatoTest and NashTest

The need of mass screening for significant liver injury for providing prognostic information in patients with NAFLD is increasing due to the epidemics of obesity and diabetics.19

Two recent studies92,96 (see Chapter 7) proved the efficacy of the screening with the new generation tests of FibroMax (in the combination FibroTest, SteatoTest and NashTest) patients with metabolic risk factors in particular hyperlipidemics and diabetic subjects. 6-7,9 These tests will allow better identification and estimation of the severity of those patients at risk (fibrosis and NASH) and reassurance for patients without fibrosis or NASH among those with steatosis.

Figure 10. Proposed screening strategy of advanced fibrosis, steatosis and NASH in NAFLD patients.

Type2 DB >45yrs Overweight, Metabolic

sd. Hyperlipidemics Screening FibroMAX

F0F1, No NASH

No or minimal fibrosis Absent NASH

F2, borderline NASH

Septal fibrosis Borderline NASH

F3F4, NASH Pre-cirrhosis/cirrhosis Present NASH

Excluded High risk of

false positive / negative

Every 5 years FibroMAX :

FibroTest SteatoTest + NashTest

Every year FibroMAX: FibroTest

SteatoTest + NashTest Reducing risk factors

Every year FibroMAX: Fibroscopy (OV) ECHO (nodule) αFP


39

9.2. Best management of the patients with excessive alcohol intake. FibroMAX pack: FibroTest, SteatoTest and AshTest

Chronic alcoholic liver disease (ALD) affects millions of individuals worldwide and is a major cause of liver transplantation and death. Those ALD patients at the highest risk to develop complications and end-stage liver disease include patients with cirrhosis and alcoholic steato-hepatitis (ASH) 72, 83-87. ASH is a necrotizing inflammatory lesion that in its severe form (Maddrey-DF >32) is associated with high mortality 88 despite corticosteroid treatment. 89-90 The use of non-invasive biomarkers validated for the prediction of fibrosis5 and ASH8 is expected to increase the proportion of patients with severe ASH treated with with corticosteroids without liver biopsy. (Figure 11). Figure 11. Proposed screening strategy of severe alcoholic steatohepatitis (ASH) in excessive drinkers. Panel A. ASH screening and treatment strategy. Panel B. Fibrosis screening strategy in ALD patient.

Excessive drinker SCREENING ASH with

FibroMAX AshTest

AshTest <0.50

AshTest >0.50 Severe ASH No Biopsy



No need of specific treatment

(corticosteroids)

Maddrey-DF <32 Benefit-risk of corticosteroids?

Maddrey-DF >32 Specific treatment:

corticosteroids

Excessive drinker SCREENING FibroMAX

FibroTest + SteatoTest + AshTest if suspicion of ASH

F0F1

No or minimal fibrosis

F2, borderline NASH

Septal fibrosis

F3F4, NASH Pre-cirrhosis/cirrhosis



Every 5 years FibroMAX :

Every year FibroMAX

Every year FibroMAX: Fibroscopy (OV) ECHO (nodule) αFP


40

9.3. Best management of the patients with chronic viral hepatitis 1,72

FibroMAX pack: FibroTest, SteatoTest and ActiTest Figure 12. Recommended algorithm for FibroTest-ActiTest-FIBROSURE™ or alternative prescription.

Treatment

Severe-Fibrosis Cirrhosis:

FibroTest >0.58

or Severe Activity:

ActiTest >0.60

Treatment

or Follow-Up

FibroTest-ActiTest

every 6 months

Moderate Fibrosis

FibroTest 0.32-0.58

Moderate Activity

ActiTest 0.37-0.60

Follow-up

FibroTest-ActiTest

every 12 months

No or Minimal Fibrosis

FibroTest 0.00-0.31

No or Minimal Activity

ActiTest 0.00-0.36

No Biopsy

Yes

No risk of false positive

No risk of false negative

Treatment

Or

Follow-up

Risk of false positive

Risk of false negative

FibroTest-ActiTest

Interpretable?

Genotype

Viral Load

FibroTest-ActiTest

HCV RNA detectable

No (extreme values)

Biopsy? Fibroscan?

< 5%of cases

WARNING95% of cases

OK


41

10. References

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46

80. Ratziu V, Bonyhay L, Di Martino V, Charlotte F, Cavallaro L, Sayegh-Tainturier MH, Giral P, Grimaldi A, Opolon P, Poynard T: Survival, liver failure, and hepatocellular carcinoma in obesity-related cryptogenic cirrhosis. Hepatology 2002, 35:1485-93. 81. Ratziu V, Giral P, Charlotte F, Bruckert E, Thibault V, Theodorou I, Khalil L, Turpin G, Opolon P, Poynard T: Liver fibrosis in overweight patients. Gastroenterology 2000, 118:1117-1123. 82. Talwalkar JA: Motion – all patients with NASH need to have a liver biopsy: arguments for the motion. Can J Gastroenterol 2002, 16:718-21. 83. Diehl AM. Liver disease in alcohol abusers: clinical perspective. Alcohol 2002; 27: 7–11. 84. Maher JJ. Alcoholic steatosis and steatohepatitis. Semin Gastrointest Dis 2002; 13: 31–39. 85. Chikritzhs TN, Jonas HA, Stockwell TR, Heale PF, Dietze PM. Mortality and life-years lost due to alcohol: a comparison of acute and chronic causes. Med J Aust 2001; 174: 281–284. 86. Menon KV, Gores GJ, Shah VH. Pathogenesis, diagnosis, and treatment of alcoholic liver disease. Mayo Clin Proc 2001; 76: 1021–1029. 87. Tome S, Lucey MR. Current management of alcoholic liver disease. Aliment Pharmacol Ther 2004; 19: 707–714. 88. Orrego H, Blake JE, Blendis LM, Medline A. Prognosis of alcoholic cirrhosis in the presence and absence of alcoholic hepatitis. Gastroenterology 1987;92:208–214. 89. Mathurin P, Duchatelle V, Ramond MJ, Degott C, Bedossa P, Erlinger S, et al. Survival and prognostic factors in patients with severe alcoholic hepatitis treated with prednisolone. Gastroenterology 1996; 110:1847–1853. 90. Mathurin P, Mendenhall CL, Carithers Jr RL, Raymond MJ, Maddrey WC, Garstide P, et al. Corticosteroids improve short-term survival in patients with severe alcoholic hepatitis (AH): individual data analysis of the last three randomized placebo controlled double blind trials of corticosteroids in severe AH. J Hepatol 2002; 36: 480–487. 91. Menon KV, Gores GJ, Shah VH. Pathogenesis, diagnosis, and treatment of alcoholic liver disease. Mayo Clin Proc 2001;76: 1021–1029. 92. Ratziu V, Giral P, Munteanu M, Messous D, Mercadier A, Bernard M, Morra R, Imbert-Bismut F, Bruckert E, Poynard T. Screening for liver disease using noninvasive biomarkers (FibroTest-SteatoTest-NashTest -FIBROSURE™) in patients with hyperlipidemia. Aliment Pharmacol Ther. 2007; 25: 207-218 Bruckert E, Giral P, Ratziu V, Poynard T, Chapman MJ, Opolon P, Turpin G. A constellation of cardiovascular risk factors is associated with hepatic enzyme elevation in hyperlipidemic patients. Metabolism 51:1071-1076, 2002 93. Poynard T, Munteanu M, Imbert-Bismut F, Charlotte F, Messous D, Dominique Thabut D, Thibaut V, Benhamou Y, Ratziu V. Prospective analysis of discordant results between biochemical markers and biopsy in patients with chronic hepatitis C. Clin Chem 50:1344-1355, 2004 94. Ngo Y, Munteanu M, Messous D, Charlotte F, Imbert-Bismut F, Thabut D, Lebray P, Thibault V, Benhamou Y, Moussalli J, Ratziu V, Poynard T. A prospective analysis of prognostic value of biomarkers (FibroTest) in patients with chronic hepatitis C (CHC) Clin Chem. 2006; 52: 1887-96. 95. Jacqueminet S, Morra R, Munteanu M, Devers L, Messous D, Bernard M, Jeanne S, Sachon C, Heurtier A, Bonnefont-Rousselot D, Mercadier A, Imbert Bismut F, Ratziu V, Grimaldi A, Poynard T. Efficacy of screening for advanced fibrosis (AF) in diabetic patients (DB) using non-invasive biomarkers: FibroTest-FibroMAX and Fibroscan. J Hepatol. 2006; 44 : S260 (Abstract)

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