METHOD OF DIAGNOSIS OF LIVER STEATOSIS

Abstract

The present invention relates to new methods for assessing the presence of nonalcoholic steatohepatitis (steatosis) in a patient, using functions combining biological markers without bilirubin and Body Mass Index being used as markers in the function.

Claims

1. An in vitro method for diagnosing and treating a patient for liver steatosis comprising: (a) combining the values of at least three biological markers selected from the group consisting of: the amount of biochemical markers as measured circulating in the blood, serum, plasma, of the subject, and optionally physical characteristics of a patient, in a function, in order to obtain an end value, wherein the function includes the values measured for circulating Alpha-2-macroglobulin, ApoA1, GGT (gammaglutamyl transpeptidase), Haptoglobin, alanine transaminases (ALT), aspartate transaminases (AST), fasting glucose, total cholesterol and triglycerides, (b) optionally, comparing the end value to predetermined values, (c) determining presence of steatosis based on the end value calculated in (a), with the proviso that Body Mass Index and level of bilirubin in the subject are not used as biological markers in (a), and (d) treating the subject depending on its level of steatosis.

2. The in vitro method of claim 1, wherein circulating biochemical markers are furthers elected from the group consisting of -globulin, albumin, 1-globulin, 2-globulin, -globulin, IL10, TGF-1, apoA2, apoB, cytokeratin 18, platelets number, prothrombin level, hyaluronic acid, urea, N-terminal of type III pro-collagen, tissue inhibitor metalloproteinase type-1 (TIMP-1), type IV collagen (Coll IV), osteoprotegerin, miRNA122, cytokeratin-18, serum amyloid A (SAA), alpha-1-antitrypsin (isoform 1), fructose-bisphosphate aldolase A, Fructose-bisphosphate aldolase B, fumarylacetoacetase, transthyretin, PR02275, C-reactive protein (isoform 1), leucine-rich alpha-2-glycoprotein, serpin A11, DNA-directed RNA polymerase I subunit RPA1, obscurin (isoform 1), alpha-skeletal muscle actin, aortic smooth muscle actin, alkaline phosphatase, uncharacterized protein C22orf30 (isoform 4), serum amyloid A2 (isoform a), apolipoprotein apolipoprotein E, apolipoprotein A-II, polymeric immunoglobulin receptor, von Willebrand factor, aminoacylase-1, G-protein coupled receptor 98 (isoform 1), paraoxonase/arylesterase 1, complement component C7, hemopexin, complement C1q subcomponent, paraoxonase/lactonase 3, complement C2 (fragment), versican core protein (isoform Vint), extracellular matrix protein 1 (isoform 1), E3 SUMO-protein ligase RanBP2, haptoglobin-related protein (isoform 1), adiponectin, retinol binding protein, ceruloplasmin, alpha 2 antiplasmin, antithrombin, thyroxin binding protein, protein C, alpha 2lipoprotein, tetranectin, fucosylated A2M, fucosylated haptoglobin, fucosylated apoA1, and carbohydrate deficient transferrin.

3. The in vitro method of any one claim 1, wherein the function includes at least one variable chosen from the group consisting of gender and age of the subject.

4. The in vitro method of claim 1, wherein the function includes the values measured for circulating Alpha-2-macroglobulin, ApoA1, GGT, Haptoglobin, ALT, AST, fasting glucose, total cholesterol, triglycerides, as well as the values corresponding to the age and sex of the patient.

5. The in vitro method of claim 1, wherein the function is a logistic function obtained by logistic regression.

6. The in vitro method of claim 5, wherein the function has been obtained by: a) evaluating the presence of steatosis in a cohort of patients, wherein the values of circulating biochemical markers are known for the patients, b) identifying by unidimensional analysis, the circulating biochemical markers for which the values differ significantly between the groups of i. patients with steatosis, and ii. patients without steatosis, c) performing a logistic regression analysis to assess the independent discriminative value of the markers identified in step b) for the occurrence of steatosis, and d) combining these identified independent factors to obtain thereby obtaining the function, by combination of these identified independent factors, wherein the identified independent factors includes the values measured for circulating Alpha-2-macroglobulin, ApoA1, GGT, Haptoglobin, ALT, AST, fasting glucose, total cholesterol and triglycerides, and wherein Body Mass Index and level of bilirubin are not combined in the function.

7. The in vitro method of claim 1, wherein the function is a0+a1Age(years)+a2ApoA1(g/l)+a3Log(A2M, g/l)+a4Log(GGT, IU/1)+a5Log(ALT, IU/1)+a6Log(AST, IU/1)+a7Log(Hapto, g/l)+a8Log(Triglycerides TG, mmol/l)+a9Log(Total Cholesterol CT, mmol/l)+a10Log(Fasting glucose, g/l)+a11Gender(0 for women, 1 for men).

8. The in vitro method of claim 7, wherein a) 4.8a05.5, b) 0.03a10.015, c) 0.9a21.2, d) 1.8a32.2, e) 1.3a41.1, f) 1.45a51.2, g) 0.5a60.7, h) 0.35a70.22, i) 1.45a81.25, j) 0.8a90.6, k) 3.55a103.35, and l) 0.35a110.55,

9. The in vitro method of claim 1, wherein the function is 5.17-0.022Age (years)+1.02ApoA1(g/l)+1.99Log(A2M, g/l)1.16Log(GGT, IU/1)1.33Log(ALT, IU/1)+0.6Log(AST, IU/1)0.29Log(Hapto, g/l)1.35Log(Triglycerides TG, mmol/l)0.68Log(Total Cholesterol CT, mmol/l)3.46Log(Fasting glucose, g/l)+0.46Gender(0 for women, 1 for men).

10. The in vitro method of claim 9, wherein the predetermined value is 0.25, and wherein the patient has nonalcoholic steatohepatitis (steatosis) if the end result is higher or equal to 0.25, and does not have nonalcoholic steatohepatitis (steatosis) if the end result is below 0.25.

11. A method for obtaining a function for identifying the presence of steatosis in a patient, wherein said function combines the values of the concentration of biochemical markers in the blood/serum or plasma of said patient and optionally the age and/or gender of the patient, comprising the steps of: a) evaluating the presence of steatosis in a cohort of patients, wherein the values of circulating biochemical markers are known for the patients, b) identifying by unidimensional analysis, the circulating biochemical markers for which the values differ significantly between the groups of i. patients with steatosis, and ii. patients without steatosis, c) identifying whether the age and/or gender of the patient differ significantly between the groups of i. patients with steatosis, and ii. patients without steatosis, d) performing a logistic regression analysis to assess the independent discriminative value of the markers identified in step b) and c) for the occurrence of steatosis, wherein Body Mass Index and level of bilirubin are not incorporated in the list of markers from which the logistic regression is performed. e) combining these identified independent factors to obtain the function, wherein the function allows for diagnosing the presence of nonalcoholic steatohepatitis (steatosis) in a subject and does not use the values of bilirubin or BMI.

12. The method of claim 11, wherein the biochemical markers of step b) are selected from the group consisting of a 2-macroglobulin (A2M), GGT (gammaglutamyl transpeptidase), haptoglobin, apolipoprotein A-I (apoA1), alanine transaminases (ALT), aspartate transaminases (AST), triglycerides, total cholesterol, fasting glucose, -globulin, albumin, 1-globulin, 2-globulin, -globulin, IL10, TGF-1, apoA2, apoB, cytokeratin 18, platelets number, prothrombin level, hyaluronic acid, urea, N-terminal of type III pro-collagen, tissue inhibitor metalloproteinase type-1 (TIMP-1), type IV collagen (Coll IV), osteoprotegerin, miRNA122, cytokeratin-18, serum amyloid A (SAA), alpha-1-antitrypsin (isoform 1), fructose-bisphosphate aldolase A, Fructose-bisphosphate aldolase B, fumarylacetoacetase, transthyretin, PR02275, C-reactive protein (isoform 1), leucine-rich alpha-2-glycoprotein, serpin A11, DNA-directed RNA polymerase I subunit RPA1, obscurin (isoform 1), alpha-skeletal muscle actin, aortic smooth muscle actin, alkaline phosphatase, uncharacterized protein C22orf30 (isoform 4), serum amyloid A2 (isoform a), apolipoprotein apolipoprotein E, apolipoprotein A-II, polymeric immunoglobulin receptor, von Willebrand factor, aminoacylase-1, G-protein coupled receptor 98 (isoform 1), paraoxonase/arylesterase 1, complement component C7, hemopexin, complement C1q subcomponent, paraoxonase/lactonase 3, complement C2 (fragment), versican core protein (isoform Vint), extracellular matrix protein 1 (isoform 1), E3 SUMO-protein ligase RanBP2, haptoglobin-related protein (isoform 1), adiponectin, retinol binding protein, ceruloplasmin, alpha 2 antiplasmin, antithrombin, thyroxin binding protein, protein C, alpha 2lipoprotein, tetranectin, fucosylated A2M, fucosylated haptoglobin, fucosylated apoA1, carbohydrate deficient transferrin, -fetoprotein (AFP), fucosylated AFP, HSP27 (heats hock protein), HSP70, Glypican-3 (GPC3), squamous cell carcinoma antigen (SCCA) and in particular SCCA-IgM IC which is a circulating immune complex composed of SCCA and IgM, Golgi protein 73 (GP73), -L-fucosidase (AFU), Des--carboxyprothrombin (DCP or P IVKA), Osteopontin (OPN), and Human Carbonyl Reductase.

13. An ex vivo method for diagnosing and treating liver disease in a patient comprising: (a) combining the values of at least three biological markers selected from the group consisting of: the amount of biochemical markers as measured circulating in the blood, serum, or plasma, of the subject, and optionally physical characteristics of a patient, in a function, in order to obtain an end value, (b) optionally, comparing the end value to predetermined values, and (c) determining presence of steatosis based on the end value calculated in (a), with the proviso that Body Mass Index and level of bilirubin in the subject are not used as biological markers in (a), (d) determining the presence of liver fibrosis in the patient, (e) determining the presence of liver inflammation in the patient, (f) diagnosing a liver disease in the patient depending on the results of the above determinations, and (g) treating the liver disease depending on the results of the above determinations.

14. The method of claim 13, wherein liver fibrosis is determined by the combination of the values of biological markers in a function.

15. The method of claim 13, wherein presence of liver inflammation in the patient is determined by the combination of the values biological markers in a function.

16. The method of claim 13, wherein the level of liver fibrosis is determined by Fibrotest, which corresponds to the function: 4.467Log(Alpha2Macroglobulin(g/l))1.357Log(Haptoglobin(g/l))+1.017Log(GGT (IU/1))+0.0281Age(in years)+1.737Log(Bilirubin(mol/l))1.184ApoA1(g/l)+0.301Sex(female=0, male=1)5.540, or by ex vivo measurement of liver stiffness by Vibration Controlled Transient Elastography.

17. The method of claim 13, wherein the presence of liver inflammation in the patient is determined by the combination of the values biological markers by a function is a0+a1Age(years)+a2ApoA1(g/l)+a3Log(A2M, g/l)+a4Log(GGT, IU/1)+a5Log(ALT, IU/1)+a6Log(AST, IU/1)+a7Log(Hapto, g/l)+a8Log(Triglycerides TG, mmol/l)+a9Log(Total Cholesterol CT, mmol/l)+a10Log(Fasting glucose, g/l)+a11Gender(0 for women, 1 for men), wherein a) 4.8a05.5 b) 0.03a10.015 c) 0.9a21.2 d) 1.8a32.2 e) 1.3a41.1 f) 1.45a51.2 g) 0.5a60.7 h) 0.35a70.22 i) 1.45a81.25 j) 0.8a90.6 k) 3.55a103.35 l) 0.35a110.55.

18. The in vitro method of claim 7, wherein a) 5a05.3, b) 0.025a10.02, c) 0.95a21.05, d) 1.95a32.05, e) 1.2a41.1, f) 1.36a51.3, g) 0.55a60.65, h) 0.3a70.25, i) 1.4a81.3, j) 0.75a90.65, k) 3.5a103.4, and l) 0.4a110.5.

19. The method of claim 1, wherein the patient has steatosis and treating the patient comprises a dietary treatment to manage obesity, high blood sugar, high blood lipids, or excess iron levels.

20. The method of claim 1, wherein the patient has steatosis and treating the patient comprises administering a medicinal drug.

Description

DESCRIPTION OF THE FIGURES

[0195] FIG. 1: Populations used for construction, validation and target.

[0196] FIG. 2: Non-inferiority of SteatoTest-2 compared to the original SteatoTest for the diagnostic of Steatosis>=5%. [0197] Legends: .sup.1 NASH patients were analyzed at 24 weeks' biopsy as steatosis <5% were absent at baseline biopsy (72 cases) according to inclusion criteria in the trial. Only 3 out of 63 cases analyzed at 24 weeks had no steatosis (<5%). Due to this very small sample size the power of the comparison is weak. For the integrated analyses all 72 cases with baseline biopsies were included. [0198] .sup.2 The AUROCs were highly decreased by a spectrum effect in chronic hepatitis C, as cases with minimal steatosis (1-4%) represented 732/918 (80%) of S0 as defined by CRN score. Unfortunately, these minimal steatosis grades were not described in metabolic disease. [0199] .sup.3 The AUROCs were increased by a spectrum effect of the control group as the blood donors had much lower steatosis tests medians than controls with biopsy, increasing the specificity. The prevalence of steatosis was not dramatically increased. [0200] .sup.4 The AUROCs were highly impacted by a dramatic increase of the prevalence of controls, despite their higher tests medians, as the prevalence of steatosis was divided by two in comparison with the integrated population with biopsies. [0201] .sup.5 Adding the blood donor's subset did not change significantly the AUROCs.

[0202] FIG. 3: Decrease of steatosis, NASH and fibrosis biomarkers in the selonsertib trial. SteatoTest-2 decrease by 0.02 vs +0.04 (P=0.03), SteatoTest 0.014 vs +0.014 (P=0.02), NashTest-2 0.06 vs +0.01, and FibroTest 0.02 vs +0.02 (P=0.04), in patients who improved NAS vs those who did not improved at 24 weeks.

EXAMPLES

Example 1. Summary

[0203] Methods. Five different subsets of 2,997 patients with biopsies were evaluated for test construction and validation, and four to assess the prevalence of steatosis in target populations with increasing risks of steatosis. The performance of the SteatoTest-2 was compared to the reference test, using the non-inferiority test (0.10 margin) and the Lin concordance coefficient. [0204] Results. The AUROCs of the SteatoTest-2 were non-inferior to the reference test (P<0.001). AUROCs varied in the SteatoTest-2 and the reference test according to subsets and the prevalence of steatosis, with 0.772 (95% CI 0.713-0.820) vs 0.786 (0.729-0.832) in the 2,997 cases with biopsy and 0.822 (0.810-0.834) vs 0.868 (0.858-0.878) in the 5,776 cases including healthy subjects without risk factors of steatosis as controls, respectively. The Lin coefficient was highly concordant (P<0.001), from 0.74 (0.74-0.74) in presumed NAFLD to 0.91 (0.89-0.93) in the construction subset. [0205] Conclusions. The SteatoTest-2 is simpler and non-inferior to the first generation SteatoTest for the diagnosis of steatosis, without the limitations of body mass index and bilirubin.

Example 2. Patients and Methods

Study Design

[0206] The primary aim of the study that prospectively analyzed patient subsets (FIG. 1), was to construct a new SteatoTest-2 that was simplified, highly concordant with and not inferior to the first generation SteatoTest, with greater applicability and less risk of false positives in the general population. The SteatoTest-2 was also assessed in a prospective trial of selonsertib for the treatment of NASH based on paired biopsies before and after treatment.

[0207] The secondary aim was to assess the performance of SteatoTest-2 when combined with NashTest-2 as disclosed in WO2018050804, in non-invasive algorithms reproducing the histological NASH-algorithms (CRN or FLIP), which needed the presence of steatosis for the diagnostic of NASH (Poynard al, Eur J Gastroenterol Hepatol. 2018; 30:384-391; Poynard et al, Eur J Gastroenterol Hepatol. 2018; 30:569-577).

[0208] The variability of SteatoTest-2 AUROCs was evaluated in relation to the prevalence of steatosis, the spectrum effect, degrees of inflammation, stages of fibrosis, fasting glucose, and BMI. Finally, the SteatoTest-2 was validated for the CRN-grades of moderate and marked steatosis (European Association for the Study of the Liver (EASL); European Association for the Study of Diabetes (EASD); European Association for the Study of Obesity (EASO). EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease. J Hepatol. 2016; 64:1388-402).

Patients and Controls

[0209] A total of nine different subsets of individual data were included. All data were previously published (FIG. 1). The first was the construction subset of the original test (C1) including 307 cases with different causes of steatosis, and controls (6. Poynard et al. Comp Hepatol 2005; 4:10). Four subsets were used for validation (V1) including 600 patients with NAFLD (Munteanu et al., Aliment Pharmacol Ther 2016; 44:877-889); (V2) 481 obese patients (Poynard et al, PLoS One. 2012; 7:e30325); (V3) 72 with NASH (Loomba et al, Hepatology. 2017); and (V4) 1,537 with chronic hepatitis C (Poynard et al, J Hepatol. 2011; 54:227-235). These five subsets had biopsies. Finally, four subsets without biopsies were used to assess the prevalence of steatosis presumed by both SteatoTest-2 and SteatoTest, in targeted subjects with increasing risks of steatosis; (T1) 327 blood donors (Jacqueminet et al, Clin Gastroenterol Hepatol. 2008; 6:828-31); (T2) 7,416 healthy volunteers (Poynard et al, BMC Gastroenterol 2010; 10:40); (T3) 359 type2-diabetics (Jacqueminet et al, op. cit.) and (T4) 133,045 patients with NAFLD (Munteanu et al, 2016, op. cit.). Controls with a low or very low risk are described in the statistics section.

Histological References

[0210] All biopsies were scored by experienced pathologists, blinded to historical biopsy reports, test results, and other clinical data. The FLIP-CRN scoring system, known in the art, was used for the main endpoint. The steatosis score (S) assesses and rates the quantity of large or medium-sized lipid droplets from 0 to 3, except for foamy microvesicles, (S0: <5%; S1: 5-33%, mild; S2: >33-66%, moderate; S3: 66%, marked). Activity grade (A, from 0 to 4) is the unweighted addition of hepatocyte ballooning (0-2) and lobular inflammation (0-2). Cases with A0 (A=0) have no activity; A1 (A=1) mild activity; A2 (A=2) moderate activity; A3 (A=3) severe activity and A4 (A=4) very severe activity. Fibrosis stage (F) was assessed by the following score: stage 0 (F0)=none; stage 1 (F1)=1a or 1b perisinusoidal zone 3 or 1c portal fibrosis; stage 2 (F2)=perisinusoidal and periportal fibrosis without bridging; stage 3 (F3)=bridging fibrosis and stage 4 (F4)=cirrhosis. To reduce interobserver variability and standardize the reading based on the new SAF-FLIP histological classification, reports reviewed by members of the FLIP Pathology Consortium (Frederic Charlotte, for C1, V1, V2, Pierre Bedossa for C1, V1, V4 Dina Tianakos for V1) or CRN (Zack Goodman for V1, and V3) were used.

Blood Tests

[0211] The FibroTest, ActiTest and original SteatoTest are patented as In Vitro Diagnostic Multivariate Index Assays for the diagnosis of METAVIR fibrosis stages, including cirrhosis, for SAF-equivalent activity and for SAF-equivalent steatosis grades, respectively (Bedossa et al, Hepatology 2012; 56: 1751-9). A quantitative NashTest-2 was constructed and internally validated in 1081 patients at risk of metabolic liver disease (Poynard et al, Eur J Gastroenterol Hepatol. 2018; 30:569-577). These tests are exclusively available online and include clinical security algorithms. The recommended cutoffs were the same, whatever the chronic liver disease. Analyzers and kits were those validated and recommended by BioPredictive, and all controls assays were performed in the reference biochemistry department of Piti Salptrire Hospital.

[0212] The original SteatoTest, recommended in recent NAFLD guidelines, was used as the comparator (European Association for the Study of the Liver (EASL), op. cit.). The SteatoTest-2 was constructed by regression analysis without BMI or total bilirubin as components, but with AST. Thus, the new test included the following ten components in its patented formula: alpha2-macroglobulin, apolipoprotein A1, haptoglobin, GGT, ALT, AST, total cholesterol, and fasting glucose, adjusted by age and gender.

Statistical Analysis

[0213] The protocol and analyses followed FibroSTARD recommendations adapted for steatosis. The limitations of the FLIP and CRN standard definitions of metabolic liver disease and their impact on the construction of non-invasive tests were previously discussed (Poynard et al, Eur J Gastroenterol Hepatol. 2018; 30:384-391; Poynard et al, Eur J Gastroenterol Hepatol. 2018; 30:569-577). These limitations include the presence of appropriate histological controls in only 2.2% (13/576) of cases without steatosis and without inflammatory activity in the reference study of the CRN-group.

[0214] SteatoTest-2 was constructed retrospectively (C1) based on the same subset used for the construction of the original SteatoTest, from a sample size of 307 cases with all components. To ensure appropriate power for the non-inferiority tests between the AUROCs of the new test and the reference test, as well as for the correlation of concordance in the different data subsets, more than 2000 subjects at risk of metabolic or virologic steatosis, with centralized biopsies were included. Two validation subsets, V3 and V4, had not been previously published for assessing the performance of SteatoTest.

[0215] For the main endpoint of non-inferiority, empirically estimated AUROCs were directly compared, without using non-binary-AUROCs because the same patients underwent both the new and reference tests simultaneously in each data subset. The primary endpoint was to compare test results in all included cases with biopsies for the diagnosis of all grades of steatosis (from S1 to S3 CRN grades) to the absence of steatosis (S0).

[0216] To prevent the limitations of previous studies of biomarkers, which have included fewer than 30 cases without steatosis, a large number of controls were discussed, 158 cases with histologically proven grade CRN-S0 from validation subsets (V1-V4), and 2,779 controls of T1 (=207) and T2 (n=2,562) subsets without biopsy (FIG. 2). These controls without biopsy, were defined on the same criteria than those used for the controls of studies which assessed the performances of localized proton magnetic resonance spectroscopy (MRS), by hepatic triglyceride content (HTGC): no identifiable risk factors, a BMI below 25 kg/m.sup.2, no diabetes, fasting glucose below 6.1 mmol/L, minimal alcohol consumption (20 g for women, 30 g for men) and no known liver disease. Secondary endpoints were AUROCs with specificity assessed in three control groups, biopsy+T1-controls, biopsy+T2-controls and biopsy+T1+T2-controls)

[0217] Cutoffs were based on the method used for MRS. In that study, the 95% percentile of HTGC, assessed in 345 controls with no risk of steatosis, was 5.56%. This corresponds to a hepatic level of 55.6 mg/g and is considered the cutoff for the upper limit of normal (ULN) on MRS, and a reference for the absence of steatosis (grade S0). In our study a cutoff was chosen that optimized a high negative predictive value (at least 90%) of ST2 for the diagnosis of steatosis 5%. Based on the usual range of the prevalence of steatosis (17-46%) in adults, it was chosen 18.1% (95% Cl 17.2%-18.9%; 1336/7395) as the predetermined prevalence to determine the negative predictive value of the new cutoff of SteatoTest-2. This prevalence was previously assessed in consecutive healthy volunteers, representative of the French population aged 40 years or older.

[0218] The performances of SteatoTest-2 were compared to the reference SteatoTest in the C1 to V4 subsets, using the non-inferiority test (0.10 margin) of the difference between AUROCs predicting NAS-CRN steatosis grades 1-to-3 versus grade 0 (no steatosis or less than 5% of hepatocytes). Sensitivity analyses were performed to evaluate the influence of factors of variability (inflammation grade, fibrosis stage, obesity, and fasting glucose with two cutoffs 6.1 for insulin resistance and 7.0 for diabetes type-2).

[0219] To ensure that the prediction of the grades of steatosis was similar in all the subsets by the Steatotest-2 and the SteatoTest, the level and significance of the Lin concordance coefficient was assessed between the two tests.

[0220] The medians and the interquartile distribution of all tests were graphically represented according to histological scores and control subsets. The Tukey-Kramer's test compared all pairs simultaneously with confidence intervals of mean differences and P-Values. Notched box plots were constructed using the formula: median(1.57IQR/.sup.n). If the notches of two boxes did not overlap, the medians were significantly different.

[0221] In patients in the selonsertib trial, liver biopsies, and serum markers including the SteatoTest, SteatoTest-2, NashTest-2, and FibroTest, were performed at baseline and at 24 weeks of treatment. The differences in paired test results at baseline and at 24 weeks of treatment were compared between patients without (nonresponders) and with (responders) a histological improvement, defined as 1-point improvement in the NAFLD Activity Score. All analyses were blinded to treatment effect as set out in the protocol.

[0222] The influence of the definition of steatosis (presumed by the SteatoTest2) was assessed on the prevalence of NASH, presumed by the NashTest-2 in the subsets of patients without biopsies, based on the standard CRN algorithm (steatosis 5%) or the FLIP algorithm for significant activity (FLIP score A2) as previously published. The aim, for use in large populations, was to identify a sensitive cutoff for the SteatoTest-2 for subjects with at least steatosis grade S1 and to identify those cases with clinically significant NASH, that is, at least grade N2 in the CRN or the FLIP scoring system.

[0223] All statistical analyses were performed using NCSS-12.0 and R.

Example 3. Results

Characteristics of Included Subjects

[0224] A total of 2,997 patients with biopsies and histological scores were assessed by the SAF scoring system. Although the characteristics of included subjects have already been published in previous publications, the value of the present integrated database was the wide range of characteristics, making it possible to evaluate the robustness of the blood tests according to factors of variability. The IQR of age was between 34 and 61 years old. The prevalence of histological steatosis ranged from 7.3% to 24.5%, NASH from 13.7% to 100%, cirrhosis from 0% to 42.6%, the prevalence of T2-diabetes from 7.5% to 70.8%, and obesity (BMI30) from 13.6% to 100%. The subset of obese subjects was younger, with a higher percentage of women, and a lower prevalence of advanced fibrosis than the other subsets.

New SteatoTest-2

[0225] Unlike the SteatoTest, the SteatoTest-2 did not include BMI or total bilirubin, but did include AST, with different independent coefficients for the remaining nine components.

[0226] The clinical characteristics and grades of steatosis and NASH as well as the stages of fibrosis presumed by the blood tests in the four targeted subsets with increasing risks of steatosis were recorded. As expected, the prevalence of steatosis presumed by the SteatoTest increased from 15.5% in blood donors to 84.4% in subjects who underwent a FibroTest.

[0227] The AUROCs of the SteatoTest-2 were non-inferior to the reference SteatoTest, which was the comparator. All non-inferiority tests were significant (P<0.001) (FIG. 2). Results of the AUROCs for the SteatoTest 2 and SteatoTest varied depending on the control groups and the prevalence of steatosis: 0.772 (95% Cl 0.713-0.820) and 0.786 (0.729-0.832), respectively in the 2,997 cases with biopsy (prevalence of steatosis 62%) to 0.822 (0.810-0.834) and 0.868 (0.858-0.878), respectively in the 5,776 cases including controls without risk factors of steatosis and without biopsy (prevalence of steatosis 32%), (FIG. 2).

[0228] The influence of the selection of controls on SteatoTest-2 AUROCs was evaluated. AUROCs varied from 0.734 (95% Cl 0.715-0.751) with controls-S0-biopsy to 0.822 (0.810-0.834) with controls-S0-biopsy-T1-T2. As the spectrum of cases with steatosis were always defined by biopsy (stages S1 to S3), the sensitivity of the test did not vary. Therefore, the AUROCs were influenced by both the spectrum of the control group which was directly related to the test specificity, but also by the change in the prevalence of steatosis which varied from 32 to 62%. When controls-T1 (n=207, median SteatoTest-2=0.15) were added to the controls-S0-biopsy, the AUROC increased slightly from 0.734 (0.715-0.751) to 0.767 (0.750-0.782), and the prevalence of steatosis decreased from 62% to 58%. When controls-T2 (n=2,562 median SteatoTest-2=0.30) were added to the controls-S0-biopsy, there was both a marked increase in the AUROC 0.815 (0.803-0.827) as well as a marked decrease in steatosis prevalence from 62% to 34%.

[0229] The Lin coefficient was highly concordant (P<0.001), in all subsets and was between 0.74 (0.74-0.74) in A4 and 0.91 (0.89-0.93) in C1.

Cutoffs for the SteatoTest-2

[0230] The upper 95% percentile (ULN) in the Steato-Test-2 was 0.40 in 177 blood donors with BMI<25 and fasting glucose<6.1. This cutoff was chosen as the ULN for ST2 to predict the presence of at least 5% of steatosis. The following cut offs were recommended according to median values and 95% confidence intervals: S1>=0.40, S2>=0.55, S3>=0.62.

[0231] The sensitivity for the integrated database with biopsies using the 0.40 cutoff was 79% (77-85) with a 92% predictive value, when adjusted for the predetermined prevalence of 18%. The corresponding specificity was 50% (47-53).

Comparison Between Presumed Prevalences of NASH According to CRN or FLIP Simplified Algorithms

[0232] The prevalence of steatosis in the NAFLD subset, was 84.7%. The prevalence of clinically significant NASH (moderate or severe) was 72.3% (434/600) using the CRN-algorithm and 80.3% (482/600) using the FLIPA2-algorithm, which was significantly (P=0.001) 8.0% (95% Cl 3-13%) different.

[0233] The prevalence of steatosis in the subset of obese subjects, was 65.5%. The prevalence of clinically significant NASH was 16.6% (80/481) using the CRN-algorithm and 18.1% (87/481) using the FLIP-A2-algorithm, which was non-significantly 1.5% (4%;6%; P=0.55) different.

[0234] All included patients in the NASH trial subset had histological steatosis and NASH at inclusion, whatever the CRN or FLIP-simplified definition, and all had presumed steatosis by the SteatoTest-2.

[0235] The prevalence of steatosis in the blood donor subset was 15.5%. The prevalence of clinically significant NASH (moderate or severe) was 3.1% (10/322) using the CRN-algorithm and 5.3% (17/322) using the FLIPA2-algorithm, which was a non-significant (P=0.17) 2.2% (1.2; 6.8%) difference.

[0236] The prevalence of steatosis in the subset of healthy volunteers was 50.1%. The prevalence of clinically significant NASH (moderate or severe) was 22.5% (1,667/7,416) using the CRN-algorithm and 29.9% (2,220/7,416) using the FLIPA2-algorithm, which was a significant (P<0.001) 7.5% (6.0; 8.9%) difference.

[0237] The prevalence of steatosis in the subset of patients with diabetes was 85.2%. The prevalence of clinically significant NASH (moderate or severe) was 41.8% (150/359) using the CRN-algorithm and 44.6% (160/359) using the FLIPA2-algorithm, which was a non-significant (P=0.45) 2.8% (4.7; 10.3%) difference.

SteatoTest-2, NashTest-2, FibroTest Improvements in the Selonsertib Trial (FIG. 3)

[0238] All 72 patients with NASH and stage 2 (35%) or 3 (65%) fibrosis were included. At baseline, all patients had a NAS score >5 (100%). At 24 weeks, NAS improved by at least one point in 49% of patients. The SteatoTest-2 decreased by 0.02 vs +0.04 (P=0.03) (FIG. 3.A) in patients who improved compared to those who did not, respectively, the SteatoTest by 0.014 vs +0.014 (P=0.02) (FIG. 3.B), the NashTest-2 0.06 vs +0.01 (FIG. 3.C), and FibroTest 0.02 vs +0.02 (P=0.04) (FIG. 3.D). Despite the narrow range of NASH disease severity (only grade 2 and 3), the AUROC for predicting an improvement in NAS by NashTest-2 was 0.700 (95% Cl 0.543, 0.809; p=0.003).

Sensitivity Analyses

[0239] The AUROCs of SteatoTest-2 were non-inferior to the reference SteatoTest, whatever the associated inflammatory activity, fibrosis stage, fasting glucose level, or obesity. The only exception was the small subset of patients with hepatitis C without activity, in which only 11 cases had steatosis. Statistical comparisons were not possible in two subsets due to a 98% prevalence of steatosis, that is, in obese patients with advanced fibrosis and with fasting glucose>=7 mmol/L.

Association with Grades of CRN Steatosis.

[0240] Significant differences were found in all different stages of disease by histological grade with the SteatoTest-2. The medians were 0.40, 0.53, 0.60 for S0, S1, S2, and S3 respectively.

Example 4. Discussion

[0241] In this study is described the construction and characterization of a new blood test for the diagnosis of steatosis and the validation that its results are not inferior to the reference SteatoTest, a recognized comparator (European Association for the Study of the Liver, op. cit.). This new test has the advantage of not including body mass index or total bilirubin in its components, which are two causes of significant variability.

Non-Inferiority and Concordance with the Comparator

[0242] It is confirmed that Steato-test2 was non-inferior to the comparator for the most frequent causes of liver steatosis, in patients with metabolic liver disease (overweight, diabetes type 2, dyslipidemia) as well as in those with chronic hepatitis C, with biopsies. To evaluate its specificity, it was also demonstrated the non-inferiority of this new test in subsets with a lower risk of steatosis, including the general population and very low risk groups such as blood donors, healthy volunteers and hepatitis C without steatosis. Furthermore, the highly significant quantitative concordance between these two tests was confirmed in all subsets.

[0243] The comparison of AUROCs demonstrated non-inferiority after the major sources of variability were taken into account, in particular, the prevalences of steatosis, stages of fibrosis, grades of inflammatory activity as well as the prevalence of diabetes, obesity, and increase in fasting glucose with standard cutoffs (6.1 and 7.0 mmol/L).

Choices and Impact of Controls

[0244] This study emphasized the importance of control (without steatosis) and case (with steatosis) selection in studies evaluating the AUROCs of biomarkers. The construction of this new test took into account the methodological limitations of previous studies, concerning sample size as well as the definitions of steatosis and controls and their impact on AUROCs.

[0245] Due to the ethical limitations of performing biopsies in healthy controls or in cases with metabolic liver disease and normal liver function tests, the same inclusion criteria as those used for MRS were used as our reference to define the presence or absence of steatosis and define the ULN in SteatoTest-2. It is felt that these criteria should be recommended in guidelines to standardize the methodology when constructing steatosis biomarkers, thus preventing artificial disagreement.

[0246] The selection of patients with steatosis in whom biopsy is indicated based on MRS or not, can significantly influence the AUROCs in biomarker studies. If a study is designed with MRS first, then biopsy is only performed in subjects with steatosis on MRS, and controls to evaluate specificity will be those patients with steatosis on MRS but not on biopsy. This is obviously rare and does not correspond to a frequent context of use. Thus, these studies cannot reliably assess specificity and AUROCs for steatosis. This strategy is also questionable to assess the value of NASH biomarkers because ballooning and lobular inflammation may be present with or without steatosis <5% at biopsy. Due to these limitations, it was decided to assess the specificity of steatosis biomarkers in large samples. Like the first generation SteatoTest, all available biopsies from 498 patients with chronic viral hepatitis were included, as well as 1,537 subjects with no risk of steatosis as controls, using the ULN for MRS as the basis of our ULN (Szczepaniak et al, Am J Physiol Endocrinol Metab. 2005; 288:462-8).

[0247] In this study the influence of the arbitrary selection of controls when evaluating steatosis non-invasive tests by AUROCs was demonstrated. Without changing the test sensitivity AUROCs artificially varied from 0.734 with biopsy-controls to 0.822 with biopsy+T1+T2-controls. The results were influenced by both the spectrum of the control group, but also by the change in the prevalence of steatosis varying from 32 to 62% according to the selection of cases without steatosis. As the prevalence of steatosis varies between 20 and 50%, presumed by MRS, the performances of new biomarkers of steatosis must be assessed according to these sources of variability, and avoiding indirect comparison of AUROCs.

Performance for Marked (grade S2) and Severe (S3) CRN Grades of Steatosis

[0248] It was also shown, for the first time, that SteatoTest-2 significantly discriminates among the four stages of the CRN score. Although the semi-quantitative correlations and significant differences among histological stages in our study are not similar to the results of MRS, which has a better correlation with percentages of hepatocyte steatosis than any blood test, the aim was to validate a robust test without the practical limitations of MRS. While the main value of SteatoTest-2 is its sensitivity and associated high negative predictive value (92%) for the diagnosis of steatosis of at least 5%, there was also a significant correlation between test values and histological grades of steatosis. Furthermore, the clinical value of the AUROC (0.603; 0.577-0.229; P<0.001 vs random) for the diagnosis of S2S3 vs S1 is limited. Indeed, this AUROC was assessed in a narrow range of steatosis spectrum, without S0 controls, or controls with no risk of steatosis.

Validation in NASH Paired Biopsies (FIG. 3)

[0249] It was demonstrated for the first time in a phase-2 trial of selonsertib, that the changes in the three non-invasive tests, SteatoTest-2, NashTest-2 and FibroTest, can reliably predict improvement in the NAS score in patients with NASH. This confirmed that the paired SteatoTest-2 detected an improvement in steatosis in patients with moderate and severe grades of steatosis, which was also observed in biopsies and MRS.

[0250] Despite the retrospective design, the SteatoTest-2 was clearly non-inferior to and highly concordant with the reference SteatoTest which has been extensively used and validated by guidelines for the diagnostic of steatosis.

[0251] In conclusion, the new multi-analyte SteatoTest-2 simplifies, and was found to be non-inferior to, the reference SteatoTest for the diagnosis of steatosis, without the variability induced by body mass index and the risk of false positives related to unconjugated bilirubin.