Methods for diagnosing liver fibrosis

Abstract

The present invention relates to a method for identifying NAFLD patients who are likely to develop liver fibrosis within 52 weeks.

Description

Technical Field

[0001] The present invention relates to a method for identifying patients with non-alcoholic fatty liver disease (NAFLD) who are likely to exhibit advanced liver fibrosis within 52 weeks.

Background Art

[0002] Abnormal and excessive deposition of the extracellular matrix is a characteristic of all fibrosis, including liver, lung, kidney, or heart fibrosis. A number of challenges are presented due to the extent of the affected organs, the nature of the progression of the fibrogenic process, the large number of affected patients, and the absence of symptoms until the disease becomes life-threatening.

[0003] When the liver is damaged, a fibrous layer is formed, which becomes scar tissue in the liver. This initial stage of damage is called fibrosis.

[0004] There are several types of liver diseases that can cause fibrosis. These include: · Autoimmune hepatitis · Bile duct obstruction · Non-alcoholic fatty liver disease, which includes non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) · Hepatitis B and C · Alcoholic liver disease · Hepatitis D: This type of hepatitis can also cause cirrhosis and is often seen in people who already have hepatitis B. · Damage to the bile ducts that perform the function of discharging bile: An example of such a condition is primary biliary cirrhosis. · Disorders that affect the body's ability to handle iron and copper: Two examples are hemochromatosis and Wilson's disease. · Pharmaceuticals (acetaminophen, some antibiotics, and some antidepressants can lead to cirrhosis).

[0005] The most common cause of liver fibrosis is non-alcoholic fatty liver disease (NAFLD), while the second is alcoholic liver disease due to long-term excessive alcohol consumption.

[0006] Liver damage can have the following ranges: * Mild to slight damage * Mild to moderate damage (fibrosis) * Moderate to severe damage (fibrosis to compensated cirrhosis) * Severe to liver failure (decompensated cirrhosis)

[0007] "Liver fibrosis" refers to the presence of fibrous connective tissue when examining sections of liver biopsy stained with H&E, trichrome, or picrosirius red staining. In the context of the present invention, the term "fibrosis stage" represents the location and degree of fibrosis in histological examination, as follows: Perisinusoidal or periportal fibrosis 1 Mild perisinusoidal fibrosis (zone 3) 1a Moderate perisinusoidal fibrosis (zone 3) 1b Portal / peri-portal fibrosis 1c Perisinusoidal and portal / peri-portal fibrosis 2 Bridging fibrosis 3 Cirrhosis 4 (Kleiner et al., Design and Validation of a Histological Scoring System for Nonalcoholic Fatty Liver Disease Hepatology, 2005; 41: 1313 - 1321). In many cases, until the liver damage becomes severe enough (cirrhosis), fibrosis is asymptomatic or nearly so, and when it becomes severe enough, patients may experience severe fatigue, loss of appetite, difficulty in clear thinking, confusion, abdominal distension (ascites) and lower limb swelling, skin itching, and bleeding in the digestive tract, etc.

[0008] Mild liver damage from fibrosis can be reversed if the cause is identified and eliminated before the damage becomes too extensive. However, if scarring persists for a long period, fibrosis becomes permanent. The damage continues, forming bands throughout the liver, destroying the liver's internal structure, impairing its regenerative capacity, and leading to decreased liver function and cirrhosis.

[0009] Complications from liver injury (scar tissue) can impair liver function. Scar tissue is replaced by healthy liver cells necessary for liver function. Scar tissue obstructs blood flow to the liver. If the liver does not receive enough blood, cells die, and more scar tissue is formed.

[0010] The more scar tissue that forms, the greater the potential for the most severe complications.

[0011] Ultimately, if a person's fibrosis progresses to cirrhosis and liver failure, they may experience the following complications: Ascites (severe fluid accumulation in the abdominal cavity) Hepatic encephalopathy (accumulation of waste products that cause confusion) hepatorenal syndrome Portal hypertension Varicose vein bleeding.

[0012] The most significant complications of liver fibrosis can be cirrhosis or severe scarring that damages the liver to the point of causing disease. Typically, this takes a long time to develop, for example, more than 10 or 20 years.

[0013] Cirrhosis is one of the leading causes of death worldwide. It can lead to cancerous liver tumors and liver failure, sometimes requiring a liver transplant. Therefore, it is crucial for individuals to receive diagnosis and treatment for liver fibrosis as early as possible, before it progresses to cirrhosis. An estimated 6-7 percent of the world's population has liver fibrosis, and these individuals are unaware of it because they experience no symptoms.

[0014] Since this disease can potentially recover if diagnosed early enough, or at least its consequences can be limited, it is considered extremely important to provide the medical field with suitable means that enable such early, rapid, and accurate diagnosis.

Prior Art Documents

Patent Documents

[0015]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0016]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0017] Several attempts have been made to propose non-invasive methods for diagnosing and determining the severity of liver fibrosis, but currently, the best method for evaluating the stage of fibrosis remains histological analysis of liver biopsy. However, liver biopsy has several obvious drawbacks. Firstly, the material collected by liver biopsy represents only a very small portion of the liver being diagnosed, thus raising questions about whether the collected sample represents the overall state of the organ. Furthermore, liver biopsy is an extremely invasive procedure that can be troublesome, worrying, and painful for the patient, and raises concerns about morbidity and death. Finally, considering the above, liver biopsy cannot be reasonably proposed as a standard method for determining whether or not fibrosis is present.

[0018] Due to these shortcomings of biopsy-based diagnosis, non-invasive methods for detecting NASH (non-alcoholic steatohepatitis) have been actively developed. For example, WO2017046181 and WO2017167934 offer non-invasive diagnosis based on the measurement of levels of circulating biomarkers.

[0019] Another option is an imaging test known as transient elastography. This test measures how stiff the liver is. In cases of liver fibrosis, the liver hardens due to scarring cells. This test uses low-frequency sound waves to measure how stiff the liver tissue is. However, it can have false positives, as liver tissue can sometimes appear stiff. [Means for solving the problem]

[0020] This invention relates to a method for identifying a subpopulation of individuals with NAFLD who are at risk of progressing to fibrosis (F≧3) within 52 weeks. Individuals thus identified are designated as “fast progressors.”

[0021] The method of the present invention has better prognostic values ​​than other tests available in the art for identifying rapidly progressing patients, such as FIB-4 (Fibrosis 4), ELF (Enhanced Liver Fibrosis), and NFS (NAFLD Fibrosis Score), as will be shown in the following experimental section.

[0022] In certain embodiments, the method of the present invention further includes the step of measuring liver fibrosis of the subject using a physical method. In particular, the method may include the step of measuring liver stiffness of the subject. In even more specific embodiments, liver stiffness is determined by the step of measuring the difference in the velocity of elastic shear wave propagation in the liver. [Modes for carrying out the invention]

[0023] According to the present invention, the terms "fibrosis," "fibrosis," and "fibrous disorder," as well as their morphological variations (declination), refer to a pathological condition characterized by excessive deposition of fibrous connective tissue in the liver. More specifically, fibrosis is a pathological process involving persistent fibrous scar formation and excessive production of extracellular matrix by connective tissue in response to tissue damage. Physiologically, the deposition of connective tissue can lead to the loss of structure and function of the underlying organ or tissue.

[0024] According to the present invention, the term "non-alcoholic steatohepatitis," or NASH, refers to a condition characterized by fatty liver, ballooning hepatocyte degeneration, and coexisting hepatitis on histological examination, after excluding excessive alcohol intake and other liver diseases such as viral hepatitis (HCV, HBV). According to the present invention, the term "steatosis" refers to a process representing the abnormal accumulation of lipids or fats within the liver. According to the present invention, the term "ballooning hepatocyte degeneration" is usually defined as hypertrophy of cells with thinned cytoplasm and a diameter 1.5 to 2 times that of normal hepatocytes, at the level of a light microscope based on hematoxylin and eosin (H&E) staining. It more generally refers to the process of hepatocyte cell death. According to the present invention, the term "intralobular inflammation" refers to the presence of intralobular inflammatory lesions (groups of inflammatory cells) on microscopic examination of liver biopsy sections stained with hematoxylin and eosin (H&E).

[0025] According to the present invention, the "NAFLD activity score" or "NAS" refers to the sum of the following scores: steatosis score, hepatocyte ballooning degeneration score, and intralobular inflammation score: S: Fatification score: 0: <5%; 1: 5-33%; 2: 34-66%; and 3: >66%; LI: Intralobular inflammation score (lesion / 20x field of view): 0: none; 1: <2; 2: 2-4, and 3: >4; HB: Balloon-like degeneration score: 0: None; 1: Few; 2: Many cells / Significant balloon-like degeneration.

[0026] Therefore, NASH refers to a state of NAFLD characterized by the following liver biopsy score: NAS ≥ 3 (having at least 1 on the steatosis score, at least 1 on the intralobular inflammation score, and at least 1 on the hepatocyte ballooning degeneration score).

[0027] More severe forms of NASH are also characterized by a higher score on one of the S, LI, and HB scores, and / or the presence of liver fibrosis.

[0028] As described above, the range of fibrosis stages is from F=0 (or F0), i.e., no fibrosis, to F4, i.e., cirrhosis. In the context of the present invention, fibrosis stages of F3 or higher (i.e., F3 or F4) are referred to herein as "progressive fibrosis."

[0029] Subjects identified as rapidly progressing by the method of the present invention may have fibrosis stages 0, 1, or 2, and conditions selected from the group consisting of NAFLD, non-alcoholic steatohepatitis (NASH), proliferative fibrosis, biliary obstruction, alcohol or drug-induced hepatic fibrosis, cirrhosis, infection-induced hepatic fibrosis, particularly viral infection-induced hepatitis such as hepatitis A, B, or C, radioactive or chemotherapeutic agent-induced fibrosis, chronic fibrotic cholangitis, e.g., primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC), biliary atresia, hemoglobinosis, Wilson's disease, and drug-induced hepatic fibrosis. In certain embodiments, subjects identified as rapidly progressing by the method of the present invention may have NASH and fibrosis stages 0, 1, or 2. More specifically, the present invention provides a method for determining the likelihood that such subjects will progress to fibrosis within 52 weeks from this determination.

[0030] The method of the present invention measures the levels of at least four circulating markers from a blood-derived sample from a subject. The at least four circulating markers are hsa-miR34a, alpha-2 macroglobulin (A2M), YKL40, and glycated hemoglobin (HbA1c).

[0031] The levels of these markers are measured in samples derived from the subject's blood, such as blood, serum, or plasma, particularly platelet-free plasma, such as cell-free, citrate-induced platelet-free plasma samples. In certain embodiments, the levels of hsa-miR34a, A2M, and YKL40 are measured from one or more serum samples from the subject. In another specific embodiment, the level of HbA1c is measured from a blood sample of the subject.

[0032] In certain embodiments, the levels of hsa-miR34a, A2M, YKL40, and HbA1c are measured as described in WO2017167934.

[0033] In all embodiments and variations disclosed herein, it should be understood that hsa-miR34a may more specifically be hsa-miR34a-5p. In this application, the combination of hsa-miR34a-5p, alpha-2 macroglobulin (A2M), YKL40, and glycated hemoglobin (HbA1c) is also referred to as NIS4™.

[0034] In certain embodiments, the levels of circulatory markers measured herein are used in a logistic function for calculating a score, as provided, for example, in application WO2017167934. Briefly, the logistic function may be determined by data obtained from a reference subject with NASH that has progressed from the F0, F1, or F2 stage at inclusion to fibrosis progression. Such data may be obtained by: Measurement of the first set of circulating marker levels when the reference subject was in the F0, F1, or F2 stage as determined by liver biopsy, and Measurement of circulating levels in the same reference subject performed 52 weeks after the first set of measurements, provided that the reference subject was F≧3 when determined by another liver biopsy.

[0035] Those skilled in the art can use the information provided in the experimental section of this specification and the teachings of WO2017167934 to determine the logistic function and the cutoffs related to the required sensitivity, specificity, positive predictive value and / or negative predictive value of the test.

[0036] According to a particular embodiment, the score is defined as a logistic function derived from the bootstrap model:

[0037]

number

[0038] During the ceremony: Y = k + a*A + b*B + c*C + d*D During the ceremony: S is the score; A is the serum level (Cq) of hsa-miR-34a (especially hsa-miR-34a-5p); B is the serum level (g / L) of alpha-2 macroglobulin; C is the serum level (pg / ml) of YKL-40. D represents the HbA1c level (percentage) (for example, if the measured HbA1c percentage is 10%, then D is equal to 10); k is a constant of the logistic function. a is a coefficient related to the serum level of hsa-miR-34a (especially hsa-miR-34a-5p); b is a coefficient related to serum levels of alpha-2 macroglobulin; c is a coefficient related to the serum level of YKL-40; d is a coefficient related to the HbA1c level.

[0039] In a further specific embodiment derived from the bootstrap model as described in the experimental section of patent application WO2017167934: k is a number that falls between 9.51 and 34.37; a is a number that falls between -1.17 and -0.47; b is a number that falls between 0.02 and 0.84; c is a number that falls between 6.10E-06 and 2.09E-05; d is a number that falls between 0.07 and 0.89.

[0040] As stated above, those skilled in the art can use the information provided herein and the teachings of WO2017167934 to determine the logistic function and the cutoffs related to the required sensitivity, specificity, positive predictive value and / or negative predictive value of the test. In a particular embodiment, the low cutoff is 0.36 and the high cutoff is 0.63. As shown in the experimental section, in this particular embodiment, subjects with a score lower than 0.36 can be identified (excluded) as having a low likelihood of progressing to fibrosis, with only a 2% probability of progression; in contrast, those with a score of 0.63 or higher can be identified (confirmed) as having a high likelihood of progressing to fibrosis, with a 38% probability of progressing to fibrosis at 52 weeks (positive predictive value of 38% (23-56)), a specificity of 88% (81-92), and a sensitivity of 69% (44-86).

[0041] In certain embodiments, the method further includes the step of measuring liver fibrosis of the subject using a physical method. This measurement can be performed by several methods well known in the art. Exemplary methods include, but are not limited to, medical imaging and / or clinical measurements. In certain embodiments, the physical method is an elastic modulus measurement method. More specifically, the elastic modulus measurement method can be selected from the group consisting of acoustic irradiance impulse imaging (ARFI imaging), transient elastography (TE), and MRI stiffness. In certain embodiments of the present invention, the physical method is transient elastography, which measures the difference in the velocity of elastic shear wave propagation in the liver. According to a preferred method, transient elastography (FIBROSCAN®, etc.), a technique used to evaluate the stiffness or rigidity of the liver, is used, which is measured in kilopascals (kPa) and is a non-invasive technique related to fibrosis. The results of TE (FIBROSCAN® results, etc.) may be in the range of 2.5 kPa to 75 kPa. 90–95% of healthy subjects without liver disease will have liver stiffness measurements of less than 7.0 kPa. In certain embodiments, the method may include a step of measuring the liver stiffness of the subject. In further specific embodiments, liver stiffness is measured by a step of measuring the difference in the velocity of elastic shear wave propagation in the liver.

[0042] The present invention will be further described with reference to the following non-limiting embodiments. [Examples]

[0043] (Example 1) Histological progression of fibrosis and cirrhosis in NASH. This analysis was performed on a subpopulation from the Phase 2 clinical trial GOLDEN-505 (NCT01694849), which included 161 patients with a NAFLD activity score (NAS) of 3 or higher and fibrosis stage 0–2, who underwent biopsy at both baseline and at the end of the trial (week 52) and had histologically confirmed NASH. GOLDEN-505 was a multicenter, randomized, double-blind, placebo-controlled trial to evaluate the efficacy and safety of once-daily ellafibranol (1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]propa-2-en-1-one) in patients with non-alcoholic steatohepatitis (NASH).

[0044] To monitor the histological progression of the disease, liver biopsies taken at enrollment and at the end of the one-year treatment period were used for histological examination and scoring of lesions. Histological scoring, following the NASH-CRN system (NASH Clinical Research Network), was centralized and performed by pathologists.

[0045] We compared patient characteristics between patients with rapidly progressing NASH (NAS≧3) who had F0-F2 fibrosis at 52 weeks and those who did not progress to F≧3 (n=145).

[0046] To determine the NIS4® score, hsa-miR-34a-5p, alpha-2-macroglobulin, YKL-40, and hemoglobin A1c were quantified before calculating the algorithm.

[0047] In short, patient blood was collected in serum separation tubes (SSTs) to measure YKL-40, hsa-miR-34a-5p levels, and alpha-2 macroglobulin A2M. Blood was also collected in EDTA collection tubes to measure HbA1c. YKL40 (also called CHI3L1) was quantified by ELISA (Human Chitinase 3-like 1 Immunoassay Quantikine® ELISA catalog number DC3L10). Values ​​were expressed as ng / mL. Alpha-2 macroglobulin levels were measured by turbidimetric method using a BN II system (Siemens Healthcare). Values ​​were expressed as g / L. HbA1c was measured by ion-exchange high-performance liquid chromatography (HPLC) (Menarini HA-8160 HbA1c automated analyzer) and reported as a percentage of total hemoglobin.

[0048] Total RNA containing stored miRNA was extracted from 100 μl individual serum samples using the miR-VanaParis extraction kit (AM1556, Ambion, Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. To monitor extraction efficiency and minimize sample variability, i) synthetic nematode (C. elegans) miR-39 [3,125 fmole] (MSY0000010, Qiagen, Venlo, The Netherlands) was added to each sample before RNA extraction, and ii) standard serum with known miR-34a Cq values ​​was processed simultaneously with the sample being tested. Subsequently, a washing step was performed using miR-VanaParis washing solution (8680G & 8543G14 Ambion), followed by centrifugation to avoid ethanol introduction. Total RNA containing miRNA was eluted into DNase / RNase-free water by centrifugation and immediately stored at -80°C until use. 5 μl of fixed total RNA from serum samples, or synthetic hsa-miRNA-34a (single-stranded sequence = 5'Phos-UGGCAGUGUCUUAGCUGGUUGU-3' (SEQ ID NO: 1); Integrated DNA Technologies) diluted to 3.125 fmol / mL (used for calibration curve construction and miR-34a copy number calculation), were simultaneously reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (4366597, Applied Biosystems). The reverse transcription reaction was performed in 15 μL of a final mixture containing 10 μL of TaqMan MicroRNA Assay 5X and incubated in an Applied Biosystems GeneAmp® PCR System 9400 thermocycler. The cDNA was stored at -20°C in low-adsorption tubes until further use.

[0049] Mature miRNA expression was quantified according to the manufacturer's instructions using Taqman miRNA RT-qPCR Assay 20X and TaqMan Universal Master Mix II, no Uracil-N-Glycosidase (UNG) 2X (Applied Biosystems). 5 μL of fixed total RNA was used as a template for qPCR assays using the CFX96™ Real-Time System. The hsa-miR-34a-5p TaqMan assay was used. RT products from synthetic miRNA were serially diluted, and PCR was performed on all samples (standard and serum-derived RNA). A dual calibration curve was performed and used to convert Cq data to copies / μL. The Cq determination mode was regression. Transcript abundance is expressed as Cq.

[0050] Report the mature miRNA sequence and Taq Man assay ID in the table below:

[0051] [Table 1]

[0052] The data used to construct the algorithm was in Cq format.

[0053] Next, the NIS4(trademark) score, defined as a logistic function with serum levels of has-miR-34a-5p expressed in Cq units, was calculated as provided in application WO2017167934.

[0054] To compare NIS4 (trademark) with other non-invasive scores, the following non-invasive scores, stratified by established clinical cutoffs, were evaluated and used as predictive models: • Fibrosis-4 [FIB-4; age, AST, ALT, platelet count] • NAFLD Fibrosis Score [NFS; Age, BMI, IGF / Diabetes status, AST, ALT, Platelet count, Albumin] • Enhanced liver fibrosis [ELF(trademark); hyaluronic acid (HA), procollagen III amino-terminal peptide (PIIINP), and matrix metalloproteinase tissue inhibitor (TIMP-1)].

[0055] Using the Chi-2 or Wilcoxon p-value, data and scores from patients who progressed to F≧3 were compared with data from patients who did not progress to F≧3.

[0056] [Table 2A]

[0057] [Table 2B]

[0058] Patients with "rapidly progressing" disease tend to have worse clinical and biochemical characteristics compared to patients with non-rapidly progressing disease (Table 1 (Table 2)), and these characteristics include: • Older age • Obesity, type 2 diabetes mellitus (T2DM), and metabolic complications including hypertension are common. • High levels of ALT and AST • High average non-invasive scores (FIB-4, NFS, NIS4®, and ELF®) Next, patients whose fibrosis progression had progressed were classified according to their score zones.

[0059] If the score exceeded the high cutoff for confirmation, it was determined to be in the high score zone; if the score was between the high and low cutoffs, it was defined as the undetermined zone; and if the score was lower than the low cutoff for exclusion, it was determined to be in the low score zone. To enable actual clinical use, the low cutoff for exclusion was set to less than 0.36, in which case the sensitivity was 81.5%, the specificity was 63%, and the NPV was 77.9%. In addition, the high cutoff for enabling confirmation was set to 0.63 or higher, in which case the specificity was 87.1%, the sensitivity was 50.7%, and the PPV was 79.2% (Harrison et al. (The Lancet Gastroenterology & Hepatology, Vol. 5, No. 11, pp. 970-985, November 1, 2020; online publication August 3, 2020)).

[0060] The NIS4® score was calculated according to the method provided in WO2017167934 and Harrison et al. (The Lancet Gastroenterology & Hepatology, Vol. 5, No. 11, pp. 970-985, November 1, 2020; online publication August 3, 2020). Briefly, the logistic function derived from the bootstrap model is defined by the following features, fitted to the desired specificity, sensitivity, PPV, and NPV for the low and high cutoffs defined above:

[0061]

number

[0062] During the ceremony: Y = k + a*A + b*B + c*C + d*D During the ceremony: S is the NIS4(trademark) score; A is the serum level (Cq) of hsa-miR-34a (especially hsa-miR-34a-5p); B is the serum level (g / L) of alpha-2 macroglobulin; C is the serum level (pg / ml) of YKL-40. D represents the HbA1c level (percentage) (for example, if the measured HbA1c percentage is 10%, then D is equal to 10); k is a number that falls between 9.51 and 34.37; a is a number that falls between -1.17 and -0.47; b is a number that falls between 0.02 and 0.84; c is a number that falls between 6.10E-06 and 2.09E-05; d is a number that falls between 0.07 and 0.89.

[0063] As shown in Table 2 (Table 3), the absolute number and relative percentage of patients with NASH (NAS≧3) and those who progressed from F0-F2 fibrosis to fibrosis progression (F≧3) ("rapidly progressing") were quantified for each test / score zone of NIS4 (trademark), FIB-4, NFS, and ELF.

[0064] [Table 3]

[0065] In NIS4® and ELF®, the proportion of patients classified as "rapidly progressing" (low-to-intermediate score zone to high score zone) increased progressively. NIS4® classified more "rapidly progressing" patients in the high score zone (69%) compared to ELF® (56%). In contrast, FIB-4 and NFS classified the majority of "rapidly progressing" patients in the low or intermediate score zone.

[0066] The probability of progression to fibrosis at 52 weeks was calculated for each baseline score range (Table 3) (Table 4).

[0067] [Table 4]

[0068] In this cohort, the low score zone of the assessed tests was generally associated with a lower risk of progression to fibrosis (F≧3) (0-6% at 52 weeks). Conversely, the high score zone generally represented the highest risk of progression beyond 52 weeks.

[0069] Finally, the clinical performance of each test in identifying rapidly progressing patients was calculated using high cutoff values ​​(Table 4 (Table 5)). Diagnostic indices (sensitivity, specificity, positive predictive value / PPV, negative predictive value / NPV) are shown along with the 95% CI (Fleiss, 2003) calculated using an asymptotic formula based on a normal approximation for the binomial distribution.

[0070] All statistical analyses were performed using R version 3.4.1 (R Core Team, 2017).

[0071] [Table 5]

[0072] Overall, NIS4™ has the most balanced profile, with the highest sensitivity (Se=69-83%) and equally high specificity (Sp=88-89%) among the tests evaluated (Se=0%-67%).

[0073] NIS4 ≥ 0.63 accurately identified most (69%) of the rapidly progressing population compared to FIB-4 ≥ 2.67 (25%), NFS ≥ 0.676 (13%), and ELF ≥ 9.8 (56%).

[0074] In conclusion, NIS4® can identify a clinical subpopulation of patients with rapid progression who are highly likely to develop fibrosis within one year. In addition, NIS4® has good prognostic utility in identifying patients with rapid progression compared to FIB-4, ELF, and NAFLD fibrosis scores.

Claims

1. A method to assist in identifying a subject as having NAFLD with a high probability of progressing to liver fibrosis within 52 weeks, wherein the method comprises measuring the levels of hsa-miR-34a, alpha-2 macroglobulin (A2M), YKL-40, and glycated hemoglobin (HbA1c) in a blood sample from the subject, the measured levels being used in a logistic function to calculate a score S, the score S being calculated using the following logistic function: [Math 1] During the ceremony: Y = k + a*A + b*B + c*C + d*D The calculation is performed according to the method, which is derived from the bootstrap model. During the ceremony: S is the score; A is the serum level (Cq) of hsa-miR-34a; B is the serum level (g / L) of alpha-2 macroglobulin; C is the serum level (pg / ml) of YKL-40. D represents the HbA1c level (percentage); k is a constant of the logistic function. a is a coefficient related to the serum level of hsa-miR-34a; b is a coefficient related to serum levels of alpha-2 macroglobulin; c is a coefficient related to the serum level of YKL-40; d is a coefficient related to the level of HbA1c, Here: k is a number that falls between 9.51 and 34.37; a is a number that falls between -1.17 and -0.47; b is a number that falls between 0.02 and 0.84; c is a number that falls between 6.10E-06 and 2.09E-05; d is a number that falls between 0.07 and 0.

89. A score of S above the cutoff value of 0.63 indicates a high probability of progression to fibrosis within 52 weeks. method.

2. The method according to claim 1, comprising measuring the levels of hsa-miR-34a-5p, HbA1c, YKL-40, and A2M, where A is the serum level of hsa-miR-34a-5p and a is a coefficient related to the serum level of hsa-miR-34a-5p.

3. The method according to claim 1 or 2, wherein the subject has NAFLD having a fibrosis stage of 0, 1, or 2.

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