CN101027561A - Method for diagnosing liver fibrosis - Google Patents

Abstract

The invention concerns a method for the detection of the presence and/or the severity of a liver disease in a patient comprising measuring in an isolated sample TIMP-1 (tissue inhibitor of metalloproteinase I), ferritin , at least one additional parameter selected from the group consisting of A2M (alpha-2-macroglobulin) and PI (prothrombin index) and optionally measuring at least one additional biochemical or clinical parameter and diagnosing the presence and/or severity of a liver disease based on the presence or measured levels of these parameters. The method can be used for monitoring therapeutic treatment of liver fibrosis and staging of liver fibrosis.

Description

Methods for diagnosing liver fibrosis

field of invention

The present invention relates to the fields of hepatology and liver fibrosis. It particularly relates to a set of serological markers which can be used for diagnosing liver fibrosis, especially for diagnosing liver fibrosis caused by chronic HCV infection. These markers can be used to monitor therapeutic treatment of liver fibrosis.

Background of the invention

Fibrotic liver disease ranks as the eighth most common cause of mortality worldwide, accounting for 1.3 million deaths per year (Murray and Lopez, 1997, Lancet 349, 1269-1276). The cellular mechanisms of fibrosis are complex. Activation of normally quiescent hepatic stellate cells into proliferating ones in response to liver injury such as that caused by chronic hepatitis C virus (HCV) infection, hepatitis B virus (HBV) infection, alcoholic or fatty liver disease, drug-induced liver disease, or incipient biliary cirrhosis Myofibroblasts. These cells produce extracellular matrix proteins and release tissue inhibitors of metalloproteinases, which bind and inactivate the metalloproteinases responsible for scar degradation. As a result, liver function is impaired through increased production of tissue and protein-like collagen and decreased degradation of these compounds, accumulation of fibrosis and scarring (McHutchinson 2004, CME Newsletter Tx Reporter Gastroenterology, 2-4).

Whereas liver fibrosis is a reversible process causing accumulation of extracellular matrix, cirrhosis is an irreversible process characterized by the formation of nodules in a thick band of matrix completely surrounding the parenchyma. If left untreated, liver fibrosis leads to cirrhosis and possibly cancer. For these reasons, timely and accurate diagnosis of liver fibrosis is essential for effective medical management.

Liver biopsy is still considered the so-called gold standard for detecting fibrosis and inflammation. Liver biopsy is recommended to grade and stage disease, confirm diagnosis and establish a baseline against which to demonstrate improvement or disease progression, and to help gauge prognosis and need for treatment (McHutchinson, see above; for review, see Gebo et al. 2002 Hepatology 36, 161-172).

There are various histological grading systems that have been used to semiquantify the extent of liver fibrosis and inflammation in chronic hepatitis C patients. One of the most commonly used grading systems is the METAVIR system (Bedossa et al., 1994, Hepatlogy, 20, 15-20). METAVIR divides liver fibrosis into 5 stages from F0 to F4. F0 indicates no fibrosis and F1 corresponds to mild fibrosis (portal vein fibrosis but no septum). Moderate to severe fibrosis is classified as F2 to F4 (F2: few septa, F3: many septa but no cirrhosis), with stage F4 corresponding to the final stage of cirrhosis. Fibrosis is considered to be clinically apparent from F ≥ 2.

However, there are several shortcomings in the application of liver biopsy to diagnose and grade fibrosis. Liver fibrosis is often subject to sampling error, such that a small fraction of the sample does not reflect the true situation in the entire liver. Thus, not an accurate marker of a dynamic process of continuous degradation. Furthermore, pathologists are often inconsistent in their judgment of data from histological samples, with intra- and inter-observer variability occurring in 10% to 20% of biopsies (Cadranel et al. 2000, Hepatology 32, 477- 481).

Liver biopsy is an invasive and painful procedure for the patient. It is also associated with a risk of post-sampling bleeding and other complications. Additionally and in part due to expected complications following patient hospitalization, this is a costly approach.

Liver fibrosis is a major complication of chronic HCV infection, leading to cirrhosis and decompensated liver disease. Targeted studies to detect the onset and progression of fibrosis are therefore necessary for the effective management of these patients. Evaluation of progressive fibrosis is best accomplished with non-invasive tests that can distinguish intermediate stages of fibrosis. Various single marker and marker panel algorithms have been published, but no valid single biomarker or biomarker score that can reliably predict fibrosis is currently available (>80% diagnostic accuracy). Further research into the development of non-invasive dynamic measures of liver fibrosis was strongly encouraged by the National Institute of Health Consensus Development Conference in 2002. In particular, studies of liver biopsy alternatives should provide sufficient detail on biopsy methods (average size of biopsy samples; histologically well-characterized quantitative maps) to convince readers of the suitability of reference standards. sex. Liver biopsies are strongly dependent on optimal performance criteria and may lead to misclassification of histological stage due to inter-observer variability and too small sample size (<10 mm).

Extensive research exists on biochemical or serological markers that reflect the fibrotic process in liver disease and can serve as a surrogate for liver biopsy. In recent years, several non-invasive or minimally invasive biochemical and serological markers have been investigated to aid in the diagnosis of liver disease. In particular, marker compositions have been used to classify patients according to their stage or degree of fibrosis.

US6,631,330 discloses using at least 4 kinds selected from α-2-macroglobulin, aspartate aminotransferase, γ-glutamyl transpeptidase, γ-globulin, total bilirubin, albumin, Combination of biochemical markers for α1-globulin, α2-globulin, haptoglobin, β-globulin, apoA1, IL-10, TGF-β1, apoA2 and ApoB. The obtained values for 4 of these markers were combined mathematically to determine the presence of liver fibrosis. Using this marker panel, a diagnostic accuracy of about 80% can be obtained.

International patent application WO2003/073822 describes a method of diagnosing the presence or severity of liver fibrosis in a patient. The method uses a combination of at least three markers, alpha-2-macroglobulin, hyaluronic acid, and tissue inhibitor of metalloproteinase 1 (TIMP-1). Using this method, a diagnostic accuracy of about 80% can be obtained (Mc Hutchinson, 2004, supra).

There is a need to create non-invasive or minimally invasive methods to achieve higher diagnostic accuracy in liver fibrosis assays and to classify and differentiate fibrosis in a more reliable manner than hitherto known in the prior art. The different stages make it possible to monitor the clinical development of fibrosis during treatment. Furthermore, such methods should be suitable for continuous testing on automated analyzers.

Description of the invention

This problem is solved by the method according to the invention. The method for detecting the presence and/or severity of liver disease in a patient comprises the steps of:

a) obtaining an isolated sample from said patient,

b) measuring TIMP-1 (Tissue Inhibitor of Metalloproteinase I) in said sample,

c) measuring ferritin in said sample,

d) measuring at least one other parameter selected from A2M (alpha-2-macroglobulin), PI (prothrombin index) in said sample,

e) optionally measuring at least one other biochemical or clinical parameter in said sample,

f) Diagnosing the presence and/or severity of liver disease based on the presence or measured levels of TIMP-1, ferritin and parameters measured according to steps d) and e).

The present invention allows a positive distinction between F0/F1 fibrosis and F2/F3/F4 stages. In addition, therapy monitoring as a control of medical treatment of liver disease can be performed by the method of the present invention.

The methods of the invention are highly correlated with the Metavir stage of well-characterized liver fibrosis. A particular advantage of the method of the present invention compared to prior art methods is the use of quantitative panels to minimize misclassification of pathological observations and errors of statistical models.

The methods of the present invention include non-invasive methods that closely agree with the severity of fibrosis measured by several methods: liver biopsy and more methods such as measuring the area of fibrosis.

The method of the present invention is based on a statistically correlated cohort of patient specimens with well-characterized liver fibrosis (covering the full range of Metavir stages) and patient specimens without liver fibrosis due to histological determination (Metavir evaluation: 0). The initial selection criterion for samples was the Metavir score. This reference was confirmed in a double evaluation and in an optimized manner using specimens larger than 15 mm in size.

The method of the invention allows the absence of prediction of fibrosis with a diagnostic accuracy (DA) of at least 82%, preferably at least 84%. The method of the present invention represents an alternative to biopsy, since arbitrary misclassification of fibrosis stage and in part leads to pain and health risks for the patient, even though the reference standard is not the gold standard for liver fibrosis.

This method allows the study of fibrosis development and progression, providing efficient monitoring of chronic HCV patients. Disease monitoring of chronic HCV patients can be performed at short intervals compared to biopsies. This method allows monitoring the success of anti-fibrotic therapy.

This method also allows the study of the development and progression of fibrosis in patients with chronic liver injury. This is a relatively common disorder with minimal symptoms, yet carries considerable long-term risk of morbidity and mortality, defined pathologically by ongoing hepatic necrosis and hepatic inflammation, often accompanied by fibrosis. HCV is the most common form of chronic liver injury. This approach can be applied to more forms of chronic liver injury: alcoholic steatohepatitis (ASH), alcoholic fatty liver disease (AFLD), nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease (NAFLD). The methods of the invention can be used to monitor the severity of NASH and NAFLD. They can be used to diagnose viral such as hepatitis A, hepatitis B, hepatitis C or D virus or human immunodeficiency virus (HIV) hepatitis, chronic persistent or chronic active hepatitis, autoimmune liver disease such as autoimmune hepatitis and drug-induced liver disease; Hepatic fibrosis in individuals with primary biliary cirrhosis, biliary atresia, liver disease caused by medical treatment, or congenital liver disease. The invention can be used to monitor the treatment of drugs with a risk of liver disease. The method can be used to diagnose the presence or severity of fibrosis and to monitor fibrosis associated with various fibrotic disorders, not limited to the liver: pulmonary fibrosis, renal fibrosis, prostate fibrosis and breast fibrosis and fibrosis in another disorder.

According to the present invention, the preferred parameter combination is TIMP-1, ferritin and A2M (also known as SNIFF 3a, SNIFF is the French abbreviation of score non-invasif de fibrose du foie; in English: non-invasive evaluation of liver fibrosis) , with 82.6% diagnostic accuracy; TIMP-1, ferritin, and PI (SNIFF 3), with 84% diagnostic accuracy; and TIMP-1, ferritin, PI, PLT, urea, age, with 84.7% for all diagnostic accuracy. See also Table 3 for these preferred combinations.

In the sense of the present invention, certain terms and expressions are to be understood as follows:

Diagnostic accuracy (DA) is the accuracy of the test itself. This means the percentage of all tests that were true positive or true negative. The higher the diagnostic accuracy, the more reliable the test's results. DA was calculated as the sum of true positives and true negatives divided by the total number of sample results and was influenced by the incidence of fibrosis in the analyzed population.

The cut-off value is the arithmetically calculated concentration of a single biomarker or a combination of several biomarkers used to distinguish between healthy and diseased states. In the understanding of the present invention, cut-off is a value of 0.5. If the value is higher than or equal to 0.5 (≥0.5), it means that Metavir stage F2 has been achieved and is used to distinguish no or mild fibrosis (Metavir stage F0 or F1) from clinically evident fibrosis CFS (Metavir stage F2 , F3, F4).

Positive predictive value (PPV) is the positive percentage of true positive tests.

Negative predictive value (NPV) means the percentage of negative tests that are true negative.

The score means an arithmetic combination of several biomarkers associated with fibrosis. In particular, the score used here has a range of 0 (minimal fibrosis) to 1 (CSF: clinically significant fibrosis).

AUROC means the area under the receiver operating characteristic curve. In these curves, sensitivity is plotted against the inverse of specificity. An area under the ROC curve of 1.00 represents ideal 100% sensitivity and 100% specificity. The greater the slope at the beginning of the curve, the better the correlation between the sensitivity and specificity of the test.

Sensitivity is the probability of producing a positive test in a patient with a disease or risk factor or other health condition.

Specificity is the probability of a negative test yielding an unaffected patient.

TIMP-1 (Tissue Inhibitor of Metalloproteinase I) is a sialoglycoprotein of 184 amino acids with a molecular weight of 28.5 kDa (see, e.g., Murphy et al. Biochem J. 1981, 195, 167-170), which inhibits metalloproteinases, Such as interstitial collagenase MMP-1 or matrix enzyme or gelatinase B. In the understanding of the present invention, the term TIMP-1 includes proteins having significant structural homology to human TIMP-1, inhibiting the proteolytic activity of metalloproteases. Antibodies that specifically detect epitopes of TIMP-1 can be used to detect the presence of human TIMP-1. TIMP-1 can also be determined by detection of associated nucleic acids, such as the corresponding mRNA.

Ferritin is a large molecule with a molecular weight of at least 440 kD, depending on iron content, and consists of a protein shell of 24 subunits (apoferritin) and an iron core containing an average of about 2500 Fe ³⁺ ions (in the liver and spleen of ferritin). Ferritin readily forms oligomers. At least 20 isoferritins can be distinguished by means of isoelectric focusing. This microscopic heterogeneity is caused by differences in the content of acidic H and weakly basic L subunits. Basic isoferritin is responsible for the function of long-term iron storage and is mainly found in the liver, spleen and bone marrow.

Measurement of ferritin is a suitable method for determining the status of iron metabolism. Ferritin determination at the start of treatment provides a representative measure of body reserves. Clinically, a limit value of about 20 ng/ml has proven useful in the detection of pre-latent iron deficiency. This value provides a reliable indication of depletion of iron stores that can be mobilized for hemoglobin synthesis. Latent iron deficiency was defined as below the limit of 12 ng/ml. For manifestations of iron overload in the body, a limit value above 400 ng/ml is considered useful.

For the detection of ferritin, a traditional sandwich immunoassay can be used, in which two ferritin-specific antibodies are used to form the sandwich complex in the test. One of the antibodies is bound to the solid phase and the other carries a label whose signal is used as a means of detecting the presence of ferritin.

PI (Prothrombin Index) is used to detect disturbances in the coagulation system and can be determined by adding thromboplastin to a plasma sample and measuring the clotting time in seconds (called Quick-time). Relate this value to an international normalized scale that includes a correction factor that takes into account the sensitivity of the thromboplastin used.

A2M (alpha-2-macroglobulin) is a conserved protein that is abundantly present in plasma and acts as a protease-binding protein to remove active proteases from tissue fluids. Instead of inactivating the catalytic activity of proteases, A2M works by physically trapping target proteases by folding around them. Proteases trapped by A2M are thus sterically prevented from cleaving their substrate proteins. In the sense of the present invention, A2M can be detected by immunoassays using specific antibodies according to test formats known to those skilled in the art. A2M can also be determined by detection of associated nucleic acids such as the corresponding mRNA.

According to the invention, other biochemical or clinical parameters may be determined. Other biochemical parameters can be any parameter directly or indirectly related to the metabolism or structure of the liver, such as urea, GGT (gamma-glutamyl transpeptidase), hyaluronate, AST (aspartate aminotransferase) , MMP-2 (matrix metalloproteinase-2), ALT (alanine aminotransferase), PIIINP (N-terminal propeptide of type III procollagen), bilirubin, haptoglobin, ApoA1, PLT (platelet count ). Hepcidin or adiponectin can also be measured.

Hepcidin is a liver protein originally identified as a circulating antimicrobial peptide. It is an important player in the flow of iron from the body to the absorptive cells of the intestine. Adiponectin is secreted by adipocytes and circulates in relatively high systemic concentrations to affect metabolic function. Decreased serum adiponectin levels imply increased disease risk, such as the severity of nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH).

Other clinical parameters can be determined, such as age, sex, weight, nutritional habits of the patient.

Urea, GGT (gamma-glutamyl transferase), hyaluronate, AST (aspartate amino transferase) and ALT (alanine aminotransferase), MMP-2, PIIINP, bilirubin, haptoglobin, ApoA1, hepcidin and adiponectin. Where applicable, hybridization techniques for the detection of nucleic acids specific for an analyte or parameter (eg, corresponding mRNA) can also be used to determine the parameter.

PLT (platelet count) is the platelet count and is determined by counting the platelets using a commercially available counter.

The present invention uses the determination of a number of parameters. Therefore, the determination of those biochemical and serological parameters according to the invention which can be performed in a test format using a solid phase is preferably carried out on a miniaturized matrix based test system as described in US2003/0017616 or WO99/67643. These test systems have a plurality of spatially defined test zones, each of which can be used to detect a single specific analyte or parameter. Thus, multiple analytes can be detected in a single round of testing.

The term solid-phase defined test zone is understood to mean a test zone comprising a solid-phase defined region, which is preferably spatially separated from another test zone by an inner region. The defined test area preferably has a diameter of 10 μm to 1 cm, particularly preferably 10 μm to 5 mm. Most preferred are miniaturized test areas with a diameter of 10 μm to 2 mm. Preference is given to solid phases with several test zones, also known as matrix systems. Such matrix systems are described, for example, in Ekins and Chu (Clin. Chem. 37, 1995, 1955-1967) and U.S. Patent nos. 5,432,099, 5,516,635 and 5,126,276. As mentioned before, the advantage of matrix systems is that several analytes and controls can be measured simultaneously on one sample. The use of control zones to detect non-specific binding and/or interfering samples can significantly increase the reliability of results, especially with miniaturized matrix test systems.

In the present invention, by using such a matrix-based test system, for example, the detection of TIMP-1, A2M and ferritin and possibly other biochemical parameters can be performed simultaneously.

According to the invention, a solid phase is any conventional support for detection methods, preferably a non-porous support, for example a support with a plastic, glass, metal or metal oxide surface. Porous supports such as test strips are also suitable. Spatially separated areas (test fields) are located on this support. Receptors immobilized on a solid phase are applied to these test areas. Immobilization of solid phase receptors by known methods, such as by direct absorptive binding, by covalent binding or by binding of high affinity pairs, such as streptavidin (or avidin)/biotin , antigen/antibody or sugar/lectin. The presence and/or amount of an analyte in a sample can be determined by specific binding of a component of the detection medium, eg the analyte or analyte analogue to be determined, to a solid phase receptor.

The detection of the analyte and - where appropriate - the detection of the presence of interfering reactions is achieved in the method according to the invention in a known manner by using suitable labeling groups, for example fluorescent labeling groups. Alternatively, the interaction of components of the detection medium with the test zone and any control zone can also be detected using suitable solid phases, for example by determining the layer thickness of the corresponding zone by plasmon resonance spectroscopy.

Using a matrix system in which several analytes of a sample are detected simultaneously, it is preferred to apply a universal labeling group capable of detecting several different analytes simultaneously to different test zones. An example of such a universal labeling group is a labeling group carrying a receptor that can specifically interact with a complementary receptor on a test reagent, e.g. for the analyte to be assayed or for an analyte analogue. Soluble receptors (such as antibody/antigen or streptavidin/biotin, etc.).

The term sample means a biological specimen containing or supposed to contain at least one marker according to the invention. For example blood samples, serum, urine, saliva, synovial fluid or liver tissue can be used. Fluid samples can be diluted prior to analysis, if desired.

To obtain results useful for diagnosing diseases, arithmetic algorithms known to those skilled in the art are used. The obtained data are combined and evaluated by statistical methods such as logistic binary regression to obtain a score.

Figure 1 shows raw data measured on 120 patients suffering from HCV infection.

The present invention is further illustrated by the following examples:

Example

All tests were performed using commercially available test kits and according to the manufacturer's instructions listed below.

Table 1

biomarker method supplier AST, ALT clinical blood chemistry Roche Diagnostics GmbH Mannheim, Germany GGT clinical blood chemistry Roche Diagnostics GmbH Mannheim, Germany Bilirubin clinical blood chemistry Roche Diagnostics GmbH Mannheim, Germany Urea clinical blood chemistry Roche Diagnostics GmbH Mannheim, Germany A2M Turbidimetry Dade Behring Marburg GmbH Apo A1 Turbidimetry Dade Behring Marburg GmbH Platelets platelet count Bayer Diagnostics PI solidification time Diagnostica Stago Hyaluronate Elisa Corgenix Inc. PIIINP RIAs Cis Bio International YKL-40 Elisa Quidel Corporation TIMP1 Elisa Amersham Pharmacia MMP2 Elisa Amersham Pharmacia

Figure 1 shows raw data measured on samples of 120 patients suffering from HCV infection. To obtain data, the test kits listed above were used.

Diagnostic accuracy and AUROC values are listed in Table 2. It can be seen that each individual marker achieved a DA of less than 80%.

Table 2

biomarker Diagnostic accuracy Correlation AUROC % p r p c p A2M 76.7 < ^10-4 0.523 < ^10-4 0.800 < ^10-4 TIMP1 72.3 <10 ^-4 0.663 < ^10-4 0.813 <10 ^-4 Ferritin 71.7 < ^10-4 0.433 < ^10-4 0.771 < ^10-4 HA 71.7 0.002 0.561 < ^10-4 0.762 <10 ^-4 Platelets 70.8 < ^10-4 -0.523 <10 ^-4 0.259 < ^10-4 AST 69.2 <10 ^-4 0.444 < ^10-4 0.782 < ^10-4 Prothrombin index 69.2 <10 ^-4 -0.444 <10 ^-4 0.265 < ^10-4 GGT 67.5 0.002 0.229 0.012 0.721 <10 ^-4 MMP2 67.2 < ^10-4 0.451 < ^10-4 0.711 <10 ^-4 ALT 66.7 0.002 0.311 0.001 0.696 < ^10-4 YKL 40 65.3 0.001 0.480 <10 ^-4 0.661 0.002 age 62.5 0.001 0.345 <10 ^-4 0.706 <10 ^-4 P3P 62.5 0.02 0.337 < ^10-4 0.626 0.019 Bilirubin 61.7 0.02 0.107 0.247 0.628 0.017 haptoglobin 61.7 0.01 -0.285 0.002 0.356 0.007 ApoA1 60.0 0.03 -0.229 0.012 0.361 0.009 gender 53.3 0.37 - - - - Urea 51.7 0.28 -0.058 0.527 0.470 0.572

Table 3 shows the comparison of DA/AUROC with state-of-the-art methods. Compared with the methods of US6,631,330 and WO2003/073882, it was shown by binary logistic regression that the method of the present invention has higher diagnostic accuracy for clinically significant fibrosis.

table 3

method selected mark nvar nPts R ² DA AUROC PLT, PI, urea, ferritin, age, TIMP 6 118 0.689 84.7 TIMP, ferritin, PI 3 119 0.629 84.0 0.904 TIMP, ferritin, A2M 3 118 82.6 0.886 WO2 003/073882 TIMP, HA, A2M 3 118 80.7 0.898 US6,631,330 (Fibrotest) A2M, age, haptoglobin, Apo, bilirubin, GGT, sex 7 120 0.518 80.8 0.857 US6,631,330 A2M, Age, Apo, GGT 4 120 0.487 77.5 0.859

Claims (3)

1. detect the existence of hepatopathy among the patient and/or the method for the order of severity, described method comprises:

A) obtain sample from described patient,

B) TIMP-1 (tissue depressant of metalloproteinases I) in the described sample of measurement,

C) ferritin in the described sample of measurement,

D) at least one is selected from other parameters of A2M (α-2-macroglobulin) and PI (prothrombin index) in the described sample of measurement,

E) randomly measure in the described sample at least one other biochemistry or clinical parameter,

F) based on TIMP-1, ferritin with according to step d) and e) existence of measured parameter or the existence and/or the order of severity that the measurement level is come diagnosis of liver disease.

2. according to the method for claim 1, described method is the treatment processing that is used to monitor liver fibrosis.

3. according to the method for claim 1, described method is to be used for liver fibrosis classification.