HK1110112A - Method for diagnosing liver fibrosis - Google Patents

Description

Method for diagnosing liver fibrosis

Technical Field

The present invention relates to the field of hepatology and liver fibrosis. In particular to a group of serological markers that can be used for diagnosing liver fibrosis, in particular for diagnosing liver fibrosis due to chronic HCV infection. These markers can be used to monitor therapeutic treatment of liver fibrosis.

Background

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-. The cellular mechanism of fibrosis is complex. In response to liver injury caused by, for example, chronic Hepatitis C Virus (HCV) infection, Hepatitis B Virus (HBV) infection, alcoholic or fatty liver disease, drug-induced liver disease, or primary biliary cirrhosis, normally quiescent hepatic stellate cells are activated into proliferating myofibroblasts. These cells produce extracellular matrix proteins and release tissue inhibitors of metalloproteinases, which bind to and inactivate metalloproteinases that cause scar degradation. As a result, liver function is impaired by increased production of tissue and protein-like collagen and decreased degradation of these compounds, fibrosis and scar accumulation (McHutchinson2004, CME Newsletter Tx reportermabrasionlogy, 2-4).

While liver fibrosis is a reversible process that causes the accumulation of extracellular matrix, cirrhosis is an irreversible process characterized by the formation of nodules in thick bands of matrix completely surrounding the parenchyma. If left untreated, liver fibrosis leads to cirrhosis, possibly with cancer. For these reasons, liver fibrosis and sometimes accurate diagnosis are essential for effective medical treatment.

Liver biopsy is still currently considered to be the so-called gold standard for detecting fibrosis and inflammation. Liver biopsies are recommended to rate and stage disease, confirm diagnosis and establish benchmarks against which to demonstrate improvement or disease progression, aid in determining prognosis and need for treatment (McHutchinson, supra; for review, see Gebo et al, 2002 Hepatology36, 161-172).

There are a number of histological grading systems that have been used to semi-quantify the degree of liver fibrosis and inflammation in chronic hepatitis c patients. One of the most commonly used hierarchical systems is the METAVIR system (Bedossa et al, 1994, hepatology, 20, 15-20). METAVIR divides liver fibrosis into 5 stages F0 to F4. F0 indicates no fibrosis, and F1 corresponds to mild fibrosis (portal fibrosis without septum). Moderate to severe fibrosis was classified as F2 to F4 (F2: few septa, F3: many septa but no cirrhosis), stage F4 corresponding to the final stage of cirrhosis. Fibrosis is considered clinically evident starting from F.gtoreq.2.

There are several drawbacks in the use of liver biopsy to diagnose and grade fibrosis. Liver fibrosis is subject to sampling errors, so that a small portion of samples cannot reflect the true condition of the whole liver. Thus, it is not an accurate marker of a dynamic process that is continuously degrading. In addition, pathologists are often inconsistent when they judge the data of histological samples, with intra-observer and inter-observer variability in 10% to 20% biopsies (Cadranel et al 2000, Hepatology 32, 477-.

Liver biopsy is an invasive and painful procedure for the patient. But also the risk of bleeding and other complications after sampling. In addition and partly due to expected post-complication hospitalization of the patient, this is a costly approach.

Liver fibrosis is a major complication of chronic HCV infection, leading to the development of cirrhosis and decompensated liver disease. Therefore, directed studies to detect the development and progression of fibrosis are essential for effective control of these patients. Assessment of progressive fibrosis is best accomplished using a non-invasive test that can distinguish between intermediate stages of fibrosis. Various single markers and marker panel algorithms have been disclosed, but no effective single biomarker or biomarker score is currently available that can reliably predict fibrosis (> 80% diagnostic accuracy). Further research into the Development of non-invasive dynamic measures of liver fibrosis was strongly encouraged in 2002 by the National Institute of health consensus Development Conference. In particular, studies on liver biopsy alternatives should provide sufficient detail on the biopsy procedure (average size of biopsy samples; histologically well characterized quantitative maps) to convince the reader of the suitability of the reference standard. Liver biopsy strongly depends on optimized performance criteria and may lead to misclassification of histological stages due to inter-observer variability and too small sample sizes (< 10 mm).

There is extensive research on biochemical or serological markers that reflect the fibrotic process in liver disease and can be used as an alternative to liver biopsy. In recent years, several non-invasive or minimally invasive biochemical and serological markers have been studied to aid in the diagnosis of liver disease. In particular, labeled compositions have been used to classify patients according to their stage or extent of fibrosis.

US6,631,330 discloses the use of a combination of at least 4 biochemical markers selected from the group consisting of alpha-2-macroglobulin, aspartate aminotransferase, gamma-glutamyl transpeptidase, gamma-globulin, total bilirubin, albumin, alpha 1-globulin, alpha 2-globulin, haptoglobin, beta-globulin, apoA1, IL-10, TGF-beta 1, apoA2 and ApoB. The values obtained for 4 of these markers were mathematically combined to determine the presence of liver fibrosis. Using this marker set, 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 the binding of at least three markers, alpha-2-macroglobulin, hyaluronic acid and a 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 a higher diagnostic accuracy in liver fibrosis determination than known hitherto in the prior art and to classify and distinguish different stages of fibrosis in a more reliable way, making it possible to monitor the clinical development of fibrosis during treatment. Furthermore, such a method should be suitable for continuous testing on an automated analyzer.

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 a liver disease in a patient, comprising the steps of:

a) obtaining an isolated sample from the patient,

b) measuring TIMP-1 (a tissue inhibitor of metalloproteinase I) in the sample,

c) measuring ferritin in the sample and measuring ferritin in the sample,

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

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

f) diagnosing the presence and/or severity of a liver disease based on the presence or measured level of TIMP-1, ferritin and the parameter measured according to steps d) and e).

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

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

The methods of the present invention include non-invasive methods that closely correspond to the severity of fibrosis as determined by several methods: liver biopsy and more methods such as determining area of fibrosis.

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

The method of the invention allows for the absence prediction of fibrosis with a Diagnostic Accuracy (DA) of at least 82%, preferably at least 84%. The method of the invention represents an alternative to biopsy, since any misclassification of the fibrosis stage leads further to pain and health risks for the patient, even if the reference standard is not the gold standard for liver fibrosis.

The method allows studying the development and progression of fibrosis, providing effective monitoring of chronic HCV patients. Disease monitoring of chronic HCV patients can be performed at short time intervals compared to biopsies. This method allows monitoring the success of anti-fibrotic treatments.

The method also allows for the study of the development and progression of fibrosis in patients with chronic liver injury. This is a relatively common disorder with minimal symptoms, and also has a significant long-term risk of morbidity and mortality, limited pathologically by ongoing hepatic necrosis and inflammation of the liver, often accompanied by fibrosis. HCV is the most common form of chronic liver injury. The method 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 method of the invention can be used to monitor the severity of NASH and NAFLD. They can be used for the diagnosis of viral, such as hepatitis A, B, C or D, or Human Immunodeficiency Virus (HIV) hepatitis, chronic persistent or chronic active hepatitis, autoimmune liver diseases, such as autoimmune hepatitis, and drug-induced liver diseases; primary biliary cirrhosis, biliary atresia, liver disease resulting from medical treatment, or liver fibrosis in individuals with congenital liver disease. The present invention may be used to monitor treatment of drugs at risk for liver disease. The method can be used for diagnosing the presence or severity of fibrosis and for monitoring fibrosis, where fibrosis is associated with various fibrotic disorders, not limited to liver: pulmonary fibrosis, renal fibrosis, prostate fibrosis and breast fibrosis, and fibrosis in another disorder.

According to the invention, the preferred combination of parameters is TIMP-1, ferritin and A2M (also known as SNIFF 3a, a French abbreviation for score non-invasive de fibrose du fooe; English: non-invasive assessment of liver fibrosis) with a diagnostic accuracy of 82.6%; TIMP-1, ferritin and PI (SNIFF 3), with a diagnostic accuracy of 84%; and TIMP-1, ferritin, PI, PLT, urea, age with diagnostic accuracy of 84.7%. These preferred combinations can also be seen in table 3.

In the sense of the present invention, specific terms and expressions should be understood as follows:

diagnostic Accuracy (DA) is the accuracy of the test itself. This means the percentage of all tests that are truly positive or truly negative. The higher the diagnostic accuracy, the more reliable the test results. DA was calculated as the sum of true positive and true negative divided by the total number of sample results and was affected by the incidence of fibrosis in the population analyzed.

Cut-off values are the arithmetically calculated concentrations of a single biomarker or a combination of several biomarkers for distinguishing 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 (. gtoreq.0.5), this means that Metavir stage F2 is reached for distinguishing between no or mild fibrosis (Metavir stage F0 or F1) and clinically significant fibrotic CFS (Metavir stage F2, F3, F4).

Positive Predictive Value (PPV) is the percentage of positive in a true positive test.

Negative Predictive Value (NPV) means the percentage of negatives in a true negative test.

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

AUROC means the area under the receiver operating characteristic curve. Of these curves, sensitivity was plotted against the reciprocal of specificity. The area under the 1.00ROC curve represents the ideal 100% sensitivity and 100% specificity. The larger the slope at the beginning of the curve, the better the correlation between sensitivity and specificity of the test.

Sensitivity is the probability of a positive test for a patient who develops a disease or risk factor or other health condition.

Specificity is the probability of a negative test leading to a patient who is not diseased.

TIMP-1 (tissue inhibitor of metalloproteinase I) is a 184 amino acid sialoglycoprotein having a molecular weight of 28.5kDa (see, e.g., Murphy et al Biochem J.1981, 195, 167-170) that inhibits metalloproteinases, such as interstitial collagenase MMP-1 or matrise or gelatinase B. In the sense of the present invention, the term TIMP-1 encompasses proteins having significant structural homology to human TIMP-1, inhibiting the proteolytic activity of metalloproteinases. 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 related nucleic acids, such as the corresponding mRNA.

Ferritin is a macromolecule with a molecular weight of at least 440kD, depending on the iron content, and is composed of a protein coat of 24 subunits (apoferritin) and a protein shell containing an average of about 2500 Fe³⁺Iron core formation of ions (in liver and spleen ferritin). Ferritin readily forms oligomers. At least 20 species of transferrin can be distinguished by isoelectric focusing. This microscopic heterogeneity is due to differences in acidic H and weakly basic L subunit contents. Basic transferrin is responsible for the long-term iron storage function and is found primarily in the liver, spleen and bone marrow.

The determination of ferritin is a suitable method for determining the status of iron metabolism. Ferritin assay at the start of treatment provided a representative measure of body reserves. Clinically, a limit value of about 20ng/ml has proven to be useful in the detection of iron deficiency before latency. This value provides a reliable indication of depletion of the iron reserves that can be mobilized for hemoglobin synthesis. Latent iron deficiency is defined as below the limit of 12 ng/ml. For the manifestation of iron overload in vivo, a limit value above 400ng/ml is considered useful.

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

PI (prothrombin index) is used to detect interference in the coagulation system and can be determined by the following method: thromboplastin was added to the plasma samples and the clotting time in seconds (called Quick-time) was measured. This value is linked to an international normalized ratio comprising a correction factor taking into account the sensitivity of the thromboplastin used.

A2M (alpha-2-macroglobulin) is a conserved protein that is present in large amounts in plasma as a protease binding protein to clear active proteases from tissue fluids. A2M does not inactivate the catalytic activity of the protease but acts by physically trapping the protease of interest by folding around the protease. Thus sterically preventing the protease trapped by A2M from cleaving its substrate protein. In the sense of the present invention, A2M can be detected by immunoassay using specific antibodies according to test formats known to the person skilled in the art. A2M can also be determined by detection of related nucleic acids, such as the corresponding mRNA.

Other biochemical or clinical parameters may be determined according to the invention. Other biochemical parameters may be any parameter directly or indirectly related to the metabolism or structure of the liver, such as urea, GGT (gamma-glutamyltranspeptidase), 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 determined.

Hepcidin is a liver protein originally identified as a circulating antimicrobial peptide. It is an important participant in the circulation of the body's iron accumulation to the intestinal absorptive cells. Adiponectin is secreted by adipocytes and circulates at relatively high systemic concentrations to affect metabolic function. Reduced serum adiponectin levels are indicative of an increased risk of disease, such as the severity of nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH).

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

Urea, GGT (gamma-glutamyltransferase), hyaluronate, AST (aspartate aminotransferase) and ALT (alanine aminotransferase), MMP-2, PIIINP, bilirubin, haptoglobin, ApoA1, hepcidin and adiponectin are determined by commercially available test kits by immunological or photometric methods known to those skilled in the art. Where applicable, hybridization techniques for detecting analyte-or parameter-specific nucleic acids (e.g., the corresponding mRNA) can also be used to determine the parameters.

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

The present invention uses the determination of multiple parameters. Thus, the determination of biochemical and serological parameters of the invention, which can be carried out 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 WO 99/67643. 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 defined test zone on the solid phase is understood to mean a test zone comprising a defined region of the solid phase, which is preferably spatially separated from another test zone by an inner region. The defined test field preferably has a diameter of 10 μm to 1cm, particularly preferably 10 μm to 5 mm. Most preferably a miniaturized test area having a diameter of 10 μm to 2 mm. A solid phase with several test zones is preferred, which is also referred to as a matrix system. Such matrix systems are described, for example, in Ekins and Chu (clin. chem.37, 1995, 1955-. As mentioned before, an advantage of the matrix system is that several analyte and control assays can be performed simultaneously on one sample. The use of control zones to detect non-specific binding and/or interfering samples can significantly improve the reliability of the 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, the solid phase is any conventional support for detection methods, preferably a non-porous support, e.g. a support with a plastic, glass, metal or metal-oxidized surface. Porous supports such as test strips are also suitable. Spatially separated areas (test zones) are located on the support. Receptors immobilized on a solid phase are applied to these test zones. The solid phase receptors are immobilized by known methods, for example 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 or/and amount of analyte in a sample can be determined by specific binding of a component of the detection medium, e.g., the analyte or analyte analog to be determined, to the solid phase receptor.

The detection of the analyte and, where appropriate, the presence of an interference reaction is effected in a known manner in the method according to the invention by using suitable labeling groups, for example fluorescent labeling groups. Alternatively, the interaction of the components of the detection medium with the test zone and optionally the control zone can also be detected using a suitable solid phase, for example by determining the layer thickness in the respective zone by means of plasmon resonance spectroscopy.

Using a matrix system in which several analytes of a sample are detected simultaneously, universal labeling groups capable of detecting several different analytes simultaneously are preferably applied to the different test zones. Examples of such universal labeling groups are those which carry receptors which can interact specifically with complementary receptors on the test agent, for example, soluble receptors for the analyte to be determined or for analyte analogs (e.g., antibodies/antigens 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, a blood sample, serum, urine, saliva, synovial fluid or liver tissue may be used. If desired, the fluid sample may be diluted prior to analysis.

To obtain a result that aids in the diagnosis of the disease, arithmetic algorithms known to those skilled in the art are used. The data obtained are combined and evaluated by statistical methods such as logistic binary regression to obtain a score.

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

The invention is further illustrated by the following examples:

examples

All tests were performed using commercially available test kits and according to the instructions provided by the manufacturers listed below.

TABLE 1

Biomarkers	Method	Suppliers of goods
			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	Turbidity measuring method	Dade Behring Marburg GmbH
Apo A1	Turbidity measuring method	Dade Behring Marburg GmbH
			Blood platelet	Platelet count	Bayer Diagnostics
PI	Setting time	Diagnostica Stago
			Hyaluronic acid salts	Elisa	Corgenix Inc.
PIIINP	RIA	Cis Bio International
			YKL-40	Elisa	Quidel Corpration
TIMP1	Elisa	Amersham Pharmacia
			MMP2	Elisa	Amersham Pharmacia

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

The diagnostic accuracy and AUROC values are listed in Table 2. It can be seen that each individual marker obtained less than 80% DA.

TABLE 2

Biomarkers	Accuracy of diagnosis		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
	Blood platelet	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 (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 binding	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
	Sex	53.3	0.37	-	-	-							-
Urea							51.7	0.28	-0.058	0.527	0.470	0.572

Table 3 shows a comparison of DA/AUROC with the prior art methods. The method of the invention has shown to have higher diagnostic accuracy of clinically significant fibrosis by binary logistic regression compared to the methods of US6,631,330 and WO 2003/073882.

TABLE 3

Method	Selected indicia	nvar	nPts	R2	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. A method of detecting the presence and/or severity of a liver disease in a patient, the method comprising:

a) obtaining a sample from the patient and,

b) measuring TIMP-1 (a tissue inhibitor of metalloproteinase I) in the sample,

c) measuring ferritin in the sample and measuring ferritin in the sample,

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

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

f) diagnosing the presence and/or severity of a liver disease based on the presence or measured level of TIMP-1, ferritin and the parameter measured according to steps d) and e).

2. The method according to claim 1, which is a therapeutic treatment for monitoring liver fibrosis.

3. The method according to claim 1, for grading liver fibrosis.