JP2012103211A - Method of detecting the occurrence of liver cancer or predicting risk thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and kit for detecting the occurrence of liver cancer in a hepatopathy patient and predicting a risk thereof quickly and with high specificity.SOLUTION: The method analyzes an amount and/or enzyme activity of a von Willebrand factor-degrading enzyme in a sample. The kit is a latex reagent which includes an antibody specifically bonding to a von Willebrand factor-degrading enzyme (preferably, a polyclonal antibody) or a fragment thereof, and preferably carries the antibody or fragment.

Description

  The present invention relates to a method and a kit for detecting or predicting liver cancer, characterized by analyzing the amount and / or enzyme activity of a von Willebrand factor (VWF) degrading enzyme. The present invention also relates to a latex reagent for analyzing von Willebrand factor degrading enzyme using a polyclonal antibody that specifically binds to von Willebrand factor degrading enzyme, for detecting or predicting the onset of liver cancer. The method and kit of the present invention are used to detect the onset of liver cancer and to grasp the possibility of the onset in advance. The “analysis” in the present specification includes both “measurement” for quantitatively or semi-quantitatively determining the amount of the analysis target substance and “detection” for determining the presence or absence of the analysis target substance. included.

  Liver cancer includes primary liver cancer made from hepatocytes and metastatic liver cancer in which other early-stage cancer metastasizes. In addition, primary liver cancer includes hepatocellular carcinoma that can be formed into hepatocytes and cholangiocellular carcinoma that can be formed into cholangiocytes in the liver. Refers to hepatocellular carcinoma, which accounts for the majority of primary liver cancer. Although the number of deaths due to hepatocellular carcinoma has increased rapidly since the late 1970s, most of them are deaths from hepatitis C. The number of deaths from all cancers is third in men and fourth in women, with around 40,000 people dying from the disease each year. Examining patients with liver cancer, many have chronic liver damage, 70% of whom have cirrhosis and 25% have chronic hepatitis. In Japan, about 70% of hepatocellular carcinomas are caused by hepatitis C and 20% are caused by hepatitis B. Therefore, about 70% of patients with cancer are viral hepatitis. Of liver cirrhosis. The causes are hepatitis B and C viruses, and it is thought that as the virus activates and hepatocytes repeatedly inflame, abnormalities in genes occur and cancer cells are produced. ing.

  Liver cancer is a very recurrent cancer, even if the first treatment appears to be complete. (1) 90% or more patients have chronic hepatitis or cirrhosis due to hepatitis B or C virus infection, so there is a high risk of new cancer in the remaining liver. 2) It is considered that liver cancer has the property of entering a nearby blood vessel at a relatively small stage (3 to 5 cm) and causing metastasis to the surroundings and other organs. In fact, recurrence in the liver in which 90% or more of the recurrence sites of liver cancer remain (residual liver recurrence), and recurrence frequency is much higher in the same organ than breast cancer and stomach cancer. Even with hepatectomy, which is the treatment that can most completely remove cancer cells, the probability of cancer recurrence by three or five years after treatment is as high as 70% or more. Currently, blood tests, X-ray tests, and ultrasonic tests using tumor markers such as α-fetoprotein (AFP) are performed as methods for predicting or detecting the onset of liver cancer.

  AFP is a type of protein found in fetal serum that disappears after birth but increases when it becomes liver cancer. It is measured together with blood biochemical tests such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and is used as a screening test for liver cancer. In liver cancer with confirmed AFP, the numerical value will drop if there is an effect of treatment, so it is an indispensable test for follow-up of treatment and discovery of recurrence. The standard value of AFP is 20 ng / mL or less (RIA solid phase method). If there is chronic liver injury and the AFP value is 200 to 400 ng / mL, the possibility of liver cancer is high, and if it is 400 to 1000 ng / mL or more, it is considered very suspicious. However, since some AFPs do not become positive even in liver cancer, it is known that it is difficult to identify a cancer simply by showing a high AFP value. Therefore, there is a demand for a marker that can correctly detect the early onset or recurrence of liver cancer or predict risk at an earlier stage.

  ADAMTS13 is a specific cleavage enzyme for VWF (von Willebrand factor). A zinc-type metalloprotease with a molecular weight of 190 kDa, which specifically cuts the VWF Try842-Met843 bond to a large molecular weight UL-VWFM (Unusually large-VWFM) up to the molecular size of VWFM (VWF multimer) Cleavage and further control of its activity. When ADAMTS13 activity or amount is normal, VWFM can be properly cleaved to become a hemostatic factor, but when ADAMTS13 is deficient or low activity, VWFM is not cleaved, which causes platelet thrombus. This representative disease was thrombotic thrombocytopenic purpura (TTP), but various cases have subsequently shown that a decrease in ADAMTS13 activity or amount is not specific to TTP, and plasma ADAMTS13 activity in cirrhosis. Decreases as the severity of the liver increases, but significantly decreases to the extent comparable to TTP at the end stage (Non-patent Document 1), ADAMTS13 activity in severe alcoholic hepatitis complicated with multiple organ failure and acute liver failure It has been reported that a marked decrease and a marked increase in UL-VWFM are closely related to the development of liver damage and the onset of multiple organ failure (Non-patent Document 2).

  On the other hand, the present inventors show that blood activity decreases in rat acute liver injury in which stellate cells are selectively impaired (Non-patent Document 3), while blood activity in the process of liver fibrosis in which stellate cells proliferate. It was found that it is enhanced (Non-patent Document 4), and it was revealed that stellate cells are important for the regulation of blood ADAMTS13 activity. ADAMTS13 is mainly produced in liver hepatic stellate cells (formerly Ito cells). Since stellate cells are present in the Disse space and stick to the outside of sinusoidal vascular endothelial cells, they are considered to be important for regulating the microcirculation of the liver. The stellate cells are also called fat storage cells, have lipid droplets containing vitamin A, and have stellate cell processes. As the liver fibrosis, stellate cells decrease in lipid droplets (and vitamin A also decreases), change their shape, resemble fibroblasts and myofibroblasts, and increase the production of extracellular matrix such as collagen. It can be seen. This suggests that in recent years, it is related to liver damage accompanied by liver fibrosis.

  By the way, as a method for measuring the VWF cleavage activity of ADAMTS13, there is SDS-agarose gel electrophoresis. However, this measurement method requires 3 days to calculate the result, and is not suitable for the “simple and rapid test method” that is currently required in clinical tests. Accordingly, the inventors have developed an ADAMTS13 antigen amount measurement reagent (ADAMTS13 ELISA measurement kit; Mitsubishi Chemical Medience) using a sandwich ELISA measurement method using two types of monoclonal antibodies (Patent Document 1). The correlation coefficient between this ELISA measurement method reagent and the activity value obtained by SDS-agarose gel electrophoresis was very good at 0.997, and it was possible to measure ADAMTS13 amount more easily and quickly. . In addition, although an ADAMTS13 activity measurement kit (ADAMTS13-act-ELISA kit; Kainos) using a monoclonal antibody against a portion of a VWF73 synthetic peptide that is exposed by being cleaved by ADAMTS13 in the sample is also commercially available, all are measured by ELISA. Since this method requires multiple human operations and the time required for measurement is 3.5 hours, further improvement is necessary, and the development of a simpler and more reproducible measurement method Is waiting.

International Publication No. WO2005 / 062054 Pamphlet

Uemura M, et al., Alcohol Clin Exp Res. 2005; 29 (12 Suppl): 264S-271S. Uemura M, et al., Thromb Haemost. 2008; 99 (6): 1019-1029. Kume Y, et al., FEBS Lett, 2007; 581 (8): 1631-1634. Watanabe N, et al., Thromb Haemost. 2009; 102: 389-396.

  As described above, it is difficult to detect the onset of liver cancer and predict the risk with high specificity using the current marker AFP or the like. An object of the present invention is to provide a method and a kit for detecting the onset of liver cancer and predicting the risk in a liver disorder patient quickly and with high specificity.

  In view of the above situation, the present inventors have intensively studied and found a marker that can detect the onset of liver cancer early and with high specificity. That is, it was discovered that ADAMTS13 can predict the onset and risk of developing liver cancer. This will be described in detail in the examples, but will be briefly described below. Paying attention to the fact that ADAMTS13 is produced in hepatic stellate cells, the expression of ADAMTS13 in patients with type B or C hepatopathy was examined, and it was found that there was a correlation between the liver fibrosis marker in the patient and the expression of ADAMTS13. It was. However, although it was observed that ADAMTS13 expression was increased between the patient and a healthy person, the increase was not so high. Conventionally, it is considered that liver fibrosis occurs as a result of hepatitis over many years as a soil that is likely to produce liver cancer. And even if the degree of liver fibrosis is the same, it is generally considered that liver cancer does not develop with high efficiency. Therefore, the present inventor conducted the following examination as to whether ADAMTS13 expression is related to the onset of liver cancer and prediction of its risk. That is, as a result of examining the relationship between the onset of liver cancer within 3 years and the expression of ADAMTS13 in the patient, when the expression level of ADAMTS13 was significantly higher than the conventional blood liver cancer marker, hepatic cancer was developed. I found out. Furthermore, for patients who have B-type or C-type hepatopathy after liver cancer treatment and have relapsed liver cancer within one year, ie, recurrence of liver cancer and the amount of ADAMTS13 and enzyme As a result of examining the relationship of activity, it was found that liver cancer recurs when the amount of ADAMTS13 and the enzyme activity are significantly higher than those of conventional blood liver cancer markers. From the above, it has been found that ADAMTS13 is a marker for detecting carcinogenesis and recurrence of liver cancer or predicting risk. Based on this finding, the significance of ADAMTS13 as a specific marker for detection of liver cancer onset or risk prediction is established, and ADAMTS13 is analyzed, and detection of liver cancer onset or risk prediction is characterized. The present invention relating to a rapid and simple test method and kit for ADAMTS13 was completed.

  The present invention further provides an immunoturbidimetric method for measuring ADAMTS13 levels. That is, the conventional method for analyzing ADAMTS13 has been focused on measuring the low amount of ADAMTS13, and the measurement range in which a sample can be measured as it is is about 100% that a healthy person shows. According to the present invention, the significance of measuring the amount of ADAMTS13 at a high concentration (which may exceed 150%) has been found. To measure the high concentration, an apparent value is obtained by, for example, diluting a sample. However, the measurement accuracy of the high concentration was poor. The second invention of the present invention was made in order to provide a rapid and simple method that can measure the amount of ADAMTS13 over a wide range from a low concentration to a high concentration, instead of the conventional enzyme immunoassay method that emphasizes the measurement of low values. It is. As a result of diligent investigations to solve the above problems, it was found that the presence or amount of ADAMTS13 in plasma can be detected by loading latex particles with an anti-ADAMTS13 polyclonal antibody. The present invention has been accomplished based on these findings.

That is, the present invention
[1] A method for detecting or predicting the onset of liver cancer, comprising analyzing the amount and / or enzyme activity of von Willebrand factorase in a sample;
[2] The method of [1], wherein the onset of the liver cancer is relapse;
[3] The method of [1] or [2], wherein the sample is blood;
[4] The method according to any one of [1] to [3], wherein the sample is derived from a patient having liver damage;
[5] The method according to any one of [1] to [4], wherein the amount and / or enzyme activity of the von Willebrand factor-degrading enzyme is higher than that of a healthy person.
[6] The method according to any one of [1] to [5], wherein the analysis of von Willebrand factorase is performed by an immunological method;
[7] A method of analyzing the amount and / or enzyme activity of von Willebrand factor-degrading enzyme in a sample for detection of liver cancer development or prediction of risk;
[8] A kit for detecting or predicting the risk of developing liver cancer, comprising an antibody or a fragment thereof that specifically binds to von Willebrand factorase;
[9] The kit of [8], which is a latex reagent for ADAMTS13 analysis;
About.

  By analyzing ADAMTS13 in a hepatic disorder patient, it is possible to specifically detect the onset of liver cancer or predict the risk at an early stage. Further, according to the ADAMTS13 measurement method using the latex reagent of the present invention, it is possible to measure the ADAMTS13 antigen amount with high reproducibility and high accuracy in a short time (for example, 10 to 20 minutes) without the need for pretreatment of the specimen. it can. The ADAMTS13 measurement method of the present invention is a useful means for detecting the onset of liver cancer or predicting risk in a case of chronic liver injury requiring detection of a high concentration of ADAMTS13.

2 is a calibration curve of the latex reagent of the present invention prepared in Example 1. FIG. It is a graph which shows the cumulative morbidity by the Kaplan-Meier method regarding cancer cell carcinoma onset in patients with chronic type B or type C liver injury.

[1] Detection Method of Liver Cancer Onset or Risk Prediction Method of the Present Invention In the method of the present invention, it is possible to detect the onset of liver cancer or predict risk by analyzing ADAMTS13 in a sample. A known method can be used as long as ADAMTS13 can be analyzed, and examples thereof include a method of measuring the amount (concentration) or enzyme activity of ADAMTS13.

  For example, detection of risk of liver cancer and prediction of risk are performed using as an index that the amount of ADAMTS13 or enzyme activity in a subject is higher than the amount of ADAMTS13 or enzyme activity in a group of healthy subjects. Specifically, for example, the occurrence of liver cancer is detected or the risk is predicted or detected when the value is significantly higher than the average value (Mean) + SD of ADAMTS13 in the group of healthy people. As shown in the examples described later, for example, when the patient's ADAMTS13 amount or ADAMTS13 activity value is equal to or higher than a threshold for determination with a healthy subject group or a negative target disease group (for example, a liver disorder patient), onset of liver cancer It can be determined that there is a high possibility of detection or onset. For example, regarding the onset of liver cancer, the threshold for determination with a healthy person group of ADAMTS13 amount or ADAMTS13 activity value can be 97.6%, and the threshold for determination with a liver disorder patient can be 107.2%. Moreover, regarding the recurrence of liver cancer, the threshold for determination with respect to the ADAMTS13 activity value or ADAMTS13 amount non-recurrence group can be set to 116.8% or 118.9%, respectively. The threshold for determination is expected to change depending on various conditions, for example, the underlying disease, sex, age, etc., but those skilled in the art will appropriately select an appropriate population corresponding to the subject and select from that population. A normal value range or a threshold for determination can be determined by performing statistical processing on the obtained data. The negative target disease group includes patients with liver damage.

  The disease whose detection or risk can be predicted by the method of the present invention includes liver cancer. Preferably, it is a liver cancer that develops from liver damage (ie, chronic hepatitis or cirrhosis), and particularly preferably a hepatocyte cancer.

  The target (subject) to which the method of the present invention can be applied is not limited as long as it is the purpose of detecting the onset of liver cancer or predicting the risk, but in particular, a liver disorder patient with a high risk of developing liver cancer, Preferably, patients with chronic liver disorder or cirrhosis, more preferably, patients with chronic type B or chronic type C liver disorder. Liver disorder may also refer to hepatitis. In addition, a patient after liver cancer treatment such as radiofrequency treatment is a subject for detecting recurrence of liver cancer or predicting risk.

  Examples of the test sample include various body fluids such as blood, cell tissue fluid, lymph fluid, thymic fluid, ascites fluid, amniotic fluid, gastric fluid, urine, pancreatic fluid, bone marrow fluid, and saliva. In particular, blood in plasma or serum form is preferred. The plasma is preferably citrate plasma or heparin plasma.

  As a method for measuring the amount (concentration) of ADAMTS13 of the present invention, a known immunoassay method such as a sandwich ELISA method, and as a method for measuring ADAMTS13 enzyme activity, a synthetic peptide of VWF73 is cleaved by ADAMTS13 in a sample and exposed. And a method using a monoclonal antibody against the portion to be treated. All of these methods are methods for detecting the presence of ADAMTS13 in a sample. In the present invention, since it is necessary to measure ADAMTS13 at a high concentration, an immunoturbidimetric method that can measure the amount of ADAMTS13 at a high concentration quickly and easily is particularly preferable. Specifically, the latex reagent mentioned later is mentioned.

[2] Latex reagent of the present invention The latex reagent of the present invention is an immunoreagent for measuring ADAMTS13, in particular, measuring the amount of ADAMTS13 antigen. A polyclonal antibody that binds to ADAMTS13 is immobilized on latex particles. is there.

<1> Solid phase carrier used for measurement of ADAMTS13 When using a latex agglutination immunoassay, for example, as a solid phase carrier for supporting an antibody, latex, gelatin, liposome, erythrocyte, silica, styrene-butadiene copolymer Particles such as coalescence, alumina, magnetic particles or ceramics can be used. Among them, latex particles are preferably used, such as polystyrene latex, styrene / divinylbenzene copolymer, acrylic acid / styrene copolymer, styrene / maleic acid copolymer, styrene / methacrylic acid copolymer, styrene / acrylic acid. Examples thereof include copolymers such as acid and alkyl acrylate, and copolymers of vinyl acetate and acrylic acid. The particle size of the latex particles is preferably about 0.1 to 1.0 μm. The particle size in the case of using magnetic latex is preferably about 0.5 to 3.0 μm. These carrier particles are loaded with an anti-ADAMTS13 antibody using a physical adsorption method, a chemical bonding method, or the like. By reacting the antibody supported on this solid phase carrier with the sample and forming an immune complex, an aggregate is formed, and measured by an optical method using transmitted light or scattered light, or judged visually. You can do it. When using magnetic latex, the supported antibody and sample are reacted to form an immune complex, and then labeled antibody (eg, alkaline phosphatase or horseradish peroxidase) is reacted to form a magnetic latex-sample. Forming a complex of labeled antibodies; What is necessary is just to measure the light-emission amount after adding a substrate (for example, CDP-sta, luminol etc.) to this.

<2> Antibody used for detecting the presence or amount of ADAMTS13 The latex reagent of the present invention is obtained by immobilizing an anti-ADAMTS13 antibody that binds to ADAMTS13 on the latex particles. The anti-ADAMTS13 antibody can be obtained by immunizing mammals such as rats, guinea pigs, rabbits, mice, goats, sheep, horses, cows, etc. with ADAMTS13 according to a conventional method. Further, the anti-ADAMTS13 antibody is prepared by electroporating a plasmid DNA obtained by incorporating a gene site for ADAMTS13 described in International Publication WO02 / 088366A1 into a pCAG vector (Niwa H, et al., Gene 1991; 108: 193-199.). It is possible to obtain it by introducing it by the rectification method. As anti-ADAMTS13 antibody immobilized on latex particles, anti-ADAMTS13 antiserum obtained by collecting blood from an immunized animal and separating the serum can also be used in the present invention, but IgG itself may be used. A fragment obtained by fragmenting into F (ab ′) ₂ , Fab, Fab ′ or the like using a digestive enzyme such as pepsin or papain or a reducing agent such as dithiothreitol or mercaptoethanol can also be used. Other classes of antibodies such as IgM can be used with similar treatments. The anti-ADAMTS13 antibody can be purified by, for example, the method of Nisonoff et al. (Nisonoff, A. Methods in Medical Research, Eisen HN (ed). Year Book Medical Publishers, Chicago, (1964) 10, 134-141).

  The method for immobilizing the anti-ADAMTS13 antibody on the latex particles is not particularly limited. For example, the physical adsorption method using physical adsorption caused by mixing the latex particles and the antibody, a coupling agent such as carbodiimide, A chemical bonding method is used in which the carboxyl group or amino group on the particle surface is chemically bonded to the antibody molecule. Alternatively, antibody molecules may be bound to latex particles through spacer molecules. Furthermore, after binding an antibody to another protein such as albumin using a chemical binding method, the protein may be physically or chemically immobilized on latex particles.

  As blocking agents, those conventionally used such as bovine serum albumin (BSA) and casein may be used, or synthetic blocking agent block masters (JSR Corporation) and lipiders that have been recently released as substitutes for BSA. (NOF Corporation) can be used.

  The immunoassay method of the present invention is a method for measuring the presence or amount of ADAMTS13 in a sample by latex aggregation using the latex reagent. That is, when a latex reagent is added to the ADAMTS13-containing sample solution, latex particles are bound and aggregated via the anti-ADAMTS antibody. By measuring the aggregation of the latex particles, the presence or amount of ADAMTS13 can be measured. The latex agglutination can be detected by the slide latex agglutination method, but the increase in absorption of light from the visible light to the near infrared region (400 to 2400 nm, preferably 600 to 1000 nm) is measured over time using a spectrophotometer. It is possible to measure accurately by performing photometry. As an apparatus for performing such a measurement, an LPIA-A700 latex agglutination fully automatic measuring machine manufactured by Mitsubishi Chemical Medience Corporation, a COBAS FARA apparatus and a COBAS MIRA apparatus manufactured by Roche Diagnostics Systems, and a Hitachi 7170 analyzer manufactured by Hitachi, Ltd. And Toshiba's TBA-120FR analyzer. The reaction between the sample and the latex reagent is performed in a liquid such as physiological saline or buffer. In order to suppress non-specific binding between the latex reagent and the protein in the sample, an adsorption inhibitor such as a surfactant may be added to the reaction solution.

<3> Method for detecting the presence or amount of ADAMTS13 in the sample of the present invention In order to detect the presence or amount of ADAMTS13, a calibration curve may be prepared using an ADAMTS13 standard having a known amount. The calibration curve is obtained, for example, by plotting the concentration of the ADAMTS13 standard product against the amount of latex particle aggregation. A sample is appropriately diluted stepwise, latex particles are added to the diluted solution, latex agglutination is measured, and the amount (concentration) of ADAMTS13 in the sample can be known from the obtained measurement value and calibration curve.
Using the antibodies and immunological techniques detailed in <1> and <2> above, the presence or amount of ADAMTS13 in the sample is detected, and the onset or risk of liver cancer in the patient is predicted.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

<< Example 1: Anti-ADAMTS13 antibody-binding latex reagent >>
(1) Preparation of anti-ADAMTS13 antibody An anti-ADAMTS13 polyclonal antibody has a gene site for ADAMTS13 in a pCAG vector (Niwa H, et al., Gene 1991; 108: 193-199.) As described in International Publication WO2004 / 029242. The plasmid DNA obtained by integration was obtained by introducing it into rabbits by electroporation. Next, according to a known method, blood was collected from the immunized rabbit, serum was separated to prepare anti-ADAMTS13 antiserum, and F (ab ′) ₂ was further prepared.

(2) Preparation of anti-ADAMTS13 antibody-bound latex reagent Polystyrene latex (JSR) having an average particle size of 0.322 μm was dissolved in a 3.5 mg / mL 1-ethyl-3- (3-methylaminopropyl) carbodiimide hydrochloride solution. The carboxyl group was activated, and after washing, the latex was suspended in a 50 mmol / L 2-morpholinoethanesulfonic acid (pH 6.0) buffer. Next, the anti-ADAMTS13 polyclonal antibody prepared in the above (1) was added to a concentration of 0.3 mg / mL and allowed to react for 1 hour. After washing, blocking was performed with a 0.5% block master solution (JSR) for 30 minutes to prepare an anti-ADAMTS13 antibody-sensitized latex reagent.

(3) Method of using anti-ADAMTS13 antibody-binding latex reagent The amount of ADAMTS13 using an anti-ADAMTS13 antibody-binding latex reagent was measured using a latex agglutination fully automatic measuring machine LPIA-A700 (Mitsubishi Chemical Medience). A test sample containing ADAMTS13 or 4 μL of a standard product containing ADAMTS13 was added to 144 μL of MOPS buffer and mixed, followed by reaction at 37 ° C. for 10 minutes. After adding 64 μL of the latex reagent obtained above, the immune reaction was observed by measuring the increase in near-infrared (800 nm) absorption at 37 degrees for 10 minutes. As a standard, a series of healthy human pooled plasma diluted with bovine serum albumin (BSA) -added Tris buffer was prepared and used. A calibration curve having good linearity depending on the concentration was obtained (FIG. 1). It was confirmed that measurement was possible from 0% to 200%. The minimum detection sensitivity of this reagent was 2%.

<< Example 2: Examination of development of liver cancer in hepatitis cases >>
Plasma ADAMTS13 activity was measured using a commercially available ADAMTS13 activity measurement kit (ADAMTS13-act-ELISA kit; Kainos) in 21 cases of chronic type B liver injury and 60 cases of chronic type C liver injury. Mean value SD of plasma ADAMTS13 in patients with chronic type B and type C hepatopathy is 114.0 ± 45.4%, data of healthy people group (published in the Kainos pamphlet): mean value ± SD = 97.6 ± 18 Compared with 0.0%, the ADAMTS13 activity value was significantly higher (110% or more) in patients with hepatic impairment. Next, Spearman's rank correlation coefficient between a marker conventionally used as an index for diagnosis of liver cancer or liver fibrosis and ADAMTS13 activity value was obtained. The value of each marker was determined as an average value ± SD. The results are shown in Table 1. Plasma ADAMTS13 activity is determined by serum AST (aspartate aminotransferase), ALT (alanine aminotransferase) and AFP (α-fetoprotein), as well as liver hardness by FibroScan (registered trademark) (ECHOSENS), APRI (Aspartate aminotransferase), which is a progression marker of cirrhosis. to platelet ratio index: a significant correlation with the ratio of AST to platelet). No correlation was found between the amount of albumin, which is a marker for liver synthesis, the number of platelets, which is a marker for liver fibrosis, and DCP (des-γ-carboxy prothrombin), which is a marker for hepatocellular carcinoma. From these results, it was found that there is a correlation between liver damage and plasma ADAMTS13 activity. Measurement of markers other than ADAMTS13 was performed according to a known method.

  Furthermore, when follow-up was conducted for 3 years, hepatocellular carcinoma developed in 10 cases, and the cumulative incidence by Kaplan-Meier method was 4.9% for 1 year, 9.1% for 2 years, and 3 years for 3 years. 11.1% (FIG. 2). Comparison of plasma ADAMTS13 activity measured in the above and known liver cancer or liver fibrosis marker in 10 patient groups who developed hepatocellular carcinoma and 71 patient groups who did not develop hepatoma Were examined by t-test. As a result, there was no significant difference between the groups with and without hepatocellular carcinoma in the known liver cancer or liver fibrosis markers excluding liver hardness, but plasma ADAMTS13 activity It was found that there was a significant difference between the group with and without cancer onset (Table 2). Specifically, plasma ADAMTS13 activity was 107.2 ± 42.8 (%) in the group without hepatocellular carcinoma and 161.9 ± 33.8 (%) in the group with onset, and the P value was determined by analysis. There was a significant difference between the two groups at 0.05% or less. Moreover, the liver hardness value by FibroScan was also significantly different from the onset of hepatocellular carcinoma.

  In the above data, or univariate analysis and even multivariate analysis, it was shown that the plasma ADAMTS13 activity value is a significant risk factor for the development of hepatocellular carcinoma. Therefore, ADAMTS13 was independent of liver hardness. It was found that it can be evaluated as a risk factor. This suggested that, for example, the threshold for the onset of hepatocellular carcinoma with plasma ADAMTS13 activity was 107.2%.

  If hepatitis is compared to "fire", the fibrosis measurement by FibroScan shows the degree of liver fibrosis, that is, the "history" of how much the previous fire has burned the liver. From the obtained knowledge, it is considered that the expression of ADAMTS13 shows not only the degree of liver fibrosis but also the wound healing mechanism as an inflammatory reaction, that is, the momentum of fire at that time. And when ADAMTS13 expression is high, that is, from an example in which hepatitis is more active, it is considered that liver cancer occurs with high efficiency. Based on the above, liver hardness measurement cannot predict the risk of liver cancer at an early stage, but by examining the expression of ADAMTS13, the degree of liver fibrosis is the same among patients with liver damage. Even in this case, the risk of developing liver cancer can be predicted specifically at an early stage, which can be said to be very useful.

  Further, FibroScan for measuring liver hardness is an expensive and special echo device, and there is a problem that it cannot be measured at any facility. On the other hand, the ability to use ADAMTS13 with a blood sample that can be easily handled as a marker leads to early detection of liver cancer or prediction of risk. Specifically, for example, by first analyzing ADAMTS13, if ADAMTS13 expression is high, it is predicted that the risk of developing liver cancer is high, and further follow-up with echo equipment, CT, etc. Liver cancer can be detected at an early stage.

<< Example 3: Examination of liver cancer recurrence in liver cancer cases >>
Among cases hospitalized with radiofrequency therapy for hepatocellular carcinoma, 14 cases with chronic type B liver injury and 83 cases with chronic type C liver injury were treated with plasma ADAMATS13 and known liver cancer or liver after the treatment. Fibrosis markers were analyzed, and then the relationship with the presence or absence of liver cancer recurrence was analyzed by t-test within one year. The value of each marker was determined as an average value ± SD. Liver cancer recurrence was targeted at cancer that had newly developed in another location, except for cancer that remained after treatment (local recurrence).

  Analysis of ADAMTS13 was performed as follows. The amount of ADAMTS13 antigen was measured with the latex reagent prepared in Example 1, and ADAMTS13 activity was measured with the ADAMTS13 activity measurement kit (ADAMTS13-act-ELISA; Kainos). For the latex reagent, the sample was measured according to Example 1 with the stock solution. In the activity measurement kit, each sample was diluted to be in a measurable range and measured according to the attached protocol.

  As a result, there is no significant difference in the measured value of the blood marker for known liver cancer or liver fibrosis between the group with and without recurrence of hepatocellular carcinoma, but the plasma ADAMTS13 amount and activity are A significant difference was observed between the group with and without hepatocellular carcinoma recurrence (Table 3). Specifically, plasma ADAMTS13 activity was 116.8 ± 28.5 (%) in the group without recurrence of hepatocellular carcinoma, and 130.0 ± 30.8 (%) in the group with recurrence. The value was significantly different at 0.05% or less. The plasma ADAMTS13 level was 118.9 ± 35.4 (%) in the group without recurrence of hepatocellular carcinoma, and 134.3 ± 36.1 (%) in the group with recurrence. The value was significantly different at 0.05% or less. From the above data, it was shown that ADAMTS13 is a risk prediction marker for liver cancer recurrence. This suggests that, for example, the plasma ADAMTS13 activity hepatocellular carcinoma recurrence risk threshold is 116.8%, and the plasma ADAMTS1 amount hepatocellular carcinoma recurrence risk threshold is 118.9%. It was done.

  The present invention can be used for detection of liver cancer development or risk prediction.

Claims (9)

  A method for detecting or predicting the risk of developing liver cancer, comprising analyzing the amount and / or enzyme activity of von Willebrand factorase in a sample.   The method of claim 1, wherein the onset of liver cancer is relapse.   The method according to claim 1 or 2, wherein the sample is blood.   The method according to any one of claims 1 to 3, wherein the sample is derived from a patient having liver damage.   The method according to any one of claims 1 to 4, wherein the amount and / or enzyme activity of the von Willebrand factorase is higher than that of a healthy person.   The method according to any one of claims 1 to 5, wherein the analysis of von Willebrand factor-degrading enzyme is performed by an immunological method.   A method for analyzing the amount and / or enzyme activity of von Willebrand factor-degrading enzyme in a sample for detection of liver cancer development or prediction of risk.   A kit for detecting the risk of developing liver cancer or predicting risk, comprising an antibody or a fragment thereof that specifically binds to von Willebrand factorase.   The kit according to claim 8, which is a latex reagent for ADAMTS13 analysis.

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