JP7239139B2 - Methods for diagnosing liver cirrhosis, methods for diagnosing complications of nonalcoholic steatohepatitis and hepatocellular carcinoma, and methods for diagnosing complications of nonalcoholic steatohepatitis and esophagogastric varices - Google Patents

Description

Article 30, Paragraph 2 of the Patent Act applies. Supplementary issue (General Assembly)}, page A100” and April 19, 2018, at the 104th General Assembly of the Japanese Society of Gastroenterology <Keio Plaza Hotel (2-2-1 Nishi-Shinjuku, Shinjuku-ku, Tokyo) > Announced at

The present invention provides a method for diagnosing liver cirrhosis, a method for diagnosing complications of non-alcoholic steatohepatitis and hepatocellular carcinoma, a method for diagnosing complications of non-alcoholic steatohepatitis and esophagogastric varices, and a method for diagnosing complications of liver fibrosis. It relates to a severity determination method.

(Chronic Liver Disease and Cirrhosis)
In chronic liver disease, intrahepatic inflammation and hepatocellular injury persist, accompanied by progression of hepatic fibrosis, leading to cirrhosis in the terminal state (see Non-Patent Document 1). Liver cirrhosis develops various serious complications such as esophageal varices rupture due to portal hypertension, and progresses to liver failure.
In addition, liver cirrhosis is a high risk condition for developing hepatocellular carcinoma. For these reasons, liver cirrhosis poses serious problems of significant deterioration of quality of life and deterioration of prognosis. Chronic liver diseases include hepatitis B virus, hepatitis C virus, alcoholic liver disease, non-alcoholic steatohepatitis, autoimmune hepatitis, primary biliary cholangitis, and the like.

(non-alcoholic fatty liver disease)
Non-alcoholic fatty liver disease (NAFLD) is a disease that presents with macrovesicular fatty deposits similar to those of alcoholic hepatitis in the absence of a history of alcohol consumption. With the increase in obesity and metabolic syndrome population, there are many NAFLD patients not only in Japan but all over the world.
NAFLD includes simple steatosis (SS) without hepatitis, and nonalcoholic steatohepatitis (NASH), which is accompanied by inflammation and hepatocellular damage and progresses to cirrhosis.

(non-alcoholic steatohepatitis)
Non-alcoholic steatohepatitis (NASH) is progressive and can lead to cirrhosis and hepatocellular carcinoma, and liver fibrosis is associated with the progression of the disease. However, there are no serological markers that can accurately evaluate the diagnosis of NASH or the progression of pathology, and pathological evaluation is considered to be the most accurate.

(prior art)
The following prior art is known.
In Patent Document 1, "Including the step of measuring one or more factors selected from the group consisting of sCD40, HMGB1, sPLA2groupIIA and sPLA2 activity in a biological sample of a subject as a marker, for assisting detection of non-alcoholic steatohepatitis method”. However, the non-alcoholic steatohepatitis-related marker described in Patent Document 1 is different from the measurement target of the present invention, and was obtained by comparing non-alcoholic steatohepatitis and healthy subjects who do not have simple steatosis. It is.

WO2011/046204

Amico, G.D. et al. Natural history and prognostic indicators of survival incirrhosis: A systematic review of 118 studies.44, 217-231 (2006)

The present inventors have discovered a method for diagnosing liver cirrhosis, a method for diagnosing complications of non-alcoholic steatohepatitis and hepatocellular carcinoma, a method for diagnosing complications of non-alcoholic steatohepatitis and esophagogastric varices, or liver fibrosis. The task was to develop a method for judging the severity of

In order to solve the above problems, the present inventors found that AKR1B10 obtained from gene expression analysis is serologically useful for diagnosing liver cirrhosis and complications of non-alcoholic steatohepatitis and evaluating the progression of liver fibrosis. Confirmed that there is. Furthermore, it was confirmed that the combination of AKR1B10 and M2BPGi is useful for predicting NASH complications and evaluating progression of liver fibrosis. This completes the present invention.

The present invention consists of the following.
1. A method for diagnosing liver cirrhosis or an auxiliary method for diagnosing liver cirrhosis, comprising the step of measuring the protein concentration or gene expression level of AKR1B10 in a biological sample.
2. 2. The method according to the preceding item 1, further comprising the step of measuring a change in the sugar chain structure of M2BP (M2BPGi).
3. 3. The method according to item 1 or 2, wherein the biological sample is a sample isolated from a non-alcoholic steatohepatitis patient.
4. 4. The method according to any one of the preceding items 1 to 3, wherein the biological sample is serum.
5. A method for diagnosing complications of non-alcoholic steatohepatitis and hepatocellular carcinoma, comprising the step of measuring the protein concentration or gene expression level of AKR1B10 in a biological sample and the sugar chain conformation change of M2BP (M2BPGi), or Diagnostic aid method.

6. 6. The method according to 5 above, wherein the biological sample is a sample isolated from a non-alcoholic steatohepatitis patient.
7. 7. The method according to the preceding item 5 or 6, wherein the biological sample is serum.
8. Diagnosis of complications of non-alcoholic steatohepatitis and esophagogastric varices, including the step of measuring the protein concentration or gene expression level of AKR1B10 and/or the carbohydrate conformational change of M2BP (M2BPGi) in a biological sample methods or diagnostic aids.
9. 9. The method according to item 8, wherein the biological sample is a sample isolated from a non-alcoholic steatohepatitis patient.
10. 12. The method according to item 10 or 11, wherein the biological sample is serum.
11. A method or auxiliary method for determining the severity of liver fibrosis, comprising: (1) measuring the protein concentration or gene expression level of AKR1B10 and/or the sugar level of M2BP in a serum sample isolated from a patient with non-alcoholic steatohepatitis; and (2) correlating the protein concentration or gene expression level measured in (1) above with the stage of liver fibrosis.
12. 11. The preceding item 11, wherein the correlation between the measured gene protein concentration or gene expression level and liver fibrosis in (2) is performed by comparing the measured protein concentration or gene expression level with a cutoff value. described method.
13. A diagnostic agent for liver cirrhosis, comprising an anti-AKR1B10 antibody or antibody fragment and/or a reagent for detecting changes in the sugar chain structure of M2BP (M2BPGi).
14. A diagnostic agent for complications of non-alcoholic steatohepatitis and hepatocellular carcinoma, comprising an anti-AKR1B10 antibody or antibody fragment and a detection reagent for M2BP sugar chain conformation change (M2BPGi).
15. A diagnostic agent for complications of non-alcoholic steatohepatitis and esophagogastric varices, comprising an anti-AKR1B10 antibody or antibody fragment and/or a detection reagent for M2BP sugar chain structural change (M2BPGi).

The present invention provides a method for diagnosing liver cirrhosis, a method for diagnosing complications of non-alcoholic steatohepatitis and hepatocellular carcinoma, a method for diagnosing complications of non-alcoholic steatohepatitis and esophagogastric varices, and a method for diagnosing complications of liver fibrosis. A severity determination method can be provided.

AKR1B10 values by liver fibrosis stage. AKR1B10 index of liver fibrosis stage 4. M2BPGi values for each liver fibrosis stage. M2BPGi index of liver fibrosis stage 4. Presence or absence of esophagogastric varices and AKR1B10 index. Comparison by AKR1B10 cutoff value and M2BPGi cutoff value of liver fibrosis stage 4. A(-)M(-) and A(+)M(+) indicate the both-negative and both-positive groups of AKR1B10 and M2BPGi, respectively. Comparison between a group positive for both AKR1B10 and M2BPGi in liver fibrosis stage 4 and a group positive for M2BPGi alone. M(-) and M(+) indicate the M2BPGi single-negative group and single-positive group, respectively. Association evaluation between AKR1B10 and M2BPGi and NASH-HCC complications1. Evaluation of association between AKR1B10 and M2BPGi and NASH-esophagogastric varices complications. Evaluation of association between AKR1B10 and M2BPGi and NASH-HCC complications2. The horizontal axis of the graph indicates the interval from HCC diagnosis (Days) with the day of diagnosis of HCC as the 0th day. The vertical axis of the graph indicates the survival rate (Overall survival, %) in each period when the number of survivors on day 0 in each group is taken as 100%. Patients at risk below the graph indicates the number of follow-up patients on days 0, 500, 1000, 1500, 2000 and 2500.

(Measurement object of the present invention)
The measurement target (marker) used in the present invention consists of AKR1B10 or a combination of AKR1B10 and M2BPGi.

(AKR1B10: Aldo-keto reductase family 1 member B10)
AKR1B10 is a member of the aldo-keto reductase (AKR) superfamily and is a NADPH-dependent reductase. In addition, AKR1B10 is involved in cancer cell proliferation through the reduction metabolism of retinoids and isoprenoids, etc., and is involved in the detoxification of carbonyl compounds produced by oxidative stress-induced lipid peroxidation (survival of cancer cells and resistance to anticancer drugs). Involved. In addition, AKR1B10 was first reported to be highly expressed in non-small cell lung cancer, and has been reported to be highly expressed in many cancer types, including liver cancer, bile duct cancer, and uterine cancer. ing.

(M2BPGi: Mac-2 binding protein glycan isomer)
M2BPGi (Mac-2 binding protein glycosylation isomer) has been studied as a fibrosis marker for liver, chronic hepatitis B, etc.
M2BPGi can be measured using a detection reagent for the change in sugar chain structure of M2BP (M2BPGi). As a detection reagent for M2BPGi, a commercially available reagent may be used, for example, HISCL ^™ M2BPGi ^™ reagent of Mac-2 binding protein (M2BP) glycosylation isomer kit (Sysmex Corporation).

(Measurement object of the present invention)
The subject of measurement (marker) of the present invention is a gene (DNA, cDNA, RNA, mRNA as well as DNA, cDNA, RNA, mRNA, etc., if detection, quantification, and/or activity can be measured in a biological sample (biological sample) obtained from a subject. (including fragments thereof), proteins (including fragments thereof), and protein activity can be measured. Preferably, protein concentration, RNA concentration, mRNA concentration, and more preferably protein concentration are measured.

(Method of the present invention)
The present invention is directed to the following methods for diagnosing cirrhosis or assisting diagnosis, methods for diagnosing complications of non-alcoholic steatohepatitis or assisting diagnosis, and methods for determining the severity of liver fibrosis.
o A diagnostic method or diagnostic aid for liver cirrhosis comprising the step of measuring the protein concentration or gene expression level of AKR1B10 in a biological sample.
o A method for diagnosing liver cirrhosis or an auxiliary method for diagnosing liver cirrhosis, which comprises the step of measuring the protein concentration or gene expression level of AKR1B10 in a biological sample and the sugar chain conformational change of M2BP (M2BPGi).
o A method for diagnosing complications of non-alcoholic steatohepatitis and hepatocellular carcinoma, comprising the step of measuring the protein concentration or gene expression level of AKR1B10 in a biological sample and the sugar chain conformation change of M2BP (M2BPGi) or diagnostic aids.
〇Measurement of complications of non-alcoholic steatohepatitis and esophagogastric varices, including the step of measuring the protein concentration or gene expression level of AKR1B10 and/or the glycan conformational change of M2BP (M2BPGi) in biological samples diagnostic methods or diagnostic aids;
o A method for determining the severity of liver fibrosis, comprising: (1) AKR1B10 protein concentration or gene expression level and/or M2BP sugar chain structure change in a serum sample isolated from a patient with non-alcoholic steatohepatitis (M2BPGi), and (2) correlating the protein concentration or gene expression level measured in (1) above with the stage of liver fibrosis.

The measurement method in the step of measuring the sugar chain structural change (M2BPGi) of M2BP is not particularly limited as long as it can measure the sugar chain structural change (M2BPGi) of M2BP. It can be measured using a detection reagent.

The severity of liver fibrosis can be pathologically evaluated by Brunt classification (Grading, Staging), Matteoni classification, and NAFLD activity score, preferably by Brunt classification (stage 1 to stage 4). can be evaluated with

(non-alcoholic steatohepatitis)
In the present invention, non-alcoholic steatohepatitis (NASH) refers to hepatitis caused by accumulation of fat in the liver not caused by alcohol, but is different from simple fatty liver.

(Subject of the present invention)
Subjects targeted by the present invention include mammals in general (including humans, cats, dogs, and horses), healthy subjects, patients with non-alcoholic steatohepatitis, people suspected of having hepatitis, and those with hepatitis. People who will develop in the future, patients with liver cirrhosis, people who are suspected of having cirrhosis, people who will develop liver cirrhosis in the future, patients with complications of non-alcoholic steatohepatitis and hepatocellular carcinoma, people with suspected complications Persons with future complications, patients with complications of non-alcoholic steatohepatitis and esophagogastric varices, persons with suspected complications, persons with future complications, fatty liver patients, fatty liver including those suspected of having fatty liver disease in the future, but preferably patients with non-alcoholic steatohepatitis.

(Sample of the present invention)
The sample of the present invention is not particularly limited as long as it contains genes and/or proteins to be measured (markers), preferably blood, blood components, extracellular vesicles in blood, serum, and more preferably Serum.

(Protein measurement/detection method)
When the measurement target (marker) of the present invention is a protein, it is not particularly limited, but a known protein measurement method (detection, quantification, activity) can be used. For example, antibodies, aptamers, etc. that specifically bind to proteins can be used. Examples of antibodies include monoclonal antibodies, polyclonal antibodies, single-chain antibodies, humanized antibodies, chimeric antibodies, and those capable of simultaneously recognizing two epitopes. Bifunctional antibodies that can be used can be exemplified. These antibodies can be made using the antigen according to conventional protocols. Aptamers are nucleic acid molecules that specifically bind to molecules such as proteins and amino acids.
ELISA (Enzyme-Liked Immunosorbent Assay) method, immunoprecipitation method, electrochemiluminescence method when measuring, detecting or quantifying a polypeptide or protein present in a test sample using an antibody that specifically binds to the above protein , RIA (radioimmunoassay) method, fluorescent antibody method and the like can be used, but ELISA method is preferably used.

(Gene measurement/detection method)
When the measurement target (marker) of the present invention is a gene, the method for measuring and detecting the gene includes a Northern marker using a labeled nucleotide, a labeled cDNA or a labeled RNA for a probe that specifically binds to the gene. A blotting method, a dot blotting method, a method for directly measuring mRNA, or the like can be used. Examples of the PCR method include real-time PCR method, competitive PCR method, and the like.
Further examples include DNA microarrays, DNA chips, antibody arrays, and the like. A DNA microarray or DNA chip on which at least one nucleotide or cDNA of the gene is immobilized is used. The nucleotide or cDNA may be a part corresponding to part of the gene.

(Cutoff value of the present invention)
According to the present invention, the “cutoff value for each measurement target (marker) of the subject-derived biological sample” can be set in advance. "Cut-off value of each measurement target (marker) of the subject-derived biological sample" is the average value of each measurement target (marker) in the non-alcoholic steatohepatitis patient-derived biological sample having Staging stage 4 of the Brunt classification, Average value of each measurement target (marker) in biological samples derived from non-alcoholic steatohepatitis patients with Brunt's staging stages 1 to 3, or average of each measurement target (marker) in biological samples derived from healthy subjects can be calculated from the values.
As a method for setting the cutoff value, the average value of each measurement target (marker) in a biological sample derived from a non-alcoholic steatohepatitis patient with Brunt staging stage 4, and the Brunt staging stage Calculate from the average value of each measurement object (marker) in biological samples derived from non-alcoholic steatohepatitis patients with 1 to 3 and / or the average value of each measurement object (marker) in biological samples from healthy subjects . Usually, a preset cutoff value (furthermore, within ±70% of standard deviation, within ±50% of standard deviation, within ±30% of standard deviation, within ±20% of standard deviation, or within ±50% of standard deviation) can be determined to be positive.
In addition, another cut-off value setting method is based on the values obtained by measuring each measurement target (marker) of the subject-derived biological sample in subjects and / or subjects with a history of non-alcoholic steatohepatitis. , ROC (Receiver Operating Characteristic) curves are generated using commercially available statistical analysis software to determine optimal sensitivity and specificity. For example, it is possible to set a cut-off value that gives priority to higher sensitivity for purposes such as primary screening, and increases specificity for examination purposes.

(Indicator of liver cirrhosis)
The present invention is useful for patients with liver cirrhosis (patients with non-alcoholic steatohepatitis having stage 4 of Brunt classification staging) and people without liver cirrhosis (in particular, simple fatty liver, healthy subjects, staging stage of Brunt classification). In order to distinguish non-alcoholic steatohepatitis patients with 1 to 3), a preset "cutoff value for each measurement target (marker) of a subject-derived biological sample" can be used as an index of liver cirrhosis.
For example, the measured value is a preset "subject-derived biological sample (marker) cutoff value (further, standard deviation within ± 70%, standard deviation within ± 50%, standard deviation within ± 30%, standard Deviation within ±20%, or standard deviation within ±50%)”, if liver cirrhosis will develop in the future, is currently developing, or is progressing (possible), or prognosis after onset is likely to be defective.
The cutoff value of serum AKR1B10 protein concentration, which is an indicator of liver cirrhosis (furthermore, standard deviation within ± 70%, standard deviation within ± 50%, standard deviation within ± 30%, standard deviation within ± 20%, or standard deviation within ± Within 50%) is not particularly limited, but may be, for example, 0.50, 0.80, 1.00, 1.10, 1.20, 1.30, 1.40, 1.50, 2.00, 2.50, or any value therebetween, preferably 1.30 is. The cutoff value of the change in the sugar chain structure of serum M2BP (serum M2BPGi), which is an indicator of liver cirrhosis, is not particularly limited. It can be any number therebetween, preferably 3.11.

(Index of complications of non-alcoholic steatohepatitis and hepatocellular carcinoma)
The present invention relates to patients with complications of non-alcoholic steatohepatitis and hepatocellular carcinoma and those without such complications (in particular, patients with non-alcoholic steatohepatitis who have not developed hepatocellular carcinoma, In order to distinguish between non-alcoholic steatohepatitis and hepatocellular carcinoma complications, the preset "cut-off value for each measurement target (marker) of the subject-derived biological sample" is used to distinguish between simple fatty liver, healthy subjects, etc. can be used as an indicator of
For example, if the measured value is equal to or greater than the preset "cutoff value for each measurement target (marker) of the subject-derived biological sample", complications of non-alcoholic steatohepatitis and hepatocellular carcinoma will develop in the future. , current onset, progression (possibility), or prognosis after onset is likely to be poor.
The cut-off value of serum AKR1B10 protein concentration as an indicator of complications of non-alcoholic steatohepatitis and hepatocellular carcinoma (standard deviation within ±70%, standard deviation within ±50%, standard deviation within ±30%) within ±20% of standard deviation, or within ±50% of standard deviation) is not particularly limited, but for example, 0.50, 0.80, 1.00, 1.10, 1.20, 1.30, 1.40, 1.50, 2.00, 2.50, or between them may be any numerical value, preferably 1.30. The cut-off value of the sugar chain structure change of serum M2BP (serum M2BPGi), which is an indicator of complications of non-alcoholic steatohepatitis and hepatocellular carcinoma, is not particularly limited, but is, for example, 1.50, 2.00, 2.80, 2.90, It may be 3.00, 3.11, 3.20, 3.30, 4.00, 5.00 or any number therebetween, preferably 3.11.

(Index of Complications of Nonalcoholic Steatohepatitis and Esophagogastric Varices)
The present invention is useful for patients with complications of nonalcoholic steatohepatitis and esophagogastric varices and those without such complications (in particular, patients with nonalcoholic steatohepatitis who have not developed esophagogastric varices, In order to distinguish between simple fatty liver, healthy subjects, etc.), the preset "cutoff value of each marker in the subject-derived biological sample" is used as an index of complications of non-alcoholic steatohepatitis and esophagogastric varices. be able to.
For example, if the measured value is equal to or greater than the preset "cutoff value for each marker in the subject-derived biological sample", complications of non-alcoholic steatohepatitis and esophagogastric varices will develop in the future, or are currently occurring. It can be determined that the disease is present, progressing (possible), or that the prognosis after onset is likely to be poor.
Cut-off value of protein concentration of serum AKR1B10, which is an indicator of complications of non-alcoholic steatohepatitis and esophagogastric varices (standard deviation within ±70%, standard deviation within ±50%, standard deviation within ±30%) within ±20% of standard deviation, or within ±50% of standard deviation) is not particularly limited, but for example, 0.50, 0.80, 1.00, 1.10, 1.20, 1.30, 1.40, 1.50, 2.00, 2.50, or between them may be any numerical value, preferably 1.30. The cutoff value of the sugar chain structure change of serum M2BP (serum M2BPGi), which is an indicator of complications of non-alcoholic steatohepatitis and esophagogastric varices, is not particularly limited, but is, for example, 1.50, 2.00, 2.80, 2.90, It may be 3.00, 3.11, 3.20, 3.30, 4.00, 5.00 or any number therebetween, preferably 3.11.

(Index of severity of liver fibrosis)
In the present invention, a preset "cutoff value for each subject-derived biological sample (marker) to be measured" can be used as an index of the severity of liver fibrosis.
For example, when the measured value is equal to or greater than the preset "cutoff value for each measurement target (marker) of the subject-derived biological sample", the severity of liver fibrosis, i.e., Brunt classification (Grading, Staging), Matteoni classification, classification evaluated by NAFLD activity score, preferably classification evaluated by Brunt classification Staging can be determined to be serious, more preferably Brunt classification Staging will reach stage 4 in the future, liver fibrosis It can be determined that the transformation is progressing (possible) or that it is currently in stage 4.
In addition, in the treatment evaluation of non-alcoholic steatohepatitis, complications of non-alcoholic steatohepatitis and hepatocellular carcinoma, or complications of non-alcoholic steatohepatitis and esophagogastric varices, Cut-off value for each marker of the biological sample (furthermore, standard deviation within ±70%, standard deviation within ±50%, standard deviation within ±30%, standard deviation within ±20%, or standard deviation within ±50%) In the following cases, it can be determined that the current treatment method is preferable (effective, continued, good prognosis).
The cutoff value of the protein concentration of serum AKR1B10, which is an index of the severity of liver fibrosis, is not particularly limited, but an index of stage 4 of Staging of the Brunt classification may be used, for example, Staging stage of the Brunt classification 4 and other Stages (Stages 1-3) and may be 0.50, 0.80, 1.00, 1.10, 1.20, 1.30, 1.40, 1.50, 2.00, 2.50, or any number in between. , preferably 1.30. The cutoff value of the sugar chain structure change of serum M2BP (serum M2BPGi), which is an index of the severity of liver fibrosis, is not particularly limited, but an index of Staging stage 4 of the Brunt classification may be used. For example, 1.50, 2.00, 2.80, 2.90, 3.00, 3.11, 3.20, 3.30, 4.00, 5.00, or their It may be any number between, preferably 3.11.
The cutoff value of serum AKR1B10 protein concentration, which serves as an indicator for stage 1 of Staging in the Brunt classification, may be calculated from the numerical value of stage 1 and the numerical value in the adjacent stage (stage 2), and is not particularly limited. , 0, 0002, or any number in between. The cutoff value of the sugar chain structure change of serum M2BP (serum M2BPGi), which is an indicator of stage 1 of Staging in the Brunt classification, may be calculated from the numerical value of stage 1 and the numerical values in the adjacent stages, and is not particularly limited. , for example, 0, 0.860, or any number therebetween.
The cut-off value of serum AKR1B10 protein concentration, which is an indicator of stage 2 of staging in Brunt's classification, may be calculated from the numerical value of stage 2 and the numerical values in the adjacent stages (stages 1 and 3), and is not particularly limited. , for example, 0.002, 0.003, or any number therebetween. The cutoff value of the sugar chain structure change of serum M2BP (serum M2BPGi), which is an indicator of stage 2 of staging in Brunt's classification, may be calculated from the numerical value of stage 2 and the numerical values in the adjacent stages, and is not particularly limited. , for example, 0.860, 2.870, or any number therebetween.
The cut-off value of serum AKR1B10 protein concentration, which is an indicator of stage 3 of Staging in the Brunt classification, may be calculated from the numerical value of stage 3 and the numerical values in adjacent stages (stages 2 and 4), and is not particularly limited. , for example, 0.003, 1.300. The cutoff value of the sugar chain structure change of serum M2BP (serum M2BPGi), which is an indicator of stage 3 of staging in the Brunt classification, may be calculated from the numerical value of stage 3 and the numerical values in the adjacent stages, and is not particularly limited. , for example, 2.870, 4.300, or any number therebetween.

The method for diagnosing liver cirrhosis or the method for assisting diagnosis of the present invention has, for example, the following steps.
(1) Measure each marker AKR1B10 or AKR1B10 and M2BPGi in a biological sample obtained from a subject.
(2) Compare the measured value of each marker with an index of liver cirrhosis.
(3) Liver cirrhosis is determined when the measured value of each marker is higher than the index.
As an indicator, it is preferable to adopt the average value of each marker in a biological sample derived from a patient with non-alcoholic steatohepatitis having Staging stage 4 according to Brunt's classification.

The method for diagnosing complications of non-alcoholic steatohepatitis and hepatocellular carcinoma or the method for assisting diagnosis of the present invention includes, for example, the following steps.
(1) Measure each marker, AKR1B10 and M2BPGi, in a biological sample obtained from a subject.
(2) Compare the measured value of each marker with an index of complications of non-alcoholic steatohepatitis and hepatocellular carcinoma.
(3) When the measured value of each marker is higher than the index, it is judged to be a complication of non-alcoholic steatohepatitis and hepatocellular carcinoma.
As an indicator, it is preferable to adopt the average value of each marker in a biological sample derived from a patient with non-alcoholic steatohepatitis having Staging stage 4 according to Brunt's classification.

The prognostic examination method for complications of non-alcoholic steatohepatitis and hepatocellular carcinoma of the present invention has, for example, the following steps.
(1) Measure each marker, AKR1B10 and M2BPGi, in a biological sample obtained from a subject.
(2) Compare the measured value of each marker with an index of complications of non-alcoholic steatohepatitis and hepatocellular carcinoma.
(3) When the measured value of each marker is higher than the index, the prognosis is judged to be poor.
As an indicator, it is preferable to adopt the average value of each marker in a biological sample derived from a patient with non-alcoholic steatohepatitis having Staging stage 4 according to Brunt's classification.

(prognosis)
The “prognosis” of the present invention means the survival rate after diagnosis by the diagnostic method or diagnostic aid method of the present invention, preferably the diagnostic method or aid for diagnosis of complications of non-alcoholic steatohepatitis and hepatocellular carcinoma of the present invention. Survival rate after diagnosis by the method, more preferably about 500 days, about 1000 days, about 1500 days after diagnosis by the diagnostic method or diagnostic aid method for complications of non-alcoholic steatohepatitis and hepatocellular carcinoma of the present invention , about 2000 days, about 2500 days, about 3000 days survival rate, more preferably about 500 after diagnosis by the diagnostic method or diagnostic aid method for complications of non-alcoholic steatohepatitis and hepatocellular carcinoma of the present invention means the complication-specific survival rate in days, about 1000 days, about 1500 days, about 2000 days, about 2500 days, about 3000 days.

The method for diagnosing or assisting diagnosis of complications of nonalcoholic steatohepatitis and esophagogastric varices of the present invention includes, for example, the following steps.
(1) Measure each marker AKR1B10 or AKR1B10 and M2BPGi in a biological sample obtained from a subject.
(2) Compare the measured values of each marker with indicators of complications of non-alcoholic steatohepatitis and esophagogastric varices.
(3) When the measured value of each marker is higher than the index, it is judged to be a complication of non-alcoholic steatohepatitis and esophagogastric varices.
As an indicator, it is preferable to adopt the average value of each marker in a biological sample derived from a patient with non-alcoholic steatohepatitis having Staging stage 4 according to Brunt's classification.

The method for determining the severity of liver fibrosis of the present invention has, for example, the following steps.
(1) Measure each marker AKR1B10 or AKR1B10 and M2BPGi in a biological sample obtained from a subject.
(2) Compare the measured value of each marker with an index of the severity of liver fibrosis.
(3) When the measured value of each marker is higher than the index, liver fibrosis is judged to be severe (progressing).
As an indicator, it is preferable to adopt the average value of each marker in a biological sample derived from a patient with non-alcoholic steatohepatitis having Staging stage 4 according to Brunt's classification.

The therapeutic evaluation determination method for non-alcoholic steatohepatitis of the present invention has, for example, the following steps.
(1) Each marker, AKR1B10 or AKR1B10 and M2BPGi, is measured in a biological sample obtained from a patient undergoing treatment for non-alcoholic steatohepatitis.
(2) Compare the measured value of each marker with an index for treatment evaluation of non-alcoholic steatohepatitis.
(3) If the measured value of each marker is lower than the index, it is determined that the treatment is good.
As an indicator, it is preferable to adopt the average value of each marker in a biological sample derived from a patient with non-alcoholic steatohepatitis having Staging stage 4 according to Brunt's classification.

(Diagnostic agent for liver cirrhosis)
The present invention also targets liver cirrhosis diagnostic agents containing anti-AKR1B10 antibodies or antibody fragments and/or reagents for detecting changes in the sugar chain structure of M2BP (M2BPGi).

(Diagnostic agent for complications of non-alcoholic steatohepatitis and hepatocellular carcinoma)
The present invention also targets a diagnostic agent for complications of non-alcoholic steatohepatitis and hepatocellular carcinoma, including an anti-AKR1B10 antibody or antibody fragment and a detection reagent for M2BP sugar chain conformation change (M2BPGi).

(Diagnostic agent for complications of non-alcoholic steatohepatitis and esophagogastric varices)
The present invention also targets a diagnostic agent for complications of non-alcoholic steatohepatitis and esophagogastric varices, which contains an anti-AKR1B10 antibody or antibody fragment and/or a reagent for detecting changes in the sugar chain structure of M2BP (M2BPGi).

(Hepatic fibrosis severity determination kit)
The kit for determining the severity of liver fibrosis of the present invention comprises an anti-AKR1B10 antibody or antibody fragment, or an anti-AKR1B10 antibody or antibody fragment and a detection reagent for M2BP sugar chain structural change (M2BPGi).

(Non-alcoholic steatohepatitis treatment evaluation determination kit)
The therapeutic assessment and determination kit for non-alcoholic steatohepatitis of the present invention comprises an anti-AKR1B10 antibody or antibody fragment, or an anti-AKR1B10 antibody or antibody fragment and a detection reagent for M2BP sugar chain conformational change (M2BPGi).

The above antibody may be a monoclonal antibody, and may be obtained by a method known per se, such as the method of Kohler and Milstein (Kohler G, Milstein C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-497. ).
In addition, the antibody fragment may have any structure as long as it can substantially maintain the antigen-recognition function of the antibody. For example, it preferably has an antigen-recognition site (light chain variable region sequence, heavy chain variable region sequence). .

EXAMPLES The present invention will be described below with reference to Examples, but the present invention is not limited to these Examples.

(subject)
METHODS: Seventy-one patients with histologically diagnosed nonalcoholic steatohepatitis (NASH) who underwent liver biopsies during the decade from 2006 to 2015 were included. The patients' median age was 69 years and median BMI was 27.8. Patient characteristics, including laboratory values for platelets, AST/ALT, total bilirubin, albumin, type IV collagen 7s and P-III-P are shown in Table 1.

(Histopathological diagnosis)
In order to evaluate the degree of histological fibrosis by histopathological diagnosis, the disease stage was determined by the Brunt classification.
As a result, 11 were stage 1, 12 were stage 2, 25 were stage 3, and 23 were stage 4. Thirty-two patients had hepatocellular carcinoma (HCC) and 33 had varicose veins when they were diagnosed with NASH.

(ELISA measurement of serum AKR1B10 and serum M2BPGi)
Subject serum AKR1B10 (ng/mL) and serum M2BPGi (cut-off index: COI) were measured by known ELISA.
When comparing the AKR1B10 levels for each stage of liver fibrosis by the Kruskal-Wallis test, it increased as the disease stage progressed, and a significant difference was observed between the groups. (Fig. 1).
The cutoff value of AKR1B10 in the stage 4 group was 1.30, the sensitivity was 65%, the specificity was 97%, and the AUC was 0.842 (Fig. 2).
M2BPGi, which is often used as a liver fibrosis marker in recent years, was also evaluated by the Kruskal-Wallis test, and as with AKR1B10, it tended to increase as the disease stage progressed (Fig. 3). In stage 4 patients with advanced liver fibrosis, the cutoff value for the area under the ROC curve was 3.11, the sensitivity was 82%, the specificity was 89%, and the AUC was 0.922 (Fig. 4).
Liver function indices (main serum test values) such as serum AKR1B10, serum M2BPGi, FIB-4 index, APRI, etc. are shown in Table 2.

From the above, serum AKR1B10 correlated with hepatic fibrosis (stage) in NASH cases, and was particularly high in stage 4 cases. Thus, serum AKR1B10 was associated with histologic disease progression in NASH patients. Serum M2BPGi also correlated with the development of liver fibrosis in NASH cases.

(Relationship evaluation between AKR1B10 and esophagogastric varices complications)
Regarding the prediction of the development of esophagogastric varices (Varix) by AKR1B10, we investigated the relationship between AKR1B10 and esophagogastric varices complications.
First, we compared the serum AKR1B10 levels for each stage of liver fibrosis in each subgroup of 33 patients with esophagogastric varices and 30 patients without esophagogastric varices. It was 0.55, significantly higher than the median value of 0.02 in patients without esophagogastric varices (Fig. 5). It was considered necessary to keep in mind the complication of esophagogastric varices in patients with high levels. The cut-off value for the area under the ROC curve was 0.17, with a sensitivity of 60% and a specificity of 83%.

(Relationship evaluation between AKR1B10 and M2BPGi and liver fibrosis stage 4)
A combined analysis of two liver fibrosis markers, AKR1B10 and M2BPGi, was considered.
The subjects were divided into a negative group and a positive group by the cutoff value of 1.30 for AKR1B10 and the cutoff value for M2BPGi of 3.11 according to the area under the ROC curve in patients with advanced liver fibrosis in stage 4, and both AKR1B10 and M2BPGi were both negative and both by Fisher test. The positive groups were compared. As a result, the ratio of liver fibrosis stage 4 was significantly higher in the both-positive group than in the both-negative group (Fig. 6).
Furthermore, the AKR1B10- and M2BPGi-positive group had a significantly higher ratio of liver fibrosis stage 4 than the M2BPGi-only positive group (Fig. 7). In the M2BPGi single-positive group, there were cases with high values even in liver fibrosis stage 3.
From the above results, it was confirmed that the combination of AKR1B10 and M2BPGi enhances the ability to diagnose liver fibrosis stage 4 as compared to the case of M2BPGi alone.

(Relationship evaluation between AKR1B10 and M2BPGi and NASH-HCC complications 1)
The rate of complication of NASH and HCC was also compared between a group negative for both AKR1B10 and M2BPGi and a group positive for both by Fisher's test. As a result, the NASH-HCC complication rate was significantly higher in the AKR1B10 and M2BPGi-positive group compared to the both-negative group, and the combination of the two markers is a useful evaluation index for predicting NASH-HCC complications. A possibility was suggested (Fig. 8).

(Relationship evaluation between AKR1B10 and M2BPGi and NASH-esophagogastric varices complications)
The rate of complications of NASH and esophagogastric varices was also compared between the group negative for both AKR1B10 and M2BPGi and the group positive for both, using Fisher's test. As a result, the AKR1B10- and M2BPGi-positive group had a significantly higher complication rate than the both-negative group, and the combination of the two markers is a useful evaluation index for predicting complications of NASH-esophagogastric varices. A possibility was suggested (Fig. 9).

(Relationship evaluation between AKR1B10 and M2BPGi and NASH-HCC complications 2)
The course of NASH-HCC complications was observed for about 8 years, and the survival rate (Overall survival) of the group negative for both AKR1B10 and M2BPGi was compared with the other group. As a result, the both AKR1B10 and M2BPGi positive group (AKR1B10 high + WFA(+)- M2BP high) had a significantly worse prognosis than the others (Others). It was suggested that it may be a useful evaluation index for predicting the prognosis of HCC complications (Fig. 10).

Based on the above, combined evaluation of serum AKR1B10 and serum M2BPGi can predict complications of HCC and esophagogastric varices in addition to liver fibrosis. The combination of serum AKR1B10 and serum M2BPGi can predict the prognosis of NASH-HCC complications.
Serum AKR1B10 alone had some predictive power for NASH-esophagogastric varices complications. By combining serum AKR1B10 and serum M2BPGi, advanced liver fibrosis can be diagnosed more effectively than serum M2BPGi alone. Furthermore, the combination of serum AKR1B10 and serum M2BPGi can be a useful marker for predicting not only progression of hepatic fibrosis but also hepatocellular carcinoma and esophagogastric varices.

In the present invention, a method for diagnosing liver cirrhosis, a method for diagnosing complications of non-alcoholic steatohepatitis and hepatocellular carcinoma, a method for diagnosing complications of non-alcoholic steatohepatitis and esophagogastric varices, or a method for diagnosing complications of liver fibrosis. A severity determination method can be provided.

Claims (5)

Non-alcoholic steatohepatitis and hepatocellular carcinoma, including the step of measuring the expression level of protein concentration of AKR1B10 in serum isolated from patients with non-alcoholic steatohepatitis and the sugar chain conformation change of M2BP (M2BPGi) Diagnostic aid for complications.
A method for assisting diagnosis of complications of non-alcoholic steatohepatitis and esophagogastric varices, comprising the step of measuring the expression level of protein concentration of AKR1B10 in serum.
A method for assisting determination of severity of liver fibrosis, comprising: (1) expression level of AKR1B10 protein concentration and/or change in sugar chain structure of M2BP (M2BPGi ) and (2) correlating the expression level of the protein concentration measured in (1) above with stage 4 of liver fibrosis.
4. The method according to claim 3, wherein correlating the measured expression level of protein concentration and liver fibrosis in (2) is performed by comparing the measured expression level of protein concentration with a cutoff value.
A diagnostic agent for complications of non-alcoholic steatohepatitis and esophagogastric varices, which is used in the method according to claim 2 , comprising an anti-AKR1B10 antibody or antibody fragment detection reagent.

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