Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57505—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the blood, e.g. leukaemia
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6806—Determination of free amino acids
  • G01N33/6812—Assays for specific amino acids

Definitions

• the present invention relates to methods for assisting in the diagnosis of hematological tumors, methods for obtaining data for diagnosing hematological tumors, and kits for these methods.
• Non-Patent Document 1 it has been reported that in myelodysplastic syndromes, the blood concentration of tryptophan (Trp) decreases, while its metabolite kynurenine (Kyn) increases, which can be used as a biomarker to aid in the diagnosis of myelodysplastic syndromes. It is known that it can be obtained (Non-Patent Document 1).
• Non-Patent Document 1 only mentions the relationship between an increase in Trp metabolites and myelodysplastic syndrome, which is a specific hematological tumor, and does not discuss its application to the diagnosis of other hematological tumors. Not done.
• the present invention provides a method for assisting in diagnosing hematological tumors with excellent accuracy (sensitivity and specificity), a method for obtaining data for diagnosing hematological tumors with excellent accuracy (sensitivity and specificity), and methods for using these methods.
• the challenge is to provide a kit for this purpose.
• Trp and the Kyn pathway which is the metabolic pathway of Trp, Trp, KA (kynurenic acid), which is a metabolite generated from Trp, AA (anthranilic acid), Kyn (kynurenine), 3-HAA (3-hydroxyanthranilic acid) and 3-HKyn (3-hydroxykynurenine).
• AA anthranilic acid
• Kyn kynurenine
• 3-HAA 3-hydroxyanthranilic acid
• 3-HKyn 3-hydroxykynurenine
• Trp Tryptophan TDO: Tryptophan 2,3-dioxygenase IDO: Indoleamine 2,3-dioxygenase N-fKyn: N-formylkynurenine FA: Formamidase Kyn: Kynurenine KAT: Kynurenine aminotransferase ⁇ KG: ⁇ -ketoglutaric acid Glu: Glutamic acid KA: Kynurenic acid KNase: Kynureninase AA: Anthranilic acid 3-HAA: 3-hydroxyanthranilic acid KMO: Kynurenine 3-monooxygenase 3-HKyn: 3-hydroxykynurenine Representative embodiments of the present invention are as follows.
• ⁇ 1> Based on the decrease in the amount of kynurenic acid in the sample derived from the subject, or from the amount of anthranilic acid, kynurenine, 3-hydroxyanthranilic acid, 3-hydroxykynurenine, and tryptophan in the sample derived from the subject. using as an indicator the ratio of the amount of at least one selected from the group consisting of the amount of kynurenic acid, Methods to aid in the diagnosis of hematologic malignancies. ⁇ 2> The method for assisting in the diagnosis of hematological tumors according to ⁇ 1>, using the above ratio as an index.
• ⁇ 3> The method for assisting in the diagnosis of hematologic tumors according to ⁇ 1> or ⁇ 2>, including the following (1), (2), and (3): (1) At least one amount selected from the group consisting of A, which is the amount of kynurenic acid in the subject-derived sample, and the amount of anthranilic acid, the amount of kynurenine, the amount of 3-hydroxyanthranilic acid, the amount of 3-hydroxykynurenine, and the amount of tryptophan.
• A which is the amount of kynurenic acid in the subject-derived sample, and the amount of anthranilic acid, the amount of kynurenine, the amount of 3-hydroxyanthranilic acid, the amount of 3-hydroxykynurenine, and the amount of tryptophan.
• Measuring B which is (2) (i) Calculate B/A, which is the ratio of the above B to the above A obtained in (1) above, or (ii) The ratio of the above A to the above B obtained in the above (1) Calculating A/B which is (3) To provide an index for diagnosing whether a subject has a hematological tumor based on the results of (2) above.
• ⁇ 5> Diagnosis of a hematological tumor according to ⁇ 3>, wherein (2) is calculating the ratio of the amount of kynurenic acid to the amount of anthranilic acid, or the ratio of the amount of anthranilic acid to the amount of kynurenic acid. How to help.
• ⁇ 6> The method for assisting in the diagnosis of a hematological tumor according to any one of ⁇ 1> to ⁇ 5>, wherein the hematological tumor is a lymphoid hematological tumor or a myeloid hematological tumor.
• ⁇ 7> The method for assisting diagnosis of a hematological tumor according to any one of ⁇ 1> to ⁇ 6>, wherein the hematological tumor is a lymphoid hematological tumor.
• the lymphoid hematological tumor is adult T-cell leukemia, diffuse large B-cell lymphoma, extranodal NK/T-cell lymphoma, nasal follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, mantle cell lymphoma, or A method for assisting in the diagnosis of a hematological tumor according to ⁇ 6> or ⁇ 7>, which is Hodgkin's lymphoma.
• Method for assisting in the diagnosis of hematologic tumors including the following (1) and (2): (1) A group consisting of A, which is the amount of kynurenic acid in the subject-derived sample, or A, which is the amount of kynurenic acid, and the amount of anthranilic acid, the amount of kynurenine, the amount of 3-hydroxyanthranilic acid, the amount of 3-hydroxykynurenine, and the amount of tryptophan.
• measuring B which is at least one quantity selected from; (2) (i) Use A obtained in (1) above as data for diagnosing a hematologic tumor; (ii) B/A, which is the ratio of B above to A obtained in (1) above; or (iii) calculate A/B, which is the ratio of the above A to the above B obtained in (1) above, and use it as data for diagnosing a hematological tumor.
• the data shall be used to carry out the following actions.
• a kit for how to obtain data The method for assisting in diagnosing a hematological tumor according to any one of ⁇ 1> to ⁇ 9>, including a mobile phase and a column, or for diagnosing a hematological tumor according to ⁇ 10>.
• kit In order to carry out the method for assisting in the diagnosis of hematological tumors according to any one of ⁇ 1> to ⁇ 9>, or the method for obtaining data for diagnosing hematological tumors according to ⁇ 10>, including kit.
• a method for assisting diagnosis of hematological tumors with excellent accuracy sensitivity and specificity
• a method for obtaining data for diagnosing hematological tumors with excellent accuracy sensitivity and specificity
• methods thereof A kit for the method is provided.
• auxiliary method of the present invention uses a decrease in the amount of KA in a sample derived from a subject as an indicator, or It includes using as an index the ratio of the amount of KA to at least one amount selected from the group consisting of the amount of AA, the amount of Kyn, the amount of 3-HAA acid, the amount of 3-HKyn, and the amount of Trp in the source sample.
• the present inventors found that the amount of KA in the specimen was decreased in the hematological tumor group compared to the healthy group, and the amount of other metabolites such as Kyn, 3-HAA, and AA was decreased. It was discovered that the amount was increasing, and the present invention was completed. The details of the auxiliary method of the present invention will be explained below.
• the hematologic tumor according to the present invention refers to a state in which hematopoietic cells (eg, hematopoietic stem cells) have become cancerous. Furthermore, the above-mentioned hematological tumors can be classified into lymphoid hematological tumors or myeloid hematological tumors, depending on the origin of the hematopoietic cells.
• Lymphoid hematological tumors are cancerous cells differentiated from lymphoid hematopoietic stem cells, and specifically include adult T-cell leukemia (ATLL), diffuse large cell type B Diffuse large B-cell lymphoma (DLBCL), extranodal NK/T-cell lymphoma (ENKL), follicular lymphoma (FL), mucosa-associated lymphoid tissue lymphoma ( MALT: mucosal-associated lymphoid tissue lymphoma), mantle cell lymphoma (MCL), Hodgkin lymphoma (HL), non-Hodgkin's lymphoma, NK/T cell lymphoma (NK/T) -cell lymphoma) and B-cell acute lymphoblastic leukemia (B-ALL).
• ATLL adult T-cell leukemia
• DLBCL diffuse large cell type B Diffuse large B-cell lymphoma
• ENKL extranodal NK/T
• myeloid hematological tumors are cancerous cells differentiated from myeloid hematopoietic stem cells, and include, for example, chronic myelomonocytic leukemia (CMMoL), myelodysplasia, These include myelodysplastic syndromes (MDS), chronic myelogenous leukemia (CML), and acute myelogenous leukemia (AML).
• CMMoL chronic myelomonocytic leukemia
• MDS myelodysplastic syndromes
• CML chronic myelogenous leukemia
• AML acute myelogenous leukemia
• the auxiliary method of the present invention is useful in assisting in the diagnosis of lymphoid hematologic tumors or myeloid hematologic tumors, and is particularly useful in assisting in the diagnosis of lymphoid hematologic tumors.
• the auxiliary method of the present invention can be applied to adult T-cell leukemia, diffuse large B-cell lymphoma, extranodal NK/T-cell lymphoma, nasal follicular lymphoma, and mucosa-associated lymphoid tissue lymphoma. , mantle cell lymphoma, or Hodgkin's lymphoma.
• a subject according to the present invention means a human suffering from a hematological tumor or a human at risk of suffering from a hematological tumor.
• the sample according to the present invention may be any sample derived from the above-mentioned subject, and specifically includes, for example, serum, plasma, whole blood, urine, saliva, cerebrospinal fluid, tissue fluid, sweat, tears, amniotic fluid, Examples include biological samples such as bone marrow fluid, pleural fluid, ascites fluid, indirect fluid, aqueous humor, and vitreous humor. Blood-derived samples such as serum, plasma, and whole blood, urine, and cerebrospinal fluid are preferred; Blood-derived samples such as blood are more preferred, and serum is particularly preferred.
• the method of obtaining (collecting) the sample according to the present invention from the subject is not particularly limited, and may be performed by, for example, obtaining (collecting) the sample from the subject based on a method known per se. Then, separation, concentration, purification, etc. may be carried out according to methods known per se.
• the sample may be one that has just been collected from the subject, or one that has been stored. Note that any method for preserving the sample may be used as long as it is a method commonly used in this field.
• the auxiliary method of the present invention includes at least one amount selected from the group consisting of the amount of AA, the amount of Kyn, the amount of 3-HAA acid, the amount of 3-HKyn, and the amount of Trp and the amount of KA in the sample derived from the subject. Preferably, it includes using the proportion of .
• ⁇ Method for measuring KA amount, AA amount, Kyn amount, 3-HAA amount, 3-HKyn amount and Trp amount KA amount, AA amount, Kyn amount, 3-HAA amount, 3-HKyn amount in the auxiliary method of the present invention As a method for measuring the Trp amount, any method that is normally used in this field may be used. Specifically, for example, (a) a method using chromatography or electrophoresis, (b) a method using affinity and (c) a method using an amino acid analyzer. Preferably, (a) a method using chromatography or electrophoresis or (b) a method using a substance having an affinity for (a) ) A method using chromatography or electrophoresis is more preferred.
• the “amount” in the above KA amount, AA amount, Kyn amount, 3-HAA amount, 3-HKyn amount, and Trp amount refers to absolute values such as capacity, mass, etc., or concentration, ionic strength, absorbance, fluorescence intensity, turbidity, etc. It may be any relative value.
• the reagents used to measure the KA amount, AA amount, Kyn amount, 3-HAA amount, 3-HKyn amount, and Trp amount may be any reagent that is normally used in this field, and its concentration, pH, may be within the range normally used in this field. Further, the conditions and operating method of the device may be any as long as they are commonly used in this field.
• Method using chromatography or electrophoresis consists of a column [ODS (octadecylsilyl) column, etc.], a detector (fluorescence detector, electrochemical detector, ultraviolet-visible spectrometer, etc.), Inject the mobile phase (organic solvent mobile phase such as acetonitrile, methanol, tetrahydrofuran, isopropanol, ethanol, aqueous mobile phase such as water, phosphoric acid, formic acid, acetic acid, etc.) and sample into a device equipped with a detector, etc.), This may be done by separating and detecting KA, AA, Kyn, 3-HAA, 3-HKyn, or Trp. Note that the specific method of using chromatography or electrophoresis may be performed in accordance with a method known per se.
• the method using chromatography or electrophoresis includes, for example, a method using high performance liquid chromatography (HPLC), a method using gas chromatography (GC), a method using liquid chromatography mass spectrometer (LC), and a method using gas chromatography (GC).
• HPLC high performance liquid chromatography
• GC gas chromatography
• LC liquid chromatography mass spectrometer
• GC gas chromatography
• /MS capillary electrophoresis mass spectrometer
• CE/MS capillary electrophoresis mass spectrometer
• GC/MS gas chromatography mass spectrometer
• ICP/MS inductively coupled plasma mass spectrometer
• LC/MS/MS liquid chromatography tandem mass spectrometer
• GC/MS/MS gas chromatography tandem mass spectrometer
• a method using high performance liquid chromatography (HPLC) or a method using a liquid chromatography tandem mass spectrometer (LC/MS/MS) is preferred.
• any method known per se can be used. Examples of the method include ultraviolet-visible absorbance detection, diode array detection, fluorescence detection, or differential refractive index detection, among which ultraviolet-visible absorbance detection or fluorescence detection is preferred.
• GC gas chromatography
• Method using a substance that has affinity is performed by using a substance that has affinity for each of KA, AA, Kyn, 3-HAA, and Trp. Ru.
• enzyme-linked immunosorbent assay ELISA method
• enzyme immunosorbent assay EIA method
• radioimmunoassay RIA method
• fluorescence immunoassay FIA method
• chemiluminescence immunoassay method CLIA method
• a substance that has an affinity for KA is brought into contact with KA, and the substance that has an affinity for KA is brought into contact with KA.
• This may be done by forming a complex and measuring the amount of each complex.
• the amount of AA, the amount of Kyn, the amount of 3-HAA, the amount of 3-HKyn, and the amount of Trp can also be measured according to the above method.
• a substance that has an affinity for the above KA a substance that has an affinity for AA, a substance that has an affinity for Kyn, a substance that has an affinity for 3-HAA, a substance that has an affinity for 3-HKyn.
• substances having affinity for Trp and substances having affinity for Trp include antibodies, lectins, nucleic acids, etc., and anti-KA antibodies, anti-AA antibodies, anti-Kyn antibodies, and anti-3 antibodies, respectively.
• - HAA antibodies, anti-3-HKyn antibodies and anti-Trp antibodies are preferred.
• the above antibody may be either a polyclonal antibody or a monoclonal antibody, and these may be used alone or in combination.
• the above-mentioned antibody may be an antibody fragment such as Fab, F(ab') 2 , Fv, or sFv, or a synthetic antibody such as a diabody, triabody, or tetrabody.
• the above-mentioned antibody may be a commercially available antibody or one prepared according to a method known per se.
• anti-KA antibody anti-AA antibody, anti-Kyn antibody, anti-3-HAA antibody, anti-3-HKyn antibody, and anti-Trp antibody according to a method known per se, for example, "immunoassay” (edited by Biochemical Measurement Study Group, Kodansha, 2014) or the method described in Special Publication No. 2018-501202.
• the anti-KA antibody, anti-AA antibody, anti-Kyn antibody, anti-3-HAA antibody, anti-3-HKyn antibody, and anti-Trp antibody may be labeled with a labeling substance.
• the labeling substances include enzymes such as horseradish peroxidase (HRP), bovine small intestine alkaline phosphatase, and ⁇ -galactosidase, 99m Tc, 131 I, 125 I, 14 C, 3 H, 32 P, Radioactive isotopes such as 35 S, fluorescent substances such as fluorescein, fluorescein isothiocyanate (FITC), 4-methylumbelliferone, rhodamine, or derivatives thereof, luminescent substances such as luciferin, luminol, ruthenium complexes, and phenol.
• HRP horseradish peroxidase
• FITC fluorescein isothiocyanate
• rhodamine 4-methylumbelliferone
• luminescent substances such
• the labeling substance may be bound to the anti-KA antibody, anti-AA antibody, anti-Kyn antibody, anti-3-HAA antibody, anti-3-HKyn antibody, and anti-Trp antibody by a method known per se. .
• the amount of a complex between a substance that has an affinity for KA and AA, the amount of a complex between a substance that has an affinity for AA and AA, and the amount of a complex between a substance that has an affinity for Kyn and Kyn Amount of complex, amount of complex between substance having affinity for 3-HAA and 3-HAA, amount of complex between substance having affinity for 3-HKyn and 3-HKyn, and Trp Any method that can measure the amount of the above-mentioned complex may be used as a method for measuring the amount of a complex between Trp and a substance that has an affinity for KA or KA.
• detection may be performed by detecting signals derived from a substance having an affinity for 3-HKyn, a labeling substance bound to Trp, or a substance having an affinity to Trp.
• the signals derived from the above-mentioned labeling substances may be detected by a method known per se.
• the labeling substance is an enzyme
• a conventional immunoassay method such as "Enzyme immunoassay” (Protein Nucleic Acid Enzyme Special Volume No. 31, edited by Tsunehiro Kitagawa, Toshio Minamibara, Akio Tsuji, and Eiji Ishikawa, 51- 63, Kyoritsu Shuppan, 1987), etc.
• the measurement of the radioactive substance can be carried out according to the conventional method used in, for example, RIA (Radio Immunoassay).
• RIA Radio Immunoassay
• measurements can be performed by appropriately selecting and using measurement equipment such as immersion type GM counters, liquid scintillation counters, well type scintillation counters, and HPLC counters (for example, in medical chemistry experiments).
• the labeling substance is a fluorescent substance
• the measurement can be carried out according to the method described in ⁇ Science Inc., 1983.''
• a conventional method using a measuring instrument such as a photo counter such as ⁇ enzyme immunoassay'' (Protein Nucleic Acid The measurement may be performed according to the method described in Enzyme Bessatsu No.
• the ratio of the amount of KA to at least one amount selected from the group consisting of the amount of AA, amount of Kyn, amount of 3-HAA, amount of 3-HKyn, and amount of Trp is as follows: (i) B/A, which is the ratio of B, which is at least one amount selected from the group consisting of AA amount, Kyn amount, 3-HAA amount, 3-HKyn amount, and Trp amount, to A, which is the KA amount; or (ii) A, which is the ratio of A, which is the amount of KA, to B, which is at least one amount selected from the group consisting of the amount of AA, the amount of Kyn, the amount of 3-HAA, the amount of 3-HKyn, and the amount of Trp. /B can be mentioned.
• a specific example of a method for measuring the amount of KA, amount of AA, amount of Kyn, amount of 3-HAA, amount of 3-HKyn, or amount of Trp is as follows. , 3-HKyn amount and Trp amount”.
• Examples of methods for determining the above-mentioned "ratio of the amount of KA to at least one amount selected from the group consisting of the amount of AA, the amount of Kyn, the amount of 3-HAA, the amount of 3-HKyn, and the amount of Trp" include the following method. It will be done.
• B/A which is the ratio of B, which is at least one amount selected from the group consisting of AA amount, Kyn amount, 3-HAA amount, 3-HKyn amount, and Trp amount, to A, which is the KA amount;
• (i) Calculate the ratio of the AA amount to the KA amount (AA/KA) by dividing the AA amount, Kyn amount, 3-HAA amount, 3-HKyn amount, or Trp amount by the KA amount, or (ii) By dividing KA by the amount of AA, the amount of Kyn, the amount of 3-HAA, the amount of 3-HKyn, or the amount of Trp, the ratio of the amount of KA to the amount of AA (KA/AA), the ratio of the amount of KA to the amount of Kyn (KA/ Kyn), the ratio of the amount of KA to the amount of 3-HAA (KA/3-HAA), the ratio of the amount of KA to the amount of 3-HKyn (KA/3-HKyn), or the ratio of the amount of KA to the amount of Trp (KA/Trp) Calculate.
• the above-mentioned "ratio of the amount of KA to at least one amount selected from the group consisting of the amount of AA, the amount of Kyn, the amount of 3-HAA, the amount of 3-HKyn, and the amount of Trp" includes AA/KA, KA /AA, KA/Kyn, KA/3-HAA, KA/Trp is preferred, AA/KA, KA/AA, KA/Kyn, KA/3-HAA, or KA/Trp is more preferred, AA/KA, KA /AA, KA/Kyn, or KA/3-HAA are more preferred, and AA/KA and KA/AA are particularly preferred.
• the auxiliary method of the present invention preferably includes the following (1), (2), and (3).
• (1) A which is the amount of kynurenic acid in the subject-derived sample, and at least one amount selected from the group consisting of the amount of anthranilic acid, the amount of kynurenine, the amount of 3-hydroxyanthranilic acid, the amount of 3-hydroxykynurenine, and the amount of tryptophan.
• Measuring B which is (2) (i) Calculate B/A, which is the ratio of the above B to the A obtained in (1) above, or (ii) Calculate the ratio of the above A to the above B obtained in (1) above. calculating a certain A/B; (3) To provide an index for diagnosing whether a subject has a hematological tumor based on the results of (2) above.
• a specific example of a method for measuring the amount of KA, AA amount, Kyn amount, 3-HAA amount, 3-Hkyn amount or Trp amount according to (1) is as follows. , 3-HAA amount, 3-Hkyn amount, and Trp amount”.
• details of the method of calculating B/A or A/B related to (2) and preferred specific examples thereof are also described in the above "AA amount, Kyn amount, 3-HAA amount, 3-Hkyn amount and Trp amount.
• (3) of the auxiliary method of the present invention described above is to provide an index for diagnosing whether a subject has a hematological tumor based on the results of (2) of the present invention.
• the amount of kynurenic acid, the above-mentioned B/A or A/B is preferable.
• the index may be an index obtained by processing these values, such as by comparing a plurality of these values.
• the auxiliary method of the present invention further includes comparing the index provided in (3) (hereinafter sometimes abbreviated as "subject-derived value”) with a preset reference value (cutoff value). It is preferable to include. Specifically, the auxiliary method of the present invention preferably further includes comparing at least one of the above (A/B) and the above (B/A) with a reference value (cutoff value).
• the reference value when making a determination based on the value of AA (nmol/L)/KA (nmol/L) is preferably 0.20 to 1.00, more preferably 0.30 to 0.90.
• the reference value when making a determination based on the value of KA (nmol/L)/AA (nmol/L) is preferably 3.0 to 5.0, more preferably 3.20 to 4.00.
• the above reference values may differ depending on the subject's race, area of living, lifestyle, etc.
• KA, KA/Trp, KA/Kyn, or KA/3-HAA For example, when making a determination based on the value of KA, KA/Trp, KA/Kyn, or KA/3-HAA, (i) If the value derived from the subject is below a preset standard value (cutoff value, etc.), there is a possibility that the subject may be suffering from a hematological tumor, or (ii) If the value derived from the subject exceeds a preset standard value (cutoff value, etc.), it is possible to make a judgment such as "the subject is not likely to be suffering from a hematological tumor.” Alternatively, it is possible to make a determination such as "there is a low risk that the patient is suffering from a hematologic tumor.”
• the reference value when determining based on the value of KA (nmol/L) is preferably 30.00 to 50.00 (nmol/L), and 31.00 to 43.00 (nmol/L). is more preferable.
• the reference value when making a determination based on the value of KA (nmol/L)/Trp ( ⁇ mol/L) is preferably 0.75 to 0.85, more preferably 0.76 to 0.81.
• the reference value when making a determination based on the value of KA (nmol/L)/Kyn ( ⁇ mol/L) is preferably 15.00 to 28.00, more preferably 16.00 to 25.00.
• the reference value when making a determination based on the value of KA (nmol/L)/3-HAA (nmol/L) is preferably 1.90 to 3.50, more preferably 2.00 to 3.00. However, these reference values may differ depending on the subject's race, area of living, lifestyle, etc.
• the above reference values are based on the values derived from subjects suffering from hematological tumors and the amount of kynurenic acid in samples derived from healthy individuals or according to the present invention (3) using samples derived from healthy individuals. It can be determined based on statistical analysis such as ROC (Receiver Operating Characteristic) curve analysis using the index obtained by the method (hereinafter sometimes abbreviated as the value derived from a healthy individual).
• ROC Receiveiver Operating Characteristic
• the sensitivity or specificity of the above reference value is, for example, 60% or more, preferably 70% or more, more preferably 80% or more, and still more preferably 90% or more.
• the value derived from the subject and the value derived from a healthy individual are compared, and (i) the value derived from the subject is higher than the value derived from a healthy individual.
• it is possible to make a judgment such as "there is a possibility that the subject is suffering from a hematological tumor, or there is a high possibility that the subject is suffering from a hematological tumor," and (ii) the values derived from the subject and those of healthy subjects can be determined.
• a judgment can be made such as ⁇ There is no risk that the subject is suffering from a hematological tumor, or there is a low risk that the subject is suffering from a hematological tumor.'' .
• the value derived from the subject and the value derived from a healthy person are compared, and (i) the value derived from the subject is compared to the value derived from a healthy person.
• the subject may be suffering from a hematological tumor, or there is a high risk that the subject is suffering from a hematological tumor," and (ii) the value derived from the subject. If there is no significant difference between the values obtained from healthy subjects and the values derived from healthy individuals, a judgment such as "there is no risk that the subject is suffering from a hematological tumor or there is a low risk that the subject is suffering from a hematological tumor" is made. can be lowered.
• the value derived from the subject and the value derived from a healthy person are compared, (i) If the value derived from the subject is lower than the value derived from a healthy individual, a judgment such as "the subject may be suffering from a hematological tumor or there is a high risk that the subject is suffering from a hematological tumor" should be made.
• the value derived from the subject at a certain time and the value derived from the subject at a different time are compared, and the above value is determined.
• the value derived from the subject at a certain time and the value derived from the subject at a different time are compared in the same subject, and the above-mentioned
• the degree of progression and malignancy of a hematological tumor it is possible to diagnose the prognosis after surgery.
• the value derived from the subject at a certain time and the value at a different time By comparing the value derived from the subject and evaluating the presence or absence of the above value and/or the degree of increase or decrease, it is possible to diagnose the degree of progression and malignancy of a hematological tumor, and to diagnose the prognosis after surgery.
• the method of obtaining data for diagnosing hematological tumors of the present invention preferably includes the following (1) and (2).
• measuring B which is at least one quantity selected from; (2) (i) Use A obtained in (1) above as data for diagnosing a hematological tumor; (ii) Calculate B/A, which is the ratio of B to A in (1) above. , as data for diagnosing hematological tumors, or (iii) calculating A/B, which is the ratio of A to B obtained in (1) above, and diagnosing hematological tumors.
• the details of (1) in the method of obtaining data for diagnosing hematological tumors of the present invention are the same as the details of (1) described above in the method of assisting the diagnosis of hematological tumors of the present invention.
• the details of (2) in the method for obtaining data for diagnosing hematological tumors of the present invention are as follows, except that (A) itself may be used as data for diagnosing hematological tumors.
• the details are the same as in (2) above in the method for assisting tumor diagnosis.
• the data obtained by the method of obtaining data for diagnosing hematological tumors of the present invention can be compared, for example, with the above-mentioned reference value (cutoff value), or between a value derived from a subject and a value derived from a healthy individual. or to compare data from the same subject at different time points.
• a first aspect of the kit of the present invention is a kit for carrying out the method of assisting in the diagnosis of hematological tumors of the present invention or the method of obtaining data for diagnosing hematological tumors of the present invention
• This kit includes a phase and a column (hereinafter sometimes abbreviated as "first kit").
• the amount of KA, AA, Kyn, 3-HAA, 3-HKyn or Trp can be measured by, for example, HPLC, LC/MS or LC/MS/MS, and the present invention
• the method of assisting in the diagnosis of hematological tumors of the present invention or the method of obtaining data for diagnosing hematological tumors of the present invention can be carried out.
• the mobile phase is not particularly limited and may be any mobile phase known in the art, including organic solvent mobile phases such as acetonitrile, methanol, tetrahydrofuran, isopropanol, and ethanol, water, phosphoric acid, formic acid, acetic acid, etc. Examples include aqueous mobile phases.
• the kit of the present invention may further contain a protein removal buffer.
• the above-mentioned protein removal buffer is not particularly limited and may be any protein removal buffer known in the art, but includes buffers containing acids such as perchloric acid, trichloroacetic acid, and metaphosphoric acid, acetone, acetonitrile, and methanol. , a buffer containing an organic solvent such as ethanol, and the like. Examples of the column include, but are not limited to, an ODS (octadecylsilyl) column and the like.
• the kit according to the second aspect of the present invention is a kit for carrying out the method of assisting diagnosis of hematological tumors of the present invention, or the method of obtaining data for diagnosing hematological tumors of the present invention, comprising: A substance that has an affinity for kynurenic acid, or a substance that has an affinity for kynurenic acid, a substance that has an affinity for anthranilic acid, a substance that has an affinity for kynurenine, 3-hydroxyanthranil
• a kit comprising at least one substance selected from the group consisting of a substance that has an affinity for acids, a substance that has an affinity for 3-hydroxykynurenine, and a substance that has an affinity for tryptophan.
• the amount of KA, AA, Kyn, 3-HAA, 3-HKyn or Trp can be measured by the above-mentioned “(b) method using a substance with affinity", The method of assisting in diagnosing hematological tumors of the present invention or the method of obtaining data for diagnosing hematological tumors of the present invention can be carried out.
• the kit of the present invention when it is simply described as “the kit of the present invention", it refers to both the above-mentioned first kit and second kit.
• a substance that has an affinity for KA, a substance that has an affinity for AA, a substance that has an affinity for Kyn, and an affinity for 3-HAA which are included in the second kit according to the present invention.
• Specific examples of the substance having an affinity for 3-HKny, the substance having an affinity for Trp, and the like include antibodies, lectins, nucleic acids, etc. Preferred are AA antibody, anti-Kyn antibody, anti-3-HAA antibody, anti-3-HKyn antibody and anti-Trp antibody.
• the second kit according to the present invention includes reagents commonly used in this field, such as buffers, detergents, reaction promoters, stabilizers such as sugars, proteins, salts, and surfactants, preservatives, A liquid for diluting a sample, an immobilized solid phase such as an antigen-immobilized solid phase, an antibody-immobilized solid phase, a secondary antibody labeled with a labeling substance or its antibody fragment, a reagent for detecting a labeled substance, etc. It may contain substances that do not inhibit the stability of coexisting reagents and the like. In addition, the concentration and pH may be within the range normally used in this field.
• immobilized solid phases such as the above antigen-immobilized solid phase and antibody-immobilized solid phase
• magnetic particles such as magnetic silica particles, polystyrene, polycarbonate, polyvinyltoluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, and gelatin.
• KA, AA, Kyn, 3-HAA, 3-HKyn or Trp or anti-KA antibody, anti-AA antibody, anti-Kyn antibody, anti-3-HAA antibody to materials such as agarose, cellulose, polyethylene terephthalate, glass, ceramic, etc.
• Any immobilized antibody or antibody fragment thereof, such as anti-3-HKyn antibody and anti-Trp antibody may be used.
• the materials for the immobilized solid phase may be manufactured by a method known per se or may be commercially available.
• magnetic particles such as the above magnetic silica particles by a method known per se
• it can be produced by the method described in International Publication No. 2012/173002.
• the secondary antibody labeled with the above labeling substance or its antibody fragment is an antibody that binds to each of anti-KA antibody, anti-AA antibody, anti-Kyn antibody, anti-3-HAA antibody, anti-3-HKyn antibody, and anti-Trp antibody. or an antibody fragment thereof.
• the labeling substance and the method of binding the labeling substance in the secondary antibody labeled with the above labeling substance or its antibody fragment please refer to the above “KA amount, AA amount, Kyn amount, 3-HAA amount, 3-HKyn amount.
• the method is as explained in the section "(b) Method using a substance with affinity” in “Method for Measuring Trp Amount", and preferred examples, specific examples, etc. are also the same.
• the above reagent for detecting a labeled substance is used when antibodies such as anti-KA antibody, anti-AA antibody, anti-Kyn antibody, anti-3-HAA antibody, anti-3-HKyn antibody, and anti-Trp antibody are labeled with a labeling substance.
• a label in an antibody or an antibody fragment thereof such as anti-KA antibody, anti-AA antibody, anti-Kyn antibody, anti-3-HAA antibody, anti-3-HKyn antibody, and anti-Trp antibody labeled with the above-mentioned labeling substance, and/or the above-mentioned label.
• substrates for absorbance measurement such as tetramethylbenzidine and ortho-phenylenediamine, fluorescent substrates such as hydroxyphenylpropionic acid and hydroxyphenylacetic acid, and luminescent substances such as luminol.
• substrates for absorbance measurement such as tetramethylbenzidine and ortho-phenylenediamine, fluorescent substrates such as hydroxyphenylpropionic acid and hydroxyphenylacetic acid, and luminescent substances such as luminol.
• Substances include reagents for measuring absorbance such as 4-nitrophenyl phosphate, fluorescent substrates such as 4-methylumbelliferyl phosphate, and the like.
• the kit of the present invention may contain instructions for carrying out the auxiliary method of the present invention.
• the above-mentioned "instructions” refer to the instruction manual, package insert, etc. of the kit of the present invention that substantially describes the features, principles, operating procedures, determination procedures, etc. of the auxiliary method of the present invention in text and/or diagrams; Pamphlet (leaflet), etc.
• the kit of the present invention includes, for example, (i) a kit in which details of the principles, operating procedures, etc. of the above-mentioned (1) and (2) of the auxiliary method of the present invention are described; ) In the auxiliary method of the present invention, details of the principles, operating procedures, determination procedures, etc. of the above (1), (2), and (3) above are described.
• the hematological tumor diagnostic aid device includes at least (1) a measuring section and (2) a processing section. Furthermore, it may include (3) a determination section, (4) an output section, and (5) an input section.
• the measurement unit in the device according to the present invention is configured to measure the amount of KA or a substance selected from KA, AA, Kyn, 3-HAA, 3-HKyn, and Trp in a sample derived from a subject.
• examples include devices used in methods using chromatography or electrophoresis, devices used in methods using substances with affinity, and the like.
• the processing unit in the apparatus according to the present invention is configured to control the amount of AA, the amount of Kyn, the amount of 3-HAA, the amount of 3-HKyn, and the amount of Trp with respect to the (i) KA amount A measured in the (1) measuring unit.
• (ii) Calculate B/A, which is the ratio of B, which is at least one amount selected from the group consisting of the amount of AA, the amount of Kyn, the amount of 3-HAA, the amount of 3-HKyn, and the amount of Trp. It is configured to calculate A/B, which is the ratio of A, which is the above-mentioned KA amount, to B, which is at least one amount selected from the group.
• the (3) determination unit in the apparatus according to the present invention is configured to determine whether or not the subject has a hematological tumor based on the calculation result obtained by the (2) processing unit.
• the (4) output unit in the device according to the present invention is configured to output the calculation result obtained by the (2) processing unit and/or (3) the determination result obtained by the determination unit.
• the input section in the device receives the operation of the operator and inputs the information to the (1) measurement section or/and (2) the processing section.
• the processing section is configured to transmit a signal for activating the processing unit.
• the auxiliary method of the present invention can be carried out simply, in a short time, and with high precision.
• the method for treating hematologic tumors according to the present invention includes the following (1-1), (1-2), and (1-3). is preferred.
• (1-1) Measuring the amount of KA in a sample derived from a subject, (1-2) Determining whether the subject has a hematological tumor based on the results of (1-1) above, and (1-3) Determining whether the subject has a hematological tumor based on the results of (1-2) above.
• Appropriate treatment should be provided to subjects who are determined to be at risk of developing hematologic malignancies or at high risk of developing hematological tumors.
• the treatment method according to the present invention preferably includes the following (2-1), (2-2), and (2-3).
• (2-2) (i) Ratio of B, which is at least one amount selected from the group consisting of AA amount, Trp amount, Kyn amount, 3-HAA amount, and 3-HKyn amount, to A, which is the above KA amount.
• Appropriate treatment in (1-3) or (2-4) of the treatment method according to the present invention includes supportive therapy such as blood transfusion therapy and iron removal therapy, and administration of anticancer drugs, immunosuppressants, etc.
• supportive therapy such as blood transfusion therapy and iron removal therapy
• anticancer drugs immunosuppressants, etc.
• examples include drug therapy, hematopoietic stem cell transplantation, etc.
• sample, blood tumor, each step, etc. in the treatment method according to the present invention are as explained in ⁇ Auxiliary method of the present invention>, and preferred examples, specific examples, etc. are also the same.
• Example 1 Analysis of Trp and its metabolites in the hematological tumor group and the healthy group
• Samples and hematological tumor group Among the hematological examination patients, 112 patients who were classified into the following classification types by the doctor's diagnosis were included in the hematological tumor group. That is, based on the hematopoietic tumor clinical practice guidelines, the serum of a person diagnosed by a doctor as having the following disease was used as a sample.
• ATLL Adult T-cell leukemia/lymphoma
• DLBCL Diffuse large B-cell lymphoma
• ENKL Extranodal NK/T-cell lymphoma
• nasal type FL Follicular lymphoma
• MALT Mucosa-associated lymphoid tissue lymphoma
• MCL Mantle cell lymphoma
• HL Hodgkin lymphoma
• Trp and its metabolites in serum derived from hematological tumor group and healthy group Serum concentration of Trp and its metabolites (Kyn, 3-HAA, KA, AA) derived from the hematological tumor group and healthy group was measured by the following method.
• Protein removal was performed by adding 25 ⁇ L of 10% perchloric acid to 100 ⁇ L of serum from the blood tumor group and healthy group. Next, after centrifugation at 14,000 rpm and 4°C for 10 minutes, 50 ⁇ L of the supernatant was injected into a high-performance liquid chromatography system [LC-20AD (manufactured by Shimadzu Corporation)], and an ultraviolet-visible spectrometer [SPD-20A (manufactured by Shimadzu Corporation)] was injected into the supernatant.
• LC-20AD high-performance liquid chromatography system
• SPD-20A ultraviolet-visible spectrometer
• Trp and Kyn in the supernatant were measured using a fluorescent detector [RF-10AXL (manufactured by Shimadzu Corporation)], and 3-HAA, KA, and AA in the supernatant were measured using a fluorescence detector [RF-10AXL (manufactured by Shimadzu Corporation)].
• Results Table 1 shows the measurement results (average value and standard deviation) of the healthy group and hematological tumor group obtained in (2) above, the P value obtained by significance determination, and the presence or absence of significant difference. Furthermore, using the measured values of the healthy group and hematological tumor group obtained in (2) above, the amount of AA relative to the amount of Trp (AA/Trp), the amount of AA relative to Kyn (AA/Kyn), and the amount of AA relative to the amount of 3-HAA were determined.
• the P values indicating the significance of the increase in Kyn and 3-HAA levels in the hematological tumor group were 3.71 ⁇ 10 -3 and 5.92 ⁇ 10 -4 , respectively [Table 4 (B ), (C)].
• the P value indicating the significance of these increases is about one-tenth or more of the P value indicating a significant difference between a decrease in the value of KA alone and an increase in the value of AA alone. Therefore, it was found that the amount of KA and the amount of AA are more useful as indicators for distinguishing between the blood tumor group and the healthy group, and the amount of KA is particularly useful.
• AA/KA, KA/AA, KA/AA, KA/Kyn, KA/3-HAA, and KA/Trp were all below 0.001 [Tables 2 and 3 (I-1) ⁇ (L)].
• AA/KA and KA/AA have the lowest P values of 2.39 ⁇ 10 -22 , and the P values for AA/KA and KA/AA are significantly decreased for all hematological tumor classification types. Got it [Table 4 (I-1) and (I-2)].
• KA and Trp-related substances i.e., AA/KA, KA/AA, KA/Kyn, KA/3-HAA, KA/Trp, differentiate the hematological tumor group from the healthy group.
• AA/KA, KA/AA, KA/Kyn, and KA/3-HAA were found to be useful, with AA/KA and KA/AA being the most useful.
• the method for assisting in the diagnosis of hematological tumors of the present invention it is possible to diagnose hematological tumors with excellent accuracy (sensitivity and specificity).

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