WO2023013673A1 - Method and reagent for detection of thromboembolism associated with malignant tumor - Google Patents
Method and reagent for detection of thromboembolism associated with malignant tumor Download PDFInfo
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Definitions
- the present invention relates to a detection method and detection reagent for malignant tumor-associated thromboembolism that targets tissue factor pathway inhibitor 2 (TFPI2).
- TFPI2 tissue factor pathway inhibitor 2
- Venous thromboembolism is a general term for a series of pathologies consisting of deep vein thrombosis (DVT) and pulmonary embolism (PE). Venous thromboembolism is known to be a complication in cancer patients, and careful handling is required as cancer associated thrombosis (CAT). Among gynecologic cancers, ovarian cancer is said to be frequently associated with venous thromboembolism, and in particular, it has been shown that ovarian clear cell carcinoma is associated with a high frequency of VTE (Non-Patent Document 1).
- D-dimer In diagnosing malignant tumors, if malignant tumor-related thromboembolism is suspected, D-dimer is measured, and if positive, imaging tests such as ultrasonography and contrast-enhanced CT are performed to confirm the diagnosis of thromboembolism.
- Malignant tumor-associated thromboembolism D-dimer is a product produced by the degradation of stabilized fibrin by plasmin in the blood coagulation/fibrinolysis system, and is a general term for mixtures having a D-dimer structure (Non-Patent Document 2).
- D-dimer is a marker with high specificity, and is useful not only for diagnosing DVT and PE, but also for judging the duration and termination of anticoagulant therapy and evaluating the possibility of recurrence. It is shown. On the other hand, it has been reported that cancer patients, pregnant women, and postoperative patients with accelerated coagulation show high values of D-dimer (Non-Patent Document 3).
- Tissue factor pathway inhibitor 2 (TFPI2) is the same protein as placental protein 5 (PP5) and is a placenta-derived serine protease inhibitor containing three Kunitz-type protease inhibitor domains.
- TFPI2 is specifically produced from clear cell cancer cell lines in ovarian cancer cell lines, and gene expression in ovarian cancer patient tissues is specifically improved only in clear cell cancer patients (Patent Document 1). ), disclosed a method for detecting ovarian clear cell carcinoma by measuring TFPI2 in blood (Patent Documents 2 and 3, Non-Patent Documents 4 and 5).
- Non-Patent Documents 6 to 10 Non-Patent Documents 6 to 10
- TFPI2 protein in body fluids could be applied to detect malignant tumor-associated thromboembolism.
- An object of the present invention is to provide a method for detecting malignant tumor-related thromboembolism and a reagent that can be used in the method.
- the present inventors found that blood TFPI2 levels in various malignant tumors were significantly improved in the VTE-complicated patient group compared to the non-VTE-complicated group.
- the present invention has been completed based on the idea that VTE associated with malignant tumors can be detected. That is, the present invention includes the following aspects. [1] A method for detecting malignant tumor-related thromboembolism, comprising measuring the amount of TFPI2 in a specimen. [2] The method of [1], wherein malignant tumor-associated thromboembolism is detected when the measured TFPI2 amount exceeds a preset reference value.
- An antibody that binds to an antigenic determinant in the region from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue in the amino acid sequence of SEQ ID NO: 1 for which the amount of TFPI2 is measured The method according to any one of [1] to [3], which is carried out by an antigen-antibody reaction using [5] The method of [4], wherein the antibody recognizes Kunitz domain 1 of TFPI2.
- [6] The method according to any one of [1] to [3], wherein the measurement is performed using mass spectrometry.
- the malignant tumor is ovarian cancer, lung cancer, gastrointestinal cancer, hematopoietic cancer, breast cancer, or uterine cancer.
- Malignant tumor-related including an antibody that binds to an antigenic determinant in the region from aspartic acid at residue 23 to histidine at residue 131 or cysteine at residue 130 in the amino acid sequence shown in SEQ ID NO: 1 Reagent for detecting thromboembolism.
- the present invention provides a method for detecting malignant tumor-related thromboembolism simply and with high accuracy, and a reagent that can be used for the method.
- FIG. 10 is a diagram showing box plots of TFPI2 values or D-dimer values in malignant tumors in a group with VTE complication and a group without VTE complication.
- the vertical axis represents the blood amount of each.
- FIG. 10 is a diagram showing box plots (Box Plots) of TFPI2 values or D-dimer values in a group with VTE complication and a group without VTE complication grouped by FIGO classification.
- the vertical axis represents the blood amount of each.
- the vertical axis represents the blood amount of each.
- FIG. 13 shows Receiver Operating Characteristic (ROC) curves for VTE-combined and non-VTE-combined groups.
- the vertical axis represents sensitivity, and the horizontal axis represents 1-specificity.
- the vertical axis represents D-dimer values, and the horizontal axis represents TFPI2 values.
- FIG. 10 is a diagram showing box plots of TFPI2 values or D-dimer values in borderline malignant tumor patients in the group with VTE complication and the group without VTE complication.
- the vertical axis represents the blood amount of each.
- FIG. 10 is a diagram showing box plots (Box Plots) of TFPI2 values in a group with VTE complication and a group without VTE complication by cancer type.
- the vertical axis represents the amount of TFPI2 in blood.
- the first aspect of the present invention is a method for detecting malignant tumor-related thromboembolism, which includes measuring the amount of TFPI2 in a specimen. This is a method based on the elevated presence of TFPI2 in malignant tumor-associated thromboembolism patient biological samples such as blood in malignant tumors. Measurement of the amount of TFPI2 in a specimen is usually performed in vitro. By this method, malignant tumor-associated thromboembolism can be detected with high accuracy, as shown in Examples described later.
- the method of the present invention includes up to the step of detecting malignant tumor-related thromboembolism, and does not include the final decision regarding the diagnosis of malignant tumor-related thromboembolism. Physicians refer to the results of detection by the method of the present invention, etc. to diagnose malignant tumor-associated thromboembolism and formulate treatment strategies.
- the malignant tumor-associated thromboembolism of the present invention there is no particular limitation on the malignant tumor that occurs in association with thromboembolism (underlying disease), and may be a so-called malignant tumor itself or a borderline malignant tumor.
- ovarian cancer is preferred, and ovarian borderline malignancy is also preferred.
- clear cell carcinoma, serous carcinoma, or endometrioid carcinoma are preferred.
- endometrioid carcinoma is preferable because, as shown in Examples below, a tendency for TFPI2 to rise was observed in the group with concurrent VTE, but this was not observed in D-dimer.
- ovarian cancer In addition to ovarian cancer, lung cancer, gastrointestinal cancer (esophageal and/or stomach cancer, liver or pancreatic cancer, colon cancer, etc.), hematopoietic cancer, breast cancer, or uterine cancer are preferred.
- gastrointestinal cancer esophageal and/or stomach cancer, liver or pancreatic cancer, colon cancer, etc.
- hematopoietic cancer breast cancer, or uterine cancer
- the type of thromboembolism is not particularly limited, but venous thromboembolism is preferred.
- TFPI2 to be measured in the present invention is not particularly limited, for example, intact TFPI2 (hereinafter also referred to as "I-TFPI2”), TFPI2 processing polypeptide (hereinafter also referred to as "NT-TFPI2”), or both There may be.
- SEQ ID NO: 1 shows the amino acid sequence based on human TFPI2 cDNA.
- the signal peptide is from the initiation methionine to the glycine at the 22nd residue.
- “Intact TFPI2” refers to a peptide represented by residues 23 to 235 of the amino acid sequence of SEQ ID NO:1.
- NT-TFPI2 refers to a peptide fragment containing Kunitz domain 1 located on the N-terminal side of intact TFPI2, as described in Patent Document 3. More specifically, NT-TFPI2 is a peptide comprising at least a sequence from aspartic acid at residue 23 to histidine at residue 131 or cysteine at residue 130 in the amino acid sequence of SEQ ID NO: 1, or A peptide containing an amino acid sequence that has 80% or more identity with the sequence. Said identity is preferably 90% or more, more preferably 95% or more.
- this polypeptide may be a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and/or added to the above sequence.
- the term "several" means preferably 2 to 20, more preferably 2 to 10, still more preferably 2 to 5.
- Patient-derived specimens (test samples) in the present invention include blood components such as whole blood, blood cells, serum, and plasma, cell or tissue extracts, urine, cerebrospinal fluid, peritoneal washings, ascites, cystic fluid, and the like. be done. It is preferable to use body fluids such as blood components and urine as specimens because it can be performed simply and non-invasively. Considering the ease of sample collection and versatility for other test items, blood components are used as specimens. is particularly preferred.
- the dilution rate of the sample may be appropriately selected from no dilution to 100-fold dilution according to the type and condition of the sample to be used.
- the method for detecting TFPI2 with an existing thromboembolism marker or diagnostic imaging such as ultrasonic echo, CT or MRI.
- the combination method is not particularly limited.
- a method for detecting TFPI2 and an existing venous thromboembolism marker are performed on the specimen to be measured at the same time or separately.
- Method for detecting embolism For the specimen to be measured, first apply an existing venous thromboembolism marker, detect TFPI2 for the specimen determined to be negative as a result, and if TFPI2 is positive, super Imaging tests such as sonography and contrast-enhanced CT are performed to detect thromboembolism. Method (A) is more preferable because venous thromboembolism can be rapidly detected by simultaneous measurement.
- the venous thromboembolism marker to be detected by the malignant tumor-related thromboembolism detection method of the present invention may be appropriately selected from conventionally known markers.
- D-dimer which is most widely used as a marker for venous thromboembolism, is particularly preferable as a marker for use in the method for detecting malignant tumor-related thromboembolism of the present invention in that its clinical utility has been established.
- the conventionally known markers detected in the method for detecting malignant tumor-related thromboembolism of the present invention may be one type, or two or more types.
- the timing of sample collection in the present invention is not particularly limited.
- specimens collected at any stage such as before and after a definitive diagnosis, before and after the start of treatment, can be used at any time from preoperative when a detailed examination is performed when a malignant tumor-related thromboembolism is suspected by imaging diagnosis, etc., to follow-up observation. Even if there is, it can be subjected to the method of the present invention.
- the amount of TFPI2 may be the amount of intact TFPI2, the amount of NT-TFPI2, or the total amount of intact TFPI2 and NT-TFPI2. It is more preferable from the viewpoint of compatibility with high sensitivity and specificity.
- the reference value used for determination may be either a measured value or a converted density value.
- the converted concentration value refers to a value converted from a measured value based on a calibration curve prepared using TFPI2 as a standard sample.
- the reference value (Cutoff value) for determining malignant tumor-related thromboembolism is determined by measuring malignant tumor-related thromboembolism and non-malignant tumor-related thromboembolism, respectively, and determining the optimal sensitivity and It can be appropriately set to a measured value that indicates specificity.
- the reference value (Cutoff value) of TFPI2 may be set at 280 pg/mL as shown in Examples below, but is not limited thereto.
- the amount of NT-TFPI2 or the amount of intact TFPI2 in the specimen may be measured individually, or the values may be totaled to obtain the total amount.
- the total amount of NT-TFPI2 and intact TFPI2 in the specimen may be measured with a measurement system capable of measuring at once.
- the amount of NT-TFPI2 may be measured indirectly from the total amount of both measurements and the amount of intact TFPI2 alone.
- the method for measuring the amount of NT-TFPI2 and/or the amount of intact TFPI2 is not particularly limited.
- a method using an antigen-antibody reaction using an antibody that recognizes NT-TFPI2 and/or intact TFPI2, and a method using mass spectrometry can be exemplified.
- (b) A method using surface plasmon resonance, in which a sample is brought into contact with a chip on which an antibody that recognizes an object to be measured is immobilized, and a signal dependent on binding between the antibody and the object to be measured is detected.
- (c) A fluorescence polarization immunoassay method that uses a fluorescence-labeled antibody that recognizes a target to be measured, and utilizes an increase in the degree of fluorescence polarization due to binding between the antibody and the target to be measured.
- (d) Using two types of antibodies (one of which is a labeled antibody) having different antigenic determinants, which are antibodies that recognize the target to be measured, to form a tripartite complex between the two antibodies and the target to be measured sandwich method.
- Specific methods for measuring the amount of NT-TFPI2 and/or the amount of intact TFPI2 using an antigen-antibody reaction include the following.
- a method of measuring the total amount of NT-TFPI2 and intact TFPI2 using an antibody that recognizes both NT-TFPI2 and intact TFPI2 (NT+I-TFPI2 measurement system).
- the antibody that recognizes both NT-TFPI2 and intact TFPI2 is from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue in the TFPI2 amino acid sequence represented by SEQ ID NO: 1.
- it is an antibody that binds to an antigenic determinant within the region of , more preferably an antibody that has an antigen recognition site that binds to an antigenic determinant within Kunitz domain 1 of TFPI2.
- sandwich method described above two types of antibodies having different antigenic determinants are usually used.
- (B) A method of measuring the amount of intact TFPI2 alone using an antibody that recognizes intact TFPI2 but does not recognize NT-TFPI2 (I-TFPI2 measurement system).
- the antibody that recognizes intact TFPI2 but does not recognize NT-TFPI2 is preferably an antibody that has an antigen recognition site that binds to an antigenic determinant within Kunitz domain 3 of TFPI2.
- (D) A method of measuring the amount of NT-TFPI2 alone using an antibody that recognizes NT-TFPI2 but does not recognize intact TFPI2.
- the antibody that recognizes NT-TFPI2 but does not recognize intact TFPI2 includes, for example, an antibody that specifically recognizes the peptide sequence of the C-terminal portion of NT-TFPI2.
- the sandwich method described above for example, the antibody is used as a solid-phase antibody, and an antibody having a recognition site in Kunitz domain 1 is used as a detection antibody.
- the amount of NT-TFPI2 alone measured by the method (C) or (D) described above may be used as a criterion for determination, but the method (A) The latter is more preferable because sufficient sensitivity and specificity can be obtained even if the total amount of measured NT-TFPI2 and intact TFPI2 is used as a criterion for determination, and the antibody can be easily obtained and the measurement is simple in one step. .
- An antibody that recognizes NT-TFPI2 and/or intact TFPI2 is an NT-TFPI2 polypeptide or protein, an oligopeptide consisting of an intact TFPI2 polypeptide or a partial region of a TFPI2 protein, an intact or partial region of an NT-TFPI2 polypeptide or a TFPI2 protein can be obtained by immunizing an animal with a polynucleotide or the like that encodes as an immunogen.
- the protein, oligopeptide or polypeptide does not reflect the tertiary structure of TFPI2 in vivo, or the structure may change during the preparation process.
- the antibody obtained may not have high specificity or binding strength to the desired in vivo TFPI2, and even if a measurement system is constructed using this antibody, it may not be included in the sample. It may not be possible to accurately quantify the TFPI2 concentration in the sample.
- an expression vector containing a polynucleotide encoding an intact or partial region of a TFPI2 polypeptide or intact TFPI2 protein as an immunogen, the intact or partial region of the TFPI2 polypeptide or intact TFPI2 protein is expressed in the body of the immunized animal. This is more preferable because an immune response is induced and an antibody with high specificity and binding strength (that is, high affinity) to TFPI2 in the specimen can be obtained.
- Animals used for immunization are not particularly limited as long as they have the ability to produce antibodies. Mammals such as mice, rats and rabbits, which are commonly used for immunization, and birds such as chickens may be used. Furthermore, TFPI1 known as a homologue of TFPI2 is also present in blood. Therefore, it is desirable to use an antibody that specifically recognizes only TFPI2 without crossing with TFPI1.
- An antibody that recognizes TFPI2 may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
- hybridoma cells that produce antibodies that recognize TFPI2 can be appropriately selected from methods with established techniques.
- B cells are collected from an animal immunized by the method described above, the B cells and myeloma cells are fused electrically or in the presence of polyethylene glycol, and hybridoma cells that produce the desired antibody are selected using HAT medium. and monocloning the selected hybridoma cells by the limiting dilution method to establish hybridoma cells that produce a monoclonal antibody that recognizes TFPI2.
- a monoclonal antibody that recognizes TFPI2 used in the present invention may be selected based on its affinity for GPI (glycosylphosphotidylinositol)-anchored TFPI2 or secretory TFPI2 derived from the host expression system.
- GPI glycosylphosphotidylinositol
- the host is not particularly limited, and may be appropriately selected from microbial cells such as Escherichia coli and yeast, insect cells, and animal cells that are commonly used for protein expression by those skilled in the art. It is preferable to use mammalian cells as hosts, which are capable of expressing a protein having a structure similar to that of native TFPI2 by post-translational modifications such as TFPI2. Examples of mammalian cells include conventionally used human embryonic kidney-derived cell (HEK) 293T cell line, monkey kidney cell line COS7, Chinese hamster ovary (CHO) cells, cancer cells isolated from humans, and the like. mentioned.
- HEK human embryonic kidney-derived cell
- COS7 monkey kidney cell line
- COS7 Chinese hamster ovary
- Purification of the antibody used in the present invention may be carried out by appropriately selecting from established techniques. As an example, after culturing the antibody-producing hybridoma cells established by the method described above, the culture supernatant is collected, and if necessary, the antibody is concentrated by ammonium sulfate precipitation, and protein A, protein G, or protein L is immobilized. Antibodies can be purified by affinity chromatography and/or ion-exchange chromatography using a fused carrier.
- the labeled antibody used in the antigen-antibody reaction by the sandwich method described above can be obtained by labeling the antibody purified by the method described above with an enzyme such as peroxidase or alkaline phosphatase, and the labeling technology has been well established. method.
- a method for measuring the amount of TFPI2 using mass spectrometry in the method of the present invention will be specifically described below.
- major proteins such as albumin, immunoglobulin, and transferrin, which are abundant in blood, are removed with Agilent Human 14 or the like, and then further separated by ion exchange, gel filtration, reversed-phase HPLC, or the like. It is preferable to draw Alternatively, it is also possible to specifically collect only TFPI2 by an immunological technique using an anti-TFPI2 antibody.
- Measurements were performed by tandem mass spectrometry (MS/MS), liquid chromatography-tandem mass spectrometry (LC/MS/MS), matrix assisted laser desorption ionization time-of-flight mass spectrometry. , MALDI-TOF/MS), surface enhanced laser desorption ionization mass spectrometry (SELDI-MS), and the like.
- the method for detecting malignant tumor-related thromboembolism of the present invention can be applied to a method for treating malignant tumor-related thromboembolism.
- a method of treating malignancy-associated thromboembolism in a subject comprising: (i) identifying a subject as having a measured TFPI2 amount that exceeds a preset reference value; and (ii) said subject identified as having a measured TFPI2 amount that exceeds a preset reference value.
- a method includes administering a treatment to a body.
- said amount of TFPI2 is the sum of the amount of TFPI2 processing polypeptide and the amount of intact TFPI2.
- the amount of TFPI2 is preferably measured from the 23rd residue aspartic acid to the 131st residue histidine or the 130th residue cysteine in the amino acid sequence of SEQ ID NO: 1. It is performed by an antigen-antibody reaction using an antibody that binds to an antigenic determinant within the region. More preferably, said antibody is an antibody that recognizes Kunitz domain 1 of TFPI2.
- the amount of TFPI2 may be measured using mass spectrometry.
- the treatment in step (ii) includes anticoagulant therapy using anticoagulants such as heparin and warfarin, thrombolytic therapy using thrombolytic agents such as urokinase and tissue plasminogen activator, and vascular therapy using an intravascular catheter. Examples include, but are not limited to, internal therapy, surgical therapy, and the like.
- the antibody preferably contains two types of antibodies having different antigenic determinants.
- the antibody contained in the reagent of the present invention may be the antibody itself, labeled, or immobilized on a solid phase.
- the reagent of the present invention can be produced by the methods shown in (I) to (III) below.
- antibody 1 is used in immunoplates or magnetic particles.
- B/F (Bound/Free) separable carrier such as.
- the bonding method may be physical bonding using hydrophobic bonding, or chemical bonding using a linker reagent capable of cross-linking between two substances.
- (II) After binding the antibody 1 to the carrier, block the carrier surface with bovine serum albumin, skimmed milk, a commercially available immunoassay blocking agent, a chemically synthesized polymer for inhibiting protein adsorption, etc., in order to avoid non-specific binding. and use it as the primary reagent.
- the other antibody 2 is labeled, and a solution containing the resulting labeled antibody is prepared as a secondary reagent.
- Substances for labeling the antibody 2 include enzymes such as peroxidase and alkaline phosphatase, fluorescent substances, chemiluminescent substances, radioisotopes, and other substances that can be detected by a detection device, or substances that have a specific binding partner, such as avidin for biotin. etc. are preferred.
- a buffer solution in which an antigen-antibody reaction can be favorably performed such as a phosphate buffer solution or a Tris-HCl buffer solution, is preferable.
- the reagent of the present invention thus prepared may be lyophilized if necessary.
- antibody 1 is bound to the carrier and subjected to blocking treatment in the same manner as in (I) to (II) described above, and labeled antibody 2 is applied to the antibody-immobilized carrier.
- a reagent may be prepared by further adding a buffer solution containing the above.
- detection and measurement of TFPI2 by the two-step sandwich method using the reagent obtained by the above-described method may be carried out by the following methods (IV) to (VI).
- IV) The primary reagent prepared in (II) and the specimen are brought into contact with each other for a certain period of time under a certain temperature.
- the temperature may range from 4° C. to 40° C. and the reaction may be carried out for 5 minutes to 180 minutes.
- Unreacted substances are removed by B/F separation, followed by contact with the secondary reagent prepared in (III) for a certain period of time under a certain temperature to form a sandwich complex.
- the temperature may range from 4° C. to 40° C.
- the antibody bound to the carrier per reaction system in which 20 ⁇ L of the specimen is reacted with the antibody may be 100 ng to 1000 ⁇ g and the amount of labeled antibody may be 2 ng to 20 ⁇ g.
- the reagent of the present invention can be used for manual measurement, and can also be used for measurement using an automatic immunodiagnostic device.
- measurement using an automated immunodiagnostic device can be performed without being affected by endogenous measurement-interfering factors or competing enzymes contained in the sample, and TFPI2 in the sample can be quantified in a short time. ,preferable.
- the reagent for measuring the amount of TFPI2 is preferably a reagent for measuring the sum of the amount of TFPI2 processed polypeptide and the amount of intact TFPI2.
- the reagent for measuring the amount of TFPI2 is preferably an antigenic determinant in the region from aspartic acid at residue 23 to histidine at residue 131 or cysteine at residue 130 in the amino acid sequence of SEQ ID NO: 1. It is an antibody that binds, more preferably an antibody that recognizes Kunitz domain 1 of TFPI2.
- the present invention provides a malignancy of an antibody that binds to an antigenic determinant in the region from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue in the amino acid sequence shown in SEQ ID NO:1. It can also be referred to as use in the manufacture of reagents for detecting tumor-associated thromboembolism.
- the present invention provides an antibody that binds to an antigenic determinant in the region from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue in the amino acid sequence shown in SEQ ID NO: 1. Use in detecting tumor-associated thromboembolism can also be mentioned.
- a TFPI2 measurement reagent was prepared as follows using a TFPI2 antibody obtained by a DNA immunization method.
- An anti-TFPI2 monoclonal antibody (TS-TF04) was physically adsorbed to a water-insoluble ferrite-containing carrier at room temperature overnight at 100 ng/carrier, and then 100 mM Tris buffer (pH 8.0) containing 1% BSA. ) at 53° C. for 4 hours to prepare an anti-TFPI2 antibody-immobilized carrier.
- Alkaline phosphatase-labeled anti-TFPI2 antibody was prepared from anti-TFPI2 monoclonal antibody (TS-TF01) using an alkaline phosphatase labeling kit (manufactured by Dojindo Laboratories). (3) Place the 12 antibody-immobilized carriers prepared in (1) in a magnetically permeable container (capacity 1.2 mL), and then buffer containing 1 ⁇ g / mL of the alkaline phosphatase-labeled antibody prepared in (2) A TFPI2 measurement reagent was prepared by adding 100 ⁇ L of a solution (Tris buffer containing 3% BSA, pH 8.0) and freeze-drying. The prepared TFPI2 measurement reagent was hermetically sealed under nitrogen filling and stored at 4° C. until measurement.
- a solution Tris buffer containing 3% BSA, pH 8.0
- Example 2 Measurement of clinical samples Table 1 shows the details of the clinical samples used in Examples 3 to 7 below. Specimens were screened with a D-dimer of 1.0 ⁇ g/mL as a cutoff, and lower extremity vein echocardiography was performed on all cases with a D-dimer of 1.0 ⁇ g/mL or higher to confirm the presence or absence of thrombi. VTE-complicated ovarian cancer patient serum (25 cases) and non-VTE-complicated ovarian cancer patient serum (97 cases) are samples collected under the same protocol at Nara Medical University, consent of informed consent and Nara Medical University Provided with Ethics Committee approval.
- a fully automatic enzyme immunoassay device AIA-2000 (manufactured by Tosoh Corporation: manufacturing and sales notification number 13B3X90002000009) was used as an evaluation device.
- TFPI2 was measured using a fully automatic enzyme immunoassay device AIA-2000 according to the following procedure.
- Fig. 1 shows a Boxplot of blood TFPI2 values and blood D-dimer values in VTE-complicated ovarian cancer patients and non-VTE-complicated ovarian cancer patients. shown. Both TFPI2 and D-dimer were higher in the VTE concurrent group than in the non-VTE concurrent group with a statistically significant difference (Mann-Whitney U test, p ⁇ 0.0001).
- Figure 2 shows a BoxPlot of the values and blood D-dimer values.
- TFPI2 and D-dimer showed higher values with statistically significant difference in the VTE-complicated group than in the non-VTE-complicated group in both the early stage group and the progressive group.
- TFPI2 and D-dimer Comparison of TFPI2 and D-dimer in tissue types Box plots of blood D-dimer values are shown in FIG.
- TFPI2 and D-dimer showed higher values in clear cell carcinoma and serous carcinoma with a statistically significant difference in the VTE-complicated group compared to the non-VTE group.
- FIG. 4 shows the results of ROC analysis of TFPI2 and D-dimer in patients with VTE-complicated ovarian cancer and non-VTE-complicated ovarian cancer.
- the area under the curve (AUC) calculated from the ROC curve was both TFPI2 (0.7963) and D-dimer (0.8266), indicating high VTE discrimination performance. It was also shown that AUC was further increased by combining TFPI2 and D-dimer (0.8495).
- FIG. 5 shows the correlation between the blood TFPI2 level and the blood D-dimer level. It was shown that there is a low correlation between the blood TFPI2 level and the blood D-dimer level in patients with VTE-complicated ovarian cancer and non-VTE-complicated ovarian cancer.
- TFPI2 had a low sensitivity but high specificity, resulting in an accuracy rate of 0.71. From the above results, it was suggested that TFPI2 has a high degree of specificity and thus may have complementarity with D-dimer.
- Table 3 shows the details of the ovarian borderline malignant tumor samples used in Examples 9 to 11 described below. Specimens were screened with a D-dimer of 1.0 ⁇ g/mL as a cutoff, and lower extremity vein echocardiography was performed on all cases of 1.0 ⁇ g/mL or higher to confirm the presence or absence of thrombi.
- VTE-complicated ovarian borderline malignant tumor patient serum (2 cases) and non-VTE-complicated ovarian borderline malignant tumor patient serum (36 cases) are specimens collected under the same protocol at Nara Medical University, informed consent and It was provided with the approval of the Nara Medical University Ethics Committee. The apparatus for evaluation and the measurement procedure were the same as those in Example 2, and the blood D-dimer value was the measured value described in the electronic medical record immediately before the day of blood sampling.
- Example 9 Comparison of TFPI2 and D-dimer between ovarian borderline malignant tumor patients with/without VTE Blood TFPI2 levels and blood levels in patients with borderline ovarian malignancy with VTE and patients with borderline ovarian malignancy with no VTE A BoxPlot of the D-dimer values is shown in FIG. Both TFPI2 and D-dimer were higher in the VTE concurrent group than in the non-VTE concurrent group with a statistically significant difference (Mann-Whitney U test, p ⁇ 0.05).
- FIG. 7 shows the results of ROC analysis of TFPI2 and D-dimer in patients with borderline ovarian malignancy with VTE and patients with borderline ovarian malignancy with no VTE.
- the area under the curve (AUC) calculated from the ROC curve was both TFPI2 (1.0000) and D-dimer (0.9861), indicating high VTE discrimination performance.
- FIG. 8 shows the correlation between the blood TFPI2 level and the blood D-dimer level. It was shown that the correlation between the blood TFPI2 level and the blood D-dimer level is low even in patients with borderline malignant tumor of the ovary.
- Example 11 Comparison of VTE detection performance of TFPI2 and D-dimer in borderline malignant tumor of the ovary
- Table 4 shows the VTE detection performance of TFPI2 and D-dimer.
- the cutoff value of TFPI2 was set to 280 pg / mL from the results of Example 7, and the cutoff value of D-dimer was set to 1.0 ⁇ g / mL. rate was calculated.
- D-dimer has a high sensitivity, the specificity is low and the correct diagnosis rate is 0.658.
- TFPI2 had high sensitivity and specificity, and the accuracy rate was 0.921.
- the above results indicate that TFPI2 may have a higher performance than D-dimer in VTE discrimination of ovarian borderline malignant tumors.
- Example 12 Measurement of cancer patient specimens other than ovaries
- Table 5 shows the breakdown of the cancer patient specimens used in Examples 13-15.
- VTE-complicated cancer patient sera (33 cases) and non-VTE-complicated cancer patient sera (70 cases) were checked for the presence or absence of thrombus from clinical information provided by the distributors, PROMEDDX, Precision For Medicine, and BioIVT.
- PROMEDDX Precision For Medicine
- BioIVT BioIVT.
- the evaluation device and measurement procedure are the same as in Example 2.
- TFPI2 Comparison of TFPI2 in VTE-complicated/non-complicated cancer groups by cancer type
- BoxPlots of blood TFPI2 values of VTE-complicated and non-VTE-complicated patients are shown in FIG.
- TFPI2 had statistically significant differences in lung, esophageal and/or gastric, liver or pancreatic, colorectal, hematopoietic, breast, and uterine cancers with VTE compared to non-VTE groups. High values were shown (Mann-Whitney U test).
- ROC analysis between VTE-complicated/non-complicated groups by TFPI2 in non-ovarian cancer patients As for non-ovarian cancer patients, the results of ROC analysis of TFPI2 in VTE-complicated and non-VTE concomitant patients are shown in Fig. 10 . .
- the area under the curve (AUC) calculated from the ROC curve was lung cancer (1.0000), esophageal and/or gastric cancer (0.9722), liver or pancreatic cancer (0.9821), colon cancer (1.0000). , hematopoietic cancer (0.8438), breast cancer (0.8333), and uterine cancer (1.0000).
- Example 15 VTE detection performance of TFPI2 in patients with cancer other than ovary
- Table 6 shows the VTE detection performance of TFPI2 in each cancer patient other than ovary.
- the cutoff value of TFPI2 was set to 280 pg/mL from the results of Example 7, and sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were calculated.
- the sensitivity and specificity of VTE detection by TFPI2 showed high values regardless of cancer types, and lung cancer, esophageal and / or stomach cancer, liver or pancreatic cancer, colon cancer, hematopoietic cancer, breast cancer, uterine cancer It showed a high accuracy rate in all cancers.
- the present invention provides a method for detecting malignant tumor-related thromboembolism with a blood test that is simple and imposes relatively little burden on the patient. This is expected to contribute to improving the diagnostic accuracy of malignant tumor-related thromboembolism through a simple and highly accurate blood test, and is very useful industrially.
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Abstract
The present invention addresses the problem of providing: a method for detecting thromboembolism associated with a malignant tumor; and a reagent which can be utilized in the method. Provided is a method for detecting thromboembolism associated with a malignant tumor, the method being characterized by measuring the amount of TFPI2 in a specimen from a patient. An antibody capable of specifically recognizing NT-TFPI2 or intact TFPI2 is contained in a reagent for detecting thromboembolism associated with a malignant tumor.
Description
本発明は、組織因子経路インヒビター２（Ｔｉｓｓｕｅ Ｆａｃｔｏｒ Ｐａｔｈｗａｙ Ｉｎｈｉｂｉｔｏｒ ２；ＴＦＰＩ２）を測定対象とする悪性腫瘍関連血栓塞栓症の検出方法及び検出試薬に関する。
The present invention relates to a detection method and detection reagent for malignant tumor-associated thromboembolism that targets tissue factor pathway inhibitor 2 (TFPI2).
静脈血栓塞栓症（Ｖｅｎｏｕｓ ｔｈｒｏｍｂｏｅｍｂｏｌｉｓｍ，ＶＴＥ）は、深部静脈血栓症（Ｄｅｅｐ ｖｅｉｎ ｔｈｒｏｍｂｏｓｉｓ，ＤＶＴ）と肺塞栓症（Ｐｕｌｍｏｎａｒｙ ｅｍｂｏｌｉｓｍ，ＰＥ）から成る一連の病態の総称である。静脈血栓塞栓症はがん患者で合併することが知られており、悪性腫瘍関連血栓症（Ｃａｎｃｅｒ ａｓｓｏｃｉａｔｅｄ ｔｈｒｏｍｂｏｓｉｓ，ＣＡＴ）としてその取扱いには注意が必要とされている。婦人科がんにおいて卵巣がんでは静脈血栓塞栓症の合併が多いとされ、特に卵巣明細胞がんではＶＴＥの合併頻度が高いことが示されている（非特許文献１）。
Venous thromboembolism (VTE) is a general term for a series of pathologies consisting of deep vein thrombosis (DVT) and pulmonary embolism (PE). Venous thromboembolism is known to be a complication in cancer patients, and careful handling is required as cancer associated thrombosis (CAT). Among gynecologic cancers, ovarian cancer is said to be frequently associated with venous thromboembolism, and in particular, it has been shown that ovarian clear cell carcinoma is associated with a high frequency of VTE (Non-Patent Document 1).
悪性腫瘍の診断において、悪性腫瘍関連血栓塞栓症を疑った場合はＤ－ｄｉｍｅｒを測定し、陽性であれば超音波検査や造影ＣＴ等の画像検査を行い、血栓塞栓症の診断を確定する。悪性腫瘍関連血栓塞栓症Ｄ－ｄｉｍｅｒは血液凝固／線溶系において安定化フィブリンがプラスミンによって分解されて生じる産物であり、Ｄ－ｄｉｍｅｒ構造を有する混合物の総称である（非特許文献２）。Ｄ－ｄｉｍｅｒは高い特異度を有するマーカーであり、ＤＶＴやＰＥの除外診断用途だけでなく、抗凝固療法の継続期間や終了時期の判断、再発可能性の評価を行う上でも有用であることが示されている。一方、凝固系が亢進しているがん患者や妊婦、手術後患者などではＤ－ｄｉｍｅｒが高値を示すことが報告されている（非特許文献３）。
In diagnosing malignant tumors, if malignant tumor-related thromboembolism is suspected, D-dimer is measured, and if positive, imaging tests such as ultrasonography and contrast-enhanced CT are performed to confirm the diagnosis of thromboembolism. Malignant tumor-associated thromboembolism D-dimer is a product produced by the degradation of stabilized fibrin by plasmin in the blood coagulation/fibrinolysis system, and is a general term for mixtures having a D-dimer structure (Non-Patent Document 2). D-dimer is a marker with high specificity, and is useful not only for diagnosing DVT and PE, but also for judging the duration and termination of anticoagulant therapy and evaluating the possibility of recurrence. It is shown. On the other hand, it has been reported that cancer patients, pregnant women, and postoperative patients with accelerated coagulation show high values of D-dimer (Non-Patent Document 3).
組織因子経路インヒビター２（ＴＦＰＩ２）は、胎盤タンパク質５（Ｐｌａｃｅｎｔａｌ Ｐｒｏｔｅｉｎ ５；ＰＰ５）と同一のタンパク質であり、３つのクニッツ型プロテアーゼインヒビタードメインを含む胎盤由来セリンプロテアーゼインヒビターである。ＴＦＰＩ２は卵巣がん細胞株において明細胞がん細胞株から特異的に産生され、卵巣がん患者組織における遺伝子発現は明細胞がん患者のみで特異的に向上することを明らかにし（特許文献１）、血中ＴＦＰＩ２の測定により卵巣明細胞がんを検出する方法を開示した（特許文献２および３、非特許文献４および５）。
Tissue factor pathway inhibitor 2 (TFPI2) is the same protein as placental protein 5 (PP5) and is a placenta-derived serine protease inhibitor containing three Kunitz-type protease inhibitor domains. TFPI2 is specifically produced from clear cell cancer cell lines in ovarian cancer cell lines, and gene expression in ovarian cancer patient tissues is specifically improved only in clear cell cancer patients (Patent Document 1). ), disclosed a method for detecting ovarian clear cell carcinoma by measuring TFPI2 in blood ( Patent Documents 2 and 3, Non-Patent Documents 4 and 5).
ＴＦＰＩ２は多くのがん種でＴＦＰＩ２ ＤＮＡプロモーターにメチル化修飾を受けていることが明らかとなり、メチル化修飾を指標とした遺伝子診断ターゲットとしての臨床研究が進められている（非特許文献６～１０）。しかし今日まで、体液中のＴＦＰＩ２タンパク質が悪性腫瘍関連血栓塞栓症の検出に適用できるかは不明であった。
It has been revealed that TFPI2 undergoes methylation modification in the TFPI2 DNA promoter in many cancer types, and clinical research is underway as a genetic diagnosis target using methylation modification as an index (Non-Patent Documents 6 to 10). ). However, to date, it was unclear whether the TFPI2 protein in body fluids could be applied to detect malignant tumor-associated thromboembolism.
本発明は、悪性腫瘍関連血栓塞栓症の検出方法、及び前記方法に利用できる試薬を提供することを課題とする。
An object of the present invention is to provide a method for detecting malignant tumor-related thromboembolism and a reagent that can be used in the method.
本発明者らは鋭意検討の結果、血中ＴＦＰＩ２値は種々の悪性腫瘍において非ＶＴＥ併発群に比べてＶＴＥ併発患者群で有意に向上することを見出し、ＴＦＰＩ２はＤ－ｄｉｍｅｒと同等の高い精度をもって悪性腫瘍と合併するＶＴＥを検出しうることに想到し、本発明を完成させた。
すなわち、本発明は、以下の態様を包含する。
［１］検体において、ＴＦＰＩ２量を測定することを含む、悪性腫瘍関連血栓塞栓症を検出する方法。
［２］前記ＴＦＰＩ２量の測定値が、予め設定した基準値を超えた場合に、悪性腫瘍関連血栓塞栓症が検出されたとする、［１］に記載の方法。
［３］前記ＴＦＰＩ２量が、ＴＦＰＩ２プロセシングポリペプチド量及びインタクトＴＦＰＩ２量の合計である、［１］又は［２］に記載の方法。
［４］前記ＴＦＰＩ２量の測定が、配列番号１のアミノ酸配列の２３残基目のアスパラギン酸から１３１残基目のヒスチジン又は１３０残基目のシステインまでの領域内の抗原決定基に結合する抗体を用いた抗原抗体反応により行われるものである、［１］～［３］のいずれかに記載の方法。
［５］前記抗体が、ＴＦＰＩ２のクニッツドメイン１を認識する抗体である、［４］に記載の方法。
［６］質量分析法を用いて測定を行う、［１］～［３］のいずれかに記載の方法。
［７］悪性腫瘍が、卵巣癌、肺がん、消化器がん、造血器がん、乳がん、又は子宮がんである、［１］～［６］のいずれかに記載の方法。
［８］配列番号１に示すアミノ酸配列の２３残基目のアスパラギン酸から１３１残基目のヒスチジン又は１３０残基目のシステインまでの領域内の抗原決定基に結合する抗体を含む、悪性腫瘍関連血栓塞栓症を検出するための試薬。 As a result of intensive studies, the present inventors found that blood TFPI2 levels in various malignant tumors were significantly improved in the VTE-complicated patient group compared to the non-VTE-complicated group. The present invention has been completed based on the idea that VTE associated with malignant tumors can be detected.
That is, the present invention includes the following aspects.
[1] A method for detecting malignant tumor-related thromboembolism, comprising measuring the amount of TFPI2 in a specimen.
[2] The method of [1], wherein malignant tumor-associated thromboembolism is detected when the measured TFPI2 amount exceeds a preset reference value.
[3] The method of [1] or [2], wherein the amount of TFPI2 is the sum of the amount of TFPI2 processing polypeptide and the amount of intact TFPI2.
[4] An antibody that binds to an antigenic determinant in the region from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue in the amino acid sequence of SEQ ID NO: 1 for which the amount of TFPI2 is measured The method according to any one of [1] to [3], which is carried out by an antigen-antibody reaction using
[5] The method of [4], wherein the antibody recognizes Kunitz domain 1 of TFPI2.
[6] The method according to any one of [1] to [3], wherein the measurement is performed using mass spectrometry.
[7] The method according to any one of [1] to [6], wherein the malignant tumor is ovarian cancer, lung cancer, gastrointestinal cancer, hematopoietic cancer, breast cancer, or uterine cancer.
[8] Malignant tumor-related, including an antibody that binds to an antigenic determinant in the region from aspartic acid at residue 23 to histidine at residue 131 or cysteine at residue 130 in the amino acid sequence shown in SEQ ID NO: 1 Reagent for detecting thromboembolism.
すなわち、本発明は、以下の態様を包含する。
［１］検体において、ＴＦＰＩ２量を測定することを含む、悪性腫瘍関連血栓塞栓症を検出する方法。
［２］前記ＴＦＰＩ２量の測定値が、予め設定した基準値を超えた場合に、悪性腫瘍関連血栓塞栓症が検出されたとする、［１］に記載の方法。
［３］前記ＴＦＰＩ２量が、ＴＦＰＩ２プロセシングポリペプチド量及びインタクトＴＦＰＩ２量の合計である、［１］又は［２］に記載の方法。
［４］前記ＴＦＰＩ２量の測定が、配列番号１のアミノ酸配列の２３残基目のアスパラギン酸から１３１残基目のヒスチジン又は１３０残基目のシステインまでの領域内の抗原決定基に結合する抗体を用いた抗原抗体反応により行われるものである、［１］～［３］のいずれかに記載の方法。
［５］前記抗体が、ＴＦＰＩ２のクニッツドメイン１を認識する抗体である、［４］に記載の方法。
［６］質量分析法を用いて測定を行う、［１］～［３］のいずれかに記載の方法。
［７］悪性腫瘍が、卵巣癌、肺がん、消化器がん、造血器がん、乳がん、又は子宮がんである、［１］～［６］のいずれかに記載の方法。
［８］配列番号１に示すアミノ酸配列の２３残基目のアスパラギン酸から１３１残基目のヒスチジン又は１３０残基目のシステインまでの領域内の抗原決定基に結合する抗体を含む、悪性腫瘍関連血栓塞栓症を検出するための試薬。 As a result of intensive studies, the present inventors found that blood TFPI2 levels in various malignant tumors were significantly improved in the VTE-complicated patient group compared to the non-VTE-complicated group. The present invention has been completed based on the idea that VTE associated with malignant tumors can be detected.
That is, the present invention includes the following aspects.
[1] A method for detecting malignant tumor-related thromboembolism, comprising measuring the amount of TFPI2 in a specimen.
[2] The method of [1], wherein malignant tumor-associated thromboembolism is detected when the measured TFPI2 amount exceeds a preset reference value.
[3] The method of [1] or [2], wherein the amount of TFPI2 is the sum of the amount of TFPI2 processing polypeptide and the amount of intact TFPI2.
[4] An antibody that binds to an antigenic determinant in the region from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue in the amino acid sequence of SEQ ID NO: 1 for which the amount of TFPI2 is measured The method according to any one of [1] to [3], which is carried out by an antigen-antibody reaction using
[5] The method of [4], wherein the antibody recognizes Kunitz domain 1 of TFPI2.
[6] The method according to any one of [1] to [3], wherein the measurement is performed using mass spectrometry.
[7] The method according to any one of [1] to [6], wherein the malignant tumor is ovarian cancer, lung cancer, gastrointestinal cancer, hematopoietic cancer, breast cancer, or uterine cancer.
[8] Malignant tumor-related, including an antibody that binds to an antigenic determinant in the region from aspartic acid at residue 23 to histidine at residue 131 or cysteine at residue 130 in the amino acid sequence shown in SEQ ID NO: 1 Reagent for detecting thromboembolism.
本発明により、悪性腫瘍関連血栓塞栓症を簡便かつ高い精度で検出する方法、及び前記方法に利用できる試薬が提供される。
The present invention provides a method for detecting malignant tumor-related thromboembolism simply and with high accuracy, and a reagent that can be used for the method.
＜１＞本発明の悪性腫瘍関連血栓塞栓症を検出する方法
本発明の第一の態様は、悪性腫瘍関連血栓塞栓症を検出する方法であり、検体においてＴＦＰＩ２量を測定することを含む。これは、悪性腫瘍において、血液等の悪性腫瘍関連血栓塞栓症患者生体試料中にてＴＦＰＩ２の存在が上昇することに基づく方法である。検体におけるＴＦＰＩ２量の測定は、通常インビトロ（ｉｎ ｖｉｔｒｏ）で行われる。この方法により、後述する実施例が示す通り、高い精度で悪性腫瘍関連血栓塞栓症を検出することができる。 <1> Method for Detecting Malignant Tumor-Related Thromboembolism of the Present Invention The first aspect of the present invention is a method for detecting malignant tumor-related thromboembolism, which includes measuring the amount of TFPI2 in a specimen. This is a method based on the elevated presence of TFPI2 in malignant tumor-associated thromboembolism patient biological samples such as blood in malignant tumors. Measurement of the amount of TFPI2 in a specimen is usually performed in vitro. By this method, malignant tumor-associated thromboembolism can be detected with high accuracy, as shown in Examples described later.
本発明の第一の態様は、悪性腫瘍関連血栓塞栓症を検出する方法であり、検体においてＴＦＰＩ２量を測定することを含む。これは、悪性腫瘍において、血液等の悪性腫瘍関連血栓塞栓症患者生体試料中にてＴＦＰＩ２の存在が上昇することに基づく方法である。検体におけるＴＦＰＩ２量の測定は、通常インビトロ（ｉｎ ｖｉｔｒｏ）で行われる。この方法により、後述する実施例が示す通り、高い精度で悪性腫瘍関連血栓塞栓症を検出することができる。 <1> Method for Detecting Malignant Tumor-Related Thromboembolism of the Present Invention The first aspect of the present invention is a method for detecting malignant tumor-related thromboembolism, which includes measuring the amount of TFPI2 in a specimen. This is a method based on the elevated presence of TFPI2 in malignant tumor-associated thromboembolism patient biological samples such as blood in malignant tumors. Measurement of the amount of TFPI2 in a specimen is usually performed in vitro. By this method, malignant tumor-associated thromboembolism can be detected with high accuracy, as shown in Examples described later.
なお、本発明の方法は、悪性腫瘍関連血栓塞栓症を検出する段階までを含むものであり、悪性腫瘍関連血栓塞栓症の診断に関する最終的な判断行為は含まれない。医師は、本発明の方法による検出結果等を参照して、悪性腫瘍関連血栓塞栓症を診断したり治療方針を立てたりする。
It should be noted that the method of the present invention includes up to the step of detecting malignant tumor-related thromboembolism, and does not include the final decision regarding the diagnosis of malignant tumor-related thromboembolism. Physicians refer to the results of detection by the method of the present invention, etc. to diagnose malignant tumor-associated thromboembolism and formulate treatment strategies.
本発明の悪性腫瘍関連血栓塞栓症において、血栓塞栓症に合併して生じる（基礎疾患となる）悪性腫瘍には特に限定は無く、いわゆる悪性腫瘍そのもの又は境界悪性腫瘍であってもよい。例えば卵巣癌が好ましく、また卵巣境界悪性腫瘍も好ましい。卵巣癌の中でも、例えば明細胞癌、漿液性癌、又は類内膜癌が好ましい。特に類内膜癌は、後述の実施例が示すように、ＶＴＥ併発群にてＴＦＰＩ２の上昇傾向が認められ、これはＤ－ｄｉｍｅｒでは認められなかったことから、好ましいものである。さらに卵巣癌以外にも、例えば肺がん、消化器がん（食道及び／若しくは胃がん、肝臓若しくは膵臓がん、大腸がん等）、造血器がん、乳がん、又は子宮がんが好ましい。
一方、本発明において、血栓塞栓症の種類には特に限定は無いが、好ましくは静脈血栓塞栓症である。 In the malignant tumor-associated thromboembolism of the present invention, there is no particular limitation on the malignant tumor that occurs in association with thromboembolism (underlying disease), and may be a so-called malignant tumor itself or a borderline malignant tumor. For example, ovarian cancer is preferred, and ovarian borderline malignancy is also preferred. Among ovarian cancers, for example, clear cell carcinoma, serous carcinoma, or endometrioid carcinoma are preferred. In particular, endometrioid carcinoma is preferable because, as shown in Examples below, a tendency for TFPI2 to rise was observed in the group with concurrent VTE, but this was not observed in D-dimer. In addition to ovarian cancer, lung cancer, gastrointestinal cancer (esophageal and/or stomach cancer, liver or pancreatic cancer, colon cancer, etc.), hematopoietic cancer, breast cancer, or uterine cancer are preferred.
On the other hand, in the present invention, the type of thromboembolism is not particularly limited, but venous thromboembolism is preferred.
一方、本発明において、血栓塞栓症の種類には特に限定は無いが、好ましくは静脈血栓塞栓症である。 In the malignant tumor-associated thromboembolism of the present invention, there is no particular limitation on the malignant tumor that occurs in association with thromboembolism (underlying disease), and may be a so-called malignant tumor itself or a borderline malignant tumor. For example, ovarian cancer is preferred, and ovarian borderline malignancy is also preferred. Among ovarian cancers, for example, clear cell carcinoma, serous carcinoma, or endometrioid carcinoma are preferred. In particular, endometrioid carcinoma is preferable because, as shown in Examples below, a tendency for TFPI2 to rise was observed in the group with concurrent VTE, but this was not observed in D-dimer. In addition to ovarian cancer, lung cancer, gastrointestinal cancer (esophageal and/or stomach cancer, liver or pancreatic cancer, colon cancer, etc.), hematopoietic cancer, breast cancer, or uterine cancer are preferred.
On the other hand, in the present invention, the type of thromboembolism is not particularly limited, but venous thromboembolism is preferred.
本発明において測定されるＴＦＰＩ２は、特に限定はなく、例えばインタクトＴＦＰＩ２（以降、「Ｉ－ＴＦＰＩ２」とも記す）、ＴＦＰＩ２プロセシングポリペプチド（以降、「ＮＴ－ＴＦＰＩ２」とも記す）、又はそれらの両方であってもよい。
配列番号１に、ヒトＴＦＰＩ２のｃＤＮＡに基づくアミノ酸配列を示す。配列番号１において、開始メチオニンから２２残基目のグリシンまではシグナルペプチドである。 TFPI2 to be measured in the present invention is not particularly limited, for example, intact TFPI2 (hereinafter also referred to as "I-TFPI2"), TFPI2 processing polypeptide (hereinafter also referred to as "NT-TFPI2"), or both There may be.
SEQ ID NO: 1 shows the amino acid sequence based on human TFPI2 cDNA. In SEQ ID NO: 1, the signal peptide is from the initiation methionine to the glycine at the 22nd residue.
配列番号１に、ヒトＴＦＰＩ２のｃＤＮＡに基づくアミノ酸配列を示す。配列番号１において、開始メチオニンから２２残基目のグリシンまではシグナルペプチドである。 TFPI2 to be measured in the present invention is not particularly limited, for example, intact TFPI2 (hereinafter also referred to as "I-TFPI2"), TFPI2 processing polypeptide (hereinafter also referred to as "NT-TFPI2"), or both There may be.
SEQ ID NO: 1 shows the amino acid sequence based on human TFPI2 cDNA. In SEQ ID NO: 1, the signal peptide is from the initiation methionine to the glycine at the 22nd residue.
「インタクトＴＦＰＩ２」とは、配列番号１のアミノ酸配列の２３残基目から２３５残基目で表されるペプチドをいう。
また、「ＮＴ－ＴＦＰＩ２」は、特許文献３に記載されるように、インタクトＴＦＰＩ２のＮ末端側に位置するクニッツドメイン１を含むペプチド断片をいう。より具体的には、ＮＴ－ＴＦＰＩ２は、配列番号１のアミノ酸配列の２３残基目のアスパラギン酸から１３１残基目のヒスチジン又は１３０残基目のシステインまでの配列を少なくとも含むペプチド、または、前記配列と８０％以上の同一性を有するアミノ酸配列を含むペプチドである。前記同一性は、好ましくは９０％以上、より好ましくは９５％以上である。また、このポリペプチドは、前記配列において１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列からなるポリペプチドであってもよい。なお、数個とは、好ましくは２～２０個、より好ましくは２～１０個、さらに好ましくは２～５個をいう。 “Intact TFPI2” refers to a peptide represented by residues 23 to 235 of the amino acid sequence of SEQ ID NO:1.
In addition, "NT-TFPI2" refers to a peptide fragment containing Kunitz domain 1 located on the N-terminal side of intact TFPI2, as described inPatent Document 3. More specifically, NT-TFPI2 is a peptide comprising at least a sequence from aspartic acid at residue 23 to histidine at residue 131 or cysteine at residue 130 in the amino acid sequence of SEQ ID NO: 1, or A peptide containing an amino acid sequence that has 80% or more identity with the sequence. Said identity is preferably 90% or more, more preferably 95% or more. Alternatively, this polypeptide may be a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and/or added to the above sequence. The term "several" means preferably 2 to 20, more preferably 2 to 10, still more preferably 2 to 5.
また、「ＮＴ－ＴＦＰＩ２」は、特許文献３に記載されるように、インタクトＴＦＰＩ２のＮ末端側に位置するクニッツドメイン１を含むペプチド断片をいう。より具体的には、ＮＴ－ＴＦＰＩ２は、配列番号１のアミノ酸配列の２３残基目のアスパラギン酸から１３１残基目のヒスチジン又は１３０残基目のシステインまでの配列を少なくとも含むペプチド、または、前記配列と８０％以上の同一性を有するアミノ酸配列を含むペプチドである。前記同一性は、好ましくは９０％以上、より好ましくは９５％以上である。また、このポリペプチドは、前記配列において１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列からなるポリペプチドであってもよい。なお、数個とは、好ましくは２～２０個、より好ましくは２～１０個、さらに好ましくは２～５個をいう。 “Intact TFPI2” refers to a peptide represented by residues 23 to 235 of the amino acid sequence of SEQ ID NO:1.
In addition, "NT-TFPI2" refers to a peptide fragment containing Kunitz domain 1 located on the N-terminal side of intact TFPI2, as described in
本発明における患者由来の検体（被検試料）は、全血、血球、血清、血漿などの血液成分、細胞または組織の抽出液、尿、脳脊髄液、腹腔洗浄液、腹水、嚢胞液などが挙げられる。血液成分や尿などの体液を検体として用いると、簡便かつ非侵襲的に行うことができるため好ましく、検体採取の容易性、他の検査項目への汎用性を考慮すると、血液成分を検体として用いるのが特に好ましい。検体の希釈倍率は無希釈から１００倍希釈の中から使用する検体の種類や状態に応じて適宜選択すればよい。
Patient-derived specimens (test samples) in the present invention include blood components such as whole blood, blood cells, serum, and plasma, cell or tissue extracts, urine, cerebrospinal fluid, peritoneal washings, ascites, cystic fluid, and the like. be done. It is preferable to use body fluids such as blood components and urine as specimens because it can be performed simply and non-invasively. Considering the ease of sample collection and versatility for other test items, blood components are used as specimens. is particularly preferred. The dilution rate of the sample may be appropriately selected from no dilution to 100-fold dilution according to the type and condition of the sample to be used.
本発明の悪性腫瘍関連血栓塞栓症において、ＴＦＰＩ２を検出する方法と既存の血栓塞栓症マーカーまたは超音波エコー、CTもしくはMRI等の画像診断とを組み合わせることが好ましい。その組み合わせ方は、特に制限されない。本発明の血栓塞栓症検出方法における、ＴＦＰＩ２を検出する方法と既存の静脈血栓塞栓症マーカーまたは画像診断との組み合わせ方の一例として、
（Ａ）測定対象検体に対し、ＴＦＰＩ２を検出する方法と既存の静脈血栓塞栓症マーカーを同時または別個に行い、少なくとも一方が陽性であれば超音波検査や造影ＣＴ等の画像検査を行い、血栓塞栓症を検出する方法
（Ｂ）測定対象検体に対し、まず既存の静脈血栓塞栓症マーカーを適用し、その結果陰性と判定された検体に対してＴＦＰＩ２を検出し、ＴＦＰＩ２が陽性であれば超音波検査や造影ＣＴ等の画像検査を行い、血栓塞栓症を検出する方法があげられるが、（Ａ）は同時測定により速やかに静脈血栓塞栓症を検出できる点でより好ましい。 In the malignant tumor-related thromboembolism of the present invention, it is preferable to combine the method for detecting TFPI2 with an existing thromboembolism marker or diagnostic imaging such as ultrasonic echo, CT or MRI. The combination method is not particularly limited. As an example of how to combine a method for detecting TFPI2 with an existing venous thromboembolism marker or diagnostic imaging in the method for detecting thromboembolism of the present invention,
(A) A method for detecting TFPI2 and an existing venous thromboembolism marker are performed on the specimen to be measured at the same time or separately. Method for detecting embolism (B) For the specimen to be measured, first apply an existing venous thromboembolism marker, detect TFPI2 for the specimen determined to be negative as a result, and if TFPI2 is positive, super Imaging tests such as sonography and contrast-enhanced CT are performed to detect thromboembolism. Method (A) is more preferable because venous thromboembolism can be rapidly detected by simultaneous measurement.
（Ａ）測定対象検体に対し、ＴＦＰＩ２を検出する方法と既存の静脈血栓塞栓症マーカーを同時または別個に行い、少なくとも一方が陽性であれば超音波検査や造影ＣＴ等の画像検査を行い、血栓塞栓症を検出する方法
（Ｂ）測定対象検体に対し、まず既存の静脈血栓塞栓症マーカーを適用し、その結果陰性と判定された検体に対してＴＦＰＩ２を検出し、ＴＦＰＩ２が陽性であれば超音波検査や造影ＣＴ等の画像検査を行い、血栓塞栓症を検出する方法があげられるが、（Ａ）は同時測定により速やかに静脈血栓塞栓症を検出できる点でより好ましい。 In the malignant tumor-related thromboembolism of the present invention, it is preferable to combine the method for detecting TFPI2 with an existing thromboembolism marker or diagnostic imaging such as ultrasonic echo, CT or MRI. The combination method is not particularly limited. As an example of how to combine a method for detecting TFPI2 with an existing venous thromboembolism marker or diagnostic imaging in the method for detecting thromboembolism of the present invention,
(A) A method for detecting TFPI2 and an existing venous thromboembolism marker are performed on the specimen to be measured at the same time or separately. Method for detecting embolism (B) For the specimen to be measured, first apply an existing venous thromboembolism marker, detect TFPI2 for the specimen determined to be negative as a result, and if TFPI2 is positive, super Imaging tests such as sonography and contrast-enhanced CT are performed to detect thromboembolism. Method (A) is more preferable because venous thromboembolism can be rapidly detected by simultaneous measurement.
本発明の悪性腫瘍関連血栓塞栓症検出方法で検出する静脈血栓塞栓症マーカーは、従来知られているマーカーから適宜選択すればよく、一例として、Ｄ－ｄｉｍｅｒ、プロトロンビン・フラグメント１＋２（Ｆ１＋２）、フィブリン分解産物（ＦＤＰ）、可溶性フィブリン（ＳＦ）、可溶性フィブリンモノマー複合体（ＳＦＭＣ）、プラスミン─α２プラスミンインヒビター複合体（ＰＰＩＣ）等が挙げられる。このうち、特に静脈血栓塞栓症マーカーとして最も汎用されているＤ－ｄｉｍｅｒは臨床的有用性が確立している点で、本発明の悪性腫瘍関連血栓塞栓症検出方法で使用するマーカーとして好ましい。また、本発明の悪性腫瘍関連血栓塞栓症検出方法において検出される従来知られているマーカーは、１種のみであってもよく、２種またはそれ以上であってもよい。
The venous thromboembolism marker to be detected by the malignant tumor-related thromboembolism detection method of the present invention may be appropriately selected from conventionally known markers. degradation products (FDP), soluble fibrin (SF), soluble fibrin monomer complex (SFMC), plasmin- α2 plasmin inhibitor complex (PPIC), and the like. Among these, D-dimer, which is most widely used as a marker for venous thromboembolism, is particularly preferable as a marker for use in the method for detecting malignant tumor-related thromboembolism of the present invention in that its clinical utility has been established. In addition, the conventionally known markers detected in the method for detecting malignant tumor-related thromboembolism of the present invention may be one type, or two or more types.
また、本発明における検体の採取時期は、特に限定されない。例えば、画像診断等で悪性腫瘍関連血栓塞栓症疑いとなって精密検査が施行される術前時から経過観察時にかけていつでもよく、確定診断前後、治療開始前後など、いずれの段階で採取した検体であっても、本発明の方法に供することができる。
In addition, the timing of sample collection in the present invention is not particularly limited. For example, specimens collected at any stage, such as before and after a definitive diagnosis, before and after the start of treatment, can be used at any time from preoperative when a detailed examination is performed when a malignant tumor-related thromboembolism is suspected by imaging diagnosis, etc., to follow-up observation. Even if there is, it can be subjected to the method of the present invention.
本発明の検出方法では、測定により得たＴＦＰＩ２量が、予め設定した基準値（Ｃｕｔｏｆｆ値）を超えた場合に、悪性腫瘍関連血栓塞栓症が検出されたと判定することが好ましい。ここで、ＴＦＰＩ２量は、インタクトＴＦＰＩ２量、ＮＴ－ＴＦＰＩ２量、又はインタクトＴＦＰＩ２量及びＮＴ－ＴＦＰＩ２量の合計のいずれでもよいが、インタクトＴＦＰＩ２量及びＮＴ－ＴＦＰＩ２量の合計が測定のしやすさと十分な感度・特異度との両立の観点からより好ましい。
In the detection method of the present invention, it is preferable to determine that malignant tumor-related thromboembolism has been detected when the amount of TFPI2 obtained by measurement exceeds a preset reference value (Cutoff value). Here, the amount of TFPI2 may be the amount of intact TFPI2, the amount of NT-TFPI2, or the total amount of intact TFPI2 and NT-TFPI2. It is more preferable from the viewpoint of compatibility with high sensitivity and specificity.
判定に用いる基準値は、測定値もしくは換算濃度値のいずれでもよい。なお、換算濃度値は、ＴＦＰＩ２を標準試料として作成された検量線に基づいて測定値から換算される値をいう。
悪性腫瘍関連血栓塞栓症と判定する基準値（Ｃｕｔｏｆｆ値）は、悪性腫瘍関連血栓塞栓症と非悪性腫瘍関連血栓塞栓症をそれぞれ測定し、受信者動作特性（ＲＯＣ）曲線解析により最適な感度と特異度を示す測定値に適宜設定することができる。例えば、ＴＦＰＩ２の基準値（Ｃｕｔｏｆｆ値）は後述の実施例で示す通り２８０ ｐｇ／ｍＬと設定してもよいがその限りではない。 The reference value used for determination may be either a measured value or a converted density value. Incidentally, the converted concentration value refers to a value converted from a measured value based on a calibration curve prepared using TFPI2 as a standard sample.
The reference value (Cutoff value) for determining malignant tumor-related thromboembolism is determined by measuring malignant tumor-related thromboembolism and non-malignant tumor-related thromboembolism, respectively, and determining the optimal sensitivity and It can be appropriately set to a measured value that indicates specificity. For example, the reference value (Cutoff value) of TFPI2 may be set at 280 pg/mL as shown in Examples below, but is not limited thereto.
悪性腫瘍関連血栓塞栓症と判定する基準値（Ｃｕｔｏｆｆ値）は、悪性腫瘍関連血栓塞栓症と非悪性腫瘍関連血栓塞栓症をそれぞれ測定し、受信者動作特性（ＲＯＣ）曲線解析により最適な感度と特異度を示す測定値に適宜設定することができる。例えば、ＴＦＰＩ２の基準値（Ｃｕｔｏｆｆ値）は後述の実施例で示す通り２８０ ｐｇ／ｍＬと設定してもよいがその限りではない。 The reference value used for determination may be either a measured value or a converted density value. Incidentally, the converted concentration value refers to a value converted from a measured value based on a calibration curve prepared using TFPI2 as a standard sample.
The reference value (Cutoff value) for determining malignant tumor-related thromboembolism is determined by measuring malignant tumor-related thromboembolism and non-malignant tumor-related thromboembolism, respectively, and determining the optimal sensitivity and It can be appropriately set to a measured value that indicates specificity. For example, the reference value (Cutoff value) of TFPI2 may be set at 280 pg/mL as shown in Examples below, but is not limited thereto.
以降、ＴＦＰＩ２の測定方法について説明する。
本発明において、検体中のＮＴ－ＴＦＰＩ２量又はインタクトＴＦＰＩ２量を個別に測定してもよく、またその値を合計して合計量としてもよい。また、検体中のＮＴ－ＴＦＰＩ２とインタクトＴＦＰＩ２の合計量を一度に測定できる測定系で測定してもよい。あるいは、後述するように、両方の測定による合計量とインタクトＴＦＰＩ２単独の測定量とから間接的にＮＴ－ＴＦＰＩ２量を測定してもよい。 Hereinafter, a method for measuring TFPI2 will be described.
In the present invention, the amount of NT-TFPI2 or the amount of intact TFPI2 in the specimen may be measured individually, or the values may be totaled to obtain the total amount. Alternatively, the total amount of NT-TFPI2 and intact TFPI2 in the specimen may be measured with a measurement system capable of measuring at once. Alternatively, as described below, the amount of NT-TFPI2 may be measured indirectly from the total amount of both measurements and the amount of intact TFPI2 alone.
本発明において、検体中のＮＴ－ＴＦＰＩ２量又はインタクトＴＦＰＩ２量を個別に測定してもよく、またその値を合計して合計量としてもよい。また、検体中のＮＴ－ＴＦＰＩ２とインタクトＴＦＰＩ２の合計量を一度に測定できる測定系で測定してもよい。あるいは、後述するように、両方の測定による合計量とインタクトＴＦＰＩ２単独の測定量とから間接的にＮＴ－ＴＦＰＩ２量を測定してもよい。 Hereinafter, a method for measuring TFPI2 will be described.
In the present invention, the amount of NT-TFPI2 or the amount of intact TFPI2 in the specimen may be measured individually, or the values may be totaled to obtain the total amount. Alternatively, the total amount of NT-TFPI2 and intact TFPI2 in the specimen may be measured with a measurement system capable of measuring at once. Alternatively, as described below, the amount of NT-TFPI2 may be measured indirectly from the total amount of both measurements and the amount of intact TFPI2 alone.
本発明の方法において、ＮＴ－ＴＦＰＩ２量及び／又はインタクトＴＦＰＩ２量を測定する方法は特に制限されない。例えば、ＮＴ－ＴＦＰＩ２及び／又はインタクトＴＦＰＩ２を認識する抗体を用いる抗原抗体反応を利用した方法や、質量分析法を利用した方法が例示できる。
（ａ）標識した測定対象及び測定対象を認識する抗体を用い、標識した測定対象及び検体に含まれる測定対象が、前記抗体に競合的に結合することを利用した競合法。
（ｂ）測定対象を認識する抗体を固定化したチップに検体を接触させ、当該抗体と測定対象との結合に依存したシグナルを検出する表面プラズモン共鳴を用いた方法。
（ｃ）蛍光標識した測定対象を認識する抗体を用い、当該抗体と測定対象とが結合することで蛍光偏光度が上昇することを利用した蛍光偏光免疫測定法。
（ｄ）測定対象を認識する抗体であって、抗原決定基の異なる２種類の抗体（うち１つは標識した抗体）を用い、当該２つの抗体と測定対象との３者の複合体を形成させるサンドイッチ法。
（ｅ）前処理として測定対象を認識する抗体により検体中の測定対象を濃縮後、その結合タンパクのポリペプチドを質量分析装置等により検出する方法。
（ｄ）、（ｅ）の方法が簡便かつ汎用性が高いが、多検体を処理する上では（ｄ）の方法が試薬及び装置に関する技術が十分確立されている点でより好ましい。 In the method of the present invention, the method for measuring the amount of NT-TFPI2 and/or the amount of intact TFPI2 is not particularly limited. For example, a method using an antigen-antibody reaction using an antibody that recognizes NT-TFPI2 and/or intact TFPI2, and a method using mass spectrometry can be exemplified.
(a) A competitive method in which a labeled target to be measured and an antibody that recognizes the target to be measured are used, and the labeled target to be measured and the target to be measured contained in a sample competitively bind to the antibody.
(b) A method using surface plasmon resonance, in which a sample is brought into contact with a chip on which an antibody that recognizes an object to be measured is immobilized, and a signal dependent on binding between the antibody and the object to be measured is detected.
(c) A fluorescence polarization immunoassay method that uses a fluorescence-labeled antibody that recognizes a target to be measured, and utilizes an increase in the degree of fluorescence polarization due to binding between the antibody and the target to be measured.
(d) Using two types of antibodies (one of which is a labeled antibody) having different antigenic determinants, which are antibodies that recognize the target to be measured, to form a tripartite complex between the two antibodies and the target to be measured sandwich method.
(e) A method of concentrating the analyte in the specimen with an antibody that recognizes the analyte as a pretreatment, and then detecting the polypeptide of the binding protein using a mass spectrometer or the like.
The methods (d) and (e) are simple and highly versatile, but the method (d) is more preferable from the viewpoint of the well-established techniques for reagents and devices for processing multiple specimens.
（ａ）標識した測定対象及び測定対象を認識する抗体を用い、標識した測定対象及び検体に含まれる測定対象が、前記抗体に競合的に結合することを利用した競合法。
（ｂ）測定対象を認識する抗体を固定化したチップに検体を接触させ、当該抗体と測定対象との結合に依存したシグナルを検出する表面プラズモン共鳴を用いた方法。
（ｃ）蛍光標識した測定対象を認識する抗体を用い、当該抗体と測定対象とが結合することで蛍光偏光度が上昇することを利用した蛍光偏光免疫測定法。
（ｄ）測定対象を認識する抗体であって、抗原決定基の異なる２種類の抗体（うち１つは標識した抗体）を用い、当該２つの抗体と測定対象との３者の複合体を形成させるサンドイッチ法。
（ｅ）前処理として測定対象を認識する抗体により検体中の測定対象を濃縮後、その結合タンパクのポリペプチドを質量分析装置等により検出する方法。
（ｄ）、（ｅ）の方法が簡便かつ汎用性が高いが、多検体を処理する上では（ｄ）の方法が試薬及び装置に関する技術が十分確立されている点でより好ましい。 In the method of the present invention, the method for measuring the amount of NT-TFPI2 and/or the amount of intact TFPI2 is not particularly limited. For example, a method using an antigen-antibody reaction using an antibody that recognizes NT-TFPI2 and/or intact TFPI2, and a method using mass spectrometry can be exemplified.
(a) A competitive method in which a labeled target to be measured and an antibody that recognizes the target to be measured are used, and the labeled target to be measured and the target to be measured contained in a sample competitively bind to the antibody.
(b) A method using surface plasmon resonance, in which a sample is brought into contact with a chip on which an antibody that recognizes an object to be measured is immobilized, and a signal dependent on binding between the antibody and the object to be measured is detected.
(c) A fluorescence polarization immunoassay method that uses a fluorescence-labeled antibody that recognizes a target to be measured, and utilizes an increase in the degree of fluorescence polarization due to binding between the antibody and the target to be measured.
(d) Using two types of antibodies (one of which is a labeled antibody) having different antigenic determinants, which are antibodies that recognize the target to be measured, to form a tripartite complex between the two antibodies and the target to be measured sandwich method.
(e) A method of concentrating the analyte in the specimen with an antibody that recognizes the analyte as a pretreatment, and then detecting the polypeptide of the binding protein using a mass spectrometer or the like.
The methods (d) and (e) are simple and highly versatile, but the method (d) is more preferable from the viewpoint of the well-established techniques for reagents and devices for processing multiple specimens.
抗原抗体反応を利用してＮＴ－ＴＦＰＩ２量及び／又はインタクトＴＦＰＩ２量を測定する方法は、具体的に以下のものが挙げられる。
（Ａ）ＮＴ－ＴＦＰＩ２とインタクトＴＦＰＩ２の両方を認識する抗体を用いて、ＮＴ－ＴＦＰＩ２及びインタクトＴＦＰＩ２の合計量を測定する方法（ＮＴ＋Ｉ－ＴＦＰＩ２測定系）。なお、前記ＮＴ－ＴＦＰＩ２とインタクトＴＦＰＩ２の両方を認識する抗体は、配列番号１で表されるＴＦＰＩ２アミノ酸配列の２３残基目のアスパラギン酸から１３１残基目のヒスチジン又は１３０残基目のシステインまでの領域内の抗原決定基に結合する抗体であることが好ましく、ＴＦＰＩ２のクニッツドメイン１内の抗原決定基に結合する抗原認識部位を有する抗体であることがさらに好ましい。また、この方法で前述したサンドイッチ法を用いる場合は、通常、前記抗体は抗原決定基の異なる２種類を用いる。 Specific methods for measuring the amount of NT-TFPI2 and/or the amount of intact TFPI2 using an antigen-antibody reaction include the following.
(A) A method of measuring the total amount of NT-TFPI2 and intact TFPI2 using an antibody that recognizes both NT-TFPI2 and intact TFPI2 (NT+I-TFPI2 measurement system). The antibody that recognizes both NT-TFPI2 and intact TFPI2 is from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue in the TFPI2 amino acid sequence represented by SEQ ID NO: 1. Preferably, it is an antibody that binds to an antigenic determinant within the region of , more preferably an antibody that has an antigen recognition site that binds to an antigenic determinant within Kunitz domain 1 of TFPI2. In addition, when the sandwich method described above is used in this method, two types of antibodies having different antigenic determinants are usually used.
（Ａ）ＮＴ－ＴＦＰＩ２とインタクトＴＦＰＩ２の両方を認識する抗体を用いて、ＮＴ－ＴＦＰＩ２及びインタクトＴＦＰＩ２の合計量を測定する方法（ＮＴ＋Ｉ－ＴＦＰＩ２測定系）。なお、前記ＮＴ－ＴＦＰＩ２とインタクトＴＦＰＩ２の両方を認識する抗体は、配列番号１で表されるＴＦＰＩ２アミノ酸配列の２３残基目のアスパラギン酸から１３１残基目のヒスチジン又は１３０残基目のシステインまでの領域内の抗原決定基に結合する抗体であることが好ましく、ＴＦＰＩ２のクニッツドメイン１内の抗原決定基に結合する抗原認識部位を有する抗体であることがさらに好ましい。また、この方法で前述したサンドイッチ法を用いる場合は、通常、前記抗体は抗原決定基の異なる２種類を用いる。 Specific methods for measuring the amount of NT-TFPI2 and/or the amount of intact TFPI2 using an antigen-antibody reaction include the following.
(A) A method of measuring the total amount of NT-TFPI2 and intact TFPI2 using an antibody that recognizes both NT-TFPI2 and intact TFPI2 (NT+I-TFPI2 measurement system). The antibody that recognizes both NT-TFPI2 and intact TFPI2 is from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue in the TFPI2 amino acid sequence represented by SEQ ID NO: 1. Preferably, it is an antibody that binds to an antigenic determinant within the region of , more preferably an antibody that has an antigen recognition site that binds to an antigenic determinant within Kunitz domain 1 of TFPI2. In addition, when the sandwich method described above is used in this method, two types of antibodies having different antigenic determinants are usually used.
（Ｂ）ＮＴ－ＴＦＰＩ２を認識せずインタクトＴＦＰＩ２を認識する抗体を用いて、インタクトＴＦＰＩ２単独の量を測定する方法（Ｉ－ＴＦＰＩ２測定系）。なお、前記ＮＴ－ＴＦＰＩ２を認識せずインタクトＴＦＰＩ２を認識する抗体は、ＴＦＰＩ２のクニッツドメイン３内の抗原決定基に結合する抗原認識部位を有する抗体であること好ましい。また、この方法で前述したサンドイッチ法を用いる場合は、通常、前記抗体は抗原決定基の異なる２種類を用い、うち少なくとも１種類はＮＴ－ＴＦＰＩ２を認識せずインタクトＴＦＰＩ２を認識する抗体を用い、もう１種類はＮＴ－ＴＦＰＩ２を認識せずインタクトＴＦＰＩ２を認識する抗体であってもＮＴ－ＴＦＰＩ２とインタクトＴＦＰＩ２の両方を認識する抗体であってもよい。
(B) A method of measuring the amount of intact TFPI2 alone using an antibody that recognizes intact TFPI2 but does not recognize NT-TFPI2 (I-TFPI2 measurement system). The antibody that recognizes intact TFPI2 but does not recognize NT-TFPI2 is preferably an antibody that has an antigen recognition site that binds to an antigenic determinant within Kunitz domain 3 of TFPI2. In addition, when using the sandwich method described above in this method, usually two types of antibodies with different antigenic determinants are used, at least one of which does not recognize NT-TFPI2 but uses an antibody that recognizes intact TFPI2, Another type may be an antibody that recognizes intact TFPI2 without recognizing NT-TFPI2, or an antibody that recognizes both NT-TFPI2 and intact TFPI2.
（Ｃ）（Ａ）のＮＴ＋Ｉ－ＴＦＰＩ２測定系で測定したＮＴ－ＴＦＰＩ２及びインタクトＴＦＰＩ２の合計量から、（Ｂ）のＩ－ＴＦＰＩ２測定系で測定したインタクトＴＦＰＩ２単独量を減じることにより、ＮＴ－ＴＦＰＩ２単独の量を算出する方法。
(C) From the total amount of NT-TFPI2 and intact TFPI2 measured by the NT + I-TFPI2 measurement system in (A), by subtracting the single amount of intact TFPI2 measured by the I-TFPI2 measurement system in (B), NT-TFPI2 How to calculate a single quantity.
（Ｄ）インタクトＴＦＰＩ２を認識せずＮＴ－ＴＦＰＩ２を認識する抗体を用いて、ＮＴ－ＴＦＰＩ２単独の量を測定する方法。なお、前記インタクトＴＦＰＩ２を認識せずＮＴ－ＴＦＰＩ２を認識する抗体は、例えば、ＮＴ－ＴＦＰＩ２のＣ末端部分のペプチド配列を特異的に認識する抗体が挙げられる。前述したサンドイッチ法を用いる場合は、例えば、当該抗体を固相抗体とし、クニッツドメイン１に認識部位を有する抗体を検出抗体とする。
本発明の血栓塞栓症を検出する方法においては、前述した（Ｃ）や（Ｄ）の方法で測定したＮＴ－ＴＦＰＩ２単独の量を判定の基準に用いてもよいが、（Ａ）の方法で測定したＮＴ－ＴＦＰＩ２及びインタクトＴＦＰＩ２の合計量を判定の基準に用いても十分な感度と特異度が得られるうえ、抗体の取得しやすさや測定が一段階で簡便なことから、後者がより好ましい。 (D) A method of measuring the amount of NT-TFPI2 alone using an antibody that recognizes NT-TFPI2 but does not recognize intact TFPI2. The antibody that recognizes NT-TFPI2 but does not recognize intact TFPI2 includes, for example, an antibody that specifically recognizes the peptide sequence of the C-terminal portion of NT-TFPI2. When the sandwich method described above is used, for example, the antibody is used as a solid-phase antibody, and an antibody having a recognition site in Kunitz domain 1 is used as a detection antibody.
In the method of detecting thromboembolism of the present invention, the amount of NT-TFPI2 alone measured by the method (C) or (D) described above may be used as a criterion for determination, but the method (A) The latter is more preferable because sufficient sensitivity and specificity can be obtained even if the total amount of measured NT-TFPI2 and intact TFPI2 is used as a criterion for determination, and the antibody can be easily obtained and the measurement is simple in one step. .
本発明の血栓塞栓症を検出する方法においては、前述した（Ｃ）や（Ｄ）の方法で測定したＮＴ－ＴＦＰＩ２単独の量を判定の基準に用いてもよいが、（Ａ）の方法で測定したＮＴ－ＴＦＰＩ２及びインタクトＴＦＰＩ２の合計量を判定の基準に用いても十分な感度と特異度が得られるうえ、抗体の取得しやすさや測定が一段階で簡便なことから、後者がより好ましい。 (D) A method of measuring the amount of NT-TFPI2 alone using an antibody that recognizes NT-TFPI2 but does not recognize intact TFPI2. The antibody that recognizes NT-TFPI2 but does not recognize intact TFPI2 includes, for example, an antibody that specifically recognizes the peptide sequence of the C-terminal portion of NT-TFPI2. When the sandwich method described above is used, for example, the antibody is used as a solid-phase antibody, and an antibody having a recognition site in Kunitz domain 1 is used as a detection antibody.
In the method of detecting thromboembolism of the present invention, the amount of NT-TFPI2 alone measured by the method (C) or (D) described above may be used as a criterion for determination, but the method (A) The latter is more preferable because sufficient sensitivity and specificity can be obtained even if the total amount of measured NT-TFPI2 and intact TFPI2 is used as a criterion for determination, and the antibody can be easily obtained and the measurement is simple in one step. .
ＮＴ－ＴＦＰＩ２及び／又はインタクトＴＦＰＩ２を認識する抗体は、ＮＴ－ＴＦＰＩ２ポリペプチドまたはタンパク質、インタクトＴＦＰＩ２ポリペプチド又はＴＦＰＩ２タンパク質の部分領域からなるオリゴペプチド、ＮＴ－ＴＦＰＩ２ポリペプチド又はＴＦＰＩ２タンパク質のインタクトまたは部分領域をコードするポリヌクレオチドなどを免疫原として、動物に免疫することで得ることができる。前記タンパク質または前記オリゴペプチドやポリペプチドは生体内のＴＦＰＩ２の立体構造を反映していない、あるいは調製する過程でその構造が変化する可能性がある。そのため、得られた抗体が、所望の生体内のＴＦＰＩ２に対して高い特異性や結合力を有さない可能性があり、本抗体を用いて測定系を構築しても結果として検体中に含まれるＴＦＰＩ２濃度を正確に定量できなくなる可能性がある。
An antibody that recognizes NT-TFPI2 and/or intact TFPI2 is an NT-TFPI2 polypeptide or protein, an oligopeptide consisting of an intact TFPI2 polypeptide or a partial region of a TFPI2 protein, an intact or partial region of an NT-TFPI2 polypeptide or a TFPI2 protein can be obtained by immunizing an animal with a polynucleotide or the like that encodes as an immunogen. The protein, oligopeptide or polypeptide does not reflect the tertiary structure of TFPI2 in vivo, or the structure may change during the preparation process. Therefore, the antibody obtained may not have high specificity or binding strength to the desired in vivo TFPI2, and even if a measurement system is constructed using this antibody, it may not be included in the sample. It may not be possible to accurately quantify the TFPI2 concentration in the
一方、免疫原としてＴＦＰＩ２ポリペプチド又はインタクトＴＦＰＩ２タンパク質のインタクトまたは部分領域をコードするポリヌクレオチドを含む発現ベクターを用いることで、免疫動物の体内でＴＦＰＩ２ポリペプチド又はインタクトＴＦＰＩ２タンパク質のインタクトまたは部分領域が発現され免疫応答が惹起されるため、検体中のＴＦＰＩ２に対して高い特異性及び結合力（すなわち高親和性）を有した抗体が得られるためより好ましい。
On the other hand, by using an expression vector containing a polynucleotide encoding an intact or partial region of a TFPI2 polypeptide or intact TFPI2 protein as an immunogen, the intact or partial region of the TFPI2 polypeptide or intact TFPI2 protein is expressed in the body of the immunized animal. This is more preferable because an immune response is induced and an antibody with high specificity and binding strength (that is, high affinity) to TFPI2 in the specimen can be obtained.
免疫に用いる動物は、抗体産生能を有するものであれば特に限定はなく、マウス、ラット、ウサギなど通常免疫に用いる哺乳動物でもよいし、ニワトリなど鳥類を用いてもよい。
さらに、血中にはＴＦＰＩ２の相同体として知られるＴＦＰＩ１も存在する。したがって、ＴＦＰＩ１と交叉せずＴＦＰＩ２のみを特異的に認識する抗体を用いることが望ましい。 Animals used for immunization are not particularly limited as long as they have the ability to produce antibodies. Mammals such as mice, rats and rabbits, which are commonly used for immunization, and birds such as chickens may be used.
Furthermore, TFPI1 known as a homologue of TFPI2 is also present in blood. Therefore, it is desirable to use an antibody that specifically recognizes only TFPI2 without crossing with TFPI1.
さらに、血中にはＴＦＰＩ２の相同体として知られるＴＦＰＩ１も存在する。したがって、ＴＦＰＩ１と交叉せずＴＦＰＩ２のみを特異的に認識する抗体を用いることが望ましい。 Animals used for immunization are not particularly limited as long as they have the ability to produce antibodies. Mammals such as mice, rats and rabbits, which are commonly used for immunization, and birds such as chickens may be used.
Furthermore, TFPI1 known as a homologue of TFPI2 is also present in blood. Therefore, it is desirable to use an antibody that specifically recognizes only TFPI2 without crossing with TFPI1.
ＴＦＰＩ２を認識する抗体は、モノクローナル抗体であってもよく、ポリクローナル抗体であってもよいが、モノクローナル抗体であるのが好ましい。
An antibody that recognizes TFPI2 may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
ＴＦＰＩ２を認識する抗体を産生するハイブリドーマ細胞の樹立は、技術が確立された方法の中から適宜選択して行えばよい。一例として、前述した方法で免疫した動物からＢ細胞を採取し、前記Ｂ細胞とミエローマ細胞とを電気的にまたはポリエチレングリコール存在下で融合させ、ＨＡＴ培地により所望の抗体を産生するハイブリドーマ細胞の選択を行い、選択したハイブリドーマ細胞を限界希釈法によりモノクローン化を行うことで、ＴＦＰＩ２を認識するモノクローナル抗体を産生するハイブリドーマ細胞を樹立することができる。
The establishment of hybridoma cells that produce antibodies that recognize TFPI2 can be appropriately selected from methods with established techniques. As an example, B cells are collected from an animal immunized by the method described above, the B cells and myeloma cells are fused electrically or in the presence of polyethylene glycol, and hybridoma cells that produce the desired antibody are selected using HAT medium. and monocloning the selected hybridoma cells by the limiting dilution method to establish hybridoma cells that produce a monoclonal antibody that recognizes TFPI2.
本発明で用いるＴＦＰＩ２を認識するモノクローナル抗体の選定は、宿主発現系に由来する、ＧＰＩ（ｇｌｙｃｏｓｙｌｐｈｏｓｐｈａｔｉｄｙｌｉｎｏｓｉｔｏｌ）アンカー型ＴＦＰＩ２または分泌型ＴＦＰＩ２に対する親和性に基づいて行えばよい。
A monoclonal antibody that recognizes TFPI2 used in the present invention may be selected based on its affinity for GPI (glycosylphosphotidylinositol)-anchored TFPI2 or secretory TFPI2 derived from the host expression system.
なお、前記宿主としては特に限定はなく、当業者がタンパク質の発現に通常用いる、大腸菌や酵母などの微生物細胞、昆虫細胞、動物細胞の中から適宜選択すればよいが、ジスルフィド結合もしくは糖鎖付加といった翻訳後修飾により、天然型のＴＦＰＩ２に近い構造を有するタンパク質の発現が可能な、哺乳細胞を宿主として用いると好ましい。哺乳細胞の一例としては、従来用いられている、ヒト胎児腎臓由来細胞（ＨＥＫ）２９３Ｔ細胞株、サル腎臓細胞ＣＯＳ７株、チャイニーズハムスター卵巣（ＣＨＯ）細胞またはヒトから単離されたがん細胞などが挙げられる。
The host is not particularly limited, and may be appropriately selected from microbial cells such as Escherichia coli and yeast, insect cells, and animal cells that are commonly used for protein expression by those skilled in the art. It is preferable to use mammalian cells as hosts, which are capable of expressing a protein having a structure similar to that of native TFPI2 by post-translational modifications such as TFPI2. Examples of mammalian cells include conventionally used human embryonic kidney-derived cell (HEK) 293T cell line, monkey kidney cell line COS7, Chinese hamster ovary (CHO) cells, cancer cells isolated from humans, and the like. mentioned.
本発明で用いられる抗体の精製は、技術が確立された方法の中から適宜選択して行えばよい。一例として、前述した方法で樹立した、抗体を産生するハイブリドーマ細胞を培養後、その培養上清を回収し、必要に応じ硫酸アンモニウム沈殿による抗体濃縮後、プロテインＡ、プロテインＧ、またはプロテインＬなどを固定化した担体を用いたアフィニティークロマトグラフィー及び／またはイオン交換クロマトグラフィーにより、抗体の精製が可能である。
Purification of the antibody used in the present invention may be carried out by appropriately selecting from established techniques. As an example, after culturing the antibody-producing hybridoma cells established by the method described above, the culture supernatant is collected, and if necessary, the antibody is concentrated by ammonium sulfate precipitation, and protein A, protein G, or protein L is immobilized. Antibodies can be purified by affinity chromatography and/or ion-exchange chromatography using a fused carrier.
なお、前述したサンドイッチ法で抗原抗体反応を行う際に用いる標識した抗体は、前述した方法で精製した抗体をペルオキシダーゼやアルカリ性ホスファターゼなどの酵素で標識すればよく、その標識も技術が十分確立された方法を用いて行えばよい。
The labeled antibody used in the antigen-antibody reaction by the sandwich method described above can be obtained by labeling the antibody purified by the method described above with an enzyme such as peroxidase or alkaline phosphatase, and the labeling technology has been well established. method.
本発明の方法において、質量分析法を利用してＴＦＰＩ２量を測定する方法について、以下に具体的に説明する。
検体が血液である場合は、前処理工程として血液に多く含まれるアルブミン、イムノグロブリン、トランスフェリン等の主要タンパク質をＡｇｉｌｅｎｔ Ｈｕｍａｎ １４等で除去した後、イオン交換、ゲル濾過または逆相ＨＰＬＣ等でさらに分画することが好ましい。または、抗ＴＦＰＩ２抗体を用いた免疫的手法によりＴＦＰＩ２のみを特異的に回収することも可能である。 A method for measuring the amount of TFPI2 using mass spectrometry in the method of the present invention will be specifically described below.
When the specimen is blood, as a pretreatment step, major proteins such as albumin, immunoglobulin, and transferrin, which are abundant in blood, are removed withAgilent Human 14 or the like, and then further separated by ion exchange, gel filtration, reversed-phase HPLC, or the like. It is preferable to draw Alternatively, it is also possible to specifically collect only TFPI2 by an immunological technique using an anti-TFPI2 antibody.
検体が血液である場合は、前処理工程として血液に多く含まれるアルブミン、イムノグロブリン、トランスフェリン等の主要タンパク質をＡｇｉｌｅｎｔ Ｈｕｍａｎ １４等で除去した後、イオン交換、ゲル濾過または逆相ＨＰＬＣ等でさらに分画することが好ましい。または、抗ＴＦＰＩ２抗体を用いた免疫的手法によりＴＦＰＩ２のみを特異的に回収することも可能である。 A method for measuring the amount of TFPI2 using mass spectrometry in the method of the present invention will be specifically described below.
When the specimen is blood, as a pretreatment step, major proteins such as albumin, immunoglobulin, and transferrin, which are abundant in blood, are removed with
測定は、タンデム質量分析（ＭＳ／ＭＳ）、液体クロマトグラフィ・タンデム質量分析（ＬＣ／ＭＳ／ＭＳ）、マトリックス支援レーザー脱離イオン化飛行時間型質量分析（ｍａｔｒｉｘ ａｓｓｉｓｔｅｄ ｌａｓｅｒ ｄｅｓｏｒｐｔｉｏｎ ｉｏｎｉｚａｔｉｏｎ ｔｉｍｅ－ｏｆ－ｆｌｉｇｈｔ ｍａｓｓ ｓｐｅｃｔｒｏｍｅｔｒｙ、ＭＡＬＤＩ－ＴＯＦ／ＭＳ）、表面増強レーザーイオン化質量分析（ｓｕｒｆａｃｅ ｅｎｈａｎｃｅｄ ｌａｓｅｒ ｄｅｓｏｒｐｔｉｏｎ ｉｏｎｉｚａｔｉｏｎ ｍａｓｓ ｓｐｅｃｔｒｏｍｅｔｒｙ、ＳＥＬＤＩ－ＭＳ）等により行うことができる。
Measurements were performed by tandem mass spectrometry (MS/MS), liquid chromatography-tandem mass spectrometry (LC/MS/MS), matrix assisted laser desorption ionization time-of-flight mass spectrometry. , MALDI-TOF/MS), surface enhanced laser desorption ionization mass spectrometry (SELDI-MS), and the like.
本発明の悪性腫瘍関連血栓塞栓症を検出する方法は、悪性腫瘍関連血栓塞栓症を治療する方法に適用することができる。すなわち、本発明により、被験体における悪性腫瘍関連血栓塞栓症を治療する方法であって、
（ｉ）ＴＦＰＩ２量の測定値が予め設定した基準値を超えるものとして被験体の同定を受ける工程、及び
（ｉｉ）ＴＦＰＩ２量の測定値が予め設定した基準値を超えるものとして同定された前記被験体に対して、治療を施す工程
を含む、方法が提供される。 The method for detecting malignant tumor-related thromboembolism of the present invention can be applied to a method for treating malignant tumor-related thromboembolism. Thus, according to the present invention, a method of treating malignancy-associated thromboembolism in a subject, comprising:
(i) identifying a subject as having a measured TFPI2 amount that exceeds a preset reference value; and (ii) said subject identified as having a measured TFPI2 amount that exceeds a preset reference value. A method is provided that includes administering a treatment to a body.
（ｉ）ＴＦＰＩ２量の測定値が予め設定した基準値を超えるものとして被験体の同定を受ける工程、及び
（ｉｉ）ＴＦＰＩ２量の測定値が予め設定した基準値を超えるものとして同定された前記被験体に対して、治療を施す工程
を含む、方法が提供される。 The method for detecting malignant tumor-related thromboembolism of the present invention can be applied to a method for treating malignant tumor-related thromboembolism. Thus, according to the present invention, a method of treating malignancy-associated thromboembolism in a subject, comprising:
(i) identifying a subject as having a measured TFPI2 amount that exceeds a preset reference value; and (ii) said subject identified as having a measured TFPI2 amount that exceeds a preset reference value. A method is provided that includes administering a treatment to a body.
前記悪性腫瘍関連血栓塞栓症を治療する方法の好ましい態様において、前記ＴＦＰＩ２量は、ＴＦＰＩ２プロセシングポリペプチド量及びインタクトＴＦＰＩ２量の合計である。
また、前記工程（ｉ）の同定において、ＴＦＰＩ２量の測定は、好ましくは、配列番号１のアミノ酸配列の２３残基目のアスパラギン酸から１３１残基目のヒスチジン又は１３０残基目のシステインまでの領域内の抗原決定基に結合する抗体を用いた抗原抗体反応により行われる。より好ましくは、前記抗体は、ＴＦＰＩ２のクニッツドメイン１を認識する抗体である。 In a preferred embodiment of the method of treating said malignant tumor-associated thromboembolism, said amount of TFPI2 is the sum of the amount of TFPI2 processing polypeptide and the amount of intact TFPI2.
In addition, in the identification of the step (i), the amount of TFPI2 is preferably measured from the 23rd residue aspartic acid to the 131st residue histidine or the 130th residue cysteine in the amino acid sequence of SEQ ID NO: 1. It is performed by an antigen-antibody reaction using an antibody that binds to an antigenic determinant within the region. More preferably, said antibody is an antibody that recognizes Kunitz domain 1 of TFPI2.
また、前記工程（ｉ）の同定において、ＴＦＰＩ２量の測定は、好ましくは、配列番号１のアミノ酸配列の２３残基目のアスパラギン酸から１３１残基目のヒスチジン又は１３０残基目のシステインまでの領域内の抗原決定基に結合する抗体を用いた抗原抗体反応により行われる。より好ましくは、前記抗体は、ＴＦＰＩ２のクニッツドメイン１を認識する抗体である。 In a preferred embodiment of the method of treating said malignant tumor-associated thromboembolism, said amount of TFPI2 is the sum of the amount of TFPI2 processing polypeptide and the amount of intact TFPI2.
In addition, in the identification of the step (i), the amount of TFPI2 is preferably measured from the 23rd residue aspartic acid to the 131st residue histidine or the 130th residue cysteine in the amino acid sequence of SEQ ID NO: 1. It is performed by an antigen-antibody reaction using an antibody that binds to an antigenic determinant within the region. More preferably, said antibody is an antibody that recognizes Kunitz domain 1 of TFPI2.
また、前記工程（ｉ）の同定において、ＴＦＰＩ２量の測定は、質量分析法を用いて行われてもよい。
前記工程（ｉｉ）の治療としては、ヘパリンやワルファリン等の抗凝固薬を用いる抗凝固療法、ウロキナーゼ、組織プラスミノーゲン活性化因子等の血栓溶解剤を用いる血栓溶解療法、血管内カテーテルを用いる血管内治療法、外科手術療法等が挙げられるが特に限定されない。 In addition, in the identification of step (i), the amount of TFPI2 may be measured using mass spectrometry.
The treatment in step (ii) includes anticoagulant therapy using anticoagulants such as heparin and warfarin, thrombolytic therapy using thrombolytic agents such as urokinase and tissue plasminogen activator, and vascular therapy using an intravascular catheter. Examples include, but are not limited to, internal therapy, surgical therapy, and the like.
前記工程（ｉｉ）の治療としては、ヘパリンやワルファリン等の抗凝固薬を用いる抗凝固療法、ウロキナーゼ、組織プラスミノーゲン活性化因子等の血栓溶解剤を用いる血栓溶解療法、血管内カテーテルを用いる血管内治療法、外科手術療法等が挙げられるが特に限定されない。 In addition, in the identification of step (i), the amount of TFPI2 may be measured using mass spectrometry.
The treatment in step (ii) includes anticoagulant therapy using anticoagulants such as heparin and warfarin, thrombolytic therapy using thrombolytic agents such as urokinase and tissue plasminogen activator, and vascular therapy using an intravascular catheter. Examples include, but are not limited to, internal therapy, surgical therapy, and the like.
＜２＞本発明の悪性腫瘍関連血栓塞栓症を検出するための試薬
本発明の試薬を前述したサンドイッチ法に利用する場合は、前記抗体として抗原決定基の異なる２種類の抗体を含むことが好ましい。
本発明の試薬に含まれる抗体は、抗体そのものであってもよく、標識されていてもよく、固相に固定化されていてもよい。 <2> Reagent for detecting malignant tumor-associated thromboembolism of the present invention When the reagent of the present invention is used in the above-described sandwich method, the antibody preferably contains two types of antibodies having different antigenic determinants. .
The antibody contained in the reagent of the present invention may be the antibody itself, labeled, or immobilized on a solid phase.
本発明の試薬を前述したサンドイッチ法に利用する場合は、前記抗体として抗原決定基の異なる２種類の抗体を含むことが好ましい。
本発明の試薬に含まれる抗体は、抗体そのものであってもよく、標識されていてもよく、固相に固定化されていてもよい。 <2> Reagent for detecting malignant tumor-associated thromboembolism of the present invention When the reagent of the present invention is used in the above-described sandwich method, the antibody preferably contains two types of antibodies having different antigenic determinants. .
The antibody contained in the reagent of the present invention may be the antibody itself, labeled, or immobilized on a solid phase.
本発明の試薬のうち、前述したサンドイッチ法の一態様である２ステップサンドイッチ法に利用する場合について、以下に具体的に説明する。ただし、本発明はこれに限定されるものではない。
まず、本発明の試薬は、以下の（Ｉ）から（ＩＩＩ）に示す方法で作製することができる。
（Ｉ）まず、サンドイッチ法で用いる、ＴＦＰＩ２を認識する、抗原決定基の異なる２種類の抗体（以下、「抗体１」及び「抗体２」とする）のうち、抗体１をイムノプレートや磁性粒子等のＢ／Ｆ（Ｂｏｕｎｄ／Ｆｒｅｅ）分離可能な担体に結合させる。結合方法は、疎水結合を利用した物理的結合であってもよいし、２物質間を架橋可能なリンカー試薬などを用いた化学的結合であってもよい。
（ＩＩ）担体に前記抗体１を結合させた後、非特異的結合を避けるため、担体表面を牛血清アルブミン、スキムミルク、市販のイムノアッセイ用ブロッキング剤、タンパク質吸着抑制用化学合成ポリマーなどでブロッキング処理を行い１次試薬とする。
（ＩＩＩ）他方の抗体２を標識し、得られた標識抗体を含む溶液を２次試薬として準備する。抗体２に標識する物質としては、ペルオキシダーゼ、アルカリ性ホスファターゼといった酵素、蛍光物質、化学発光物質、ラジオアイソトープなどの検出装置で検出可能な物質、又はビオチンに対するアビジンなど特異的に結合する相手が存在する物質等が好ましい。また、２次試薬の溶液としては、抗原抗体反応が良好に行える緩衝液、例えばリン酸緩衝液、Ｔｒｉｓ－ＨＣｌ緩衝液などが好ましい。このようにして作製した本発明の試薬は必要に応じ凍結乾燥させてもよい。 Among the reagents of the present invention, the case where they are used in the two-step sandwich method, which is one aspect of the sandwich method described above, will be specifically described below. However, the present invention is not limited to this.
First, the reagent of the present invention can be produced by the methods shown in (I) to (III) below.
(I) First, of the two types of antibodies that recognize TFPI2 and have different antigenic determinants (hereinafter referred to as "antibody 1" and "antibody 2") used in the sandwich method, antibody 1 is used in immunoplates or magnetic particles. B/F (Bound/Free) separable carrier such as. The bonding method may be physical bonding using hydrophobic bonding, or chemical bonding using a linker reagent capable of cross-linking between two substances.
(II) After binding the antibody 1 to the carrier, block the carrier surface with bovine serum albumin, skimmed milk, a commercially available immunoassay blocking agent, a chemically synthesized polymer for inhibiting protein adsorption, etc., in order to avoid non-specific binding. and use it as the primary reagent.
(III) Theother antibody 2 is labeled, and a solution containing the resulting labeled antibody is prepared as a secondary reagent. Substances for labeling the antibody 2 include enzymes such as peroxidase and alkaline phosphatase, fluorescent substances, chemiluminescent substances, radioisotopes, and other substances that can be detected by a detection device, or substances that have a specific binding partner, such as avidin for biotin. etc. are preferred. Further, as the secondary reagent solution, a buffer solution in which an antigen-antibody reaction can be favorably performed, such as a phosphate buffer solution or a Tris-HCl buffer solution, is preferable. The reagent of the present invention thus prepared may be lyophilized if necessary.
まず、本発明の試薬は、以下の（Ｉ）から（ＩＩＩ）に示す方法で作製することができる。
（Ｉ）まず、サンドイッチ法で用いる、ＴＦＰＩ２を認識する、抗原決定基の異なる２種類の抗体（以下、「抗体１」及び「抗体２」とする）のうち、抗体１をイムノプレートや磁性粒子等のＢ／Ｆ（Ｂｏｕｎｄ／Ｆｒｅｅ）分離可能な担体に結合させる。結合方法は、疎水結合を利用した物理的結合であってもよいし、２物質間を架橋可能なリンカー試薬などを用いた化学的結合であってもよい。
（ＩＩ）担体に前記抗体１を結合させた後、非特異的結合を避けるため、担体表面を牛血清アルブミン、スキムミルク、市販のイムノアッセイ用ブロッキング剤、タンパク質吸着抑制用化学合成ポリマーなどでブロッキング処理を行い１次試薬とする。
（ＩＩＩ）他方の抗体２を標識し、得られた標識抗体を含む溶液を２次試薬として準備する。抗体２に標識する物質としては、ペルオキシダーゼ、アルカリ性ホスファターゼといった酵素、蛍光物質、化学発光物質、ラジオアイソトープなどの検出装置で検出可能な物質、又はビオチンに対するアビジンなど特異的に結合する相手が存在する物質等が好ましい。また、２次試薬の溶液としては、抗原抗体反応が良好に行える緩衝液、例えばリン酸緩衝液、Ｔｒｉｓ－ＨＣｌ緩衝液などが好ましい。このようにして作製した本発明の試薬は必要に応じ凍結乾燥させてもよい。 Among the reagents of the present invention, the case where they are used in the two-step sandwich method, which is one aspect of the sandwich method described above, will be specifically described below. However, the present invention is not limited to this.
First, the reagent of the present invention can be produced by the methods shown in (I) to (III) below.
(I) First, of the two types of antibodies that recognize TFPI2 and have different antigenic determinants (hereinafter referred to as "antibody 1" and "
(II) After binding the antibody 1 to the carrier, block the carrier surface with bovine serum albumin, skimmed milk, a commercially available immunoassay blocking agent, a chemically synthesized polymer for inhibiting protein adsorption, etc., in order to avoid non-specific binding. and use it as the primary reagent.
(III) The
なお、１ステップサンドイッチ法の場合は、前述した（Ｉ）～（ＩＩ）同様に担体に抗体１を結合させブロッキング処理を行ったものを作製し、前記抗体固定化担体に、標識した抗体２を含む緩衝液をさらに添加して試薬を作製すればよい。
In the case of the one-step sandwich method, antibody 1 is bound to the carrier and subjected to blocking treatment in the same manner as in (I) to (II) described above, and labeled antibody 2 is applied to the antibody-immobilized carrier. A reagent may be prepared by further adding a buffer solution containing the above.
次に、前述した方法で得られた試薬を用いて、２ステップサンドイッチ法でＴＦＰＩ２を検出し測定するには、以下の（ＩＶ）から（ＶＩ）に示す方法で行えばよい。
（ＩＶ）（ＩＩ）で作製した１次試薬と検体とを一定時間、一定温度のもと接触させる。反応条件は、温度４℃から４０℃の範囲で、５分から１８０分間反応させればよい。
（Ｖ）未反応物質をＢ／Ｆ分離により除去し、続いて（ＩＩＩ）で作製した２次試薬と一定時間、一定温度のもと接触させ、サンドイッチ複合体を形成させる。反応条件は、温度４℃から４０℃の範囲で、５分から１８０分間反応させればよい。
（ＶＩ）未反応物質をＢ／Ｆ分離により除去し、標識抗体の標識物質を定量し、既知濃度のＴＦＰＩ２溶液を標準とし作成した検量線により、検体中のヒトＴＦＰＩ２を定量する。
本発明の試薬に含まれる抗体等の試薬成分の量は、検体量、検体の種類、試薬の種類、測定の手法等の諸条件に応じて適宜設定すればよい。具体的には、例えば、後述するように検体として血清や血漿を２０μＬ使用して、サンドイッチ法によりＴＦＰＩ２量の測定を行う場合、当該検体２０μＬを抗体と反応させる反応系当たり、担体へ結合させる抗体量が１００ｎｇから１０００μｇであってよく、標識抗体量が２ｎｇから２０μｇであってよい。 Next, detection and measurement of TFPI2 by the two-step sandwich method using the reagent obtained by the above-described method may be carried out by the following methods (IV) to (VI).
(IV) The primary reagent prepared in (II) and the specimen are brought into contact with each other for a certain period of time under a certain temperature. As for the reaction conditions, the temperature may range from 4° C. to 40° C. and the reaction may be carried out for 5 minutes to 180 minutes.
(V) Unreacted substances are removed by B/F separation, followed by contact with the secondary reagent prepared in (III) for a certain period of time under a certain temperature to form a sandwich complex. As for the reaction conditions, the temperature may range from 4° C. to 40° C. and the reaction may be carried out for 5 minutes to 180 minutes.
(VI) Unreacted substances are removed by B/F separation, the labeled substance of the labeled antibody is quantified, and human TFPI2 in the specimen is quantified using a calibration curve prepared using TFPI2 solutions of known concentrations as standards.
The amount of reagent components such as antibodies contained in the reagent of the present invention may be appropriately set according to various conditions such as the amount of specimen, the type of specimen, the type of reagent, and the method of measurement. Specifically, for example, when using 20 μL of serum or plasma as a specimen as described later and measuring the amount of TFPI2 by the sandwich method, the antibody bound to the carrier per reaction system in which 20 μL of the specimen is reacted with the antibody The amount may be 100 ng to 1000 μg and the amount of labeled antibody may be 2 ng to 20 μg.
（ＩＶ）（ＩＩ）で作製した１次試薬と検体とを一定時間、一定温度のもと接触させる。反応条件は、温度４℃から４０℃の範囲で、５分から１８０分間反応させればよい。
（Ｖ）未反応物質をＢ／Ｆ分離により除去し、続いて（ＩＩＩ）で作製した２次試薬と一定時間、一定温度のもと接触させ、サンドイッチ複合体を形成させる。反応条件は、温度４℃から４０℃の範囲で、５分から１８０分間反応させればよい。
（ＶＩ）未反応物質をＢ／Ｆ分離により除去し、標識抗体の標識物質を定量し、既知濃度のＴＦＰＩ２溶液を標準とし作成した検量線により、検体中のヒトＴＦＰＩ２を定量する。
本発明の試薬に含まれる抗体等の試薬成分の量は、検体量、検体の種類、試薬の種類、測定の手法等の諸条件に応じて適宜設定すればよい。具体的には、例えば、後述するように検体として血清や血漿を２０μＬ使用して、サンドイッチ法によりＴＦＰＩ２量の測定を行う場合、当該検体２０μＬを抗体と反応させる反応系当たり、担体へ結合させる抗体量が１００ｎｇから１０００μｇであってよく、標識抗体量が２ｎｇから２０μｇであってよい。 Next, detection and measurement of TFPI2 by the two-step sandwich method using the reagent obtained by the above-described method may be carried out by the following methods (IV) to (VI).
(IV) The primary reagent prepared in (II) and the specimen are brought into contact with each other for a certain period of time under a certain temperature. As for the reaction conditions, the temperature may range from 4° C. to 40° C. and the reaction may be carried out for 5 minutes to 180 minutes.
(V) Unreacted substances are removed by B/F separation, followed by contact with the secondary reagent prepared in (III) for a certain period of time under a certain temperature to form a sandwich complex. As for the reaction conditions, the temperature may range from 4° C. to 40° C. and the reaction may be carried out for 5 minutes to 180 minutes.
(VI) Unreacted substances are removed by B/F separation, the labeled substance of the labeled antibody is quantified, and human TFPI2 in the specimen is quantified using a calibration curve prepared using TFPI2 solutions of known concentrations as standards.
The amount of reagent components such as antibodies contained in the reagent of the present invention may be appropriately set according to various conditions such as the amount of specimen, the type of specimen, the type of reagent, and the method of measurement. Specifically, for example, when using 20 μL of serum or plasma as a specimen as described later and measuring the amount of TFPI2 by the sandwich method, the antibody bound to the carrier per reaction system in which 20 μL of the specimen is reacted with the antibody The amount may be 100 ng to 1000 μg and the amount of labeled antibody may be 2 ng to 20 μg.
本発明の試薬は、用手法での測定にも利用可能であり、自動免疫診断装置を用いた測定にも利用可能である。特に自動免疫診断装置を用いた測定は、検体中に含まれる内在性の測定妨害因子や競合酵素の影響を受けることなく測定が可能で、かつ短時間に検体中のＴＦＰＩ２が定量可能であるため、好ましい。
The reagent of the present invention can be used for manual measurement, and can also be used for measurement using an automatic immunodiagnostic device. In particular, measurement using an automated immunodiagnostic device can be performed without being affected by endogenous measurement-interfering factors or competing enzymes contained in the sample, and TFPI2 in the sample can be quantified in a short time. ,preferable.
本発明の別の側面は、ＴＦＰＩ２量を測定する試薬の、悪性腫瘍関連血栓塞栓症を検出するための試薬の製造における使用である。ここで、前記ＴＦＰＩ２量を測定する試薬は、ＴＦＰＩ２プロセシングポリペプチド量及びインタクトＴＦＰＩ２量の合計を測定する試薬であることが好ましい。また、前記ＴＦＰＩ２量を測定する試薬は、好ましくは配列番号１のアミノ酸配列の２３残基目のアスパラギン酸から１３１残基目のヒスチジン又は１３０残基目のシステインまでの領域内の抗原決定基に結合する抗体であり、より好ましくはＴＦＰＩ２のクニッツドメイン１を認識する抗体である。
Another aspect of the present invention is the use of a reagent that measures the amount of TFPI2 in the manufacture of a reagent for detecting malignant tumor-associated thromboembolism. Here, the reagent for measuring the amount of TFPI2 is preferably a reagent for measuring the sum of the amount of TFPI2 processed polypeptide and the amount of intact TFPI2. In addition, the reagent for measuring the amount of TFPI2 is preferably an antigenic determinant in the region from aspartic acid at residue 23 to histidine at residue 131 or cysteine at residue 130 in the amino acid sequence of SEQ ID NO: 1. It is an antibody that binds, more preferably an antibody that recognizes Kunitz domain 1 of TFPI2.
従って、本発明は、配列番号１に示すアミノ酸配列の２３残基目のアスパラギン酸から１３１残基目のヒスチジン又は１３０残基目のシステインまでの領域内の抗原決定基に結合する抗体の、悪性腫瘍関連血栓塞栓症を検出するための試薬の製造における使用ともいうことができる。
Therefore, the present invention provides a malignancy of an antibody that binds to an antigenic determinant in the region from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue in the amino acid sequence shown in SEQ ID NO:1. It can also be referred to as use in the manufacture of reagents for detecting tumor-associated thromboembolism.
また、本発明は、配列番号１に示すアミノ酸配列の２３残基目のアスパラギン酸から１３１残基目のヒスチジン又は１３０残基目のシステインまでの領域内の抗原決定基に結合する抗体の、悪性腫瘍関連血栓塞栓症の検出における使用ともいうことができる。
In addition, the present invention provides an antibody that binds to an antigenic determinant in the region from aspartic acid at the 23rd residue to histidine at the 131st residue or cysteine at the 130th residue in the amino acid sequence shown in SEQ ID NO: 1. Use in detecting tumor-associated thromboembolism can also be mentioned.
以下に本発明を具体的に説明するために実施例を示すが、これら実施例は本発明の一例を示すものであり、本発明は実施例に限定されるものではない。
Examples will be shown below to specifically describe the present invention, but these examples show an example of the present invention, and the present invention is not limited to the examples.
＜実施例１＞ ＴＦＰＩ２測定試薬の調製
特許文献３の方法に従い、ＤＮＡ免疫法により得られたＴＦＰＩ２抗体を用いてＴＦＰＩ２測定試薬を以下の通り調製した。
（１）水不溶性フェライト含有担体に抗ＴＦＰＩ２モノクローナル抗体（ＴＳ－ＴＦ０４）を１００ｎｇ／担体になるように室温にて一昼夜物理的に吸着させ、その後１％ＢＳＡを含む１００ｍＭトリス緩衝液（ｐＨ８．０）にて５３℃・４時間ブロッキングを行うことで、抗ＴＦＰＩ２抗体固定化担体を調製した。
（２）抗ＴＦＰＩ２モノクローナル抗体（ＴＳ－ＴＦ０１）をアルカリフォスファターゼ標識キット（同仁化学社製）にて、アルカリフォスファターゼ標識抗ＴＦＰＩ２抗体を調製した。
（３）磁力透過性の容器（容量１．２ｍＬ）に、（１）で調製した１２個の抗体固定化担体を入れた後、（２）で調製したアルカリフォスファターゼ標識抗体を１μｇ／ｍＬ含む緩衝液（３％ＢＳＡを含むトリス緩衝液、ｐＨ８．０）１００μＬを添加し、凍結乾燥を実施することで、ＴＦＰＩ２測定試薬を作製した。なお、作製したＴＦＰＩ２測定試薬は窒素充填下にて密閉封印シールを施し、測定まで４℃で保管した。 <Example 1> Preparation of TFPI2 measurement reagent According to the method ofPatent Document 3, a TFPI2 measurement reagent was prepared as follows using a TFPI2 antibody obtained by a DNA immunization method.
(1) An anti-TFPI2 monoclonal antibody (TS-TF04) was physically adsorbed to a water-insoluble ferrite-containing carrier at room temperature overnight at 100 ng/carrier, and then 100 mM Tris buffer (pH 8.0) containing 1% BSA. ) at 53° C. for 4 hours to prepare an anti-TFPI2 antibody-immobilized carrier.
(2) Alkaline phosphatase-labeled anti-TFPI2 antibody was prepared from anti-TFPI2 monoclonal antibody (TS-TF01) using an alkaline phosphatase labeling kit (manufactured by Dojindo Laboratories).
(3) Place the 12 antibody-immobilized carriers prepared in (1) in a magnetically permeable container (capacity 1.2 mL), and then buffer containing 1 μg / mL of the alkaline phosphatase-labeled antibody prepared in (2) A TFPI2 measurement reagent was prepared by adding 100 μL of a solution (Tris buffer containing 3% BSA, pH 8.0) and freeze-drying. The prepared TFPI2 measurement reagent was hermetically sealed under nitrogen filling and stored at 4° C. until measurement.
特許文献３の方法に従い、ＤＮＡ免疫法により得られたＴＦＰＩ２抗体を用いてＴＦＰＩ２測定試薬を以下の通り調製した。
（１）水不溶性フェライト含有担体に抗ＴＦＰＩ２モノクローナル抗体（ＴＳ－ＴＦ０４）を１００ｎｇ／担体になるように室温にて一昼夜物理的に吸着させ、その後１％ＢＳＡを含む１００ｍＭトリス緩衝液（ｐＨ８．０）にて５３℃・４時間ブロッキングを行うことで、抗ＴＦＰＩ２抗体固定化担体を調製した。
（２）抗ＴＦＰＩ２モノクローナル抗体（ＴＳ－ＴＦ０１）をアルカリフォスファターゼ標識キット（同仁化学社製）にて、アルカリフォスファターゼ標識抗ＴＦＰＩ２抗体を調製した。
（３）磁力透過性の容器（容量１．２ｍＬ）に、（１）で調製した１２個の抗体固定化担体を入れた後、（２）で調製したアルカリフォスファターゼ標識抗体を１μｇ／ｍＬ含む緩衝液（３％ＢＳＡを含むトリス緩衝液、ｐＨ８．０）１００μＬを添加し、凍結乾燥を実施することで、ＴＦＰＩ２測定試薬を作製した。なお、作製したＴＦＰＩ２測定試薬は窒素充填下にて密閉封印シールを施し、測定まで４℃で保管した。 <Example 1> Preparation of TFPI2 measurement reagent According to the method of
(1) An anti-TFPI2 monoclonal antibody (TS-TF04) was physically adsorbed to a water-insoluble ferrite-containing carrier at room temperature overnight at 100 ng/carrier, and then 100 mM Tris buffer (pH 8.0) containing 1% BSA. ) at 53° C. for 4 hours to prepare an anti-TFPI2 antibody-immobilized carrier.
(2) Alkaline phosphatase-labeled anti-TFPI2 antibody was prepared from anti-TFPI2 monoclonal antibody (TS-TF01) using an alkaline phosphatase labeling kit (manufactured by Dojindo Laboratories).
(3) Place the 12 antibody-immobilized carriers prepared in (1) in a magnetically permeable container (capacity 1.2 mL), and then buffer containing 1 μg / mL of the alkaline phosphatase-labeled antibody prepared in (2) A TFPI2 measurement reagent was prepared by adding 100 μL of a solution (Tris buffer containing 3% BSA, pH 8.0) and freeze-drying. The prepared TFPI2 measurement reagent was hermetically sealed under nitrogen filling and stored at 4° C. until measurement.
＜実施例２＞ 臨床検体の測定
後述の実施例３～７で使用した臨床検体の内訳を表１に示す。検体は、Ｄ－ｄｉｍｅｒ １．０ μｇ／ｍＬをカットオフとしてスクリーニングを行い、１．０ μｇ／ｍＬ以上は全例に下肢静脈エコーをおこない血栓の有無を確認した。ＶＴＥ併発卵巣癌患者血清（２５例）と非ＶＴＥ併発卵巣癌患者血清（９７例）は奈良県立医科大学にて同一プロトコルにて収集された検体であり、インフォームドコンセントの承諾及び奈良県立医科大倫理委員会の承認を受けて提供された。 <Example 2> Measurement of clinical samples Table 1 shows the details of the clinical samples used in Examples 3 to 7 below. Specimens were screened with a D-dimer of 1.0 μg/mL as a cutoff, and lower extremity vein echocardiography was performed on all cases with a D-dimer of 1.0 μg/mL or higher to confirm the presence or absence of thrombi. VTE-complicated ovarian cancer patient serum (25 cases) and non-VTE-complicated ovarian cancer patient serum (97 cases) are samples collected under the same protocol at Nara Medical University, consent of informed consent and Nara Medical University Provided with Ethics Committee approval.
後述の実施例３～７で使用した臨床検体の内訳を表１に示す。検体は、Ｄ－ｄｉｍｅｒ １．０ μｇ／ｍＬをカットオフとしてスクリーニングを行い、１．０ μｇ／ｍＬ以上は全例に下肢静脈エコーをおこない血栓の有無を確認した。ＶＴＥ併発卵巣癌患者血清（２５例）と非ＶＴＥ併発卵巣癌患者血清（９７例）は奈良県立医科大学にて同一プロトコルにて収集された検体であり、インフォームドコンセントの承諾及び奈良県立医科大倫理委員会の承認を受けて提供された。 <Example 2> Measurement of clinical samples Table 1 shows the details of the clinical samples used in Examples 3 to 7 below. Specimens were screened with a D-dimer of 1.0 μg/mL as a cutoff, and lower extremity vein echocardiography was performed on all cases with a D-dimer of 1.0 μg/mL or higher to confirm the presence or absence of thrombi. VTE-complicated ovarian cancer patient serum (25 cases) and non-VTE-complicated ovarian cancer patient serum (97 cases) are samples collected under the same protocol at Nara Medical University, consent of informed consent and Nara Medical University Provided with Ethics Committee approval.
評価用装置は全自動エンザイムイムノアッセイ装置ＡＩＡ－２０００（東ソー社製：製造販売届出番号１３Ｂ３Ｘ９０００２０００００９）を用いた。全自動エンザイムイムノアッセイ装置ＡＩＡ－２０００によるＴＦＰＩ２の測定は、以下の手順で行った。
（１）サンプル２０μＬと界面活性剤を含む希釈液１００μＬを、実施例１で作製したＴＦＰＩ２測定試薬に自動で分注し、
（２）３７℃恒温下で１０分間の抗原抗体反応を行い、
（３）Ｂ／Ｆ分離後、界面活性剤を含む緩衝液にて８回の洗浄を行い、
（４）４－メチルウンベリフェリルリン酸塩を添加し、単位時間当たりのアルカリフォスファターゼによる４－メチルウンベリフェロン生成濃度をもって測定値（ＴＦＰＩ２ ｉｎｔｅｎｓｉｔｙ、ｎｍｏｌ／（Ｌ・ｓ））とした
市販ＴＦＰＩ２組み換えタンパク（Ｒ＆Ｄ社）を標準品として検量線を作成し、血中ＴＦＰＩ２値を算出した。血中Ｄ－ｄｉｍｅｒ値は採血日直近の電子カルテ記載の測定値を引用した。 A fully automatic enzyme immunoassay device AIA-2000 (manufactured by Tosoh Corporation: manufacturing and sales notification number 13B3X90002000009) was used as an evaluation device. TFPI2 was measured using a fully automatic enzyme immunoassay device AIA-2000 according to the following procedure.
(1) 20 μL of the sample and 100 μL of the diluted solution containing a surfactant are automatically dispensed into the TFPI2 measurement reagent prepared in Example 1,
(2) performing an antigen-antibody reaction for 10 minutes at a constant temperature of 37°C,
(3) After B/F separation,wash 8 times with a buffer solution containing a surfactant,
(4) 4-methylumbelliferyl phosphate was added, and the concentration of 4-methylumbelliferone produced by alkaline phosphatase per unit time was taken as the measured value (TFPI2 intensity, nmol/(L s)) Commercially available TFPI2 A calibration curve was prepared using the recombinant protein (R&D) as a standard, and the blood TFPI2 value was calculated. As the blood D-dimer value, the measured value described in the electronic medical record immediately before the day of blood sampling was quoted.
（１）サンプル２０μＬと界面活性剤を含む希釈液１００μＬを、実施例１で作製したＴＦＰＩ２測定試薬に自動で分注し、
（２）３７℃恒温下で１０分間の抗原抗体反応を行い、
（３）Ｂ／Ｆ分離後、界面活性剤を含む緩衝液にて８回の洗浄を行い、
（４）４－メチルウンベリフェリルリン酸塩を添加し、単位時間当たりのアルカリフォスファターゼによる４－メチルウンベリフェロン生成濃度をもって測定値（ＴＦＰＩ２ ｉｎｔｅｎｓｉｔｙ、ｎｍｏｌ／（Ｌ・ｓ））とした
市販ＴＦＰＩ２組み換えタンパク（Ｒ＆Ｄ社）を標準品として検量線を作成し、血中ＴＦＰＩ２値を算出した。血中Ｄ－ｄｉｍｅｒ値は採血日直近の電子カルテ記載の測定値を引用した。 A fully automatic enzyme immunoassay device AIA-2000 (manufactured by Tosoh Corporation: manufacturing and sales notification number 13B3X90002000009) was used as an evaluation device. TFPI2 was measured using a fully automatic enzyme immunoassay device AIA-2000 according to the following procedure.
(1) 20 μL of the sample and 100 μL of the diluted solution containing a surfactant are automatically dispensed into the TFPI2 measurement reagent prepared in Example 1,
(2) performing an antigen-antibody reaction for 10 minutes at a constant temperature of 37°C,
(3) After B/F separation,
(4) 4-methylumbelliferyl phosphate was added, and the concentration of 4-methylumbelliferone produced by alkaline phosphatase per unit time was taken as the measured value (TFPI2 intensity, nmol/(L s)) Commercially available TFPI2 A calibration curve was prepared using the recombinant protein (R&D) as a standard, and the blood TFPI2 value was calculated. As the blood D-dimer value, the measured value described in the electronic medical record immediately before the day of blood sampling was quoted.
＜実施例３＞ＶＴＥ併発群と非ＶＴＥ併発群におけるＴＦＰＩ２とＤ－ｄｉｍｅｒの比較
ＶＴＥ併発卵巣癌患者及び非ＶＴＥ併発卵巣癌患者における血中ＴＦＰＩ２値と血中Ｄ－ｄｉｍｅｒ値のＢｏｘＰｌｏｔを図１に示す。ＴＦＰＩ２とＤ－ｄｉｍｅｒは非ＶＴＥ併発群と比べてＶＴＥ併発群にていずれも統計的有意差を持って高値を示した（マン＝ホイットニーのＵ検定、ｐ＜０．０００１）。 <Example 3> Comparison of TFPI2 and D-dimer in VTE-complicated group and non-VTE-complicated group Fig. 1 shows a Boxplot of blood TFPI2 values and blood D-dimer values in VTE-complicated ovarian cancer patients and non-VTE-complicated ovarian cancer patients. shown. Both TFPI2 and D-dimer were higher in the VTE concurrent group than in the non-VTE concurrent group with a statistically significant difference (Mann-Whitney U test, p<0.0001).
ＶＴＥ併発卵巣癌患者及び非ＶＴＥ併発卵巣癌患者における血中ＴＦＰＩ２値と血中Ｄ－ｄｉｍｅｒ値のＢｏｘＰｌｏｔを図１に示す。ＴＦＰＩ２とＤ－ｄｉｍｅｒは非ＶＴＥ併発群と比べてＶＴＥ併発群にていずれも統計的有意差を持って高値を示した（マン＝ホイットニーのＵ検定、ｐ＜０．０００１）。 <Example 3> Comparison of TFPI2 and D-dimer in VTE-complicated group and non-VTE-complicated group Fig. 1 shows a Boxplot of blood TFPI2 values and blood D-dimer values in VTE-complicated ovarian cancer patients and non-VTE-complicated ovarian cancer patients. shown. Both TFPI2 and D-dimer were higher in the VTE concurrent group than in the non-VTE concurrent group with a statistically significant difference (Mann-Whitney U test, p<0.0001).
＜実施例４＞ＦＩＧＯ分類におけるＴＦＰＩ２とＤ－ｄｉｍｅｒの比較
早期群（ＦＩＧＯ Ｉ－ＩＩ期）と進行群（ＦＩＧＯ ＩＩＩ－ＩＶ期）におけるＶＴＥ併発卵巣癌及び非ＶＴＥ併発卵巣癌患者の血中ＴＦＰＩ２値と血中Ｄ－ｄｉｍｅｒ値のＢｏｘＰｌｏｔを図２に示す。ＴＦＰＩ２とＤ－ｄｉｍｅｒは早期群と進行群のいずれにおいても非ＶＴＥ併発群と比べてＶＴＥ併発群にて統計的有意差をもって高値を示した。 <Example 4> Comparison of TFPI2 and D-dimer in FIGO Classification Blood TFPI2 of patients with VTE-complicated ovarian cancer and non-VTE-complicated ovarian cancer in early group (FIGO I-II stage) and advanced group (FIGO III-IV stage) Figure 2 shows a BoxPlot of the values and blood D-dimer values. TFPI2 and D-dimer showed higher values with statistically significant difference in the VTE-complicated group than in the non-VTE-complicated group in both the early stage group and the progressive group.
早期群（ＦＩＧＯ Ｉ－ＩＩ期）と進行群（ＦＩＧＯ ＩＩＩ－ＩＶ期）におけるＶＴＥ併発卵巣癌及び非ＶＴＥ併発卵巣癌患者の血中ＴＦＰＩ２値と血中Ｄ－ｄｉｍｅｒ値のＢｏｘＰｌｏｔを図２に示す。ＴＦＰＩ２とＤ－ｄｉｍｅｒは早期群と進行群のいずれにおいても非ＶＴＥ併発群と比べてＶＴＥ併発群にて統計的有意差をもって高値を示した。 <Example 4> Comparison of TFPI2 and D-dimer in FIGO Classification Blood TFPI2 of patients with VTE-complicated ovarian cancer and non-VTE-complicated ovarian cancer in early group (FIGO I-II stage) and advanced group (FIGO III-IV stage) Figure 2 shows a BoxPlot of the values and blood D-dimer values. TFPI2 and D-dimer showed higher values with statistically significant difference in the VTE-complicated group than in the non-VTE-complicated group in both the early stage group and the progressive group.
＜実施例５＞組織型におけるＴＦＰＩ２とＤ－ｄｉｍｅｒの比較
卵巣癌のうち、明細胞癌、漿液性癌または類内膜癌におけるＶＴＥ併発卵巣癌及び非ＶＴＥ併発卵巣癌患者の血中ＴＦＰＩ２値と血中Ｄ－ｄｉｍｅｒ値のＢｏｘＰｌｏｔを図３に示す。ＴＦＰＩ２とＤ－ｄｉｍｅｒは明細胞癌および漿液性癌において非ＶＴＥ併発群と比べてＶＴＥ併発群にて統計的有意差を持って高値を示した。一方、類内膜癌ではＴＦＰＩ２のみで非ＶＴＥ併発群と比べてＶＴＥ併発群にて上昇傾向が認められた（マン＝ホイットニーのＵ検定、ｐ＝０．０５０３）。 <Example 5> Comparison of TFPI2 and D-dimer in tissue types Box plots of blood D-dimer values are shown in FIG. TFPI2 and D-dimer showed higher values in clear cell carcinoma and serous carcinoma with a statistically significant difference in the VTE-complicated group compared to the non-VTE group. On the other hand, in endometrioid cancer, only TFPI2 showed a tendency to increase in the VTE-complicated group compared to the non-VTE-complicated group (Mann-Whitney U test, p=0.0503).
卵巣癌のうち、明細胞癌、漿液性癌または類内膜癌におけるＶＴＥ併発卵巣癌及び非ＶＴＥ併発卵巣癌患者の血中ＴＦＰＩ２値と血中Ｄ－ｄｉｍｅｒ値のＢｏｘＰｌｏｔを図３に示す。ＴＦＰＩ２とＤ－ｄｉｍｅｒは明細胞癌および漿液性癌において非ＶＴＥ併発群と比べてＶＴＥ併発群にて統計的有意差を持って高値を示した。一方、類内膜癌ではＴＦＰＩ２のみで非ＶＴＥ併発群と比べてＶＴＥ併発群にて上昇傾向が認められた（マン＝ホイットニーのＵ検定、ｐ＝０．０５０３）。 <Example 5> Comparison of TFPI2 and D-dimer in tissue types Box plots of blood D-dimer values are shown in FIG. TFPI2 and D-dimer showed higher values in clear cell carcinoma and serous carcinoma with a statistically significant difference in the VTE-complicated group compared to the non-VTE group. On the other hand, in endometrioid cancer, only TFPI2 showed a tendency to increase in the VTE-complicated group compared to the non-VTE-complicated group (Mann-Whitney U test, p=0.0503).
＜実施例６＞ＲＯＣ解析によるＴＦＰＩ２とＤ－ｄｉｍｅｒの比較
ＶＴＥ併発卵巣癌及び非ＶＴＥ併発卵巣癌患者におけるＴＦＰＩ２とＤ－ｄｉｍｅｒのＲＯＣ解析結果を図４に示す。ＲＯＣ曲線から算出した曲線下面積（ＡＵＣ）はＴＦＰＩ２（０．７９６３）とＤ－ｄｉｍｅｒ（０．８２６６）といずれも高いＶＴＥの判別性能が示された。また、ＴＦＰＩ２とＤ－ｄｉｍｅｒを組み合わせることにより、ＡＵＣが更に上昇することが示された（０．８４９５）。本結果より、ＴＦＰＩ２は単独でもＤ－ｄｉｍｅｒと匹敵する良好な悪性腫瘍関連血栓塞栓症の判別性能を有しているものの、Ｄ－ｄｉｍｅｒと組み合わせることで悪性腫瘍関連血栓塞栓症の判別性能がさらに向上することが示された。
血中ＴＦＰＩ２値と血中Ｄ－ｄｉｍｅｒ値の相関を図５に示す。ＶＴＥ併発卵巣癌及び非ＶＴＥ併発卵巣癌患者における血中ＴＦＰＩ２値と血中Ｄ－ｄｉｍｅｒ値は相関が低いことが示された。 <Example 6> Comparison of TFPI2 and D-dimer by ROC Analysis FIG. 4 shows the results of ROC analysis of TFPI2 and D-dimer in patients with VTE-complicated ovarian cancer and non-VTE-complicated ovarian cancer. The area under the curve (AUC) calculated from the ROC curve was both TFPI2 (0.7963) and D-dimer (0.8266), indicating high VTE discrimination performance. It was also shown that AUC was further increased by combining TFPI2 and D-dimer (0.8495). From this result, although TFPI2 alone has a good discrimination performance of malignant tumor-related thromboembolism comparable to D-dimer, the discrimination performance of malignant tumor-related thromboembolism is further improved by combining with D-dimer. shown to improve.
FIG. 5 shows the correlation between the blood TFPI2 level and the blood D-dimer level. It was shown that there is a low correlation between the blood TFPI2 level and the blood D-dimer level in patients with VTE-complicated ovarian cancer and non-VTE-complicated ovarian cancer.
ＶＴＥ併発卵巣癌及び非ＶＴＥ併発卵巣癌患者におけるＴＦＰＩ２とＤ－ｄｉｍｅｒのＲＯＣ解析結果を図４に示す。ＲＯＣ曲線から算出した曲線下面積（ＡＵＣ）はＴＦＰＩ２（０．７９６３）とＤ－ｄｉｍｅｒ（０．８２６６）といずれも高いＶＴＥの判別性能が示された。また、ＴＦＰＩ２とＤ－ｄｉｍｅｒを組み合わせることにより、ＡＵＣが更に上昇することが示された（０．８４９５）。本結果より、ＴＦＰＩ２は単独でもＤ－ｄｉｍｅｒと匹敵する良好な悪性腫瘍関連血栓塞栓症の判別性能を有しているものの、Ｄ－ｄｉｍｅｒと組み合わせることで悪性腫瘍関連血栓塞栓症の判別性能がさらに向上することが示された。
血中ＴＦＰＩ２値と血中Ｄ－ｄｉｍｅｒ値の相関を図５に示す。ＶＴＥ併発卵巣癌及び非ＶＴＥ併発卵巣癌患者における血中ＴＦＰＩ２値と血中Ｄ－ｄｉｍｅｒ値は相関が低いことが示された。 <Example 6> Comparison of TFPI2 and D-dimer by ROC Analysis FIG. 4 shows the results of ROC analysis of TFPI2 and D-dimer in patients with VTE-complicated ovarian cancer and non-VTE-complicated ovarian cancer. The area under the curve (AUC) calculated from the ROC curve was both TFPI2 (0.7963) and D-dimer (0.8266), indicating high VTE discrimination performance. It was also shown that AUC was further increased by combining TFPI2 and D-dimer (0.8495). From this result, although TFPI2 alone has a good discrimination performance of malignant tumor-related thromboembolism comparable to D-dimer, the discrimination performance of malignant tumor-related thromboembolism is further improved by combining with D-dimer. shown to improve.
FIG. 5 shows the correlation between the blood TFPI2 level and the blood D-dimer level. It was shown that there is a low correlation between the blood TFPI2 level and the blood D-dimer level in patients with VTE-complicated ovarian cancer and non-VTE-complicated ovarian cancer.
＜実施例７＞ＴＦＰＩ２とＤ－ｄｉｍｅｒのＶＴＥ検出性能の比較
ＴＦＰＩ２とＤ－ｄｉｍｅｒのＶＴＥ検出性能を表２に示す。ＧｒａｐｈＰａｄ Ｐｒｉｓｍ ９（ＧｒａｐｈＰａｄ Ｓｏｆｔｗｒｅ Ｉｎｃ．）を用い、ＴＦＰＩ２のカットオフ値はＲＯＣ解析におけるＹｏｕｄｅｎ Ｉｎｄｅｘ（感度＋特異度－１)の最大値から２８０ｐｇ／ｍＬ、Ｄ－ｄｉｍｅｒのカットオフ値は１．０μｇ／ｍＬと設定し、感度、特異度、正診率、陽性的中率、陰性的中率を算出した。その結果、Ｄ－ｄｉｍｅｒは高い感度を有するものの低い特異度により正診率が０．５４となった。一方、ＴＦＰＩ２は低い感度ながら高い特異度により正診率が０．７１となった。
以上の結果より、ＴＦＰＩ２は高い特異度を有することからＤ－ｄｉｍｅｒと相補性・補完性を有する可能性が示唆された。 <Example 7> Comparison of VTE detection performance between TFPI2 and D-dimer Table 2 shows the VTE detection performance of TFPI2 and D-dimer. Using GraphPad Prism 9 (GraphPad Software Inc.), the cutoff value of TFPI2 is 280 pg / mL from the maximum value of Youden Index (sensitivity + specificity -1) in ROC analysis, and the cutoff value of D-dimer is 1.0 μg. / mL, and the sensitivity, specificity, accuracy rate, positive predictive value, and negative predictive value were calculated. As a result, although D-dimer has high sensitivity, the accuracy rate was 0.54 due to low specificity. On the other hand, TFPI2 had a low sensitivity but high specificity, resulting in an accuracy rate of 0.71.
From the above results, it was suggested that TFPI2 has a high degree of specificity and thus may have complementarity with D-dimer.
ＴＦＰＩ２とＤ－ｄｉｍｅｒのＶＴＥ検出性能を表２に示す。ＧｒａｐｈＰａｄ Ｐｒｉｓｍ ９（ＧｒａｐｈＰａｄ Ｓｏｆｔｗｒｅ Ｉｎｃ．）を用い、ＴＦＰＩ２のカットオフ値はＲＯＣ解析におけるＹｏｕｄｅｎ Ｉｎｄｅｘ（感度＋特異度－１)の最大値から２８０ｐｇ／ｍＬ、Ｄ－ｄｉｍｅｒのカットオフ値は１．０μｇ／ｍＬと設定し、感度、特異度、正診率、陽性的中率、陰性的中率を算出した。その結果、Ｄ－ｄｉｍｅｒは高い感度を有するものの低い特異度により正診率が０．５４となった。一方、ＴＦＰＩ２は低い感度ながら高い特異度により正診率が０．７１となった。
以上の結果より、ＴＦＰＩ２は高い特異度を有することからＤ－ｄｉｍｅｒと相補性・補完性を有する可能性が示唆された。 <Example 7> Comparison of VTE detection performance between TFPI2 and D-dimer Table 2 shows the VTE detection performance of TFPI2 and D-dimer. Using GraphPad Prism 9 (GraphPad Software Inc.), the cutoff value of TFPI2 is 280 pg / mL from the maximum value of Youden Index (sensitivity + specificity -1) in ROC analysis, and the cutoff value of D-dimer is 1.0 μg. / mL, and the sensitivity, specificity, accuracy rate, positive predictive value, and negative predictive value were calculated. As a result, although D-dimer has high sensitivity, the accuracy rate was 0.54 due to low specificity. On the other hand, TFPI2 had a low sensitivity but high specificity, resulting in an accuracy rate of 0.71.
From the above results, it was suggested that TFPI2 has a high degree of specificity and thus may have complementarity with D-dimer.
＜実施例８＞卵巣境界悪性腫瘍検体の測定
後述の実施例９～１１で使用した卵巣境界悪性腫瘍検体の内訳を表３に示す。検体はＤ－ｄｉｍｅｒ １．０μｇ／ｍＬをカットオフとしてスクリーニングを行い、１．０μｇ／ｍＬ以上は全例に下肢静脈エコーを実施して血栓の有無を確認した。ＶＴＥ併発卵巣境界悪性腫瘍患者血清（２例）と非ＶＴＥ併発卵巣境界悪性腫瘍患者血清（３６例）は奈良県立医科大学にて同一プロトコルにて収集された検体であり、インフォームドコンセントの承諾及び奈良県立医科大学倫理委員会の承認を受けて提供された。
評価用装置及び測定手順は実施例２の内容と同様であり、血中Ｄ－ｄｉｍｅｒ値は採血日直近の電子カルテ記載の測定値を引用した。 <Example 8> Measurement of ovarian borderline malignant tumor samples Table 3 shows the details of the ovarian borderline malignant tumor samples used in Examples 9 to 11 described below. Specimens were screened with a D-dimer of 1.0 μg/mL as a cutoff, and lower extremity vein echocardiography was performed on all cases of 1.0 μg/mL or higher to confirm the presence or absence of thrombi. VTE-complicated ovarian borderline malignant tumor patient serum (2 cases) and non-VTE-complicated ovarian borderline malignant tumor patient serum (36 cases) are specimens collected under the same protocol at Nara Medical University, informed consent and It was provided with the approval of the Nara Medical University Ethics Committee.
The apparatus for evaluation and the measurement procedure were the same as those in Example 2, and the blood D-dimer value was the measured value described in the electronic medical record immediately before the day of blood sampling.
後述の実施例９～１１で使用した卵巣境界悪性腫瘍検体の内訳を表３に示す。検体はＤ－ｄｉｍｅｒ １．０μｇ／ｍＬをカットオフとしてスクリーニングを行い、１．０μｇ／ｍＬ以上は全例に下肢静脈エコーを実施して血栓の有無を確認した。ＶＴＥ併発卵巣境界悪性腫瘍患者血清（２例）と非ＶＴＥ併発卵巣境界悪性腫瘍患者血清（３６例）は奈良県立医科大学にて同一プロトコルにて収集された検体であり、インフォームドコンセントの承諾及び奈良県立医科大学倫理委員会の承認を受けて提供された。
評価用装置及び測定手順は実施例２の内容と同様であり、血中Ｄ－ｄｉｍｅｒ値は採血日直近の電子カルテ記載の測定値を引用した。 <Example 8> Measurement of ovarian borderline malignant tumor samples Table 3 shows the details of the ovarian borderline malignant tumor samples used in Examples 9 to 11 described below. Specimens were screened with a D-dimer of 1.0 μg/mL as a cutoff, and lower extremity vein echocardiography was performed on all cases of 1.0 μg/mL or higher to confirm the presence or absence of thrombi. VTE-complicated ovarian borderline malignant tumor patient serum (2 cases) and non-VTE-complicated ovarian borderline malignant tumor patient serum (36 cases) are specimens collected under the same protocol at Nara Medical University, informed consent and It was provided with the approval of the Nara Medical University Ethics Committee.
The apparatus for evaluation and the measurement procedure were the same as those in Example 2, and the blood D-dimer value was the measured value described in the electronic medical record immediately before the day of blood sampling.
＜実施例９＞卵巣境界悪性腫瘍患者のＶＴＥ併発／非併発群間におけるＴＦＰＩ２とＤ－ｄｉｍｅｒの比較
ＶＴＥ併発卵巣境界悪性腫瘍患者及び非ＶＴＥ併発卵巣境界悪性腫瘍患者における血中ＴＦＰＩ２値と血中Ｄ－ｄｉｍｅｒ値のＢｏｘＰｌｏｔを図６に示す。ＴＦＰＩ２とＤ－ｄｉｍｅｒは非ＶＴＥ併発群と比べてＶＴＥ併発群にていずれも統計的有意差を持って高値を示した（マン＝ホイットニーのＵ検定、ｐ＜０．０５）。 <Example 9> Comparison of TFPI2 and D-dimer between ovarian borderline malignant tumor patients with/without VTE Blood TFPI2 levels and blood levels in patients with borderline ovarian malignancy with VTE and patients with borderline ovarian malignancy with no VTE A BoxPlot of the D-dimer values is shown in FIG. Both TFPI2 and D-dimer were higher in the VTE concurrent group than in the non-VTE concurrent group with a statistically significant difference (Mann-Whitney U test, p<0.05).
ＶＴＥ併発卵巣境界悪性腫瘍患者及び非ＶＴＥ併発卵巣境界悪性腫瘍患者における血中ＴＦＰＩ２値と血中Ｄ－ｄｉｍｅｒ値のＢｏｘＰｌｏｔを図６に示す。ＴＦＰＩ２とＤ－ｄｉｍｅｒは非ＶＴＥ併発群と比べてＶＴＥ併発群にていずれも統計的有意差を持って高値を示した（マン＝ホイットニーのＵ検定、ｐ＜０．０５）。 <Example 9> Comparison of TFPI2 and D-dimer between ovarian borderline malignant tumor patients with/without VTE Blood TFPI2 levels and blood levels in patients with borderline ovarian malignancy with VTE and patients with borderline ovarian malignancy with no VTE A BoxPlot of the D-dimer values is shown in FIG. Both TFPI2 and D-dimer were higher in the VTE concurrent group than in the non-VTE concurrent group with a statistically significant difference (Mann-Whitney U test, p<0.05).
＜実施例１０＞卵巣境界悪性腫瘍患者におけるＴＦＰＩ２とＤ－ｄｉｍｅｒの比較
ＶＴＥ併発卵巣境界悪性腫瘍患者及び非ＶＴＥ併発卵巣境界悪性腫瘍患者におけるＴＦＰＩ２とＤ－ｄｉｍｅｒのＲＯＣ解析結果を図７に示す。ＲＯＣ曲線から算出した曲線下面積（ＡＵＣ）はＴＦＰＩ２（１．００００）とＤ－ｄｉｍｅｒ（０．９８６１）といずれも高いＶＴＥの判別性能が示された。
血中ＴＦＰＩ２値と血中Ｄ－ｄｉｍｅｒ値の相関を図８に示す。卵巣境界悪性腫瘍患者においても血中ＴＦＰＩ２値と血中Ｄ－ｄｉｍｅｒ値は相関が低いことが示された。 <Example 10> Comparison of TFPI2 and D-dimer in Patients with Borderline Malignant Tumor of the Ovarian FIG. 7 shows the results of ROC analysis of TFPI2 and D-dimer in patients with borderline ovarian malignancy with VTE and patients with borderline ovarian malignancy with no VTE. The area under the curve (AUC) calculated from the ROC curve was both TFPI2 (1.0000) and D-dimer (0.9861), indicating high VTE discrimination performance.
FIG. 8 shows the correlation between the blood TFPI2 level and the blood D-dimer level. It was shown that the correlation between the blood TFPI2 level and the blood D-dimer level is low even in patients with borderline malignant tumor of the ovary.
ＶＴＥ併発卵巣境界悪性腫瘍患者及び非ＶＴＥ併発卵巣境界悪性腫瘍患者におけるＴＦＰＩ２とＤ－ｄｉｍｅｒのＲＯＣ解析結果を図７に示す。ＲＯＣ曲線から算出した曲線下面積（ＡＵＣ）はＴＦＰＩ２（１．００００）とＤ－ｄｉｍｅｒ（０．９８６１）といずれも高いＶＴＥの判別性能が示された。
血中ＴＦＰＩ２値と血中Ｄ－ｄｉｍｅｒ値の相関を図８に示す。卵巣境界悪性腫瘍患者においても血中ＴＦＰＩ２値と血中Ｄ－ｄｉｍｅｒ値は相関が低いことが示された。 <Example 10> Comparison of TFPI2 and D-dimer in Patients with Borderline Malignant Tumor of the Ovarian FIG. 7 shows the results of ROC analysis of TFPI2 and D-dimer in patients with borderline ovarian malignancy with VTE and patients with borderline ovarian malignancy with no VTE. The area under the curve (AUC) calculated from the ROC curve was both TFPI2 (1.0000) and D-dimer (0.9861), indicating high VTE discrimination performance.
FIG. 8 shows the correlation between the blood TFPI2 level and the blood D-dimer level. It was shown that the correlation between the blood TFPI2 level and the blood D-dimer level is low even in patients with borderline malignant tumor of the ovary.
＜実施例１１＞卵巣境界悪性腫瘍におけるＴＦＰＩ２とＤ－ｄｉｍｅｒのＶＴＥ検出性能の比較
ＴＦＰＩ２とＤ－ｄｉｍｅｒのＶＴＥ検出性能を表４に示す。ＴＦＰＩ２のカットオフ値は実施例７の結果から２８０ｐｇ／ｍＬ、Ｄ－ｄｉｍｅｒのカットオフ値は１．０μｇ／ｍＬと設定し、感度、特異度、正診率、陽性的中率、陰性的中率を算出した。その結果、Ｄ－ｄｉｍｅｒは高い感度を有するものの特異度が低く正診率が０．６５８となった。一方、ＴＦＰＩ２は感度、特異度ともに高く正診率が０．９２１となった。
以上の結果より、ＴＦＰＩ２は卵巣境界悪性腫瘍のＶＴＥ判別においてＤ－ｄｉｍｅｒを上回る性能を有する可能性が示された。 <Example 11> Comparison of VTE detection performance of TFPI2 and D-dimer in borderline malignant tumor of the ovary Table 4 shows the VTE detection performance of TFPI2 and D-dimer. The cutoff value of TFPI2 was set to 280 pg / mL from the results of Example 7, and the cutoff value of D-dimer was set to 1.0 μg / mL. rate was calculated. As a result, although D-dimer has a high sensitivity, the specificity is low and the correct diagnosis rate is 0.658. On the other hand, TFPI2 had high sensitivity and specificity, and the accuracy rate was 0.921.
The above results indicate that TFPI2 may have a higher performance than D-dimer in VTE discrimination of ovarian borderline malignant tumors.
ＴＦＰＩ２とＤ－ｄｉｍｅｒのＶＴＥ検出性能を表４に示す。ＴＦＰＩ２のカットオフ値は実施例７の結果から２８０ｐｇ／ｍＬ、Ｄ－ｄｉｍｅｒのカットオフ値は１．０μｇ／ｍＬと設定し、感度、特異度、正診率、陽性的中率、陰性的中率を算出した。その結果、Ｄ－ｄｉｍｅｒは高い感度を有するものの特異度が低く正診率が０．６５８となった。一方、ＴＦＰＩ２は感度、特異度ともに高く正診率が０．９２１となった。
以上の結果より、ＴＦＰＩ２は卵巣境界悪性腫瘍のＶＴＥ判別においてＤ－ｄｉｍｅｒを上回る性能を有する可能性が示された。 <Example 11> Comparison of VTE detection performance of TFPI2 and D-dimer in borderline malignant tumor of the ovary Table 4 shows the VTE detection performance of TFPI2 and D-dimer. The cutoff value of TFPI2 was set to 280 pg / mL from the results of Example 7, and the cutoff value of D-dimer was set to 1.0 μg / mL. rate was calculated. As a result, although D-dimer has a high sensitivity, the specificity is low and the correct diagnosis rate is 0.658. On the other hand, TFPI2 had high sensitivity and specificity, and the accuracy rate was 0.921.
The above results indicate that TFPI2 may have a higher performance than D-dimer in VTE discrimination of ovarian borderline malignant tumors.
＜実施例１２＞卵巣以外のがん患者検体測定
実施例１３～１５で使用したがん患者検体の内訳を表５に示す。ＶＴＥ併発がん患者血清（３３例）と非ＶＴＥ併発がん患者血清（７０例）は、販売元のＰＲＯＭＥＤＤＸ社、Ｐｒｅｃｉｓｉｏｎ Ｆｏｒ Ｍｅｄｉｃｉｎｅ社、ＢｉｏＩＶＴ社の臨床情報から血栓の有無を確認した。また、これらの検体については製品添付書類に倫理委員会承認済のプロトコルで収集されたことが明記されているものを使用した。
評価用装置及び測定手順は、実施例２と同様である。 <Example 12> Measurement of cancer patient specimens other than ovaries Table 5 shows the breakdown of the cancer patient specimens used in Examples 13-15. VTE-complicated cancer patient sera (33 cases) and non-VTE-complicated cancer patient sera (70 cases) were checked for the presence or absence of thrombus from clinical information provided by the distributors, PROMEDDX, Precision For Medicine, and BioIVT. In addition, for these specimens, we used those that clearly stated in the product attachment that they were collected according to an Ethics Committee-approved protocol.
The evaluation device and measurement procedure are the same as in Example 2.
実施例１３～１５で使用したがん患者検体の内訳を表５に示す。ＶＴＥ併発がん患者血清（３３例）と非ＶＴＥ併発がん患者血清（７０例）は、販売元のＰＲＯＭＥＤＤＸ社、Ｐｒｅｃｉｓｉｏｎ Ｆｏｒ Ｍｅｄｉｃｉｎｅ社、ＢｉｏＩＶＴ社の臨床情報から血栓の有無を確認した。また、これらの検体については製品添付書類に倫理委員会承認済のプロトコルで収集されたことが明記されているものを使用した。
評価用装置及び測定手順は、実施例２と同様である。 <Example 12> Measurement of cancer patient specimens other than ovaries Table 5 shows the breakdown of the cancer patient specimens used in Examples 13-15. VTE-complicated cancer patient sera (33 cases) and non-VTE-complicated cancer patient sera (70 cases) were checked for the presence or absence of thrombus from clinical information provided by the distributors, PROMEDDX, Precision For Medicine, and BioIVT. In addition, for these specimens, we used those that clearly stated in the product attachment that they were collected according to an Ethics Committee-approved protocol.
The evaluation device and measurement procedure are the same as in Example 2.
＜実施例１３＞がん種別のＶＴＥ併発／非併発群におけるＴＦＰＩ２の比較
卵巣以外のがん患者について、ＶＴＥ併発及び非ＶＴＥ併発患者の血中ＴＦＰＩ２値のＢｏｘＰｌｏｔを図９に示す。ＴＦＰＩ２は肺がん、食道及び／又は胃がん、肝臓又は膵臓がん、大腸がん、造血器がん、乳がん、子宮がんにおいて非ＶＴＥ併発群と比べてＶＴＥ併発群にて統計的有意差を持って高値を示した（マン＝ホイットニーのＵ検定）。 <Example 13> Comparison of TFPI2 in VTE-complicated/non-complicated cancer groups by cancer type As for cancer patients other than ovaries, BoxPlots of blood TFPI2 values of VTE-complicated and non-VTE-complicated patients are shown in FIG. TFPI2 had statistically significant differences in lung, esophageal and/or gastric, liver or pancreatic, colorectal, hematopoietic, breast, and uterine cancers with VTE compared to non-VTE groups. High values were shown (Mann-Whitney U test).
卵巣以外のがん患者について、ＶＴＥ併発及び非ＶＴＥ併発患者の血中ＴＦＰＩ２値のＢｏｘＰｌｏｔを図９に示す。ＴＦＰＩ２は肺がん、食道及び／又は胃がん、肝臓又は膵臓がん、大腸がん、造血器がん、乳がん、子宮がんにおいて非ＶＴＥ併発群と比べてＶＴＥ併発群にて統計的有意差を持って高値を示した（マン＝ホイットニーのＵ検定）。 <Example 13> Comparison of TFPI2 in VTE-complicated/non-complicated cancer groups by cancer type As for cancer patients other than ovaries, BoxPlots of blood TFPI2 values of VTE-complicated and non-VTE-complicated patients are shown in FIG. TFPI2 had statistically significant differences in lung, esophageal and/or gastric, liver or pancreatic, colorectal, hematopoietic, breast, and uterine cancers with VTE compared to non-VTE groups. High values were shown (Mann-Whitney U test).
＜実施例１４＞卵巣以外のがん患者のＴＦＰＩ２によるＶＴＥ併発／非併発群間のＲＯＣ解析
卵巣以外のがん患者について、ＶＴＥ併発及び非ＶＴＥ併発患者におけるＴＦＰＩ２のＲＯＣ解析結果を図１０に示す。ＲＯＣ曲線から算出した曲線下面積（ＡＵＣ）は、肺がん（１．００００）、食道及び／又は胃がん（０．９７２２）、肝臓又は膵臓がん（０．９８２１）、大腸がん（１．００００）、造血器がん（０．８４３８）、乳がん（０．８３３３）、子宮がん（１．００００）といずれも高いＶＴＥの判別性能が示された。本結果より、ＴＦＰＩ２は卵巣以外のがん患者においても良好な悪性腫瘍関連血栓塞栓症の検出性能を有していることが示された。 <Example 14> ROC analysis between VTE-complicated/non-complicated groups by TFPI2 in non-ovarian cancer patients As for non-ovarian cancer patients, the results of ROC analysis of TFPI2 in VTE-complicated and non-VTE concomitant patients are shown in Fig. 10 . . The area under the curve (AUC) calculated from the ROC curve was lung cancer (1.0000), esophageal and/or gastric cancer (0.9722), liver or pancreatic cancer (0.9821), colon cancer (1.0000). , hematopoietic cancer (0.8438), breast cancer (0.8333), and uterine cancer (1.0000). These results indicate that TFPI2 has a good ability to detect malignant tumor-associated thromboembolism even in patients with cancers other than those of the ovary.
卵巣以外のがん患者について、ＶＴＥ併発及び非ＶＴＥ併発患者におけるＴＦＰＩ２のＲＯＣ解析結果を図１０に示す。ＲＯＣ曲線から算出した曲線下面積（ＡＵＣ）は、肺がん（１．００００）、食道及び／又は胃がん（０．９７２２）、肝臓又は膵臓がん（０．９８２１）、大腸がん（１．００００）、造血器がん（０．８４３８）、乳がん（０．８３３３）、子宮がん（１．００００）といずれも高いＶＴＥの判別性能が示された。本結果より、ＴＦＰＩ２は卵巣以外のがん患者においても良好な悪性腫瘍関連血栓塞栓症の検出性能を有していることが示された。 <Example 14> ROC analysis between VTE-complicated/non-complicated groups by TFPI2 in non-ovarian cancer patients As for non-ovarian cancer patients, the results of ROC analysis of TFPI2 in VTE-complicated and non-VTE concomitant patients are shown in Fig. 10 . . The area under the curve (AUC) calculated from the ROC curve was lung cancer (1.0000), esophageal and/or gastric cancer (0.9722), liver or pancreatic cancer (0.9821), colon cancer (1.0000). , hematopoietic cancer (0.8438), breast cancer (0.8333), and uterine cancer (1.0000). These results indicate that TFPI2 has a good ability to detect malignant tumor-associated thromboembolism even in patients with cancers other than those of the ovary.
＜実施例１５＞卵巣以外のがん患者におけるＴＦＰＩ２のＶＴＥ検出性能
卵巣以外の各がん患者におけるＴＦＰＩ２のＶＴＥ検出性能を表６に示す。ＴＦＰＩ２のカットオフ値は実施例７の結果から２８０ｐｇ／ｍＬと設定し、感度、特異度、正診率、陽性的中率、陰性的中率を算出した。その結果、ＴＦＰＩ２によるＶＴＥ検出の感度・特異度はがん種によらず高い値を示し、肺がん、食道及び／又は胃がん、肝臓又は膵臓がん、大腸がん、造血器がん、乳がん、子宮がんのいずれにおいても高い正診率を示した。
以上の結果より、ＴＦＰＩ２は卵巣以外のがん患者においてもＶＴＥ検出において有用である可能性が示された。 <Example 15> VTE detection performance of TFPI2 in patients with cancer other than ovary Table 6 shows the VTE detection performance of TFPI2 in each cancer patient other than ovary. The cutoff value of TFPI2 was set to 280 pg/mL from the results of Example 7, and sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were calculated. As a result, the sensitivity and specificity of VTE detection by TFPI2 showed high values regardless of cancer types, and lung cancer, esophageal and / or stomach cancer, liver or pancreatic cancer, colon cancer, hematopoietic cancer, breast cancer, uterine cancer It showed a high accuracy rate in all cancers.
These results indicate that TFPI2 may be useful in detecting VTE in patients with cancers other than those of the ovary.
卵巣以外の各がん患者におけるＴＦＰＩ２のＶＴＥ検出性能を表６に示す。ＴＦＰＩ２のカットオフ値は実施例７の結果から２８０ｐｇ／ｍＬと設定し、感度、特異度、正診率、陽性的中率、陰性的中率を算出した。その結果、ＴＦＰＩ２によるＶＴＥ検出の感度・特異度はがん種によらず高い値を示し、肺がん、食道及び／又は胃がん、肝臓又は膵臓がん、大腸がん、造血器がん、乳がん、子宮がんのいずれにおいても高い正診率を示した。
以上の結果より、ＴＦＰＩ２は卵巣以外のがん患者においてもＶＴＥ検出において有用である可能性が示された。 <Example 15> VTE detection performance of TFPI2 in patients with cancer other than ovary Table 6 shows the VTE detection performance of TFPI2 in each cancer patient other than ovary. The cutoff value of TFPI2 was set to 280 pg/mL from the results of Example 7, and sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were calculated. As a result, the sensitivity and specificity of VTE detection by TFPI2 showed high values regardless of cancer types, and lung cancer, esophageal and / or stomach cancer, liver or pancreatic cancer, colon cancer, hematopoietic cancer, breast cancer, uterine cancer It showed a high accuracy rate in all cancers.
These results indicate that TFPI2 may be useful in detecting VTE in patients with cancers other than those of the ovary.
本発明により、簡便かつ患者負担が比較的少ない血液検査で悪性腫瘍関連血栓塞栓症を検出する方法が提供される。これは、簡便かつ高精度な血液検査による悪性腫瘍関連血栓塞栓症の診療精度向上への貢献が期待され、産業上非常に有用である。
The present invention provides a method for detecting malignant tumor-related thromboembolism with a blood test that is simple and imposes relatively little burden on the patient. This is expected to contribute to improving the diagnostic accuracy of malignant tumor-related thromboembolism through a simple and highly accurate blood test, and is very useful industrially.
Claims (8)
- 検体において、ＴＦＰＩ２量を測定することを含む、悪性腫瘍関連血栓塞栓症を検出する方法。 A method for detecting malignant tumor-related thromboembolism, including measuring the amount of TFPI2 in a specimen.
- 前記ＴＦＰＩ２量の測定値が、予め設定した基準値を超えた場合に、悪性腫瘍関連血栓塞栓症が検出されたとする、請求項１に記載の方法。 The method according to claim 1, wherein malignant tumor-associated thromboembolism is detected when the measured TFPI2 amount exceeds a preset reference value.
- 前記ＴＦＰＩ２量が、ＴＦＰＩ２プロセシングポリペプチド量及びインタクトＴＦＰＩ２量の合計である、請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein the amount of TFPI2 is the sum of the amount of TFPI2 processing polypeptide and the amount of intact TFPI2.
- 前記ＴＦＰＩ２量の測定が、配列番号１のアミノ酸配列の２３残基目のアスパラギン酸から１３１残基目のヒスチジン又は１３０残基目のシステインまでの領域内の抗原決定基に結合する抗体を用いた抗原抗体反応により行われるものである、請求項１～３のいずれか一項に記載の方法。 The amount of TFPI2 is measured using an antibody that binds to an antigenic determinant in the region from aspartic acid at residue 23 to histidine at residue 131 or cysteine at residue 130 in the amino acid sequence of SEQ ID NO: 1. The method according to any one of claims 1 to 3, which is carried out by antigen-antibody reaction.
- 前記抗体が、ＴＦＰＩ２のクニッツドメイン１を認識する抗体である、請求項４に記載の方法。 The method according to claim 4, wherein the antibody is an antibody that recognizes Kunitz domain 1 of TFPI2.
- 質量分析法を用いて測定を行う、請求項１～３のいずれか一項に記載の方法。 The method according to any one of claims 1 to 3, wherein the measurement is performed using mass spectrometry.
- 悪性腫瘍が、卵巣癌、肺がん、消化器がん、造血器がん、乳がん、又は子宮がんである、請求項１～６のいずれか一項に記載の方法。 The method according to any one of claims 1 to 6, wherein the malignant tumor is ovarian cancer, lung cancer, gastrointestinal cancer, hematopoietic cancer, breast cancer, or uterine cancer.
- 配列番号１に示すアミノ酸配列の２３残基目のアスパラギン酸から１３１残基目のヒスチジン又は１３０残基目のシステインまでの領域内の抗原決定基に結合する抗体を含む、悪性腫瘍関連血栓塞栓症を検出するための試薬。
Malignant tumor-associated thromboembolism comprising an antibody that binds to an antigenic determinant within the region from aspartic acid at residue 23 to histidine at residue 131 or cysteine at residue 130 in the amino acid sequence shown in SEQ ID NO: 1 A reagent for detecting
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