JP6763611B2 - How to test for asbestos exposure history or mesothelioma - Google Patents

Description

The present invention is a method for testing asbestos exposure history or mesothelioma by analyzing immune-related molecules in blood collected from a subject, asbestos exposure history or mesothelioma test reagent kit, asbestos exposure history or The present invention relates to a test program for testing mesothelioma and a test device.

This application claims the priority of Japanese application, Japanese Patent Application No. 2015-084983, which is incorporated herein by reference.

In recent years, the incidence of mesothelioma has been on the rise, and it has been clarified that the history of exposure to asbestos (asbestos) is closely related to the onset. Mesothelioma has a long incubation period of several decades, often develops 20 to 50 years after inhalation of asbestos, and no abnormal findings are observed before the onset. Subjective symptoms after onset include shortness of breath, pain below the thorax, cough, and unexplained weight loss, but there are few characteristic symptoms and early detection is difficult. The prognosis is extremely severe because many patients have progressed from the time of discovery and the deterioration is rapid. Half survival after diagnosis is 12 months with the combination of cisplatin and pemetrexade, and no chemotherapy that surpasses this treatment has been clinically applied. Pleural resection / decortication (P / D) is the mainstream surgical resection based on early diagnosis, but the presence of residual tumor is a problem in this surgical procedure. As with other malignant tumors, it is most important to make an accurate diagnosis at an early stage before selecting a treatment method for malignant mesothelioma. However, there is currently no effective early diagnostic index for malignant mesothelioma. The actual case is a specialist who is suspected of having pleural mesothelioma on a chest X-ray taken by chance for a medical examination or other illness, or to see a doctor with symptoms such as cough or respiratory failure. It is a situation to see a doctor.

Traditionally, asbestos exposure history has been confirmed by investigations based on work history and residence history, and in actual cases by detection of pleural plaque by chest X-ray examination. Exposure to asbestos results in asbestosis (a typical disease of pneumoconiosis) or lung cancer, which causes fibrosis of the lungs when exposed to relatively high concentrations. In the case of relatively low concentration exposure, pleural plaque (callus-like irregular white plate-like thickening on the parietal pleura), benign asbestos pleural effusion, or diffuse pleural thickening, which is considered to be another benign disease along with malignant mesothelioma. And so on. These are often detected by chance by chest X-ray. In addition, regarding the condition of developing malignant mesothelioma (mesothelioma-bearing cancer), the case with symptoms is examined by chest X-ray or CT / PET, and finally diagnosed by pathological diagnosis. Has been made. Not only the workers of the asbestos handling factory, but also the residents around the asbestos handling factory, the rubble disposal workers such as the earthquake, the residents around the rubble, the building demolition work workers, etc. are considered to be a high risk group of asbestos exposure. However, in the current clinical practice, the screening method for health disorders caused by asbestos is mainly by chest X-ray or CT, and in the high-risk group, as a biomarker for mesothelioma, mesothelin or mesothelin-related protein (SMRP) in serum. Measurements and the like are being carried out on a trial basis.

Screening with conventional high-risk group radiodiagnosis techniques poses exposure problems. Due to the limited frequency of radiodiagnosis, malignant mesothelioma rapidly progresses and exacerbates, limiting screening. In addition, mesothelin and SMRP are substances produced by cancer cells, and it is thought that these substances increase in the blood as the cancer cells proliferate. Therefore, it is difficult to measure a single cancer cell-producing substance as a tool for early detection aimed at early treatment of mesothelioma.

Although it is clear that mesothelioma is related to the history of asbestos exposure, there are many unclear points about the detailed pathogenic mechanism, etc., and no effective marker has been found at present. Serum TNF-α concentration can be an indicator of early diagnosis of asbestos lung-related lung cancer, MCP-1 is involved in promoting canceration of the mesothelium, CXCR3 expression in CD4-positive T cells, and CXCL10 / IP10 It is disclosed that the serum concentration can be a candidate for an index for determining the pathological condition by exposure to asbestos (Non-Patent Documents 1 to 3). In addition, it has been shown that the value indicated by the prediction formula obtained by multiple regression analysis of changes in blood cytokine concentration is higher in mesothelioma patients than in healthy subjects and pleural plaque-positive subjects (Non-Patent Document 4). ..

Based on changes in the amount of asbestos used in Japan, it is estimated that the onset of malignant mesothelioma will occur in about 40 years, and that about 100,000 people will develop mesothelioma in Japan by about 2030. Long-term observation is required as some high-risk groups are predicted and are currently exposed to asbestos. The international situation is similar, and asbestos is still being exported and imported, so the World Health Organization (WHO) is in the process of issuing a declaration to eradicate asbestos-derived health disorders.

Partanen, R., et al .: Occup. Environ. Med., 52, 316-319 (1995) Chew, S.H., et al .: Carcinogenesis, 35, 164-172 (2014) Maeda, M., et al .: Am. J. Respir. Cell Mol. Biol., 45, 795-803 (2011) Yasumitsu Nishimura: Grant-in-Aid for Scientific Research (Grants-in-Aid for Scientific Research) Research Results Report (June 11, 2012)

An object of the present invention is to provide a test method applicable to early diagnosis of mesothelioma onset, degree of mesothelioma progression, risk of mesothelioma onset / progression, determination of therapeutic effect, and prognosis diagnosis. And.

As a result of diligent studies to solve the above problems, the present inventors have obtained measured values of three or more finite number of molecules to be analyzed selected from immune-related molecules in blood collected from a subject. , Asbestos exposure history, mesothelioma, and the presence or absence of pleural plaque can be easily determined, and the present invention has been completed.

That is, the present invention comprises the following.
1. 1. Examination of asbestos exposure history or mesothelioma, including the step of obtaining measurements of three or more finite number of molecules to be analyzed selected from immune-related molecules detected in the blood using blood collected from a subject. Method.
2. 2. The asbestos exposure history or mesothelioma is determined based on the step of analyzing the obtained measured values of the analysis target molecule using a pre-prepared multiple regression equation and the results obtained using the multiple regression equation. The inspection method according to item 1 above, which further comprises a step.
3. 3. The multiple regression equation prepared in advance analyzes the measured values of immune-related molecules obtained using blood collected from healthy subjects, pleural plaque patients, and mesothelioma-bearing cancer patients, and the following (a) It was created by selecting three or more finite immune-related molecules shown in any of (c) to (c) as analysis target molecules and performing multiple regression analysis of the measured values of the analysis target molecules. Inspection method described in item 2 above:
(A) Three or more finite immune-related molecules that show differences in measured values between the group consisting of healthy subjects and pleural plaque patients and the mesothelioma-bearing cancer patient group;
(B) Three or more finite immune-related molecules that show differences in measurements between the healthy group and the group consisting of pleural plaque patients and mesothelioma-bearing cancer patients;
(C) Three or more finite number of immune-related molecules having different measured values between the pleural plaque patient group and the group consisting of healthy subjects and mesothelioma-bearing cancer patients.
4. The immune-related molecules detected in the blood include plasma cytokines, proteins expressed on the surface of immunocompetent cells of peripheral blood mononuclear cells, and genes expressed by the immunocompetent cells in the above items 1 to 3. The inspection method according to any one.
5. Cytokines in plasma are IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL. -12p40, IL-12p70, IL-13, IL-15, IL-17A, EGF, Eotaxin, G-CSF, GM-CSF, IFN-α2, IFN-γ, IP-10, MCP-1, MIP-1α , MIP-1β, TNF-α, TNF-β and VEGF,
Proteins expressed on the surface of immunocompetent cells are CD25, CD69, CD25, GITR, CTLA-4, CXCR3, IL-1R1, IL-18R1, CD8-positive cytotoxic T cell surface Includes CD25, CD69, HLA-DR, CD45RO, CXCR3, FasL, NK cell surface CD69, NKG2D, NKp46, CXCR3, FasL, and monocyte surface HLA-DR, CD80, CD86.
The genes expressed by immunocompetent cells are the Foxp3 gene, T-bet gene, GATA-3 gene, RORC gene, TNF-α gene, IFN-γ gene, TGF-β gene, IL-4 gene, etc. of CD4-positive T cells. IL-6 gene, IL-10 gene, IL-17A gene, IL-R1 gene, IL-18R1 gene, Bcl-2 gene, CD8 positive cytotoxic T cell Notch1 gene, EOMES gene, CREB1 gene, Granzyme B gene , FasL gene, IFN-γ gene, TNF-α gene, IL-6 gene, IL-1R1 gene, IL-18R1 gene, NK cell NKp46 gene, Granzyme B gene, FasL gene, TNF-α gene, IFN-γ Any of the above items 1 to 4, including genes, IL-6 genes, and monocytic TNF-α genes, IL-1β genes, IL-12p35 genes, IL-15 genes, IL-18 genes, and IL-23 genes. The inspection method according to 1.
6. Using blood collected from the subject, the measured values of IFN-α2 in plasma, TNF-α gene of CD4-positive T cells, and the molecule to be analyzed, which is MCP1 in plasma, were obtained, and the measured values were calculated by the following formula 1. To identify subjects who are likely to have mesothelioma-bearing cancer based on the M-Score, which is the result obtained by using the multiple regression equation represented by the multiple regression equation;
Measurement of molecules to be analyzed, which are CXCR3 on the surface of CD8-positive cytotoxic T cells, Granzyme B gene of CD8-positive cytotoxic T cells, and IL18R1 gene of CD8-positive cytotoxic T cells, using blood collected from subjects. It is possible that there is a history of asbestos exposure based on the A-Score, which is the result obtained by acquiring the value, measuring the measured value using the multiple regression equation represented by the following formula 2, and using the multiple regression equation. Identifying highly likely subjects; or
Using blood collected from the subjects, the TNF-α gene of CD4 positive T cells, CXCR3 on the surface of NK cells, and the measured values of the molecules to be analyzed, which are GITRs on the surface of CD4 positive T cells, were obtained, and the measured values are as follows. Based on the P-Score, which is the result obtained by using the multiple regression equation expressed by the multiple regression equation in Equation 3, it is possible that the patient has a history of asbestos exposure but is not mesothelioma-bearing cancer. Identifying high subjects;
The inspection method according to any one of the preceding paragraphs 2 to 5, including
(Formula 1) M-Score = d ₁ + a ₁ [IFN-α 2] + b ₁ [Ths_TNF-α] + c ₁ [MCP-1]
(Formula 2) A-score = d ₂ + a ₂ [CXCR3MFI_CTL] + b ₂ [CTLs_GranzymeB] + c ₂ [CTL_IL18R1]
(Formula 3) P-score = d ₃ + a ₃ [Ths_TNF-α] + b ₃ [CXCR3MFI_NK] + c ₃ [GITR_Th]
[However, a _1, b 1, _{c 1,} d ₁ in Equation 1 is arbitrary real number not zero, [IFN-α2] is IFN-[alpha] 2 concentration in plasma, [Ths_TNF-α] is a CD4-positive T cells The TNF-α gene expression level, [MCP-1], is the plasma MCP-1 concentration, a ₂ , b ₂ , c ₂ , d ₂ in Equation 2 are arbitrary non-zero real numbers, and [CXCR3MFI_CTL] is CD8. The amount of CXCR3 on the surface of positive cytotoxic T cells, [CTLs_Granzyme B] is the expression level of Granzyme B gene in CD8 positive cytotoxic T cells, and [CTL_IL18R1] is the expression level of IL18R1 gene in CD8 positive cytotoxic T cells. in _{a 3, b 3, c 3} , d 3 are arbitrary real number not zero, [Ths_TNF-α] is TNF-alpha gene expression level of CD4-positive T cells, in CXCR3 amount of [CXCR3MFI_NK] is NK cell surface Yes, [GITR_Th] is the amount of GITR on the surface of CD4-positive T cells. ]
7. If the M-Score is higher than the cutoff value, the subject is identified as likely to have mesothelioma-bearing cancer;
If the A-score is higher than the cutoff value, the subject is identified as likely to have a history of asbestos exposure; or
If the P-score is higher than the cutoff value, it is identified that the subject has a history of asbestos exposure but is likely not a mesothelioma-bearing cancer;
The inspection method according to item 6 above.
8. Reagent kit for testing asbestos exposure history or mesothelioma containing at least 3 reagents selected from reagents for obtaining measurements of immune-related molecules detected in the blood collected from a subject. ..
9. Immunorelated molecules detected in blood include plasma cytokines, proteins expressed on the surface of immunocompetent cells of peripheral blood mononuclear cells, and genes expressed by the immunocompetent cells, and the above-mentioned at least three types. The reagent is selected from the group consisting of a reagent for detecting cytokines in blood, a reagent for detecting surface proteins of immunocompetent cells, and a reagent for detecting expression of genes expressed by immunocompetent cells.
Reagents for detecting plasma cytokines include anti-IL-1α antibody-containing reagent, anti-IL-1β antibody-containing reagent, anti-IL-1ra antibody-containing reagent, anti-IL-2 antibody-containing reagent, and anti-IL-3 antibody. Reagents containing, Reagents containing anti-IL-4 antibody, Reagents containing anti-IL-5 antibody, Reagents containing anti-IL-6 antibody, Reagents containing anti-IL-7 antibody, Reagents containing anti-IL-8 antibody, Anti-IL -10 antibody-containing reagent, anti-IL-12p40 antibody-containing reagent, anti-IL-12p70 antibody-containing reagent, anti-IL-13 antibody-containing reagent, anti-IL-15 antibody-containing reagent, anti-IL-17A antibody-containing reagent Reagents, Reagents Containing Anti-EGF Antibodies, Reagents Containing Anti-Eotaxin Antibodies, Reagents Containing Anti-G-CSF Antibodies, Reagents Containing Anti-GM-CSF Antibodies, Reagents Containing Anti-IFN-α2 Antibodies, Includes Anti-IFN-γ Antibodies Reagent, Reagent containing anti-IP-10 antibody, Reagent containing anti-MCP-1 antibody, Reagent containing anti-MIP-1α antibody, Reagent containing anti-MIP-1β antibody, Reagent containing anti-TNF-α antibody, Anti-TNF- Contains reagents containing β-antibodies and reagents containing anti-VEGF antibodies
Reagents for detecting immunocompetent cell surface expression proteins include anti-CD25 antibody-containing reagent, anti-CD69 antibody-containing reagent, anti-GITR antibody-containing reagent, anti-CTLA-4 antibody-containing reagent, anti-CXCR3 antibody-containing reagent, anti-CXCR3 antibody. Reagent containing IL-1R1 antibody, Reagent containing anti-IL-18R1 antibody, Reagent containing anti-HLA-DR antibody, Reagent containing anti-CD45RO antibody, Reagent containing anti-CXCR3 antibody, Reagent containing anti-FasL antibody, Anti-NKG2D antibody Contains reagents, anti-NKp46 antibody-containing reagents, anti-CD80 antibody-containing reagents, and anti-CD86 antibody-containing reagents.
Reagents for detecting the expression of genes expressed by immunocompetent cells include reagents that hybridize to the Foxp3 gene, reagents that contain polynucleotides that hybridize to the T-bet gene, and polynucleotides that hybridize to the GATA-3 gene. A reagent containing a polynucleotide that hybridizes to the RORC gene, a reagent containing a polynucleotide that hybridizes to the TNF-α gene, a reagent containing a polynucleotide that hybridizes to the IFN-γ gene, and a reagent that hybridizes to the TGF-β gene. Reagents containing polynucleotides to hybridize, Reagents containing polynucleotides that hybridize to the IL-4 gene, Reagents containing polynucleotides that hybridize to the IL-6 gene, Reagents that contain polynucleotides that hybridize to the IL-10 gene, A reagent containing a polynucleotide that hybridizes to the IL-17A gene, a reagent containing a polynucleotide that hybridizes to the IL-R1 gene, a reagent that contains a polynucleotide that hybridizes to the IL-18R1 gene, and a reagent that hybridizes to the Bcl-2 gene. A reagent containing a polynucleotide, a reagent containing a polynucleotide that hybridizes to the Notch1 gene, a reagent containing a polynucleotide that hybridizes to the EOMES gene, a reagent containing a polynucleotide that hybridizes to the CREB1 gene, and a poly that hybridizes to the Granzyme B gene. A reagent containing a nucleotide, a reagent containing a polynucleotide that hybridizes to the FasL gene, a reagent containing a polynucleotide that hybridizes to the TNF-α gene, a reagent containing a polynucleotide that hybridizes to the IL-1R1 gene, and a reagent that hybridizes to the NKp46 gene. A reagent containing a polynucleotide that hybridizes to the IL-1β gene, a reagent containing a polynucleotide that hybridizes to the IL-1β gene, a reagent that contains a polynucleotide that hybridizes to the IL-12p35 gene, a reagent that contains a polynucleotide that hybridizes to the IL-15 gene, IL A reagent containing a polynucleotide that hybridizes to the -18 gene, a reagent containing a polynucleotide that hybridizes to the IL-23 gene,
The reagent kit for testing asbestos exposure history or mesothelioma according to item 8 above.
10. The reagent kit for testing asbestos exposure history or mesothelioma according to item 8 or 9 above, which comprises at least a combination of any one of the following reagents:
1) A combination of a reagent containing an anti-IFN-α2 antibody, a reagent containing an anti-CD4 antibody, a reagent containing a polynucleotide that hybridizes to the TNF-α gene, and a reagent containing an anti-MCP-1 antibody;
2) A combination of a reagent containing an anti-CD8 antibody, a reagent containing an anti-CXCR3 antibody, a reagent containing a polynucleotide that hybridizes to the Granzyme B gene, and a reagent containing a polynucleotide that hybridizes to the IL18R1 gene;
3) A combination of a reagent containing an anti-CD4 antibody, a reagent containing a polynucleotide that hybridizes to the TNF-α gene, a reagent containing an anti-CD56 antibody, and a reagent containing an anti-GITR antibody.
11. The measured values of three or more finite number of analysis target molecules selected from the immune-related molecules detected in the blood obtained by using the blood collected from the subject are analyzed using a multiple regression equation prepared in advance. For testing asbestos exposure history or mesothelioma, which functions a computer as a data analysis means and a determination means for determining asbestos exposure history or mesothelioma based on the results obtained using the multiple regression equation. program.
12. By analyzing the measured values of immune-related molecules obtained using blood collected from healthy subjects, pleural plaque patients, and mesothelioma-bearing cancer patients, it is shown in any of the following (a) to (c). A computer as a sorting means for selecting three or more finite number of immune-related molecules as the analysis target molecule and a means for creating a multiple regression equation by performing multiple regression analysis of the measured values of the analysis target molecule. The inspection program according to the preceding paragraph 11 for the functioning of
(A) Three or more finite immune-related molecules that show differences in measured values between the group consisting of healthy subjects and pleural plaque patients and the mesothelioma-bearing cancer patient group;
(B) Three or more finite immune-related molecules that show differences in measurements between the healthy group and the group consisting of pleural plaque patients and mesothelioma-bearing cancer patients;
(C) Three or more finite number of immune-related molecules having different measured values between the pleural plaque patient group and the group consisting of healthy subjects and mesothelioma-bearing cancer patients.
13. The measured values of three or more finite number of molecules to be analyzed selected from the immune-related molecules detected in the blood obtained by using the blood collected from the subject are analyzed using a multiple regression equation prepared in advance. A device for examining asbestos exposure history or mesothelioma, comprising data analysis means and a determination means for determining asbestos exposure history or mesothelioma based on the results obtained by using a multiple regression equation.

By the test method of the present invention, the presence or absence of asbestos exposure history, mesothelioma, and pleural plaque was obtained by acquiring the measured values of three or more finite number of molecules to be analyzed selected from immune-related molecules in the blood collected from the subject. Can be easily determined. By comprehensively judging the results of asbestos exposure history, mesothelioma, and the presence or absence of pleural plaque, early diagnosis of mesothelioma onset, degree of mesothelioma progression, risk of mesothelioma onset / progression, Judgment of therapeutic effect and prognosis can be performed quickly, easily, and with high accuracy as compared with conventional examination methods. Further, since the test method of the present invention uses blood as a sample, it is possible to continuously test and monitor a subject at high risk of mesothelioma, and early diagnosis and treatment of mesothelioma are possible. It becomes. Further, according to the examination method of the present invention, it can be performed by collecting about 10 to 20 ml of blood, and it can be performed more frequently than the conventional method by X-ray photography.

Representative examples of cell surface markers showing differences are shown. (Example 1) A representative example of the mRNA amount of the gene in which a difference is observed is shown. (Example 1) A representative example of the mRNA amount of the gene in which a difference is observed is shown. (Example 1) A representative example of plasma cytokine concentrations showing a difference is shown. (Example 1) It is a figure which shows the analysis result about M-Score. (Example 2) It is a figure which shows the analysis result about A-Score. (Example 2) It is a figure which shows the analysis result about P-Score. (Example 2) The correlation between M-Score and P-Score is shown. (Example 2) It is a figure which shows the ROC curve of each score and the cutoff value. (Example 2)

The present invention includes asbestos exposure history or mesothelioma, which comprises the step of obtaining measurements of three or more finite number of molecules to be analyzed selected from immune-related molecules detected in the blood collected from a subject. Regarding the method of examining tumors. The inspection method of the present invention provides diagnostic materials for evaluating the presence or absence of the onset of mesothelioma, the degree of progression of mesothelioma, the risk of onset / progression of mesothelioma, and the like. Pleural plaque is a bilateral irregular white plate-like thickening that occurs mainly in the parietal pleura. The pleural plaque itself is a benign lesion without pulmonary dysfunction. Although pleural plaque does not convert to mesothelioma, it is known that those with pleural plaque have a higher cumulative exposure to asbestos than those without. Although the history of asbestos exposure is thought to be closely related to the onset of mesothelioma, pleural plaque caused by asbestos exposure is not found in all cases, and pleural plaque is precancerous. Can't be caught. The test method of the present invention obtains a score regarding the presence or absence of mesothelioma-bearing cancer, a score regarding the presence or absence of asbestos exposure history, and a score when there is asbestos exposure history but not mesothelioma-bearing cancer, and the scores are used as diagnostic materials. By using it, it is possible to comprehensively predict or evaluate the risk of developing / developing mesothelioma, the degree of progression of mesothelioma, and the like.

In the present invention, the subject may be any person as long as it is the subject of the examination. The subject in the present invention may be a subject of a general medical examination, a human dock, or the like, or is a subject of a test for mesopharyngoma, for example, a test for mesencephalon, a test for early diagnosis, or a stage of disease. It may be a person to be examined for examinations such as malignancy, judgment of therapeutic effect, and prognosis.

In the present invention, blood collected from a subject is used as a test sample. In the present specification, a sample is a sample collected from a living body, and a sample that is subjected to some kind of processing on the sample and is subjected to an examination is referred to as a sample. Blood-derived samples are those obtained by treating the collected blood and separating it into plasma, those separated by peripheral blood mononuclear cells, and those separated based on the expression of various surface antigens. The method for preparing a blood-derived sample from blood can be a method known per se and is not particularly limited. For molecules produced by tumor cells and leaking into the blood, it is thought that the amount in the blood increases as the size of the tumor cells increases. Conventionally, molecules in blood have been used as an auxiliary diagnostic item at the time of discovery or as a monitor for the presence or absence of recurrence after treatment, rather than as an early diagnostic index. On the other hand, the present invention can detect the onset of malignant mesothelioma by detecting an immune-related molecule in the blood of a subject.

The immune-related molecule detected in blood is a molecule present in blood and involved in the immune system in the living body. Immunorelated molecules detected in blood include plasma cytokines, proteins expressed on the surface of immunocompetent cells of peripheral blood mononuclear cells, and genes expressed by the immunocompetent cells. The measured values of three or more finite number of molecules to be analyzed selected from the immune-related molecules detected in blood obtained in the present invention are the concentration of the molecule to be analyzed secreted in blood and the blood. The expression level of the molecule to be analyzed in the cells contained in. More specifically, the concentration of cytokines in plasma, the expression level (degree of expression) of proteins expressed on the surface of immunocompetent cells of peripheral blood mononuclear cells, and the expression level of genes expressed by the immunocompetent cells. It is selected from.

The plasma cytokine may be any cytokine contained in plasma separated from blood. Cytokines are microbiologically active proteins that are released from cells and become various cell-cell communication molecules. They bind to high-affinity receptors on the cell surface via blood and express multifaceted biological activities. .. Specific examples of cytokines in plasma include IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL. -10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17A, EGF, Eotaxin, G-CSF, GM-CSF, IFN-α2, IFN-γ, IP-10, MCP-1 , MIP-1α, MIP-1β, TNF-α, TNF-β and VEGF. The measurement of the cytokine concentration in plasma can be performed by a method known per se, and is not particularly limited.

Immunocompetent cells are cells involved in the immune response in vivo, and specifically include B cells, T cells (CD4 positive T cells (helper T cells), CD8 positive T cells (cytotoxic T cells), etc. And regulatory T cells, etc.), lymphoid cells, including natural killer (NK) cells, antigen-presenting cells, including monospheres, macrophages, and dendritic cells, neutrophils, eosinophils, basinocytes, and obesity Examples include granulocytes containing cells. In the test method of the present invention, the expression level (degree of expression) of the protein expressed on the cell surface of the immunocompetent cell or the expression level of the gene expressed in the immunocompetent cell is measured by a method known per se. It can be done and is not particularly limited. The expression level (degree of expression) of the protein expressed on the cell surface of the immunocompetent cell can be measured by staining with an antibody or the like and performing flow cytometry. The expression level of a gene expressed by an immunocompetent cell can be measured by detecting the intracellular mRNA level or protein level of the gene, and is preferably measured after stimulating the immune system.

Specific examples of proteins expressed on the surface of immunocompetent cells include CD25, CD69, CD25, GITR, CTLA-4, CXCR3, IL-1R1, IL-18R1, and CD8-positive cytotoxicity on the surface of CD4-positive T cells. CD25, CD69, HLA-DR, CD45RO, CXCR3, FasL on the surface of T cells, CD69, NKG2D, NKp46, CXCR3, FasL on the surface of NK cells, and HLA-DR, CD80, CD86 on the surface of monocytes.

Specific examples of genes expressed by immunocompetent cells include Foxp3 gene, T-bet gene, GATA-3 gene, RORC gene, TNF-α gene, IFN-γ gene, TGF-β gene, and IL of CD4-positive T cells. -4 gene, IL-6 gene, IL-10 gene, IL-17A gene, IL-1R1 gene, IL-18R1 gene, Bcl-2 gene, CD8 positive cytotoxic T cell Notch1 gene, EOMES gene, CREB1 gene , Granzyme B gene, FasL gene, IFN-γ gene, TNF-α gene, IL-6 gene, IL-1R1 gene, IL-18R1 gene, NK cell NKp46 gene, Granzyme B gene, FasL gene, TNF-α gene , IFN-γ gene, IL-6 gene, and monocytic TNF-α gene, IL-1β gene, IL-12p35 gene, IL-15 gene, IL-18 gene, IL-23 gene.

The three or more finite number of molecules to be analyzed of the present invention are preferably selected from plasma cytokines, proteins expressed on the surface of immunocompetent cells of peripheral blood mononuclear cells, and genes expressed by the immunocompetent cells. Is selected from each of the molecules specifically exemplified above. The molecule to be analyzed is preferably selected to be 3 or more in order to increase the sensitivity and specificity, and is preferably 5 or less from the viewpoint of complexity of the test and sensitivity and specificity.

In the present invention, asbestos exposure history or mesothelioma is examined based on the measured values of immune-related molecules, and the formula obtained by multivariate analysis of the measured values of immune-related molecules is used in the examination of the present invention. Can be done. As the multivariate analysis, it is preferable that the relationship between the explanatory variable and the objective variable can be used. For example, discriminant analysis, principal component analysis, factor analysis, quantification theory class 1, quantification theory class 2, quantification theory class 3, Regression analysis (MLR, PLS, PCR, logistic), multidimensional scale method, clustering with objective variables, neural network, ensemble learning method, etc. can be exemplified, and formulas can be created using free software or commercially available ones. Can be done. Of these, multiple regression analysis is particularly preferred. Using the measured values of the molecules to be analyzed selected from immune-related molecules as explanatory variables, for example, identification of subjects with mesothelioma-bearing cancer, identification of subjects with asbestos exposure history, and mesothelioma-bearing cancer with asbestos exposure history It is preferable to perform multiple regression analysis to obtain a multiple regression equation with the identification of no subject as the objective variable. Hereinafter, the present invention will be described by taking a multiple regression equation as an example.

The molecule to be analyzed can be selected as follows. First, measurements of immune-related molecules are obtained from blood collected from healthy subjects, pleural plaque patients, and mesothelioma-bearing cancer patients. Multiple regression analysis can be performed on the acquired measured values, and immune-related molecules that show differences between groups for the purpose of distinguishing can be selected as analysis target molecules. As the immune-related molecules that show differences, those that have a large influence on the distinction between the target groups are selected. The measurements of the selected immune-related molecules are used as explanatory variables in the multiple regression equation to make a distinction between the groups of interest. Further, the coefficient and the determination term (constant) in the multiple regression equation can be set by the multiple regression analysis of the measured value.

That is, a healthy person, a pleural plaque patient, and a mesothelioma-bearing cancer patient are a group of people who can be a standard for deriving a test result by the test method of the present invention. A healthy person can be defined in a broad sense, and if a person included in a healthy person has some kind of disease but is judged not to have pleural plaque and mesothelioma-bearing cancer, the healthy person Can be included in. Pleural plaque patients are a group of patients with pleural plaque and are composed of patients who do not have mesothelioma-bearing cancer. Patients with mesothelioma-bearing cancer include a group of patients diagnosed with mesothelioma-bearing cancer, a group of patients with a clear stage of mesothelioma-bearing cancer, and a group of patients with a clear malignancy of mesothelioma-bearing cancer. It may include patients who are composed and have comorbid pleural plaques. By performing multiple regression analysis using measurements obtained from healthy subjects, pleural plaque patients, and mesothelioma-bearing cancer patients, a multiple regression equation was created to distinguish between the target groups. The cutoff value of the score obtained by can be set.

In the present invention, the inspection is carried out by the following steps using the obtained multiple regression equation.
(A) A step of acquiring measured values of 3 or more finite number of molecules to be analyzed in blood using blood collected from a subject;
(B) A step of analyzing the acquired measured values of the molecule to be analyzed using a multiple regression equation created in advance, preferably a step of calculating a score for distinguishing between target groups;
(C) A step of comparing the preset cutoff value with the score calculated in (B) above;
(D) A step in which a subject is determined to belong to a specific group when the score of the multiple regression equation obtained in (B) above belongs to a specific group as compared with a preset cutoff value.

Even if the multiple regression equation is created, the measured values of immune-related molecules are additionally obtained for blood collected from healthy subjects, pleural plaque patients, and mesothelioma-bearing cancer patients, and the obtained measured values are included in the weight. By performing regression analysis, it is possible to update the selection of the molecule to be analyzed, the coefficients and determinants in the multiple regression equation, and the cutoff value for distinguishing between the target groups at any time. The update may be performed at the same time as the examination by the subject.

In the present invention, three or more finite number of immune-related molecules are selected as analysis target molecules among the groups selected from the following (a) to (c) for the purpose of distinction, and the analysis target is concerned. It is preferable that a multiple regression equation is created by performing multiple regression analysis of the measured values of the molecule.
(A) Three or more finite number of immune-related molecules showing a difference in measured values between a group consisting of healthy subjects and pleural plaque patients and a group of mesothelioma-bearing cancer patients.
(B) Three or more finite number of immune-related molecules showing a difference in measured values between the healthy subject group and the group consisting of pleural plaque patients and mesothelioma-bearing cancer patients.
(C) Three or more finite number of immune-related molecules having different measured values between the pleural plaque patient group and the group consisting of healthy subjects and mesothelioma-bearing cancer patients.

The molecule to be analyzed may be any molecule selected according to the above method, and is not particularly limited. For example, the following three combinations of the test purpose and the molecule to be analyzed are exemplified.
(1) Using blood collected from a subject, the measured values of IFN-α2 in plasma, the TNF-α gene of CD4-positive T cells, and the molecule to be analyzed, which is MCP-1 in plasma, were obtained, and the measured values were obtained. Analysis is performed using the multiple regression equation, and subjects who are likely to have mesothelioma-bearing cancer are identified based on the score (M-Score) obtained using the multiple regression equation.
(2) Using blood collected from the subject, CXCR3 on the surface of CD8-positive cytotoxic T cells, Granzyme B gene of CD8-positive cytotoxic T cells, and IL-18R1 gene of CD8-positive cytotoxic T cells. It is possible that there is a history of asbestos exposure based on the score (A-Score) obtained by acquiring the measured value of the molecule to be analyzed, measuring the measured value using the multiple regression equation, and using the multiple regression equation. Identify highly likely subjects.
(3) Using blood collected from the subject, the measured values of the TNF-α gene of CD4-positive T cells, CXCR3 on the surface of NK cells, and the molecule to be analyzed, which is the GITR on the surface of CD4-positive T cells, were obtained and measured. Based on the score (P-Score) obtained by analyzing the values using the multiple regression equation and using the multiple regression equation, subjects with a history of asbestos exposure but likely not to have mesothelioma-bearing cancer To identify.

The multiple regression equations in the above (1) to (3) are expressed by the following equations 1 to 3.
(Formula 1) M-Score = d ₁ + a ₁ [IFN-α 2] + b ₁ [Ths_TNF-α] + c ₁ [MCP-1]
(Formula 2) A-Score = d ₂ + a ₂ [CXCR3MFI_CTL] + b ₂ [CTLs_GranzymeB] + c ₂ [CTL_IL18R1]
(Formula 3) P-Score = d ₃ + a ₃ [Ths_TNF-α] + b ₃ [CXCR3MFI_NK] + c ₃ [GITR_Th]
However, in Equation 1, a ₁ , b ₁ , c ₁ , and d ₁ are non-zero real numbers, [IFN-α2] is the plasma IFN-α2 concentration, and [Ths_TNF-α] is the TNF of CD4-positive T cells. -α gene expression level, [MCP-1] is the plasma MCP-1 concentration, a ₂ , b ₂ , c ₂ , d ₂ in Equation 2 are arbitrary non-zero real numbers, and [CXCR3MFI_CTL] is CD8 positive. CXCR3 level on the surface of cytotoxic T cells, [CTLs_Granzyme B] is the Granzyme B gene expression level of CD8-positive cytotoxic T cells, [CTL_IL18R1] is the IL-18R1 gene expression level of CD8-positive cytotoxic T cells. In 3, a ₃ , b ₃ , c ₃ , and d ₃ are non-zero real numbers, [Ths_TNF-α] is the TNF-α gene expression level in CD4-positive T cells, and [CXCR3MFI_NK] is the CXCR3 level on the surface of NK cells. And [GITR_Th] is the amount of GITR on the surface of CD4-positive T cells. Specific values of a ₁ , b ₁ , c ₁ , d ₁ , a ₂ , b ₂ , c ₂ , d ₂ , a ₃ , b ₃ , c ₃ , d ₃ are set by multiple regression analysis. It should be.

The following mathematical formulas are exemplified as the multiple regression equations in the above (1) to (3).
(Formula 1) M-Score = 2.43056 + 0.00460 x [IFN-α2] + 0.56948 x [Ths_TNF-α] + 0.00075 x [MCP-1]
(Formula 2) A-Score = 0.76825 --0.00248 x [CXCR3MFI_CTL] + 0.52846 x [CTLs_GranzymeB] --0.72952 x [CTL_IL18R1]
(Formula 3) P-Score = 0.22813 --0.66572 x [Ths_TNF-α] --0.01333 x [CXCR3MFI_NK] --0.00733 x [GITR_Th]

When the subject's M-Score obtained by the above formula 1 is higher than the cutoff value, the subject is identified as having a high possibility of mesothelioma-bearing cancer, and the subject's A-Score obtained by the above formula 2 is identified. Is higher than the cut-off value, it is identified that the subject is likely to have a history of asbestos exposure, and if the subject's P-Score obtained by Equation 3 above is higher than the cut-off value, the subject is asbestos. Identified as having a history of exposure but likely not mesothelioma-bearing cancer. If a subject is identified as having a high probability of mesothelioma-bearing cancer, the subject should be promptly transferred to chest X-ray, CT / PET, and pathological diagnosis. Mesothelioma cancer is unlikely, but if a history of asbestos exposure is identified, subjects can be monitored for testing and are relatively frequent (eg 2-3 times / year). It is preferable to continuously carry out the inspection method of the present invention and observe the progress. Subjects may be excluded from close examination monitoring if they are identified as having a low likelihood of mesothelioma-bearing cancer and no history of asbestos exposure, but are originally high-risk groups 1- Monitoring by collecting blood once every two years is desirable.

The present invention relates to blood collected from a subject and contains at least three reagents selected from reagents for obtaining measurements of immune-related molecules detected in the blood, asbestos exposure history or mesothelioma. Includes test reagent kits. With the at least three or more reagents, it is possible to obtain measured values of three or more finite number of molecules to be analyzed selected from immune-related molecules detected in blood. The at least three types of reagents are a reagent for detecting a cytokine in plasma containing an antibody capable of binding to a cytokine in plasma, and a detection of an immunocompetent cell surface protein containing an antibody capable of binding to a protein expressed on the surface of the immunocompetent cell. It is selected from a reagent for detecting the expression of the gene, which comprises a reagent for use and a polynucleotide capable of detecting gene expression in immunocompetent cells.

In the present specification, an antibody capable of binding to a specific protein such as a cytokine in plasma or a protein expressed on the surface of immunocompetent cells may be an antibody known per se, or an existing general production method. Antibodies produced by or commercially available antibodies can be used. The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may be labeled with a labeling substance. Specifically, as the labeling substance, an enzyme, a radioisotope, a fluorescent dye, biotin, a dye sol, and an insoluble carrier such as colloidal gold or latex particles can be used. Further, although labeling can be performed by a known method, the labeling substance may be bound to an antibody. The above antibody is included in the detection reagent kit of the present invention as a reagent (reagent composition). Examples of the reagent containing the antibody include reagents that can be stored for a long period of time, such as in a state diluted with physiological saline or a buffer solution, or in a freeze-dried form. The reagent may be prepared by dissolving the antibody in a solution containing an additive (for example, a carrier, an excipient, a diluent, etc.), a stabilizer, or the like, and then freeze-drying the antibody. Stabilizers include monosaccharides such as glucose, disaccharides such as saccharose and maltose, sugar alcohols such as mannitol and sorbitol, neutral salts such as sodium chloride, amino acids such as glycine, polyethylene glycol and polyoxyethylene-polyoxy. Nonionic surfactants such as propylene copolymer (Pluronic) and polyoxyethylene sorbitan fatty acid ester (Tween), human glucose and the like are exemplified, and it is preferable that about 1 to 10 w / v% is added. Further, the detection reagent kit of the present invention may appropriately contain a blocking solution, a reaction solution, a reaction terminator solution and the like.

In the present specification, a polynucleotide refers to a biopolymer in which two or more nucleotides consisting of a base, a sugar, and a phosphate are bonded to each other by a phosphate ester, and includes nucleic acids such as DNA and RNA. The polynucleotides of the invention have a length of at least 15 nucleotides (bases), preferably 17 nucleotides (bases). The polynucleotide functions as a probe capable of specifically hybridizing to a specific gene, a primer capable of specifically annealing to a specific gene, and a primer capable of amplifying a nucleic acid fragment in combination with the primer. The base sequence of the polynucleotide does not have to be a sequence that is 100% complementary to the base sequence of the gene whose expression is detected, and is about 1 to 5 bases (preferably about 1 to 4 bases, more preferably 1 to 2). It may be deleted (as much as a base), or it may be replaced, inserted or added with a non-complementary base. When the polynucleotide is a primer, it is preferably about 17 to 25 nucleotides, and when it is a probe, it is preferably about 10 to 30 nucleotides. The primers may be included in the detection reagent kit of the present invention as a pair of primer sets.

Primers and probes can be designed using known primers or probe design software. Known software includes, for example, OLIGO Primer Analysis Software (Molecular Biology Insights), Beacon Designer (PREMIER Biosoft), Primer Express Software (Life Technologies), Primer3web version 4.0.0 Pick primers from a DNA sequence (http: /). /bioinfo.ut.ee/primer3/) etc. can be used. Further, the polynucleotide can be synthesized by a method known in the art as a method for synthesizing a polynucleotide, for example, a phosphotriethyl method, a phosphodiester method, or the like, using a commonly used automatic DNA synthesizer. The above-mentioned polynucleotide can be used as a solid reagent in a dry state or in an alcohol-precipitated state, or as a reagent dissolved in water, physiological saline, or a suitable buffer solution (eg, TE buffer solution, etc.). It can also be included in the detection reagent kit of the invention. The reagent may contain additives (for example, carriers, excipients, diluents, etc.), stabilizers, and the like. Specific examples of the stabilizer are as described above. In addition, the test reagent kit of the present invention may contain reagents necessary for the nucleic acid amplification reaction, such as DNA synthase, buffer solution, dNTP mix, intercalator, magnesium chloride and the like. The intercalator can detect the presence or absence of the amplification product by staining the amplification product by a nucleic acid amplification reaction such as the PCR method. Examples of the intercalator include ethidium bromide and Cybergreen. Further, for the detection of the amplified product by real-time PCR, a polynucleotide labeled with a fluorescent substance or the like may be included in the test reagent kit of the present invention.

Various reagents known per se are known for preparing blood-derived samples such as plasma, peripheral blood mononuclear cells, and cell groups separated based on the expression of various surface antigens from blood. For example, a reagent containing an anti-CD4 antibody for detecting CD4-positive T cells, a reagent containing an anti-CD8 antibody for detecting CD8-positive cytotoxic T cells, and a reagent containing an anti-CD56 antibody for detecting NK cells. Etc. are exemplified. In addition, as reagents for separating (sorting) various cell groups, reagents containing anti-CD3 antibody, reagents containing anti-CD56 antibody, reagents containing anti-CD19 antibody, reagents containing anti-CD4 antibody, reagents containing anti-CD8 antibody, Reagents containing anti-HLA-DR antibodies are exemplified. In addition, a reagent containing an anti-CD14 antibody can be used to detect monocytes. The reagent kit for testing asbestos exposure history or mesothelioma of the present invention may contain one or more of the reagents for preparing the above blood-derived samples.

Specific examples of various antibodies or polynucleotides include those described in Examples described later.

Further, in the present invention, multiple regression equations prepared in advance are used to obtain measured values of three or more finite number of molecules to be analyzed selected from immune-related molecules detected in blood, which are obtained using blood collected from a subject. Asbestos exposure history or medium, which makes a computer function as a data analysis means to analyze using the above and as a judgment means for determining asbestos exposure history or mesothelioma based on the results obtained using the multiple regression equation. It also includes a program for testing mesothelioma. The inspection program of the present invention may further function a computer as a storage means for storing measured values. The test program of the present invention further analyzes the measured values of immune-related molecules obtained using blood collected from healthy subjects, pleural plaque patients, and mesothelioma-bearing cancer patients, from the above (a). A multiple regression equation is obtained by performing multiple regression analysis on a selection means for selecting three or more finite number of immune-related molecules shown in any one of (c) as analysis target molecules and the measured values of the analysis target molecules. As a means of creating the device, it may be an inspection program for operating the computer.

Furthermore, the present invention further prepares in advance multiple regression equations of measurement values of three or more finite number of molecules to be analyzed selected from immune-related molecules detected in blood obtained by using blood collected from a subject. Asbestos exposure history or mesothelioma, provided with data analysis means for analysis using and a determination means for determining asbestos exposure history or mesothelioma based on the results obtained using the multiple regression equation. It also includes inspection equipment. The test apparatus of the present invention is for measuring the measured values of three or more finite number of molecules to be analyzed selected from immune-related molecules detected in blood obtained by using blood collected from a subject. It may be provided with a measuring means and a display means for showing a determination result based on the determination means.

The test method, test program, and test device of the present invention mainly use multi-marker tests rather than one biomarker, and relate to the condition of the subject (history of asbestos exposure and malignant mesothelioma). The state) can be reflected in more detail. This makes it possible to more accurately diagnose mesothelioma at an early stage, determine the risk of onset / progression, determine the therapeutic effect, and diagnose the prognosis.

In order to help understanding of the present invention, the present invention will be specifically described with reference to Examples below, but it goes without saying that the present invention is not limited to the present examples.

(Example 1) Measurement of immune-related molecules With the approval of the Ethics Committee of Okayama Labor Disaster Hospital and Hyogo College of Medicine Hospital, healthy volunteers (healthy individuals) (HV) and pleural plaque-positive patients shown in Table 1 below ( Blood was collected from PL) and mesothelioma-bearing cancer patients (MM), and the immune-related molecules shown in Table 2 below were analyzed.

Blood collected from the above-mentioned healthy subjects, pleural plaque-positive patients, and mesothelioma-bearing cancer patients was centrifuged to obtain plasma. About plasma Using the Human Cytokine Magnetic 30-Plex kit (Life Technologies Japan Co., Ltd.), the concentrations of 29 cytokines were measured with the Luminex machine (manufactured by Bio-Rad) using the HCYTMAG-60K-PX29 (Merck Millipore). (Plasma cytokines).

Peripheral blood mononuclear cells (PBMCs) were also isolated using the same blood by Ficoll density gradient centrifugation. The obtained PBMC is stained with an antibody according to a conventional method, and then the fluorescence intensity is measured with a flow cytometer (FACS Calibur, manufactured by BD Bioscience) to detect Monocyte region (monocytes) and CD3 + CD4 + cells (CD4 positive). Cell surface markers (Surface molecules) for each of helper T cells (Th), CD3 + CD8β + cells (CD8-positive cytotoxic T cells: CTL), and CD3-CD56 + cells (natural killer cells: NK). ) Was confirmed.

The PBMC obtained in the same manner was stained with an antibody, and then the fluorescence intensity was measured with a flow cytometer (FACS Aria, manufactured by BD Bioscience), and the Monocyte region (monocyte) and CD4 + CD8β-cell (CD4) were measured. Positive helper T cells: Th), CD4-CD8β + cells (CD8-positive cytotoxic T cells: CTL), and CD4-CD8β-CD56 + cells (natural killer cells: NK) were fractionated. For each fractionated cell group, various mRNA expression levels were measured (Fresh) without pretreatment, or various mRNA expression levels were measured after stimulation with PMA / ionomycin for 1 day (Stimulated with PMA /). IM). The mRNA expression level was measured by a conventional method using real-time PCR.

To confirm CD4 positive, CD8 positive, and CD56 positive, each cell was fractionated by staining with a monoclonal antibody against CD4, CD8, and CD56, respectively. Monocytes were classified by flow cytometer according to cell granules or intracellular structure represented by FSC and SSC. Gene expression was observed using the ^R Easy mini Kit (Qiagen) as an RNA extraction kit for RNA extraction and the Prime Script ^(R) II 1st strand cDNA Synthesis kit (TaKaRa), which is a cDNA synthesis kit, for cDNA synthesis. ..

The antibodies used to detect the membrane surface molecules are as follows. The Aria antibody is for fractionation (sorting), and the Calibur antibody is for observing the expression level.
<Aria antibody list>
BD Pharmingen FITC Mouse Anti-Human CD3 (anti-CD3 antibody)
BD Pharmingen PE Mouse Anti-Human CD56 (anti-CD56 antibody)
BD Pharmingen PE-Cy5 Mouse Anti-Human CD19 (anti-CD19 antibody)
BD Pharmingen Anti-CD4 (Human) mAb-FITC (anti-CD4 antibody)
BECKMAN COULTER Anti-CD8beta (Human) mAb-PC5 (anti-CD8 antibody)
eBioscience anti-human HLA-DR PE LN3 100 tests, (PE) (anti-HLA-DR antibody)
<Calibur antibody list>
BD Pharmingen FITC Mouse Anti-Human CD3 (anti-CD3 antibody)
BD Pharmingen PE-Cy5 Mouse Anti-Human CD56 (anti-CD56 antibody)
BECKMAN COULTER Anti-CD8beta (Human) mAb-PC5 (anti-CD8 antibody)
BECKMAN COULTER Anti-CD4 (Human) mAb-PC5 (anti-CD4 antibody)
BECKMAN COULTER Anti-CD14 (Human) mAb-FITC (anti-CD14 antibody)
eBioscience anti-human HLA-DR PE LN3 100 tests, (PE) (anti-HLA-DR antibody)
R & D SystemsBD Pharmingen PE Mouse Anti-Human CD183 (CXCR3) (Anti-CXCR3 antibody)
BD Pharmingen PE Mouse Anti-Human CD25 (anti-CD25 antibody)
BECKMAN COULTER Anti-CD86 (B7-2) (Human) mAb-PE (anti-CD86 antibody)
BECKMAN COULTER Anti-CD335 (NKp46) (Human) mAb-PE (anti-NKp46 antibody)
Bio Legend PE anti-human CD45RO Antibody (anti-CD45RO antibody)
BioLegend PE anti-human CD218a (IL-18Rα) Antibody (anti-IL-18R1 antibody)
Bio Legend Anti-Human CD69 PE (FN50) 100 tests, (PE) (Anti-CD69 antibody)
BioLegend PE anti-human CD80 (B7-1, B71) 100 tests, (PE) (anti-CD80 antibody)
Bio Legend Anti-human CD178 (CD95 Ligand) (Anti-FasL antibody)
eBioscience Anti-human CD314 (NKG2D) (anti-NKG2D antibody)
R & D Systems Anti CTLA-4, Human (Goat) (Anti-CTLA-4 antibody)
R & D Systems Anti GITR / TNFRSF18, Human (Mouse) (Anti-GITR antibody)
R & D Systems Anti IL-1 Receptor 1, Human (Goat) (Anti-IL-1R1 antibody)

The mRNA expression level was measured by the real-time PCR method using the Mx3000P QPCR System (Agilent Technologies, Inc.,) using Cybergreen. The primers used in the real-time PCR method were designed using the open source net service: Primer3web version 4.0.0 Pick primers from a DNA sequence (http://bioinfo.ut.ee/primer3/), and were designed by the Hokkaido system.・ Prepared by outsourcing to Science. The nucleotide sequences of the primers used are shown in Table 3 below. In Table 3, F: forward primer, R: reverse primer, Fresh: fresh cells, PMA / IM: cells after PMA and ionomycin stimulation, 4+: CD4 positive T cells, 8+: CD8 positive T cells, NK: NK cell, Mono: monocyte, R: receptor.

The measurement results of various molecules are shown in FIGS. 1 to 4. FIG. 1 shows a representative example of cell surface markers in which differences were observed. 2 and 3 show typical examples of gene mRNA amounts in which differences are observed. FIG. 4 shows a representative example of plasma cytokine concentrations in which differences were observed.

(Example 2) Multiple regression analysis of immune-related molecules A comprehensive multiple regression analysis was performed on the measured values of the immune-related molecules obtained in Example 1. Multiple regression analysis was performed using SPSS version 22 (IBM). An analysis was performed to discriminate the following 1) to 3) using the measured values of a total of three groups, a healthy subject group, a pleural plaque patient group, and a mesothelioma patient group.
1) Determine whether or not the cancer is mesothelioma-bearing, that is, the [healthy subject group + pleural plaque patient group] group and the [mesothelioma patient group] group.
2) Determine whether there is asbestos exposure, that is, the group of [healthy subjects] and the group of [pleural plaque patients + mesothelioma patients].
3) Asbestos exposure but not mesothelioma-bearing cancer, that is, the [pleural plaque patient group] group and the [healthy subject group + mesothelioma patient group] are discriminated.

A prediction formula (multiple regression formula) for calculating the score for discriminating the above 1) to 3) was constructed as follows by multiple regression analysis.
1) Identification and prediction formula for subjects with mesothelioma-bearing cancer by multiple regression analysis
M-Score = 2.43056 + 0.00460 x [IFN-α2] + 0.56948 x [Ths_TNF-α] + 0.00075 x [MCP-1]
[IFN-α2]: Plasma interferon (IFN) α2 concentration
[Ths_TNF-α]: TNF-α gene expression level in CD4-positive T cells
[MCP-1]: Plasma MCP-1 concentration 2) Identification and prediction formula for subjects with a history of asbestos exposure by multiple regression analysis
A-Score = 0.76825 --0.00248 x [CXCR3MFI_CTL] + 0.52846 x [CTLs_GranzymeB] --0.72952 x [CTL_IL18R1]
[CXCR3MFI_CTL]: Expression level of CXCR3 (a type of chemokine receptor) on the cell surface of CD8-positive cytotoxic T cells
[CTLs_Granzyme B]: Gene expression level of Granzyme B in CD8-positive cytotoxic T cells
[CTL_IL18R1]: IL-18R1 gene expression level in CD8-positive cytotoxic T cells 3) Identification and prediction formula for subjects with a history of asbestos exposure by multiple regression analysis but non-mesothelioma-bearing cancer
P-Score = 0.22813 --0.66572 x [Ths_TNF-α] --0.01333 x [CXCR3MFI_NK] --0.00733 x [GITR_Th]
[Ths_TNF-α]: TNF-α gene expression level in CD4-positive T cells
[CXCR3MFI_NK]: Expression level of CXCR3 on the cell membrane surface of NK cells
[GITR_Th]: Expression level of GITR on the surface of CD4-positive T cell membrane Of the above, the gene expression level is the mRNA expression level, and [Ths_TNF-α] and [CTLs_Granzyme B] are mRNA expression in cells after PMA / ionomycin stimulation. Is.

The measured values of each molecule to be analyzed used in the above prediction formula showed fluctuations among the groups. However, some markers could not be used alone. By calculating the scores of the measured values of the three molecules to be analyzed using the above prediction formula, subjects with mesothelioma-bearing cancer, asbestos exposure history, and asbestos exposure history but non-mesothelioma-bearing cancer were selected. It was found that the discrimination can be performed easily and accurately (FIGS. 5 to 7).
FIG. 8 shows the correlation between M-Score and P-Score. It was found that both showed a significantly negative correlation. Since the M-Score and P-Score of mesothelioma-bearing cancer patients and pleural plaque-positive patients are significantly different, it was considered that the onset of mesothelioma can be predicted using these scores.
FIG. 9 shows the results of ROC analysis (Receiver Operating Characteristic curve) for the sensitivity and specificity of M-Score, P-Score, and A-Score. In the multiple regression analysis using the immune-related molecules of the present invention, it was considered that the sensitivity and specificity were superior to those of a single biomarker. In addition, it was found that the test method of the present invention can set a cut-off value for discriminating mesothelioma-bearing cancer, asbestos exposure history, and asbestos-bearing history but non-mesothelioma-bearing cancer.

As described in detail above, the present invention is accurate by performing multiple regression analysis on an immune-related molecule that can be easily detected using the blood of a subject by using three or more finite number of molecules to be analyzed. It is possible to determine whether or not the patient has mesothelioma, whether or not he / she has a history of asbestos exposure, and whether or not he / she has a history of asbestos exposure but does not have mesothelioma-bearing cancer. In Japan, it is predicted that asbestos-induced mesothelioma will develop in about 100,000 people until around 2030. The clinical application of the test method of the present invention leads to early detection and, in turn, enables early treatment by early detection. In addition, according to the method of the present invention, it is possible to perform a simple screening of asbestos exposure history or mesothelioma in the vicinity of the handling factory, the building demolition contractor, the rubble disposal worker, and the general public living in the rubble area. .. Furthermore, since many people have anxiety about asbestos, it can be adopted as an optional item for ordinary human dogs and health examinations.

Claims (3)

Using blood collected from the subject, the measured values of IFN-α2 in plasma, TNF-α gene of CD4-positive T cells, and the molecule to be analyzed, which is MCP1 in plasma, were obtained, and the measured values were calculated by the following formula 1. Analyze using the represented multiple regression equation and detect a sample that is likely to be mesothelioma-bearing cancer based on the M-Score that is the result obtained using the multiple regression equation;
Measurement of molecules to be analyzed, which are CXCR3 on the surface of CD8-positive cytotoxic T cells, Granzyme B gene of CD8-positive cytotoxic T cells, and IL18R1 gene of CD8-positive cytotoxic T cells, using blood collected from subjects. It is possible that there is a history of asbestos exposure based on the A-Score, which is the result obtained by acquiring the value, measuring the measured value using the multiple regression equation represented by the following formula 2, and using the multiple regression equation. Detecting highly potent samples ; or
Using the blood collected from the subjects, the TNF-α gene of CD4 positive T cells, CXCR3 on the surface of NK cells, and the measured values of the molecules to be analyzed, which are GITRs on the surface of CD4 positive T cells, were obtained, and the measured values are as follows. Based on the P-Score, which is the result obtained by using the multiple regression equation expressed by the multiple regression equation in Equation 3, it is possible that the patient has a history of asbestos exposure but is not mesothelioma-bearing cancer. Detecting high samples ;
Including , inspection method:
(Formula 1) M-Score = d ₁ + a ₁ [IFN-α 2] + b ₁ [Ths_TNF-α] + c ₁ [MCP-1]
(Formula 2) A-score = d ₂ + a ₂ [CXCR3MFI_CTL] + b ₂ [CTLs_GranzymeB] + c ₂ [CTL_IL18R1]
(Formula 3) P-score = d ₃ + a ₃ [Ths_TNF-α] + b ₃ [CXCR3MFI_NK] + c ₃ [GITR_Th]
[However, a _1, b 1, _{c 1,} d ₁ in Equation 1 is arbitrary real number not zero, [IFN-α2] is IFN-[alpha] 2 concentration in plasma, [Ths_TNF-α] is a CD4-positive T cells The TNF-α gene expression level, [MCP-1], is the plasma MCP-1 concentration, a ₂ , b ₂ , c ₂ , d ₂ in Equation 2 are arbitrary non-zero real numbers, and [CXCR3MFI_CTL] is CD8. The amount of CXCR3 on the surface of positive cytotoxic T cells, [CTLs_Granzyme B] is the expression level of Granzyme B gene in CD8 positive cytotoxic T cells, and [CTL_IL18R1] is the expression level of IL18R1 gene in CD8 positive cytotoxic T cells. in _{a 3, b 3, c 3} , d 3 are arbitrary real number not zero, [Ths_TNF-α] is TNF-alpha gene expression level of CD4-positive T cells, in CXCR3 amount of [CXCR3MFI_NK] is NK cell surface Yes, [GITR_Th] is the amount of GITR on the surface of CD4-positive T cells. ] Detecting samples with a high probability of mesothelioma-bearing cancer when the M-Score is higher than the cutoff value;
Detecting samples that are likely to have a history of asbestos exposure if the A-score is higher than the cutoff value; or
Detecting samples with a history of asbestos exposure but likely not mesothelioma-bearing cancer if the P-score is higher than the cutoff value;
The inspection method according to claim 1 . One of the following one at least comprises a combination of reagents, asbestos exposure history or mesothelioma test reagent kit for:
1) A combination of a reagent containing an anti-IFN-α2 antibody, a reagent containing an anti-CD4 antibody, a reagent containing a polynucleotide that hybridizes to the TNF-α gene, and a reagent containing an anti-MCP-1 antibody;
2) A combination of a reagent containing an anti-CD8 antibody, a reagent containing an anti-CXCR3 antibody, a reagent containing a polynucleotide that hybridizes to the Granzyme B gene, and a reagent containing a polynucleotide that hybridizes to the IL18R1 gene;
3) A combination of a reagent containing an anti-CD4 antibody, a reagent containing a polynucleotide that hybridizes to the TNF-α gene, a reagent containing an anti-CD56 antibody, and a reagent containing an anti-GITR antibody.