WO2014030683A1

WO2014030683A1 - Biomarker for detecting factor for anemia in anemic patient

Info

Publication number: WO2014030683A1
Application number: PCT/JP2013/072335
Authority: WO
Inventors: 杉山　大介
Original assignee: 国立大学法人九州大学
Priority date: 2012-08-21
Filing date: 2013-08-21
Publication date: 2014-02-27
Also published as: JPWO2014030683A1; JP6214537B2

Abstract

The present invention addresses the problems of providing a method for identifying the cause of anemia as MDS, and in particular of providing a method for identifying MDS as the cause in a simple manner without performing a bone marrow puncture, without the need for a blood specialist's decision, or the like. The problems are solved by a method for detecting a factor for anemia in an anemic patient, the method comprising steps (1) and (2), wherein step (1) comprises measuring the expression level of ACVR2B in polycythemia blood cells contained in peripheral blood obtained from the patient, and step (2) comprises determining that a factor of the patient's anemia is MDS if the expression level of ACVR2B measured in step (1) is greater than the expression level of ACVR2B in the polycythemia blood cells contained in peripheral blood obtained from a healthy individual.

Description

Biomarkers for detecting anemia factors in patients with anemia

The present invention relates to a biomarker for determining that an anemic patient is MDS, a method for detecting a factor of anemia in an anemic patient, a kit therefor, and a diagnostic agent.

The present invention also relates to a biomarker for determining the risk of developing leukemia in an anemic patient, a method for determining the risk of developing leukemia in an anemic patient, and a kit and diagnostic agent therefor.

Furthermore, the present invention relates to a biomarker for predicting the necessity and frequency of blood transfusion in an anemic patient, a method for predicting the necessity and frequency of blood transfusion in an anemic patient, and a kit and a diagnostic agent used therefor.

To diagnose a patient who complains of anemia, it is necessary to investigate the cause. Usually, in addition to the patient's symptom and physical condition inquiry, after making a definitive diagnosis by measuring the peripheral blood hemoglobin value in the peripheral blood, the number of blood cells such as red blood cells, white blood cells, platelets, iron, Check the amount of vitamins.

If the cause of anemia still cannot be grasped, a sample is collected from the patient by bone marrow puncture and morphological information on the multiblood cells such as hematopoietic status in the bone marrow, presence or absence of blasts, presence or absence of abnormal chromosomes, etc. They need to be examined by a skilled hepatologist who has mastered the so-called skill.
By such a method, myelodysplastic syndrome (MDS) can finally be diagnosed.

MDS is a type of blood tumor and is a disease in which the quantity and quality of blood cells are abnormal. And it is known that MDS causes a pre-leukemia state to cause hematopoietic disorder, has a poor prognosis, and shifts to acute myeloid leukemia. It is also a disease in which disorders such as blood cell differentiation and maturation are observed. Specifically, symptoms such as a decrease in the number of red blood cells (anemia state), a decrease in the number of platelets, an abnormality (decrease or increase) in the number of white blood cells are observed.

The number of patients with MDS is 300,000 worldwide, and 7,100 in Japan, and the average age is 70s and the elderly. For this reason, there is concern about an increase in the number of MDS patients because it will enter an aging society in the future.

MDS includes refractory anemia (RA), refractory cytopenia with multicytic dysplasia (RCMD), ironblastic refractory anemia with multicytic dysplasia (RCMD-RS), and increased blasts Refractory anemia with (RAEB) and the like are included. The diagnostic criteria for RA is based on the presence of blasts less than 1% in peripheral blood, less than 5% in bone marrow, and no cyclic iron blasts, with a probability of 10 becoming a leukemia from RA. %. As a method for treating RA, no treatment, follow-up, or blood transfusion treatment is mainly used, and 60 to 80% of RA patients have worsened symptoms. In the case of long-term survival, it is necessary to take measures against complications due to long-term blood transfusion, such as iron deposition in the liver. As for the diagnostic criteria for RCMD, in addition to the diagnostic criteria for RA, clear morphological dysplasia of multiblood cells is observed at 10% or more, and the probability of transition from RCMD to leukemia is 15 to 20%.

Of these MDS-related diseases, RA and RARS are mild, RCMD and RCMD-RS are moderate, and RAEB is severe.

Among such MDS, kits using mRNA encoding WT1 protein have been put on the market for diagnosis of RAEB, which is a severe myelodysplastic syndrome. However, such a kit requires a step of collecting total RNA from peripheral blood and the like, and further improvement is required for easy use in medical practice.

In addition, these kits are not suitable for diagnosis of mild cases of MDS.

Patent Document 1 discloses a method for measuring the kinase activity of activin receptor 2B. And it is disclosed that activin is a factor related to the differentiation of erythrocytes as an action exerted in vivo.

Patent Document 2 discloses a monoclonal antibody that recognizes an extracellular region of a mouse-derived activin receptor. It is also disclosed that activin is a protein that acts in the field of hematopoiesis.

Patent Document 3 discloses a method of increasing red blood cells using an activin receptor 2 antagonist. The use of such antagonists as a method for treating anemia is also disclosed.

Patent Document 4 discloses a bone marrow erythroid progenitor cell differentiation promoter comprising arginine as a main component and further containing activin.

JP 2003-250537 A JP 11-335399 A Special table 2010-513506 gazette International Patent Publication 2006/115274 Pamphlet

As described above, a method useful for determining the cause of anemia, in particular, MDS, is a cause of anemia. It is necessary to make a morphological diagnosis of the multi-blood cells. Therefore, it is very difficult to examine whether a patient who presents with anemia suffers from MDS in a general clinic where there is no facility capable of collecting a specimen from the bone marrow by bone marrow puncture and a hematologist.

As an alternative method, for example, a biomarker expressed in the process of differentiation from hematopoietic stem cells is used as an index in order to provide a method capable of easily diagnosing MDS by outsourcing a test even from a general clinic. Development of a diagnostic method for MDS is required. Thus, in the process of hematopoietic differentiation, for example, Sca-1, c-kit, CD71, Gata1, Ter119 etc. are expressed in mice, and CD34, CD71, GPA (Glycophorin A) etc. are expressed in humans. Although MDS diagnostic methods using biomarkers as biomarkers are being developed, none of them has been realized in practical use.

The present inventor has also found that it is not clear whether ACVR2B, which is one of the activin receptors known to be involved in hematopoiesis as described above, is involved in the process of hematopoietic differentiation. .

Furthermore, a kit using mRNA encoding WT1 is not simple in that it is necessary to collect total RNA from peripheral blood.

In view of the above-mentioned present situation, the main problem of the present invention is to provide a method for determining the cause of anemia, and in particular, to determine by a simple method such as not performing bone marrow puncture or requiring the determination of a hematologist Is to provide a way to do. Furthermore, the subject of the present invention includes providing a simple method for determining whether anemia patients are prone to developing leukemia. The subject of the present invention also includes a method for predicting the necessity and frequency of blood transfusion in an anemic patient.

The present inventor isolates positively stained erythroblasts, reticulocytes, and erythrocytes, which are cell groups classified into differentiation stages from hematopoietic stem cells, by experiments using Gata1-GFP transgenic mice. succeeded in.

As a result of further research on these separated cell groups, it was found that the expression level of ACVR2B is significantly different among the cell groups.

The present invention has been made on the basis of such findings, and encompasses the inventions of the following broad aspects.

Item 1 A method for detecting an anemia factor in an anemia patient comprising the following steps 1 and 2;
(1) Step 1 of measuring the expression level of activin receptor 2B (ACVR2B) in multiblood cells contained in peripheral blood obtained from the patient,
(2) When the expression level of ACVR2B measured in step 1 is greater than the expression level of ACVR2B in the polycytic cells contained in peripheral blood obtained from a healthy person, it is determined that the cause of anemia in the patient is MDS Step 2 to do.

Item 2. The method according to Item 1, wherein the multicytic cells are one or more selected from the group consisting of red blood cells, white blood cells, and platelets.

Item 3. The method according to Item 1 or 2, wherein the MDS is at least one selected from the group consisting of RA, RCMD, RCMD-RS, and RAEB.

Item 4 A method for determining the risk of developing leukemia in an anemia patient comprising the following steps 1 and 2;
[1] Step 1 of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from the patient,
[2] Step 2 for determining that the patient is at risk of developing leukemia when the expression level of ACVR2B measured in Step 1 is greater than the expression level of ACVR2B in polycytic cells contained in peripheral blood obtained from healthy subjects .

Item 5. The method according to Item 4, wherein the multicytic cells are one or more selected from the group consisting of red blood cells, white blood cells, and platelets.

Item 6. The method according to Item 4 or 5, wherein the leukemia is acute myeloid leukemia.

Item 7 A method for predicting the necessity or frequency of blood transfusion of an anemic patient comprising the following steps 1 and 2;
{1} Step 1 of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from the patient,
{2} When the expression level of ACVR2B measured in step 1 is greater than the expression level of ACVR2B in the polycytic cells contained in peripheral blood obtained from healthy subjects, blood transfusion is required for the patient, and the frequency is Step 2 for determining that the frequency is higher than the blood transfusion frequency of the patient at the time of collection.

Item 8 The method according to Item 7, wherein the multicytic cells are any one selected from the group consisting of red blood cells, white blood cells, and platelets.

Item 9. The method according to Item 7 or 8, wherein the leukemia is acute myeloid leukemia.

Item 10. A diagnostic agent for detecting an anemia factor of an anemic patient as MDS, including an anti-ACVR2B antibody.

Item 11. The diagnostic agent according to Item 10, wherein the MDS is at least one selected from the group consisting of RA, RCMD, RCMD-RS, and RAEB.

Item 12. A kit for detecting an anemia factor of an anemic patient as MDS, including an anti-ACVR2B antibody.

Item 13. The kit according to Item 12, wherein the MDS is at least one selected from the group consisting of RA, RCMD, RCMD-RS, and RAEB.

Item 14. A diagnostic agent for determining the risk of developing leukemia in anemia patients, comprising an anti-ACVR2B antibody.

Item 15. The diagnostic agent according to Item 14, wherein the leukemia is acute myeloid leukemia.

Item 16. A kit for determining the risk of developing leukemia in anemia patients, comprising an anti-ACVR2B antibody.

Item 17. The kit according to Item 16, wherein the leukemia is acute myeloid leukemia.

Item 18. A diagnostic agent for the necessity and frequency of transfusion of anemia patients, including an anti-ACVR2B antibody.

Item 19. A kit for detecting the necessity and frequency of blood transfusion of an anemia patient, including an anti-ACVR2B antibody.

Item 20. A biomarker comprising ACVR2B for detecting an anemia factor of an anemic patient as MDS.

Item 21. The biomarker according to Item 20, wherein the MDS is at least one selected from the group consisting of RA, RCMD, RCMD-RS, and RAEB.

Item 22. A biomarker comprising ACVR2B for determining the risk of developing leukemia in anemia patients.

Item 23. The biomarker according to Item 22, wherein the leukemia is acute myeloid leukemia.

Item 24. A biomarker for predicting the necessity and frequency of blood transfusion of an anemic patient, comprising ACVR2B.

The effects of the present invention are described below. However, it goes without saying that the present invention is not limited to the invention having all of the effects described below, and is an invention having at least one of the effects.

By using the method, diagnostic agent, kit, or biomarker according to the present invention, it is excellent in that it can be diagnosed without performing bone marrow puncture, which is conventionally performed in diagnosing MDS and burdens the patient. ing. And it is very excellent also in the point that the diagnosis of MDS is possible without using a hematologist with advanced technology.

The method, diagnostic agent, kit, or biomarker according to the present invention is particularly useful for determining the cause of anemia by MDS and a simple method. This is excellent in that it is possible to obtain sufficient and simple patient evidence necessary to introduce an outpatient in a general clinic to a specialist.

In particular, since anemia patients visit outpatients almost every day, it is very useful in diagnosing very serious diseases such as MDS at an early stage.

Furthermore, the method, diagnostic agent, kit, or biomarker of the present invention is effectively used for predicting progression from MDS to leukemia.

It is known that MDS is likely to progress to leukemia. As a result, when leukemia develops, leukemia cells proliferate violently in the bone marrow and cannot normally produce blood cells, and as a result, red blood cells can be made. It is also known that anemia is caused by disappearance, and blood transfusion is required for the treatment of MDS. In addition, since MDS is impaired in differentiation into red blood cells, maturation, etc., blood transfusion is also necessary for treatment of MDS itself.

From the above, the method, diagnostic agent, kit, or biomarker of the present invention is very useful in predicting the necessity and frequency of blood transfusion.

The figure which shows the result of having fractionated the blood cell of each differentiation stage. The figure which shows the result of having measured the quantity of mRNA of Acvr2b (human protein homolog name: ACVR2B) expressed in the cell population of a normal erythroblast, a reticulocyte, and an erythrocyte. The figure which shows the result of having measured the quantity of ACVR2B expressed in the erythrocyte contained in peripheral blood. Samples are peripheral blood obtained from healthy subjects, RA patients among MDS, and RCMD patients, and the expression level of ACVR2B in erythrocytes (marker is CD45 ⁻ / CD71 ⁻ / GPA ⁺ ) therein. Show. The figure which shows the result of having measured the quantity of ACVR2B expressed in the leukocyte contained in peripheral blood. Samples are peripheral blood obtained from healthy subjects, RA patients among MDS, and RCMD patients, and the expression level of ACVR2B in white blood cells (marker is CD45 ⁺ / GPA ⁻ ) therein is shown. . The figure which graphed the result of having measured the quantity of ACVR2B expressed in the red blood cell (FIG. 4) and white blood cell (FIG. 5) contained in peripheral blood. The figure which shows the result of having measured the quantity of ACVR2B expressed in the erythrocyte contained in peripheral blood. Samples are peripheral blood obtained from healthy individuals, RA patients among MDS, and RCMD patients, and healthy elderly and renal anemia patients as control experiments.

Hereinafter, the present invention will be described in detail. Various techniques used for carrying out the present invention can be easily and surely implemented by those skilled in the art based on known literatures and the like, except for the technique that clearly indicates the source.

For example, Sambrook and Russell, “Molecular Cloning A LABORATORY MANUAL”, Cold Spring Harbor Laboratory Press, New York, 2001; Ausubel, F. M. Et al. “Current Protocols in Molecular Biology”, John Wiley & Sons, New York,. NY; Molecular Biology of the Cell 5E: Reference Edition Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff; Basic and Clinical Pharmacology 12 / E (LANGE Basic Science) by Bertram Katzung, Susan Masters and Anthony Trevor (Dec 13, 2011) References such as these may be referred to.

<Explanation of terms>
As used in this specification, the term “anemia” means that blood hemoglobin concentration is 13 g / dL or less for men and 12 g / dL or less for women, as shown in the WHO guidelines. The condition refers to symptoms such as palpitation, shortness of breath, dizziness, etc., but even if it is asymptomatic, it may be determined to be anemia if the blood hemoglobin concentration is as described above. The term “anemia patient” is a person who suffers from the above-mentioned symptoms of “anemia”.

As used herein, the term “factor of anemia” refers to the onset mechanism of the above-mentioned “anemia”, and can be rephrased as, for example, a disease in which anemia is cited as one of the symptoms.

Such diseases include MDS, renal anemia, aging-related anemia, iron deficiency anemia, thalassemia, ironblastic anemia, megaloblastic anemia (vitamin B12 deficiency, folate deficiency), primary Examples include bone marrow failure, inflammation, uremia, hemolytic anemia, aplastic anemia, sickle cell identification, and renal anemia.

As used herein, the term “MDS” is a type of blood tumor, called myelodysplastic syndrome, a disease in which the quantity and quality of blood cells are abnormal, differentiation of multiblood cells including red blood cells, There is a disease in which a disorder is recognized in maturity.

Specifically, MDS includes refractory anemia (RA), refractory cytopenias with multicytic dysplasia (RCMD), ironblastic refractory anemia with multicytic dysplasia (RCMD-RS), blasts Refractory anemia (RAEB) with an increase is included. As described above, these diseases are impaired in differentiation into red blood cells, maturation and the like, and finally some of them progress to leukemia described later.

These diseases can be diagnosed by examining blood cells obtained from bone marrow and / or peripheral blood, respectively, using morphological means. A specific diagnostic method may be a known method and is not particularly limited.

Among the above-mentioned diseases, RAEB is most likely to progress to leukemia.

As used herein, the term “leukemia” is classified into, for example, acute leukemia, chronic leukemia, etc., depending on its symptoms, pathological conditions, and the like. Moreover, according to another classification method, it is classified into myeloid leukemia and lymphocytic leukemia. Among the above-mentioned leukemias, acute myeloid leukemia is particularly preferable.

Regarding acute myeloid leukemia, according to a classification method called FAB classification, M0: acute undifferentiated myeloid leukemia (most undifferentiated type); M1: acute undifferentiated myeloblastic leukemia; M2: acute Differentiated myeloblastic leukemia; M3 acute promyelocytic leukemia; M4: acute myelomonocytic leukemia; M5: acute monocytic leukemia; M6: erythroleukemia; M7: acute megakaryocytic leukemia.

As used herein, the term “risk of onset” refers to the risk of developing a certain disease, and is not influenced by the result of diagnosis of whether or not the disease actually develops. .

The term “frequency” used in this specification is used as a blood transfusion frequency, and indicates the degree of whether the required number of blood transfusions is large or small. This is because the amount that is possible for a single blood transfusion has been determined, so if a large amount of blood transfusion is required, increase the number of blood transfusions and perform the necessary amount of blood transfusion. The number of blood transfusions is determined based on the fact that the period during which the effect is exerted is a maximum of 120 days.

For example, if the hemoglobin level in blood is 7 g / dL or less in a patient diagnosed with myelodysplastic syndrome clinically (body weight 60 kg), 1 transfusion can be performed as an upper limit of 2 units (280 mL). The hemoglobin value of about 3 to 1.4 g / dL increases. If the blood hemoglobin level still does not reach the desired blood hemoglobin level, or the deterioration of symptoms, i.e., red blood cell differentiation or maturation, is not improved, and the blood hemoglobin level has decreased, the period again The upper limit of 2 units of blood transfusion will be performed.

The term “ACVR2B” used herein is also referred to as activin receptor 2B. Activin receptor 2B is a receptor protein having an extracellular domain containing a cysteine-rich region from the N-terminus, a transmembrane domain, and an intracellular domain containing a serine / threonine-type kinase domain.

Activin receptor 2B includes Bone Morphogenetic Protein 7 (BMP7), Growth / Differentialation factor 5 (GDF5), Myostatin (MSTN), Inhibin α (INHA), Inhibin β A subunit (INHBA), Inhibin β B A serine / threonine kinase type receptor having inhibin β C subunit (INHBC), NODAL, TDGF and the like as ligands, and BMP receptor 1A (BMPR1A), BMP receptor 1B (BMPR1B) in response to stimulation of the ligand, A receptor that forms a complex with activin receptor 1 (ACVR1) etc. and plays a part in the function of activating the intracellular Smad cascade etc. It is over protein.

Activin receptor 2B specifically includes, for example, http: // www. uniprot. It is a protein consisting of the amino acid sequence (SEQ ID NO: 1) shown in org / uniprot / Q13705 and the like. Among such proteins, it is particularly preferable to have an extracellular domain consisting of at least the first to 119th amino acid sequences in SEQ ID NO: 1.

The ACVR2B targeted by the present invention is not limited to the above protein itself, and may be a protein with mutation (substitution, deletion, addition, etc.).

As used herein, the term “expression level” means not the expression level of mRNA but the expression level of protein translated based on mRNA.

The term “biomarker” used in the present specification refers to substances derived from living bodies such as carbohydrates, lipids, proteins, and the like, which serve as an index for quantitatively grasping biological changes in the living body. .

More specifically, the biomarker is a role as an index for diagnosing or judging the current state used for grasping the current state of diseases and the like, and a predictive diagnosis for predicting the prevention of diseases and the transition of disease states. Or, it has a role as an index for estimation.

The method of the present invention The method of the present invention includes <a method for detecting an anemia factor>, <a method for determining the risk of developing leukemia>, and <a method for predicting the necessity and frequency of blood transfusion>. .

<Method of detecting anemia factors>
The method for detecting an anemia factor of an anemic patient according to the present invention includes the following steps 1 and 2.
(1) Step 1 of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from the patient,
(2) When the expression level of ACVR2B measured in step 1 is greater than the expression level of ACVR2B in the polycytic cells contained in peripheral blood obtained from a healthy person, it is determined that the cause of anemia in the patient is MDS Step 2 to do.

(About step 1)
Step 1 is a step of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from an anemic patient.

Peripheral blood means blood existing in blood vessels. Such peripheral blood may be collected by inserting an injection needle or the like into a human blood vessel. For example, a metal needle may be used as the injection needle or the like, and an indwelling material such as SURFLOW (registered trademark) may be used. Peripheral blood may be collected by inserting a plastic injection needle into a blood vessel together with a metal needle and placing it. The collected peripheral blood may be subjected to a step of preventing coagulation as appropriate. Specifically, an anticoagulant such as heparin or EDTA may be used, but it is preferable to use EDTA which is inexpensive and highly versatile.

The polycytic cells are not particularly limited, and examples include red blood cells, white blood cells, and platelets. More preferred are red blood cells, white blood cells and the like.

Although the specific measurement method of ACVR2B is not specifically limited, For example, it can measure by using the flow cytometry method using the antibody with respect to ACVR2B. As other methods, measurement can also be performed by using ELISA method using an antibody against ACVR2B, Western blotting method, or the like.

These measurement methods can be easily measured by those skilled in the art according to known protocols.

As an antibody against ACVR2B, a commercially available antibody or an antibody prepared based on a conventional method may be used as it is, or a modified antibody obtained by adding a labeling substance to the antibody.

The origin of the antibody is not particularly limited, and examples include antibodies derived from animal species such as humans, mice, rats, chickens, goats, sheep, donkeys, llamas, ostriches, alpaca and horses.

The antibody may be a monoclonal antibody or a polyclonal antibody. The ACVR2B antibody is not particularly limited as long as it is a molecule that specifically binds to ACVR2B. Examples thereof include immunoglobulins (Ig) or antibody fragments such as scFv, F (ab ′) ₂ , minibody, and the like. Affibodies are also included in the above-described antibodies. Antisera are also encompassed by the antibodies described above.

When the antibody is an immunoglobulin, its subtype is not particularly limited, and may be any of IgA, IgD, IgE, IgG, IgM, IgY, IgW, and the like. Further, the subclass of the antibody is not limited to a specific subclass, and may be any of IgG1 to IgG as long as it is human IgG, for example.

As long as the anti-ACVR2B antibody used in Step 1 exhibits the ability to specifically bind to ACVR2B, the above-mentioned several types of antibodies may be mixed and used.

When measuring the amount of ACVR2B in Step 1, the multiblood cells are identified using an antibody against a marker that is specifically expressed in the multiblood cells. This makes it possible to measure the amount of ACVR2B in specific blood cells.

Such markers include CD3, CD4, CD5, CD10, CD13, CD19, CD20, CD23, CD33, CD34, CD38, CD45, CD45RA, CD90, IgM, HLA-DR and other leukocyte markers; CD71, GPA, etc. Red blood cell markers; platelet markers such as CD41, CD42b, CD61, CD62P, PAC-1, and the like, and these antibodies may be combined as appropriate.

In addition, antibodies against these markers are not limited to specific origin species, and may be the same as the antibodies against the above-mentioned ACVR2B, and may be appropriately labeled with a labeling substance.

The specific method for using the antibody against the marker is not particularly limited as long as it is used in accordance with a known method such as the flow cytometry method, ELISA method, Western blotting method or the like.

(About step 2)
In step 2, the expression level of ACVR2B in the polycytic cells contained in the peripheral blood of the anemic patient measured in the above (step 1) is larger than the expression level of ACVR2B in the polycytic cells contained in the peripheral blood obtained from a healthy subject In some cases, the step of determining that the anemia factor of the patient is MDS.

A healthy person is a person who is not in an anemic state as described in the term “anemia”, and whether or not he / she complains of symptoms of anemia as described above is not particularly limited in defining a healthy person. Moreover, the race, age, and gender of a healthy person are not ask | required. The “peripheral blood” obtained from healthy subjects is as described above.

The “multiple blood cell” is defined in the same manner as the multiple blood cell contained in the peripheral blood obtained from the patient to be measured in (Step 1).

The expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from healthy subjects can be determined by the same method as the ACVR2B measurement method in (Step 1) described above.

Here, the comparison between the expression level of ACVR2B in the polycytic cells contained in the peripheral blood obtained from the anemia patient measured in (Step 1) and the expression level of ACVR2B in the polycytic cells contained in the peripheral blood obtained from healthy subjects is as follows. Although not particularly limited, for example, the following comparison method may be employed.

As a first comparison method, the same method for measuring a multiblood cell sample contained in peripheral blood obtained from a healthy person as the multiblood cell sample contained in peripheral blood obtained from an anemia patient measured in (Step 1), and There is a method in which the measurement is performed under the same measurement conditions and the amount of the obtained ACVR2B is relatively compared.

As a result of the comparison, it can be determined that the greater the amount of ACVR2B, the stronger the degree of disorder such as the differentiation and maturation of multiblood cells that are the pathological conditions of MDS as described above. Can be determined.

Therefore, when the amount of ACVR2B in the multiblood cells contained in the peripheral blood obtained from the anemia patient measured in (Step 1) is greater than the amount of ACVR2B in the multiblood cells contained in the peripheral blood obtained from a healthy subject, anemia It can be determined that the cause of the patient's anemia is MDS. .

As a second comparison method, the amount of ACVR2B in a multi-blood cell sample contained in peripheral blood obtained from a healthy subject is measured in advance, and is obtained from an anemia patient measured in (Step 1) rather than the measured value. When the amount of ACVR2B in the multicytic cells in the peripheral blood obtained is high, the cause of anemia in an anemic patient can be determined as MDS.

In the second comparison method, the ACVR2B measurement method and measurement conditions in peripheral blood polycytic cells obtained from an anemic patient are the same as in the first method described above. It is the same as the measurement method and measurement conditions of ACVR2B in multiblood cells contained in blood.

It should be noted that the amount (measured value) of ACVR2B in the peripheral blood polycytic cells obtained from a healthy person measured in advance is generalized, and based on the generalized numerical value (for example, called a cut-off value) (steps). You may compare whether the amount of ACVR2B in the multiblood cell contained in the peripheral blood obtained from the anemia patient measured in 1) is large or small.

The generalized numerical value is a statistical analysis of the amount of ACVR2B in peripheral blood multi-blood cells obtained from a certain number of different or more healthy subjects, and is generally used as a value per unit peripheral blood volume or per unit multi-blood cell volume. However, the method is not limited to the above-described method, and any known analysis technique, statistical technique, or the like may be used.

<Method of determining the risk of developing leukemia>
The method for determining the risk of developing leukemia in an anemic patient of the present invention includes the following steps 1 and 2.
[1] Step 1 of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from the patient,
[2] Step 2 in which when the expression level of ACVR2B measured in Step 1 is greater than the expression level of ACVR2B in polycytic cells contained in peripheral blood obtained from healthy subjects, the patient is determined to be at risk for leukemia.

[About Step 1]
Step 1 is a step of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from an anemic patient.

This step may be performed in the same manner as (Step 1) in the above <Method for detecting anemia factor>.

[About step 2]
In the step 2, when the expression level of ACVR2B measured in the above [step 1] is larger than the expression level of ACVR2B in the multiblood cells contained in peripheral blood obtained from a healthy person, the patient is at risk of developing leukemia. It is a step of determining.

The healthy person is as described in (Step 1) in the above <Method for detecting anemia factor>.

The “multiple blood cell” is defined in the same manner as the multiblood cell contained in the peripheral blood obtained from the patient to be measured in [Step 1].

The expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from healthy subjects can be determined by the same method as the measurement method of ACVR2B in [Step 1] described above.

Here, the comparison between the expression level of ACVR2B in the polycytic cells contained in the peripheral blood obtained from the anemia patient measured in [Step 1] and the expression level of ACVR2B in the polycytic cells contained in the peripheral blood obtained from healthy subjects is as follows: What is necessary is just to employ | adopt the comparison method shown below.

As a first comparison method, the same measurement method as the multiblood cell sample contained in the peripheral blood obtained from the anemia patient measured in [Step 1], the multiblood cell sample contained in the peripheral blood obtained from a healthy person, and There is a method in which the measurement is performed under the same measurement conditions and the amount of the obtained ACVR2B is relatively compared.

As a result of comparison, the greater the amount of ACVR2B, the stronger the degree of disorder such as differentiation and maturation of the above-mentioned multiblood cells, and thus it can be determined that anemia patients are more likely to develop leukemia. 1) If the amount of ACVR2B in the polycytic cells contained in the peripheral blood obtained from the anemia patient measured in 1) is greater than the amount of ACVR2B in the polycytic cells contained in the peripheral blood obtained from a healthy subject, the anemia patient is leukemia. It can be determined that there is a risk. .

As a second comparison method, the amount of ACVR2B in a multiblood cell sample contained in peripheral blood obtained from a healthy person is measured in advance, and the obtained value is obtained from an anemia patient measured in [Step 1]. Anemia patients can be determined to be at risk of developing leukemia when the amount of ACVR2B in the multicytic cells in the peripheral blood obtained is high.

In the second comparison method, the ACVR2B measurement method and measurement conditions in peripheral blood polycytic cells obtained from an anemic patient are the same as in the first method described above. The measurement method and measurement conditions for ACVR2B in multiblood cells contained in blood are the same.

In the second comparison method, the target numerical value to be compared with the amount of ACVR2B in the peripheral blood polycytic cells obtained from the anemia patient measured in [Step 1] is the above <Method for detecting anemia factor>. The cut-off value described in the above may be used similarly.

<Method of predicting the necessity and frequency of blood transfusion>
The method for predicting the necessity and frequency of blood transfusion of an anemic patient according to the present invention includes the following steps 1 and 2.
{1} Step 1 of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from the patient,
{2} If the expression level of ACVR2B measured in step 1 is greater than the expression level of ACVR2B in the multiblood cells contained in peripheral blood obtained from a healthy subject, blood transfusion is required for the patient, and the frequency is Step 2 for determining that the blood transfusion frequency is higher than that of the patient at the time of blood collection.

{About Step 1}
Step 1 is a step of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from the patient.

{About Step 2}
Step 2 determines that transfusion is necessary for the patient when the expression level of ACVR2B measured in Step 1 above is greater than the expression level of ACVR2B in the multiblood cells contained in peripheral blood obtained from a healthy subject In both cases, the transfusion frequency is determined to be higher than the transfusion frequency of the patient at the time of collecting peripheral blood.

An anemia patient who has been determined to have a high transfusion frequency according to Step 2 and who has received 0 transfusions at the time of peripheral blood collection (no transfusion measures have been taken for anemia). Patients who have been determined to be necessary and who have received at least one transfusion treatment for anemia at the time of peripheral blood collection are determined to require more transfusions.

The “multiple blood cell” is defined in the same manner as the multiple blood cell contained in the peripheral blood obtained from the patient to be measured in {Step 1}.

The expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from healthy subjects can be determined by the same method as the measurement method of ACVR2B in {Step 1} described above.

Here, the comparison of the expression level of ACVR2B in the polycytic cells contained in the peripheral blood obtained from the anemia patient measured in {Step 1} and the expression level of ACVR2B in the polycytic cells contained in the peripheral blood obtained from healthy subjects is as follows: What is necessary is just to employ | adopt the comparison method shown below.

As a first comparison method, the same measurement method as the multiblood cell sample contained in the peripheral blood obtained from the anemia patient measured in {step 1}, the multiblood cell sample contained in the peripheral blood obtained from the healthy person, and There is a method in which the measurement is performed under the same measurement conditions and the amounts of the obtained ACVR2Bs are relatively compared.

As a result of the comparison, the greater the amount of ACVR2B, the more blood transfusion is necessary for the anemic patient, and it can be determined that the frequency is high. Therefore, the peripheral blood obtained from the anemic patient measured in {Step 1} When the amount of ACVR2B in the included polycytic cells is greater than the amount of ACVR2B in the polycytic cells contained in peripheral blood obtained from a healthy person, it may be determined that blood transfusion is necessary for anemia patients and the transfusion frequency is high it can.

As a second comparison method, the amount of ACVR2B in a multiblood cell sample contained in peripheral blood obtained from a healthy person is measured in advance, and the obtained value is obtained from an anemia patient measured in {Step 1}. When the amount of ACVR2B in the polycytic cells in the peripheral blood obtained is high, it can be determined that blood transfusion is required for anemia patients and the frequency is high.

In the second comparison method, the target numerical value to be compared with the amount of ACVR2B in the peripheral blood polycytic cells obtained from the anemia patient measured in {Step 1} is the above <Method for detecting anemia factor>. The cut-off value described in the above may be used similarly.

High transfusion frequency means more transfusions compared to the number of transfusions (including 0) at the time of peripheral blood collection, in other words, the future transfusion frequency of anemia patients Means more than the number of blood transfusions at the time of blood collection. *

Diagnostic Agent of the Present Invention The diagnostic agent of the present invention is a <diagnostic agent for detecting that the cause of anemia is MDS>, <diagnostic agent for determining the risk of developing leukemia>, and <necessity of blood transfusion> And diagnostic agents of sex and frequency thereof.

The diagnostic agent of the present invention contains an anti-ACVR2B antibody. The anti-ACVR2B antibody is the same as the antibody against ACVR2B described in detail in (Step 1) of <Method for detecting anemia factor> described above.

Even if the diagnostic agent itself is an anti-ACVR2B antibody, it may contain other components. When other components are contained, the content of the anti-ACVR2B antibody in the diagnostic agent is not particularly limited, but is usually about 0.0001 to 99.99 parts by weight per diagnostic agent. The other components contained in the diagnostic agent are known components used in the field of diagnostic agents as long as they exhibit the ability of the anti-ACVR2B antibody to specifically bind to ACVR2B, and are not particularly limited. .

<Diagnostic agent for detecting the cause of anemia as MDS>
The diagnostic agent for detecting an anemia factor as MDS is preferably used as one of the components of <kit for detecting an anemia factor as MDS> described later. And it is usefully used according to the method described in the above <Method for detecting anemia factor>.

<Diagnostic agent for determining leukemia risk>
The diagnostic agent for determining the risk of developing leukemia is suitably used as one of the components of <kit for determining the risk of developing leukemia> described later. And it is usefully used according to the method described in the above <Method for determining leukemia risk>.

<Diagnosis of necessity and frequency of blood transfusion>
The diagnostic agent for the necessity and frequency of blood transfusion is suitably used as one of the constituent elements of <kit for predicting the necessity and frequency of blood transfusion> described later. And it is usefully used according to the method described in the above <Method for predicting the necessity and frequency of blood transfusion>.

Kit of the Present Invention The kit of the present invention comprises <a kit for detecting an anemia factor as MDS>, <a kit for determining the risk of developing leukemia>, and <necessity and frequency of blood transfusion in anemia patients> A kit for predicting

The kit of the present invention contains an anti-ACVR2B antibody. The anti-ACVR2B antibody may be the same as the antibody against ACVR2B described in detail in (Step 1) of <Method for detecting anemia factor>.

The anti-ACVR2B antibody included in the kit of the present invention may include two or more types of antibodies as long as the antibody exhibits an ability to specifically bind to ACVR2B. In this case, each antibody may be filled in a separate container, or may be filled in the same container.

In addition, the above-mentioned anti-ACVR2B antibody may be included in the kit of the present invention in a mode of being adsorbed on the bottom surface of an instrument that may be included in the kit of the present invention described later.

The kit of the present invention may further contain an antibody against a marker that is specifically expressed in multiblood cells. The multiblood cell is as described in (Step 1) of <Method for detecting an anemia factor>, and such an antibody is specifically described in (Method 1 for detecting an anemia factor) (Step 1). It is described in.

Also, the kit of the present invention may include instruments mainly used for appropriate biochemical experiments. For example, a plate, a multiwell plate, a seal, a dish, a dropper, a capillary, a tube and the like can be mentioned.

Furthermore, the kit of the present invention may contain an appropriate biochemical reagent. For example, a washing solution, a staining solution, a coloring solution, a sensitizing solution, a reaction stopping solution containing a buffer solution, an appropriate surfactant and the like. Avidinized compounds, biotinylated compounds; anti-immunoglobulin antibodies, labeled anti-immunoglobulin antibodies; stabilizers, preservatives, preservatives, proteolytic enzyme inhibitors; ACVR2B standard substances and the like.

<Kit for detecting the cause of anemia as MDS>
A kit for detecting the cause of anemia as MDS includes the above-mentioned anti-ACVR2B antibody.

The kit for detecting that the cause of anemia is MDS can be particularly suitably used in the above-described <Method for detecting anemia factor of anemia patient>. Thus, the kit may include a protocol described for the method.

Furthermore, any one or more of the components included in other kits for detecting that an anemia factor is MDS may be included. The component is not particularly limited. For example, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD10 antibody, anti-CD13 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD23 antibody, anti-CD33 antibody, anti-CD33 antibody, CD34 antibody, anti-CD38 antibody, anti-CD45 antibody, anti-CD45RA antibody, anti-CD90 antibody, anti-IgM antibody, anti-HLA-DR antibody; anti-CD71 antibody, anti-GPA antibody; anti-CD41 antibody, anti-CD42b antibody, anti-CD61 antibody, anti-CD61 antibody Examples include CD62P antibody and anti-PAC-1 antibody. Etc. Among these, anti-CD45 antibody, anti-CD34 antibody, anti-GPA antibody and the like are preferable.

<Kit for determining the risk of developing leukemia>
A kit for determining the risk of developing leukemia contains the anti-ACVR2B antibody described above.

The kit for determining the risk of developing leukemia can be particularly suitably used for the above-mentioned <Method for determining risk of developing leukemia>. Thus, the kit may include a protocol described for the method.

Furthermore, any one or more of the components included in another kit for determining the risk of developing leukemia may be included. Examples of such a component include anti-Tim-3 antibody. The method for determining the risk of developing leukemia using such components may be in accordance with a known protocol when using the other kits described above.

Note that the kit for determining the risk of developing leukemia may include components of the kit for determining the type of leukemia. For example, anti-CD1a antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 Antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD10 antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD33 antibody, anti-CD34 antibody, Examples include anti-CD38 antibody, anti-CD41 antibody, anti-HLA-DR antibody, anti-CD56 antibody, anti-CD235a: GPA antibody.

The method of determining leukemia typing by using such components may follow a known protocol when using the above-described kit for determining leukemia typing.

<Kit for predicting necessity and frequency of blood transfusion>
A kit for predicting the need for transfusion and its frequency comprises the anti-ACVR2B antibody described above.

The kit for predicting the necessity and frequency of blood transfusion can be particularly suitably used in the above-described <Method for predicting the necessity and frequency of blood transfusion>. Accordingly, the kit may include a protocol describing the method, and further includes any one or more of the components included in other kits for predicting the need and frequency of transfusion. Also good. Although it does not specifically limit with a component, For example, an anti- ferritin antibody, an antifolate antibody, an anti- vitamin B12 antibody etc. are mentioned.

Biomarker of the Present Invention The biomarker of the present invention comprises <a biomarker for determining that the cause of anemia is MDS>, <a biomarker for determining the risk of developing leukemia>, and <necessity of blood transfusion> Biomarkers for predicting sex and its frequency.

<Biomarker for determining the cause of anemia as MDS>
The biomarker for determining the cause of anemia as MDS consists of ACVR2BACVR2B.

ACVR2B is as described above. Such ACVR2B is present in peripheral blood multi-cells. Peripheral blood is as described above.

The biomarker for detecting an anemia factor as MDS is usefully used according to the method described in the above <Method for detecting an anemia factor>.

<Biomarkers for determining the risk of developing leukemia>
The biomarker for determining the risk of developing leukemia consists of ACVR2B.

The biomarker for determining the risk of developing leukemia is usefully used according to the method described in the above <Method for determining risk of developing leukemia>.

<Biomarker for predicting necessity and frequency of blood transfusion>
A biomarker for predicting the need and frequency of transfusions consists of ACVR2B.

The biomarker for predicting the necessity and frequency of blood transfusion is usefully used according to the method described in the above <Method for predicting the necessity and frequency of blood transfusion>.

The present invention will be described in more detail based on the following examples. However, it goes without saying that the present invention is not limited to the following examples.

<Antibody>
The antibodies used in this example are as follows.

* In addition to the above antibodies, human ACVR2B-APC was prepared using human ACVR2B (abcam) and APC conjugation kit (Dojindo) according to a conventional method.

<Separation of blood cells at each differentiation stage>
Liver tissue was collected from a 16.5 week-old fetus of a Gata1-GFP transgenic mouse prepared according to a conventional method, and then dispersed into a cell group according to a conventional method. The obtained cell group was washed with 3 mL of PBS and then subjected to blocking treatment with 3 mL of MACS buffer on ice for 30 minutes.

Then, while still on ice, add the mouse CD45-PE, mouse Sca1-PE, mouse cKit, mouse CD71-PE, and mouse Ter119-PB, which are the antibodies shown in Table 1 above, to the cell population, and allow immunoreaction for 30 minutes. Finally, it was stained with DRAQ5 for 30 minutes while still on ice.

The cell groups subjected to these treatments were analyzed using a flow cytometry apparatus manufactured by BD Bioscience, and then fractionated into cells of various hematopoietic differentiation stages using a cell sorter apparatus. The results are shown in FIG.

In the figure, the region gated with A was a cell population of positive erythroblasts, and the region gated with B was a cell population of reticuloblasts. The lower left of the figure is a cell population of erythrocytes.

From these, cell populations of positively dyed erythroblasts and reticulocytes were sorted using a cell sorter device.

<Search for differentiation marker candidates>
The sorted positive erythroblasts and reticulocytes were subjected to the DNA array method (Agilent Technology), and the genes expressed in each cell population were analyzed. Table 2 shows genes that are expressed less in reticulocytes than in normal erythroblasts.

Acvr2b was selected from these genes, and the amount of mRNA of Acvr2b expressed in the cell populations of positive erythroblasts, reticulocytes, and erythrocytes was measured according to a conventional method. The results are shown in FIG.

2. From the results shown in FIG. 2, it has been clarified that Acvr2b is expressed in a significant amount in the positive erythroblasts, but is hardly expressed in the reticulocytes and erythrocytes. In the normal hematopoietic differentiation process, Acvr2b (human protein homolog name: ACVR2B) does not mature into normal erythrocytes, considering that the differentiation stages proceed in the order of normal erythroblasts, reticulocytes, and erythrocytes. It was suggested that it is useful as a biomarker of MDS that is not seen.

<About the expression level of ACVR2B in human peripheral blood>
Peripheral blood from healthy individuals, RA patients, and RCMD patients was collected at 1 to 2 mL, and treated with EDTA-2Na to prevent coagulation. Next, 1 × 10 ⁶ cells were taken into a tube, the peripheral blood was washed 3 times with 3 mL of PBS, and subjected to blocking treatment with 3 mL of MACS buffer for 30 minutes on ice. Thereafter, human GPA-FITC, human CD71-PE, human CD45-APC-Cy7, and human ACTR-IIB-APC were added to the peripheral blood on the peripheral blood, and an immune reaction was performed for 30 minutes. It was.

The peripheral blood treated with these treatments was analyzed using a flow cytometry apparatus manufactured by BD Bioscience. The results are shown in FIGS.

From these results, it was clear that MDS patients (RA and RCMD) clearly had higher expression levels of ACVR2B in peripheral blood erythrocytes than in healthy individuals. This was also the same for leukocytes. In addition, from FIG. 6, MDS patients have a high expression level of ACVR2B compared to elderly healthy individuals who are likely to become anemic, and anemia is caused by other factors (renal anemia). It became clear that it was higher than

From these results, when the expression level of ACVR2B in erythrocytes and / or leukocytes obtained from peripheral blood is large compared with those obtained from healthy subjects, healthy elderly people, and patients with renal anemia, MDS causes It was suggested that it was possible to determine that anemia was occurring. Moreover, since this reflects the differentiation / maturation disorder of multicytic cells, it is suggested that there is a high possibility of progressing from MDS to leukemia, particularly acute leukemia.

Claims

A method for detecting a factor of anemia in an anemia patient, comprising the following steps 1 and 2;
(1) Step 1 of measuring the expression level of activin receptor 2B (ACVR2B) in multiblood cells contained in peripheral blood obtained from the patient,
(2) When the expression level of ACVR2B measured in step 1 is greater than the expression level of ACVR2B in the polycytic cells contained in peripheral blood obtained from healthy subjects, the cause of anemia in the patient is myelodysplastic syndrome ( Step 2 for determining that it is MDS).
The method according to claim 1, wherein the MDS is at least one selected from the group consisting of RA, RCMD, RCMD-RS, and RAEB.
A method for determining the risk of developing leukemia in an anemic patient comprising the following steps 1 and 2;
[1] Step 1 of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from the patient,
[2] Step 2 for determining that the patient is at risk of developing leukemia when the expression level of ACVR2B measured in Step 1 is greater than the expression level of ACVR2B in polycytic cells contained in peripheral blood obtained from healthy subjects .
The method according to claim 3, wherein the leukemia is acute myeloid leukemia.
A method for predicting the need and frequency of blood transfusion in anemia patients, comprising the following steps 1 and 2;
{1} Step 1 of measuring the expression level of ACVR2B in multicytic cells contained in peripheral blood obtained from the patient,
{2} When the expression level of ACVR2B measured in step 1 is greater than the expression level of ACVR2B in the multiblood cells contained in peripheral blood obtained from healthy subjects, blood transfusion is necessary for the patient, and the frequency is peripheral blood Step 2 for determining that the frequency is higher than the blood transfusion frequency of the patient at the time of collection.
A diagnostic agent for detecting an anemia factor of an anemic patient as MDS, comprising an anti-ACVR2B antibody.
The diagnostic agent according to claim 6, wherein the MDS is at least one selected from the group consisting of RA, RCMD, RCMD-RS, and RAEB.
A diagnostic agent for determining the risk of developing leukemia in anemia patients, comprising an anti-ACVR2B antibody.
The diagnostic agent according to claim 8, wherein the leukemia is acute myeloid leukemia.
A diagnostic agent for the necessity and frequency of transfusion of anemia patients, comprising an anti-ACVR2B antibody.
A kit for detecting an anemia factor of an anemic patient as MDS, comprising an anti-ACVR2B antibody.
The kit according to claim 11, wherein the MDS is at least one selected from the group consisting of RA, RCMD, RCMD-RS, and RAEB.
A kit for determining the risk of developing leukemia in anemia patients, comprising an anti-ACVR2B antibody.
The kit according to claim 13, wherein the leukemia is acute myeloid leukemia.
A kit for predicting the necessity and frequency of transfusion of anemia patients, comprising an anti-ACVR2B antibody.
A biomarker for determining that an anemic patient is MDS, comprising ACVR2B.
A biomarker for determining the risk of developing leukemia in anemia patients, comprising ACVR2B.
A biomarker for predicting the necessity and frequency of transfusion of anemia patients, comprising ACVR2B.