JP6206953B2 - Method for testing the likelihood of developing adult T-cell leukemia - Google Patents

Description

  The present invention measures a soluble interleukin-2 receptor (hereinafter referred to as sIL-2R) in a sample, and adult T cells in a human T lymphotropic virus type 1 (hereinafter referred to as HTLV-1) carrier The present invention relates to a method for testing the likelihood of developing leukemia (hereinafter referred to as ATL), and a kit used in these methods.

  Human T lymphotropic virus is a type of retrovirus, and there are two types, type 1 and type 2. Of these, type 1, ie, HTLV-1, has been identified as a causative virus for adult T-cell leukemia / lymphoma by Hinuma et al. (See Non-Patent Documents 1, 2, and 3).

  HTLV-1 infection in humans is mainly vertical transmission from mother to child and horizontal transmission from husband to wife, but iatrogenic infection by blood transfusion is also known. Iatrogenic infections from blood transfusions have been prevented by examining anti-HTLV-1 (or 2) antibodies, started in 1986. From epidemiological studies of iatrogenic infection by blood transfusion, it is known that HTLV-1 infection is mediated by blood cell components. When infected with HTLV-1, an anti-HTLV-1 antibody is produced in the living body. Therefore, by measuring the anti-HTLV-1 antibody, infection of HTLV-1 can be known.

  As a method for measuring an anti-HTLV-1 antibody, a measurement method using an immunological technique is known. Gelatin particle aggregation method (PA method), fluorescent antibody method (FA method), indirect fluorescent antibody method (IF Method), chemiluminescent enzyme immunoassay (CLEIA method), Western blot method (WB method) and the like are known. According to HTLV-1 Career Guidance Guide (Ministry of Health, Labor and Welfare, Research Group “Study of HTLV-1 infection and related diseases in Japan and comprehensive measures”, 2011), general medical institutions use PA and CLEIA methods as screening tests. It is done. Even if it is determined to be positive by the screening test, a confirmation test by the WB method is performed. Further, when the determination is suspended in the confirmation test by the WB method, a test by the PCR method (polymerase chain reaction method) for detecting the HTLV-1 provirus may be performed.

  Diseases caused by HTLV-1 include, for example, adult T-cell leukemia / lymphoma that develops in the bloodstream and lymphoid organs, HTLV-1-related myelopathy that develops in the spinal cord (HAM / TSP), and in the eyeball Uveitis (HU) and the like are known.

  ATL is an independent disease caused by HTLV-1 infection. The number of HTLV-1 carriers (those who have been infected with HTLV-1 and who have not developed the disease caused by HTLV-1) is estimated to be about 1.08 million in Japan in 2008. Moreover, ATL develops at a rate of 1 in 1000 HTLV-1 carriers per year, and the ATL lifetime incidence is said to be about 5% of HTLV-1 carriers. Although the incidence is very low, once it develops, it is said to have very short and severe consequences.

  ATLs are classified into four types of clinical disease types, acute type, lymphoma type, chronic type, and smoldering type, and this clinical type is widely used in determining treatment strategies including chemotherapy. According to the ATL survey in Japan from 1990 to 1995, among the ATL2123 cases, the acute type was 1328 cases (62.6%), the lymphoma type was 505 cases (23.8%), and the chronic type was 176 cases (8.3). %) And smoldering type was 114 cases (5.4%).

  Lymphoma type and acute type are defined as high-grade ATL and have a poor prognosis (survival rate) and require immediate treatment. On the other hand, the smoldering type and the chronic type are defined as low-grade ATL, and it is a common practice to observe the course without treatment. As described above, it is said that the chronic type and the smoldering type have a higher survival rate than the acute type and the lymphoma type.

  The prognosis of ATL is said to be extremely poor. Currently, multi-drug combination therapy called mLSG15 has achieved the best survival, but the median survival is about 13 months and the 3-year survival rate is still about 24%, which is still very poor. Recently, Poterigio (registered trademark), an antibody drug targeting CCR4 (chemokine receptor 4) was approved, and it was reported that the response rate to relapsed high-grade ATL was 50% in a phase 2 study. Yes. When a sufficient effect cannot be expected with chemotherapy, bone marrow transplantation (allogeneic hematopoietic stem cell transplantation) has been actively performed, and some patients can also be expected to be cured (see Non-Patent Document 4). ).

  Studies on risk factors for the development of ATL have been conducted in several institutions. It has been gradually found that those who satisfy certain conditions among HTLV-1 carriers are likely to develop ATL, such as mother-to-child infection, sex (male), aging, specific HLA (human leukocyte antigen), Increased HTLV-1 antibody titer, increased sIL-2R, increased white blood cell count, and the like are listed as risk factors. However, it is not possible to clearly separate carriers that develop ATL from carriers that do not develop ATL. In the project “Joint Study of Predisposing Factors of ATL Development (JSPFAD)”, changes in the body from infection to onset are analyzed.

  Recently, JSPFAD has reported that an increase in the amount of HTLV-1 provirus is a risk factor based on analysis of 14 cases that developed ATL from HTLV-1 carriers (see Non-Patent Document 5). However, since the amount of HTLV-1 infected cells in the peripheral blood mononuclear cells is observed in the measurement of the amount of provirus, the onset of lymphoma type in which almost no HTLV-1 infected cells (including ATL cells) are present in the peripheral blood It is difficult to predict.

  Further, it has been reported that an antibody against Asp197-Leu216 of glycoprotein gp46 of the HTLV-1 coat can be used to determine the onset of ATL in an HTLV-1 carrier (see Patent Document 1).

  Interleukin-2 receptor (hereinafter referred to as IL-2R) is composed of three cell membrane surface proteins, α chain (CD25), β chain (CD122), and γ chain (CD132), which is cleaved by protease. SIL-2R is reported to be released into the blood. sIL-2R generally refers to the α chain cleaved by a protease. Since ATL cells have the surface traits of regulatory T cells and express CD25 (IL-2Rα chain) on the surface, sIL-2R is used as a useful indicator for ATL disease state monitoring and prognosis prediction. ing. Recently, it has also been reported that a treatment policy after blast crisis can be determined by measuring CD30 and IL-2R in samples collected from chronic or smoldering ATL patients (see Patent Document 2). Therefore, IL-2R is expected not only as a predictor of ATL onset but also as a predictor of acute transformation. However, it has not been reported so far that ATL can be predicted based on a specific concentration of sIL-2R alone.

International Publication No. 2005/076002 Pamphlet JP 2008-292474 A

Proceeding of the National Academy of Sciences of the UnitedStates of America, Vol. 78 (10), pp. 6476-6480. 1981 Proceeding of the National Academy of Sciences of the UnitedStates of America, Vol. 79, 2031-2035. 1982 Sciences, Vol.219, pp.856-859.1983 International Journal of Hematology, Vol.69 (3), pp.203-205. 1999 Blood, Vol.116, pp.1211-1219, 2010

  An object of the present invention is to improve the QOL of an HTLV-1 carrier by early diagnosis of ATL onset in an HTLV-1 carrier, and to test the ease of occurrence of ATL in an HTLV-1 carrier, and a kit for the method Is to provide.

  The present inventors diligently studied to solve this problem, and can predict the likelihood of HTLV-1 carrier onset of ATL by measuring sIL-2R in a sample collected from an HTLV-1 carrier. And the present invention was completed.

  That is, the present invention measures sIL-2R in a sample collected from [1] HTLV-1 carrier, and if the concentration of sIL-2R in the sample is 900 U / mL or more, the carrier develops ATL. It relates to a method for testing the ease of onset of ATL of the carrier by comparing with a standard that the carrier is less likely to develop ATL if it is less than 900 U / mL. As another aspect of the present invention, sIL-2R in a sample collected from an HTLV-1 carrier is measured, and if the concentration of sIL-2R in the sample is 900 U / mL or more, the carrier has ATL. If it is easy to develop and less than 900 U / mL, the data collection method for testing the carrier's likelihood of developing ATL is listed by comparing with the standard that the carrier is unlikely to develop ATL. be able to.

  The present invention also relates to a kit for testing the ease of onset of ATL of an HTLV-1 carrier, which contains a reagent for measuring [2] sIL-2R. As another aspect of the present invention, if the sIL-2R measurement reagent and the sIL-2R measurement result indicate that the concentration of sIL-2R in the sample is 900 U / mL or more, the carrier is likely to develop ATL. HTLV-1 carrier, comprising a package insert containing a description of the ease of onset of ATL of the carrier that the carrier is less likely to develop ATL if less than 900 U / mL A test for testing the ease of onset of ATL and the test for the ease of onset of ATL in HTLV-1 carriers, which contains a sIL-2R measurement reagent and no CD30 measurement reagent Can be listed.

  INDUSTRIAL APPLICABILITY According to the present invention, a method for testing the ease of occurrence of ATL in an HTLV-1 carrier and a kit for the method are provided, which enables early diagnosis of ATL onset in an HTLV-1 carrier, and the QOL of the HTLV-1 carrier. Can be improved.

The onset rate curve by a Kaplan-Meier method is shown with respect to the group whose sIL-2R density | concentration in the serum extract | collected from the HTLV-1 carrier is 900 U / mL or more, and a group below 900 U / mL.

  In the present invention, the HTLV-1 carrier means a person who is infected with HTLV-1 as described above but has not developed a disease caused by HTLV-1 such as ATL. Infection with HTLV-1 may be caused by particle agglutination assay (PA method), ELISA method, indirect fluorescence assay, fluorescent immunoassay method (FIA method), Western blotting method, etc. Known testing methods such as a method for detecting an anti-HTLV-1 antibody produced using an HTLV-1 antigen, a method for detecting HTLV-1 proviral DNA, such as a PCR (Polymerase Chain Reaction) method and a Southern blotting method However, if the primary screening by the PA method or the FIA method is positive, it is further confirmed by the World Health Organization (WHO) criteria (Weekly epidemiological record, vol. 65 (37), pp.281-283 , 1990) and the like, and when it is confirmed to be positive, it can be confirmed that the subject is an HTLV-1 carrier.

  In the present invention, the onset of ATL refers to a state in which T cells (T lymphocytes) are tumorized due to HTLV-1 and tumor cells have been detected in the blood, swollen lymph nodes, or skin lesions. In the part, it refers to a state in which tumorous T cells have been detected. Tumorized T cells can be obtained by using antibodies against tumor cell surface antigens such as CD3 and CD4 and cell analyzers such as cell sorters and flow cytometers (Proc Natl Acad Sci USA, 101, 3781-3786 (2004). ), Cancer Res, 65, 6207-6219 (2005)).

  In the present invention, the sample collected from the HTLV-1 carrier is not particularly limited as long as it is a sample collected from the carrier and can measure sIL-2R. Examples thereof include plasma and serum. It is done.

In the present invention, the test for the likelihood of ATL onset in the HTLV-1 carrier can be performed, for example, by a method comprising the following steps (1) to (5).
(1) A step of collecting a sample from an HTLV-1 carrier;
(2) A step of measuring sIL-2R in the sample using the sample collected in step (1);
(3) A step of determining the sIL-2R concentration in the sample from a calibration curve representing the relationship between the sIL-2R concentration prepared in advance and the measured value of sIL-2R, and the measured value in step (2);
(4) If the sIL-2R concentration determined in step (3) is sIL-2R concentration of 900 U / mL or more, the carrier is likely to develop ATL, and if it is less than 900 U / mL, the carrier is Comparing with criteria that ATL is unlikely to develop;
(5) A step of determining the ease of onset of ATL of the carrier by comparison with the reference in step (4).

  Measurement of sIL-2R in a sample collected from an HTLV-1 carrier can be performed by using a known method and kit for measuring the concentration of sIL-2R in a biological sample. The method is not particularly limited as long as it can measure sIL-2R in a biological sample, and examples thereof include an immunological measurement method.

  Examples of the immunoassay include any known immunoassay, such as radioimmunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), indirect fluorescence. Antibody method (Indirect Fluorescence assay), luminescence immunoassay (Luminescent immunoassay), physicochemical assay [turbidimetric immunoassay (TIA), latex agglutination (LAPIA), microparticle counting immunoagglutination (PCIA)], The Western blotting method and the like can be mentioned, but the ELISA method is preferably used [Monoclonal antibody experiment manual (Kodansha Scientific, 1987), Secondary Biochemistry Laboratory Lecture 5 Immunobiochemistry Research Method (Tokyo Kagaku Dojin, 1986)].

  In the immunological measurement method, a sandwich method, a competitive method, or the like can be used, and a homogenis method, a heterogeneous method, or the like can also be used.

  In the present invention, sIL-2R in a sample can be measured using a sIL-2R measurement reagent. Examples of the reagent for measuring sIL-2R include a solid phase in which a first antibody that specifically binds to sIL-2R is bound, and a label in which a label is bound to a second antibody that specifically binds to sIL-2R A reagent containing the second antibody can be exemplified. The antigen determining site of sIL-2R in the first antibody and the antigen determining site of sIL-2R in the second antibody may be the same or different.

Examples of the label in the labeled second antibody include enzymes such as peroxidase and alkaline phosphatase, and radioactive substances such as ¹²⁵ I.

  Examples of the bond between the antibody and the solid phase in the solid phase to which the first antibody is bound include non-covalent bonds and covalent bonds. Examples of non-covalent bonds include physical adsorption. Examples of the covalent bond include a direct bond between the antibody and the solid phase and an indirect bond between the antibody and the solid phase via a linker or the like.

  The solid phase is not particularly limited as long as the first antibody is immobilized and the solid phase enabling immunoassay of sIL-2R is used. For example, a polystyrene plate such as a microtiter plate, glass or synthetic resin Examples thereof include particles made of beads (beads), glass or synthetic resin spheres (balls), latex, magnetic particles, various membranes such as nitrocellulose membrane, synthetic resin test tubes, and the like.

  By using the solid phase to which the first antibody is bound, the unreacted substance is removed from the solid phase by washing the solid phase after the reaction between sIL-2R in the specimen and the antibody on the solid phase. This is preferable because it can be removed. By using this sIL-2R measurement reagent, sIL-2R in a sample can be measured. Examples of commercially available kits for measuring sIL-2R include cell-free N IL-2R (manufactured by Kyowa Medex), determiner CL IL-2R (manufactured by Kyowa Medex), Siemens Imrise IL-2R II (manufactured by Siemens), Siemens Imrise IL-2R II 2000 (manufactured by Siemens), IL-2R test BML (manufactured by BML) and the like.

  The sIL-2R concentration of 900 U / mL in the present invention was stimulated for 4 days with the method described in the package insert of cell-free N IL-2R, ie, 10% (W / V) interleukin 2 (IL-2) The concentration determined by the method in which the concentration of sIL-2R contained in the cell-free culture supernatant (undiluted) of normal human IL-2-dependent T cells is 1000 U / mL, or the cell-free N IL-2R A concentration determined by other measurements that correlates with the measurement. As the concentration determined by such other measurement, when converted to the sIL-2R concentration determined by the measurement using cell-free N IL-2R, the sIL-2R concentration is at least 90%, preferably at least A concentration showing 95%, more preferably 100% identity can be mentioned.

  The present invention also relates to a kit for testing the ease of onset of ATL of an HTLV-1 carrier, comprising a reagent for measuring sIL-2R. As the reagent for measuring sIL-2R used in the kit of the present invention, the aforementioned reagent for measuring IL-2R can be used. In the kit, in addition to the reagent for measuring sIL-2R, an index for determining the likelihood of ATL onset of HTLV-1 carrier (ATL onset risk), for example, the concentration of sIL-2R in the sample is 900 U If the carrier is more than / mL, the carrier is likely to develop ATL, and if it is less than 900 U / mL, it preferably includes a package insert that states that the carrier is unlikely to develop ATL. .

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to this Example.

SIL-2R concentration in 27 cases of HTLV-1 carrier The sIL-2R concentration in the plasma or serum of 27 HTLV-1 carriers not affected by disease other than HTLV-1 infection with informed consent was expressed as sIL-2R. Measurement was performed using a measurement kit [Cell-free N IL-2R (manufactured by Kyowa Medex)].

  A method for analyzing the time from a certain reference time until a certain target event occurs is called survival time analysis, and a Kaplan-Meier curve is used to estimate a survival function. In addition, in order to test whether there is a difference in survival time distribution between the two groups compared, log rank test (also called Peto-Peto test, Cochran-Mantel-Haenszel test) and generalized Wilcoxon test (Gehan-Breslow test) Test, also called Peto-Prentice test).

  In this example, the time until the onset of ATL was analyzed as follows, based on the day when the IL-2R concentration of the HTLV-1 carrier was measured.

  27 cases were divided into two groups with IL-2R concentrations of 900 U / mL or more and less than 900 U / mL, and the sIL-2R concentration on the day (reference day) when the IL-2R concentration of HTLV-1 carrier was measured, presence or absence of onset of ATL The number of days from the reference date until the onset of ATL (in the case where ATL did not develop from the reference date in cases where ATL did not develop) was examined. The results are shown in Table 1. Moreover, the incidence rate curve by the Kaplan-Meier method created based on the data of Table 1 is shown in FIG. 1, and the results of the log rank test and the generalized Wilcoxon test are shown in Table 2.

  From the results shown in Table 2, since the P value was less than 0.05 in any of the log rank test, generalized Wilcoxon test, the group having a sIL-2R concentration of 900 U / mL or more was ATL. It was shown that it is easy to develop.

  As described above, the sIL-2R concentration in the specimen of HTLV-1 carrier is measured, and if the concentration is 900 U / mL or more, ATL is likely to develop, and if it is less than 900 U / mL, it is difficult to develop ATL. By comparing with the above, it is possible to test the ease of onset of ATL of the carrier.

  According to the present invention, a method for testing the susceptibility to developing ATL in an HTLV-1 carrier and a kit for the method are provided.

Claims (2)

  When the soluble interleukin-2 receptor in a sample collected from a human T lymphocyte-tropic virus type 1 carrier is measured, and the concentration of the soluble interleukin-2 receptor in the sample is 900 U / mL or more, The carrier is likely to develop adult T-cell leukemia, and if the carrier is less than 900 U / mL, the carrier develops adult T-cell leukemia by comparing with the standard that adult T-cell leukemia is unlikely to develop. How to test ease. A reagent for measuring soluble interleukin-2 receptor (hereinafter referred to as sIL-2R) , and
If the concentration of sIL-2R in a sample collected from a human T lymphotropic virus type 1 carrier is 900 U / mL or more, the carrier is likely to develop adult T-cell leukemia , and if it is less than 900 U / mL An adult of a human T lymphocyte-tropic virus type 1 carrier, characterized in that it contains a package insert describing that the carrier is compared with a criterion that it is difficult to develop adult T-cell leukemia A kit for testing the ease of onset of T cell leukemia.