WO2017111129A1

WO2017111129A1 - Novel genetic abnormality related to acute lymphoblastic leukemia, and uses thereof

Info

Publication number: WO2017111129A1
Application number: PCT/JP2016/088570
Authority: WO
Inventors: 友介奥野; 勢二小島; 喬悟鈴木; 希川島; 由子関屋
Original assignee: 国立大学法人名古屋大学
Priority date: 2015-12-25
Filing date: 2016-12-22
Publication date: 2017-06-29
Also published as: JPWO2017111129A1

Abstract

The present invention addresses the problem of discovering a novel genetic abnormality, which is useful for the determination of treatment strategies or prognostic prediction of acute lymphoblastic leukemia, and contributing to the improvement of treatment outcomes. A genetic abnormality characterized by the formation of a fusion gene comprising the MEF2D gene and the BCL9 gene has been discovered from recurrent childhood acute lymphoblastic leukemia patients. This genetic abnormality will serve as a new indicator when determining treatment strategies.

Description

Novel genetic abnormalities associated with acute lymphoblastic leukemia and their use

The present invention relates to a novel genetic abnormality related to acute lymphoblastic leukemia. Specifically, it relates to various uses (for example, testing method, determination of treatment policy, treatment method) of the newly found MEF2D-BCL9 fusion gene. This application claims priority based on Japanese Patent Application No. 2015-255179 filed on Dec. 25, 2015, the entire contents of which are incorporated by reference.

Acute lymphoblastic leukemia (ALL) is the most common leukemia in childhood. In Japan, 500 cases occur annually, and the long-term survival rate is around 80%. As a characteristic of ALL, it is known that it develops with various genetic abnormalities (gene fusion, gene deletion / amplification, point mutation) (Non-patent Document 1). And the prognosis of life and responsiveness to various treatment methods differ depending on the difference in genetic abnormality provided. For example, ALL having an ETV6-RUNX1 fusion gene or a TCF3-PBX1 fusion gene responds well to normal chemotherapy and has a good prognosis. ALL with the BCR-ABL fusion gene was considered to have a poor prognosis, but it became clear that treatment with BCR-ABL molecular target drugs (imatinib, dasatinib, etc.) improved the prognosis. ALL with point mutations / deletions in the TP53 and IKZF1 genes and ALL with partial amplification within chromosome 21 have a poor prognosis. ALL having a point mutation in the NT5C2 gene has resistance to a specific chemotherapy (6-mercaptopurine) (Patent Document 1). Recent large-scale studies have identified a group of ALL with activated thyrone kinase mutations (due to point mutations and gene fusions) and suggested the possibility of treatment with corresponding thyrone kinase inhibitors.

International Publication No. 2014/074651 Pamphlet International Publication No. 2005/082933 Pamphlet

Although various genetic abnormalities have been discovered in ALL, there is still an example (B-other ALL) in which no genetic abnormality that predicts prognosis is found. One hypothesis explaining this may be that the genetic abnormality in ALL is still insufficiently elucidated. For example, there are many reports of analysis at the time of diagnosis, but there are few reports of analysis at a more advanced stage such as the time of recurrence. Worldwide, the long-term survival rate of ALL patients is improving to 90%, but in order to further improve treatment outcomes, new genetic abnormalities and molecules that predict response to specific chemotherapy It may be necessary to look for genetic abnormalities that are the target of targeted therapy.

Thus, it is desired to find unknown genetic abnormalities that are useful in determining treatment strategies and predicting prognosis. Accordingly, an object of the present invention is to meet such needs and contribute to the improvement of ALL treatment results.

In order to solve the above problems, the present inventors have conducted intensive studies. First, bone marrow or peripheral blood leukemia cells in 59 patients with relapsed or refractory ALL (3 infants, 6 Ph1-positive ALL, 4 T-cell ALL, 3 mature B-cell ALL) Was used for RNA sequencing. As a result, MEF2D-BCL9 fusion gene was identified in 4 cases. In these 4 cases, the age of onset is relatively high (10-13 years), relapses in a short period of time, and treatment after recurrence is difficult, and cell morphology (giant vacuoles) Have common features, such as being different from normal ALL. Furthermore, no known genetic abnormality of ALL was found in these 4 cases. That is, it was suggested that the MEF2D-BCL9 fusion gene alone defines a group of characteristic ALL. Therefore, if the presence of a genetic abnormality characterized by the formation of the fusion gene is used as an index, it can be said that the case can be identified and a more effective treatment policy can be determined. Regarding MEF2D gene, fusion with DAZAP1 gene (Patent Document 2, Non-Patent Documents 2 and 3) and fusion with CSF1R gene (Non-Patent Document 4) have been reported.

As a result of further investigation, it was possible to identify the breakpoints of the MEF2D gene and the BCL9 gene in the genome, and to establish or implement a detection method. In addition, primers were designed based on the information on the breakpoints, and the first 115 cases of acute leukemia (100 cases of B progenitor ALL, 4 cases of T-cell ALL, 10 cases of acute myeloid leukemia, 10 cases of mixed trait acute leukemia) In the case of acute mixed leukemia (1 case), the MEF2D-BCL9 fusion gene was screened, but no cases were detected, confirming that the fusion gene had a genetic abnormality characteristic to the above 4 cases.

On the other hand, further studies were conducted with the aim of establishing a new treatment strategy using the MEF2D-BCL9 fusion gene as an index. First, expression analysis of tumor cells in 4 positive cases of MEF2D-BCL9 fusion gene was performed. As a result, characteristic high expression of the HDAC9 gene known as a classic poor prognosis factor was observed. HDAC9 is a class IIa histone deacetylase and is involved in transcriptional regulation. As drugs effective for inhibiting HDAC9, HDAC inhibitors such as vorionstat, quisinostat and TMP269 have been developed. In order to examine the effectiveness of treatments using HDAC inhibitors, a drug susceptibility test using patient-derived primary cultured leukemia cells was performed. As a result, HDAC inhibitors (vorinostat, xinostat) showed significant cell growth inhibitory activity. In recent years, bortezomib, a proteasome inhibitor that is expected to be effective against treatment-resistant B-precursor ALL, showed similar activity. That is, the possibility that the HDAC inhibitor and the proteasome inhibitor are effective for the treatment of the case was shown.
The following invention is mainly based on the above-mentioned results.
[1] A method for testing acute lymphocytic leukemia comprising the following steps (1) to (3):
(1) preparing a specimen containing leukemia cells isolated from a patient with acute lymphoblastic leukemia;
(2) detecting the presence or absence of a fusion gene of the MEF2D gene and the BCL9 gene or a fusion protein encoded by the fusion gene in the specimen;
(3) A step of determining that the prognosis is poor or difficult to treat when the fusion gene or the fusion protein is detected.
[2] The test according to [1], wherein the fusion gene is formed by a chromosomal inversion in which a breakpoint is present in

intron

6 or 7 in the MEF2D gene and a breakpoint is present in exon 8 or intron 9 in the BCL9 gene. Method.
[3] The detection of the fusion gene in step (2) is from the group consisting of RT-PCR, PCR, PCR-RFLP, PCR-SSCP, RNA sequence analysis, target sequence analysis, FISH method and whole genome analysis. The test method according to [1] or [2], wherein the detection is performed by any selected detection method, and the detection of the fusion protein in step (2) is performed by an immunological measurement method.
[4] The examination method according to any one of [1] to [3], wherein the patient with acute lymphoblastic leukemia is a child.
[5] The inspection method according to any one of [1] to [4], further including the following step (4) or (4 ′):
(4) identifying a risk group to which the acute lymphoblastic leukemia patient belongs based on the determination in the step (3), and determining or changing a treatment policy;
(4 ′) A step of identifying a risk group to which the patient with acute lymphocytic leukemia belongs and determining or changing a treatment policy based on the determination in step (3) and the results of other tests.
[6] The inspection method according to [5], wherein the risk group is a high risk group.
[7] The examination method according to [5] or [6], wherein the treatment policy after the change in step (4) or (4 ′) includes a therapy that is strengthened than before the change.
[8] The therapeutic policy determined or changed in the step (4) or (4 ′) includes treatment by administration of a histone deacetylase inhibitor and / or a proteasome inhibitor, [5] or [ 6].
[9] The test method according to [8], wherein the histone deacetylase inhibitor is a histone deacetylase 9 inhibitor.
[10] The examination method according to [5] or [6], wherein the treatment policy determined or changed in step (4) or (4 ′) includes adaptation of hematopoietic stem cell transplantation.
[11] The examination method according to [10], wherein the hematopoietic stem cell transplantation is performed in a first remission period.
[12] Treatment of acute lymphocytic leukemia, comprising treating the patient with acute lymphocytic leukemia according to a treatment policy determined or changed by the test method according to any one of [5] to [11] Method.
[13] A medicament for treating a patient with acute lymphocytic leukemia characterized by formation of a fusion gene of MEF2D gene and BCL9 gene, comprising a histone deacetylase inhibitor and / or a proteasome inhibitor.
[14] The medicament according to [13], wherein the histone deacetylase inhibitor is a histone deacetylase 9 inhibitor.
[15] A therapeutically effective amount of a medicine containing a histone deacetylase inhibitor and / or a proteasome inhibitor is administered to a patient with acute lymphocytic leukemia characterized by the formation of a fusion gene between the MEF2D gene and the BCL9 gene A method for treating acute lymphoblastic leukemia.
[16] The treatment method according to [15], wherein the histone deacetylase inhibitor is a histone deacetylase 9 inhibitor.
[17] Transcription of a fusion gene between the MEF2D gene and the BCL9 gene formed by chromosomal inversion in the MEF2D gene where a breakpoint exists in

intron

6 or 7 and in the BCL9 gene where a breakpoint exists in exon 8 or intron 9 Primer set consisting of forward and reverse primers designed to specifically amplify DNA complementary to the product mRNA.
[18] The MEF2D gene has a breakpoint in

intron

6 or 7 and the BCL9 gene has a fusion gene of MEF2D gene and BCL9 gene formed by chromosomal inversion with a breakpoint in exon 8 or intron 9 Primer set consisting of forward primer and reverse primer designed for efficient amplification.
[19] A kit for detecting a genetic abnormality characterized by formation of a fusion gene of a MEF2D gene and a BCL9 gene, comprising the primer set according to [17] or [18].
[20] The detection kit according to [19], comprising the following primer set as the primer set:
A forward primer consisting of a sequence complementary to a part of exon 1 to 6 of the MEF2D gene and consisting of 13 bases or more, and a part of the exon 10 region of the BCL9 gene consisting of 13 bases or more Primer set comprising reverse primers comprising complementary sequences [21] The detection kit according to [19], wherein the primer set comprises one or more of the following (a1) to (a3):
(a1) Forward primer consisting of a sequence complementary to a part of the exon 3 to 4 region of the MEF2D gene and 13 bases or more, and a part of the exon 10 region of the BCL9 gene from 13 bases or more A primer set consisting of a reverse primer consisting of a sequence complementary to
(a2) Forward primer consisting of a sequence complementary to a part of exon 5 to 6 of the MEF2D gene and consisting of 13 bases or more, and part of the exon 10 region of the BCL9 gene from 13 bases or more A primer set consisting of a reverse primer consisting of a sequence complementary to
(A3) A part of the exon 6 region of the MEF2D gene that is complementary to a portion consisting of 13 bases or more and a part of the exon 10 region of the BCL9 gene that is a portion of 13 bases or more A primer set consisting of a reverse primer consisting of a sequence complementary to.
[22] The detection kit according to [19], comprising the following (b1) and / or (b2) as the primer set:
(b1) A part of the exon 2 region of the MEF2D gene that is complementary to a portion consisting of 13 bases or more and a part of the exon 10 region of the BCL9 gene that is a portion of 13 bases or more A primer set consisting of a reverse primer consisting of a sequence complementary to
(b2) A part of the exon 5 region of the MEF2D gene that is complementary to the portion consisting of 13 bases or more and a part of the exon 10 region of the BCL9 gene that is a portion of 13 bases or more A primer set consisting of a reverse primer consisting of a sequence complementary to.
[23] A screening method for a substance effective for the treatment of a patient with acute lymphocytic leukemia characterized by formation of a fusion gene of MEF2D gene and BCL9 gene, comprising the following steps (i) to (iii):
(i) providing a cell expressing a fusion gene of MEF2D gene and BCL9 gene;
(ii) culturing the cell in the presence of a test substance;
(iii) measuring the number of viable cells and determining the effectiveness of the test substance.
[24] A fusion gene of MEF2D gene and BCL9 gene.
[25] The fusion gene according to [24], which is generated by chromosomal inversion in which a breakpoint exists in

intron

6 or 7 in the MEF2D gene and a breakpoint exists in exon 8 or intron 9 in the BCL9 gene.
[26] The fusion gene according to [25], comprising exons 1 to 6 of the MEF2D gene and exon 10 of the BCL9 gene or a part thereof.
[27] The fusion gene according to [26], comprising the sequence of SEQ ID NO: 11 and the sequence of SEQ ID NO: 12.
[28] A fusion protein encoded by the fusion gene according to any one of [24] to [27].
[29] The fusion protein according to [28], comprising the sequence of SEQ ID NO: 13 and the sequence of SEQ ID NO: 14.
[30] DNA complementary to mRNA that is a transcription product of the fusion gene according to any one of [24] to [27].
[31] The DNA of [30], comprising the sequence of SEQ ID NO: 15 and the sequence of SEQ ID NO: 16.
[32] An antibody that recognizes a fusion protein encoded by a fusion gene of MEF2D gene and BCL9 gene.
[33] The antibody according to [32], wherein the fusion protein is a fusion protein defined in [28] or [29].

Discovery of MEF2D-BCL9 fusion gene in childhood ALL. A: The position on the chromosome of MEF2D gene and BCL9 gene. B: An example of a breakpoint that forms a fusion gene of MEF2D-BCL9. Detection of fusion gene mRNA by RT-PCR. RT-PCR results. Case 1 (upper) and case 4 (lower) are shown. Detection of breakpoints in genomic DNA. PCR results (top). Detection was attempted using primer set 4 (left) and primer set 5 (right). The genomic breakpoints in each case are shown in the bottom row. Results of expression profile analysis. Functional analysis of MEF2D-BCL9 fusion gene. HDAC9 expression levels were compared between MEF2D-BCL9 positive and negative cases (A). MELM2D-BCL9 was introduced into NALM-6, and the expression level (B) and cell proliferation rate (C) of HDAC9 were examined. Effect of molecular target drugs. Using primary culture cells established from patient leukemia cells positive for MEF2D-BCL9 fusion gene, the effect (drug sensitivity) of vorinostat (A), xynostat (B), and bortezomib (C) was tested.

1. Test Method for Acute Lymphoblastic Leukemia (ALL) The first aspect of the present invention relates to a test method for ALL. In the inspection method of the present invention, the following steps (1) to (3) are performed.
(1) preparing a specimen containing leukemia cells isolated from an ALL patient (2) detecting the presence or absence of a fusion gene of MEF2D gene and BCL9 gene or a fusion protein encoded by the fusion gene in the specimen ( 3) A step of determining that the prognosis is poor or difficult to treat when the fusion gene or the fusion protein is detected.

In step (1), prepare a sample to be used for the test. Specimens containing leukemia cells isolated from ALL patients are used. As long as leukemia cells are included, the type and origin of the specimen are not particularly limited. For example, a cell fraction (bone marrow cell) prepared from bone marrow or a cell fraction (blood cell) prepared from blood such as peripheral blood is used as a specimen. Since the test method of the present invention is particularly useful for predicting the prognosis of ALL in children, determining the treatment policy, and the like, it is preferable to use a sample derived from a child with ALL. In general, children under the age of 15 are considered children. The specimen is prepared prior to the practice of the present invention. That is, the examination method of the present invention does not include a step of isolating (collecting) bone marrow or the like for preparing a specimen from a patient.

In step (2), the presence or absence of a fusion gene (MEF2D-BCL9 fusion gene) of MEF2D gene and BCL9 gene or a fusion protein encoded by the fusion gene is detected in the sample. In the former embodiment (MEF2D-BCL9 fusion gene is detected), genomic DNA or mRNA is the detection target. When genomic DNA is targeted for detection, the presence or absence of fusion gene formation due to partial inversion of the chromosome is detected. On the other hand, when mRNA is the detection target, the presence or absence of the expression of the fusion gene is detected. The means for detecting the MEF2D-BCL9 fusion gene is not particularly limited. For example, RT-PCR (reverse transcription-polymerase chain reaction) method, PCR method, PCR-RFLP (restriction fragment fragment length polymorphism) method, PCR-SSCP (single strand strand conformation polymorphism) method, RNA sequence analysis, target sequence analysis, FISH ( Fluorescence in situ hybridization) method, whole genome analysis, Invader (registered trademark, Third Wave Technologies) method, LAMP (Loop-Mediated Isothermal Amplification) method, CGH (Comparative Genomic Hybridization) method, dot hybridization method, Northern hybridization method, etc. The detection means can be used. Extraction and purification of genomic DNA and mRNA can be performed by known methods. Various kits for preparation are commercially available, and they may be used.

The MEF2D-BCL9 fusion gene is a novel fusion gene derived from ALL patients, as shown in the Examples below. According to the study by the present inventors, it is clear that the fusion gene is formed by a chromosomal inversion in which a breakpoint exists in

intron

6 or 7 in the MEF2D gene and a breakpoint exists in exon 8 or intron 9 in the BCL9 gene. It became. Based on this information, it is possible to design and prepare primers and probes used for detection of fusion genes. Specific examples of primers that can be used in detection means using the nucleic acid amplification reaction (the above-mentioned RT-PCR method and PCR method) will be described later.

In the case of the latter mode (detection of fusion protein), for example, the fusion protein can be detected by an immunological assay. According to the immunological measurement method, rapid and sensitive detection is possible. Also, the operation is simple. In the immunoassay, an antibody against the fusion protein is used, and the fusion protein is detected using the binding property (binding amount) of the antibody as an index. Examples of immunoassays are Western blot, immunohistochemistry, fluorescence immunoassay (FIA), enzyme immunoassay (EIA), radioimmunoassay (RIA), flow cytometry (FCM) , Immunoprecipitation, immunochromatography, ELISA, and the like.

In step (3), the prognosis or treatment difficulty of the patient is evaluated based on the detection result in step (2). Specifically, when a fusion gene or fusion protein to be detected in step (2) is detected, it is determined that the prognosis is poor or treatment is difficult. Thus, genetic abnormalities characterized by the formation of the MEF2D-BCL9 fusion gene (hereinafter referred to as “genetic abnormalities of the present invention”) are used as indicators for determining the prognosis and treatment responsiveness of ALL patients. May be used). The determination here can be automatically / mechanically performed without depending on the determination of a person having specialized knowledge such as a doctor or a laboratory technician, as is apparent from the determination criteria.

By the way, as shown in Examples described later, it was suggested that the MEF2D-BCL9 fusion gene defines a group of characteristic ALL. Based on this knowledge, in one embodiment of the present invention, the following step (4) is performed following step (3).
(4) Based on the determination in step (3), identifying a risk group to which the patient with acute lymphocytic leukemia belongs, and determining or changing a treatment policy

In ALL, stratified treatment is generally performed according to risk. ALL stratification treatment generally consists of remission induction therapy, intensive therapy, central nervous system invasion prevention therapy, remission induction therapy, maintenance therapy, etc., and a treatment policy combining these therapies is set. Indications for hematopoietic stem cell transplantation are also considered when treatment alone is not possible with chemotherapy. Treatment of ALL usually begins with the initial goal of introducing remission. After successful introduction of remission, treatment is performed to further reduce the remaining tumor cells while preventing infiltration of the central nervous system and the like. Thereafter, maintenance therapy, re-induction therapy, etc. will be continued, aiming to eradicate tumor cells.

In stratified treatment of ALL, multiple risk groups are set using various indicators, and treatment is performed for each risk group according to the optimal treatment policy. Usually, a risk group to which a patient should belong is identified based on examinations before and after the start of treatment. The therapeutic effect is maximized by the stratified treatment.

The inspection method of the present invention is performed before or after the start of treatment. If it is carried out before the start of treatment, the present invention can be used to identify a provisional or definite risk group (to which an immediate or definitive treatment policy is determined). Stratified treatment protocols include, for example, age, white blood cell count, chromosomal / gene abnormalities, initial treatment (eg, methotrexate (MTX) intrathecal injection and 7-day prednisolone (PSL) administration), presence or absence of central nervous system invasion, etc. Comprehensive assessment of risk assessment, provisional risk classification, and initiation of remission induction therapy. If the test method of the present invention is carried out before the start of treatment and the results are used, cases having genetic abnormalities of the present invention can be classified into more qualified risk groups, and earlier and appropriate treatment can be performed. It becomes possible. On the other hand, if the test method of the present invention is carried out after the start of treatment, the present invention can be used for determination or change of risk groups (accordingly, subsequent treatment policy is determined). This aspect allows, for example, a review of the treatment policy if it recurs after remission. The examination method of the present invention may be performed multiple times over time to monitor the treatment effect and review the treatment policy (for example, change of risk group and change of treatment method associated therewith).

Currently, various indicators are used for stratification. The present invention provides a novel genetic abnormality that is one of useful indices for stratification. Step (4) corresponds to stratification using the genetic abnormality of the present invention as an index. The “risk group” identified in step (4) is typically a group associated with a high risk, that is, a “high risk group”. When the present invention is applied to an existing stratified treatment protocol, a corresponding risk group can be identified from among a plurality of risk groups set in the stratified treatment protocol. Below, a specific example is shown regarding this point.

In Japan, JACLS (Children's Leukemia Research Group), TCCSG (Tokyo Children's Cancer Research Group), CCLSG (Children's Cancer and Leukemia Research Group), KYCCSG (Kyushu / Yamaguchi Children's Cancer Research Group) Etc., and each created its own stratified treatment protocol. In 2010, JPLSG (Japan Pediatric Leukemia and Lymphoma Research Group) was established with the aim of establishing a standard treatment for childhood leukemia and lymphoma. In the protocol of the latest clinical trial conducted by JPLSG (JPLSG ALL-B12, a multicenter phase II and III clinical trial for childhood B precursor cell acute lymphoblastic leukemia), the standard risk group (SR group) and the intermediate risk group There are 3 groups (IR group) and high-risk group (HR group) (a control group is also set for each test group). When the present invention is applied to this protocol, for example, at the time of determination of poor prognosis or difficult treatment (step (3)), the patient is considered to belong to the “HR group” and another determination (ie, step ( When the fusion gene or fusion protein of the MEF2D gene and the BCL9 gene is not detected in 2), the patient belongs to the “SR group” or the patient belongs to the “IR group”.

On the other hand, in the previous treatment protocol (JACLS ALL-02) prepared by the above-mentioned treatment research group JACLS, standard risk group (SR group), high risk group (HR group) and super risk group (ER group) are set. It was. However, other treatment groups include T group (T-cell ALL), F group (adaptation of hematopoietic stem cell transplantation, inability to introduce remission and t (4; 11) positive ALL), and Ph1 group (Philadelphia chromosome positive ALL) Is set. In addition, special treatment is selected for infants under 1 year of age (MLL gene rearrangement positive and non-MLL gene rearrangements). If the present invention is applied to such a protocol, for example, at the time of determination of poor prognosis or treatment difficulty (step (3)), the patient belongs to the “ER group” and another determination (ie, When the fusion gene or fusion protein of MEF2D gene and BCL9 gene is not detected in step (2)), it is assumed that the patient belongs to the “HR group” or the patient belongs to the “SR group”.

As shown in the above examples, patients in whom the genetic abnormality of the present invention is detected are usually classified into high risk groups (poor prognosis, difficult treatment) among a plurality of set risk groups. The By classifying in this way, it is possible to perform an appropriate treatment (enhanced treatment) on a patient having a genetic abnormality of the present invention.

In step (4) and alternative step (4 '), the risk group to which the patient belongs is specified by using the determination in step (3) and the results of other tests together. In other words, various indices including a new index provided by the present application (that is, the genetic abnormality of the present invention) are comprehensively evaluated to identify a risk group to which the patient belongs. Other tests here include interviews, physical examination (extramedullary invasion, especially testicular invasion screening, etc.), blood tests (total blood count, blood biochemistry, cell surface marker analysis, detection of chromosomal abnormalities), Imaging (chest x-ray (eg, mediastinal mass screening)), ultrasonography, CT examination, bone marrow puncture or bone marrow biopsy (nucleated cell count, May Giemsa staining, peroxidase staining, esterase staining, cell surface Marker analysis, detection of chromosomal abnormalities, G-band staining, genetic analysis, pathological examination), cerebrospinal fluid examination, and the like. These inspections may be performed in a conventional manner. In addition, contract inspection may be used. For example, BML Inc., SRL Inc., etc. provide contract inspection services for the detection of chromosomal abnormalities, which is one of the most important tests for appropriate stratified therapy.

Instead of identifying the risk group to which the patient belongs from the existing risk group, a dedicated risk group is provided, and in step (4) or (4 '), the fusion gene or fusion protein of the MEF2D gene and the BCL9 gene is The detected patient may belong to the risk group. By providing a dedicated risk group, it is possible to treat a case of a genetic abnormality of the present invention under a more optimized treatment policy.

In ALL stratified treatment, treatment policy is set for each risk group. Therefore, by specifying the risk group to which the patient belongs, the patient's treatment policy is also determined. When the test method of the present invention is performed a plurality of times along the course of treatment, or when risk classification based on another index has already been performed, step (4) or (4 The risk group identified in ') may differ from the previous risk group (ie, the risk group is changed). In this case, the treatment policy is also changed with the change of the risk group. The treatment strategy after the change typically includes an enhanced therapy than before the change. Intensified therapy, for example, increased the number of drugs used in combination (for example, use of etoposide, ifosfamide, vindesine in high-risk groups), increased cumulative doses / number of doses of drugs (eg, vincristine, daunorubicin, Intrathecal injection of cyclophosphamide, L-asparaginase, methotrexate, cytarabine, methotrexate, cytarabine and prednisolone) chemotherapy, cranial radiation, or hematopoietic stem cell transplantation.

As shown in Examples described later, it was suggested that a histone deacetylase inhibitor and a proteasome inhibitor are effective for cases of genetic abnormalities of the present invention. Based on this finding, preferably, the treatment policy determined in step (4) or (4 ′) (also applicable to the modified treatment policy) is a histone deacetylase inhibitor or proteasome inhibitor, or Treatment by administration of both of these is included. On the other hand, cases of genetic abnormalities according to the present invention have a poor prognosis and show resistance to treatment, and therefore, more aggressive and enhanced treatment application is desired. From this point of view, preferably, the treatment policy determined in step (4) or (4 ′) (also applicable to the modified treatment policy) includes indication of hematopoietic stem cell transplantation. Usually, hematopoietic stem cell transplantation is used when chemotherapy alone cannot be cured. Hematopoietic stem cell transplantation is performed during the first remission phase or a subsequent remission phase, but is preferably the first remission phase so as to enhance the therapeutic effect by early aggressive therapeutic intervention. Examples of hematopoietic stem cell transplantation are allogeneic bone marrow transplantation, autologous bone marrow transplantation, peripheral blood stem cell transplantation, umbilical cord blood stem cell transplantation, and mini-transplantation. Although allogeneic bone marrow transplantation has few recurrences of disease, it has the problem that there are many complications (for example, GVHD) accompanying transplantation. Autologous bone marrow transplantation is an option when no matching HLA donor is found. Peripheral blood stem cell transplantation is a method in which hematopoietic stem cells in peripheral blood are collected and transplanted, and has an advantage that hematopoietic recovery after transplantation is quick. Umbilical cord blood stem cell transplantation has advantages such as no burden on the donor and early transplantation. Mini-transplantation is a method for reducing side effects by reducing the amount of pre-transplantation treatment (administration of anticancer drugs or irradiation) by using an immunosuppressant, and is also called non-myeloablative transplantation.

2. As described above, the test method of the present invention is useful for determining, changing, and reviewing the ALL treatment policy. In other words, if the test method of the present invention is applied, ALL can be treated under a more optimized treatment policy. Then, this invention provides the treatment method using the test | inspection method of this invention as another situation. In addition, when using the term “treatment” or “therapy” in the present specification, prevention, remission, prevention or cure of an illness (ALL) or a disease state is intended. Treatment after symptoms appear is aimed at reducing, ameliorating or eliminating the symptoms and / or related symptoms, or preventing exacerbations. Treatment before symptoms appear (ie, preventative treatment) typically aims to reduce the risk of symptoms appearing or to reduce the severity if symptoms appear.

In the treatment method of the present invention, an acute lymphoblastic leukemia patient is treated according to the treatment policy determined or changed by the test method of the present invention. According to the present invention, more optimized stratification treatment is possible. In particular, it is possible to provide a more appropriate treatment strategy for a case in which a genetic abnormality of the present invention has been recognized that could not be identified or distinguished until now. Specific examples of treatment in the therapeutic method of the present invention are chemotherapy, hematopoietic stem cell transplantation, and radiation therapy. If classified according to purpose, remission induction therapy, reinforcement therapy, central nervous system invasion prevention therapy, remission induction therapy, maintenance therapy and the like can be mentioned. Examples of drugs used in chemotherapy include corticosteroids (prednisolone, dexamethasone, hydrocortisone), alkylating agents (eg cyclophosphamide, ifosfamide, melphalan), antimetabolites (eg methotrexate, 6-mercapto) Purine, cytarabine, fludarabine, clofarabine), anticancer antibiotics (eg, daunorubicin, doxorubicin, pirarubicin, idarubicin, mitoxantrone), plant alkaloids (eg, vincristine, vinblastine, vindesine, etoposide), L-asparaginase. Chemotherapy is based on multi-drug combination therapy that combines several types of anticancer drugs with different mechanisms of action and side effects. Drug administration methods include intravenous injection (one-shot intravenous injection method, intravenous infusion method), subcutaneous injection, intramuscular injection, oral administration (internal use), intrathecal injection and the like.

The present invention is further based on the fact that histone deacetylase (HDAC) inhibitors and proteasome inhibitors have been suggested to be effective for cases in which the genetic abnormality of the present invention is observed, and specific cases of ALL Provide medicines for The medicament of the present invention is used for the treatment of ALL patients with a genetic abnormality characterized by the formation of the MEF2D-BCL9 fusion gene, and contains an HDAC inhibitor, a proteasome inhibitor, or both as active ingredients . The HDAC inhibitor and the proteasome inhibitor are not particularly limited. As the HDAC inhibitor, preferably, one that inhibits HDAC9 directly or through other molecules is employed. Examples of HDAC inhibitors are vorinostat, panobunostat, quisinostat, romidepsin, TMP269 (see Baas, T. Closer to class IIa HDAC inhibitors. SciBX 6 (13) 2013) . Vorinostat is a class I and class II HDAC inhibitor and is marketed under the trade name “ZOLINZA®”. Romidepsin is a class I HDAC inhibitor and is marketed under the trade name “ISTODAX (registered trademark)”. On the other hand, specific examples of proteasome inhibitors are bortezomib, carfilzomib, ixazomib. Bortezomib is a drug that is expected to have an effect on treatment-resistant B-precursor ALL in recent years.

Treatment with a therapeutically effective amount of the medicament of the present invention (medicament containing an HDAC inhibitor and / or proteasome inhibitor) can be expected as a promising therapeutic strategy for ALL patients with genetic abnormalities of the present invention.

3. Primer set and detection kit for detecting a novel genetic abnormality A further aspect of the present invention provides a primer set and a detection kit for detecting a genetic abnormality of the present invention. The primer set of the present invention is typically used in the inspection method of the present invention. The same applies to the detection kit.

<Primer set>
In one embodiment of the primer set of the present invention, the MEF2D gene and the BCL9 are formed by a chromosomal inversion in which a breakpoint exists in

intron

6 or 7 in the MEF2D gene and a breakpoint exists in exon 8 or intron 9 in the BCL9 gene. It is designed to specifically amplify DNA (cDNA) complementary to mRNA which is a fusion gene with a gene or its transcription product. Examples of the primer set of the present invention include the following.
A forward primer consisting of a sequence complementary to a part of exon 1 to 6 of the MEF2D gene and consisting of 13 bases or more, and a part of the exon 10 region of the BCL9 gene consisting of 13 bases or more Primer set consisting of reverse primers consisting of complementary sequences

Preferred examples of primer sets targeting cDNA include the following (a1) to (a3). Since the primer set targets cDNA, it is suitable for various methods such as RT-PCR, in which cDNA prepared using mRNA as a template is to be detected.
(a1) Forward primer consisting of a sequence complementary to a part of the exon 3 to 4 region of the MEF2D gene and 13 bases or more, and a part of the exon 10 region of the BCL9 gene from 13 bases or more A primer set consisting of a reverse primer consisting of a sequence complementary to a part of (a2) a forward primer consisting of a part of the exon 5-6 region of the MEF2D gene and a part complementary to a part consisting of 13 bases or more, and BCL9 Primer set consisting of a reverse primer consisting of a sequence complementary to a part of the exon 10 region of the gene that is complementary to a part consisting of 13 bases or more (a3) A part of the region of the exon 6 of the MEF2D gene consisting of 13 bases or more Forward primer consisting of a sequence complementary to the part and part of the exon 10 region of the BCL9 gene, complementary to a part consisting of 13 bases or more Primer set consisting of reverse primer consisting of a typical sequence

Specific examples of the primer set (a1) include a forward primer 5′-CATCATCGAGACCCTGAGGAAG-3 ′ (SEQ ID NO: 1) and reverse primer 5′-TGTGGGGGAGACTGTACTGG-3 ′ (SEQ ID NO: 2). . Similarly, as a specific example of the primer set (a2) above, a set of forward primer 5′-GGCGCTATGGGTCAACTGTC-3 ′ (SEQ ID NO: 3) and reverse primer 5′-CGTCCTTGAGGTACCATCGG-3 ′ (SEQ ID NO: 4) is Specific examples of the primer set of a3) include a set of forward primer 5′-GCCCGTGTCCAATCAGAGC-3 ′ (SEQ ID NO: 5) and reverse primer 5′-CCGGGCATTGTAGATTGTGC-3 ′ (SEQ ID NO: 6).

Another embodiment of the primer set targets genomic DNA, and the MEF2D gene has a breakpoint in

intron

6 or 7 and the BCL9 gene has a breakpoint in exon 8 or intron 9. Designed to specifically amplify the fusion gene of MEF2D gene and BCL9 gene. Preferable specific examples include the following (b1) and (b2). These primer sets are suitable for PCR, but are not limited to use in other nucleic acid amplification reactions.
(b1) A part of the exon 2 region of the MEF2D gene that is complementary to a portion consisting of 13 bases or more and a part of the exon 10 region of the BCL9 gene that is a portion of 13 bases or more (B2) A forward primer consisting of a sequence complementary to a part of the exon 5 region of the MEF2D gene that is complementary to a part consisting of 13 or more bases, and exon 10 of the BCL9 gene Primer set consisting of a reverse primer consisting of a sequence complementary to a part of the region of 13 and more than 13 bases

Specific examples of the primer set (b1) include a forward primer 5′-AGGCTGTGCAGAAGGTATCC-3 ′ (SEQ ID NO: 7) and reverse primer 5′-GTGCAACACATGACCGATGG-3 ′ (SEQ ID NO: 8). Similarly, as a specific example of the primer set (b2) above, a set of forward primer 5′-TTCTGTGGGCCAGAAATGGA-3 ′ (SEQ ID NO: 9) and reverse primer 5′-GGGACCCCATGAGGAGGTAT-3 ′ (SEQ ID NO: 10) can be mentioned. it can.

The length of each primer is usually 13 bases or more (upper limit is, for example, 40 bases), but preferably 15 bases to 30 bases, more preferably 18 bases to 26 bases in consideration of specificity, efficiency of amplification reaction, and the like. The base is more preferably 20 to 24 bases. The length of the DNA fragment amplified by the primer set is, for example, 200 to 2000 bases long, preferably 400 to 1500 bases long when cDNA is a target, and 500 to 500 bases long when genomic DNA is a target. The length is 12000 bases, preferably 800 to 10000 bases. Each primer sequence is complementary to the target (template) sequence, but only slightly between the primer sequence and the target sequence as long as specific hybridization occurs and the desired DNA fragment is specifically amplified. There may be minor mismatches. The degree of mismatch is 1 to several, preferably 1 to 3, and more preferably 1 to 2.

The primer can be previously labeled with a labeling substance. By using such a labeled primer, for example, detection using the labeling amount of the amplification product as an index becomes possible. 7-AAD, Alexa Fluor (registered trademark) 488, Alexa Fluor (registered trademark) 350, Alexa Fluor (registered trademark) 546, Alexa Fluor (registered trademark) 555, Alexa Fluor (registered) trademark) 568, Alexa Fluor (registered trademark) 594, Alexa Fluor (registered trademark) 633, Alexa Fluor (registered ^{trademark) 647, Cy TM 2, DsRED} , EGFP, EYFP, FITC, PerCP TM, R-Phycoerythrin, Propidium Iodide, AMCA, DAPI, ECFP, MethylCoumarin, Allophycocyanin (APC), Cy ^TM 3, Cy ^TM 5, Rhodamine-123, Tetramethylrhodamine, Texas Red (Texas Red (registered trademark)), PE, PE-Cy ^TM 5, PE-Cy ^TM 5.5, PE-Cy ^TM 7, APC-Cy ^TM 7, Oregon Green, carboxyfluorescein, carboxyfluorescein diacetate, fluorescent dyes such as quantum dots, radioisotopes such as ³² P, ¹³¹ I, ¹²⁵ I, Biotin can be exemplified, and alkaline phosphor as a labeling method. 5 'end labeling method using tase and T4 polynucleotide kinase, 3' end labeling method using T4 DNA polymerase or Klenow fragment, nick translation method, random primer method (Molecular Cloning, Third Edition, Chapter 9, Cold Spring Harbor Laboratory Press, New York).

Various software can be used for designing the primer set of the present invention. Examples of the primer design software include Primer 3, OLIGO Primer Analysis Software, Primer-BLAST, and the like. The primer can be synthesized by a known method such as a phosphodiester method.

<Detection kit>
The kit of the present invention makes it possible to perform the detection method of the present invention simply and efficiently. The detection kit of the present invention contains the primer set of the present invention as an essential element. In the case of a detection kit that targets cDNA, the primer set (a) and the primer set (b) are preferably included. Similarly, the detection kit targeting genomic DNA preferably includes the primer set (A) and the primer set (B). According to these detection kits, the coverage of cases of genetic abnormalities according to the present invention (that is, the number of cases detected as having genetic abnormalities) is increased, and the test results are more useful. .

The kit of the present invention may contain other elements. Examples of other elements are instructions on the use of primer sets, various reagents (DNA polymerase, restriction enzymes, buffers, etc.), solvents, standard specimens, reaction vessels, and other instruments. Further, it may include guidelines and explanations for determining a treatment policy.

4). Drug screening method The present invention further shows that ALL cells introduced with the MEF2D-BCL9 fusion gene expressed HDAC9 as well as MEF2D-BCL9-positive patient-derived cells and increased their proliferation rate (Examples described later). And the fact that a drug susceptibility evaluation system could be constructed using MEF2D-BCL9 positive patient-derived cells (see the Example section below), the fusion gene of MEF2D gene and BCL9 gene Provided is a screening method for substances effective in the treatment of patients with ALL who form.

In the screening method of the present invention, the following steps (i) to (iii) are performed.
(i) a step of preparing a cell expressing a fusion gene of MEF2D gene and BCL9 gene (ii) a step of culturing the cell in the presence of the test substance (iii) measuring the number of viable cells, and Step of determining effectiveness

In the screening method of the present invention, first, cells that express a fusion gene of MEF2D gene and BCL9 gene are prepared (step (i)). In other words, a MEF2D-BCL9 fusion gene positive cell is prepared. For example, an ALL cell line (for example, NALM-6, BALL-1, CCRF-CEM, Jurkat, CPT-K5) in which the MEF2D-BCL9 fusion gene is introduced and forcibly expressed is used as the cell here can do. It is also possible to use cells isolated from MEF2D-BCL9 fusion gene-positive patients, passage cells of the cells, or cell lines established from the cells.

In step (ii), the prepared cells are cultured in the presence of the test substance. The number of cells to be used is not particularly limited and can be determined in consideration of detection sensitivity, experimental equipment, and the like. For example, 1 × 10 ² to 1 × 10 ⁶ cells can be used in one screening operation.

The abundance (addition amount) of the test substance in the culture solution can be arbitrarily set, but the addition amount may be set within a range that does not have a fatal effect when normal cells are cultured under the same conditions. A person skilled in the art can set an appropriate addition amount by preliminary experiments.

The incubation time is set so that the action / effect of the test substance can be sufficiently evaluated, but is not particularly limited. For example, the culture time can be set within a range of 10 minutes to 1 month. When a substance exhibiting the desired action / effect is found, the culture time in the subsequent screening can be set based on the time required for the substance to show the action / effect.

As the test substance, organic compounds or inorganic compounds of various molecular sizes can be used. Examples of organic compounds include nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycosylglycerides, cerebrosides, etc.), prostaglandins, isoprenoids, terpenes, steroids, polyphenols, catechins, and vitamins. The test substance may be derived from a natural product or synthesized, and in the latter case, an efficient screening system can be constructed by using, for example, a combinatorial synthesis technique. In addition, cell extracts, culture supernatants, etc. may be used as test substances, or existing drugs may be used as test substances, by adding two or more kinds of test substances at the same time, You may investigate synergistic action etc.

In step (iii) following step (ii), the number of viable cells after culturing is measured, and the cell growth inhibitory activity (cytotoxic activity) of the test substance, that is, the effectiveness is determined. For example, cells cultured in the presence of the test substance (test group) and cells cultured in the absence of the test substance (control group) are prepared, and the number of viable cells is measured and compared for each group. From the comparison results, the degree to which the cell viability has changed as a result of the presence of the test substance is determined. When the number of living cells in the test group is small compared to the control group (cell viability is low), that is, when the test substance shows cell growth inhibitory activity, the test substance exhibits the genetic abnormality of the present invention. It can be determined that it is effective for the case it has. When a significant decrease in the survival rate is observed in the test group, it can be determined that the effectiveness of the test substance is particularly high. Rather than setting a control group, it is also possible to determine the effectiveness of a test substance by comparing the number of cells before and after culturing in a test group. However, more reliable results can be obtained by setting the control group as described above.

The substance (screening result) selected by the screening method of the present invention is a promising candidate (lead compound) as an active ingredient of a medicine for ALL cases having a genetic abnormality of the present invention. When the selected substance has a sufficient medicinal effect, it can be used as an active ingredient of a medicine as it is. On the other hand, even if it does not have a sufficient medicinal effect, it can be used as an active ingredient of a medicine after it has been modified by chemical modification to enhance its medicinal effect. Of course, even if it has a sufficient medicinal effect, the same modification may be applied for the purpose of further increasing the medicinal effect.

5). Fusion gene, fusion protein The present invention further provides a MEF2D-BCL9 fusion gene and a MEF2D-BCL9 fusion protein that define the genetic abnormality of the present invention. The MEF2D-BCL9 fusion gene is caused by a chromosomal inversion in which a breakpoint exists in

intron

6 or 7 in the MEF2D gene and a breakpoint exists in exon 8 or intron 9 in the BCL9 gene. The MEF2D-BCL9 fusion protein is an expression product of the fusion gene. MEF2D-BCL9 fusion gene and MEF2D-BCL9 fusion protein can define a new group of ALL, become an index when detecting the group (for example, become a detection target of the test method of the present invention), treatment It is useful in that it can be a target of the above. In addition, according to the knowledge obtained by the study of the present inventors (the column of Examples described later), it is suggested that the genetic abnormality of the present invention causes the onset of ALL.

Typically, the MEF2D-BCL9 fusion gene contains exons 1 to 6 of the MEF2D gene and exon 10 of the BCL9 gene or a part thereof. A specific example of the sequence of exons 1 to 6 of the MEF2D gene is shown in SEQ ID NO: 11. On the other hand, a specific example of the sequence of exon 10 of the BCL9 gene is shown in SEQ ID NO: 12.

MEF2D-BCL9 fusion protein is encoded by the MEF2D-BCL9 fusion gene. Accordingly, the amino acid sequence (SEQ ID NO: 13) corresponding to exons 1 to 6 of the MEF2D gene and the amino acid sequence (SEQ ID NO: 14) corresponding to exon 10 of the BCL9 gene or a part thereof are included. The amino acid sequences of specific examples of the MEF2D-BCL9 fusion protein are shown in SEQ ID NOs: 15-20.

The MEF2D-BCL9 fusion gene and MEF2D-BCL9 fusion protein can be prepared in an isolated state, for example, by separating and purifying from a patient having a genetic abnormality of the present invention. Moreover, based on the sequence information disclosed in the present specification, it may be prepared by chemical synthesis, genetic engineering techniques, or the like.

The present invention further provides cDNA complementary to mRNA which is a transcription product of the MEF2D-BCL9 fusion gene. The cDNA is particularly useful in that it serves as an index for detecting a new group of ALL (for example, a detection target of the test method of the present invention).

The cDNA of the present invention includes the sequence of exons 1 to 6 (SEQ ID NO: 21) of the MEF2D gene and the sequence of exon 10 (SEQ ID NO: 22) of the BCL9 gene or a part thereof. Specific examples of the cDNA of the present invention are shown in SEQ ID NOs: 23 to 28.

The cDNA of the present invention can be prepared by a conventional method. For example, various reagents and kits for preparing cDNA are commercially available, and the cDNA of the present invention can be easily prepared by using them.

6). Antibodies Recognizing Fusion Proteins The present invention further provides antibodies that recognize MEF2D-BCL9 fusion proteins. The antibody of the present invention is useful for detecting, for example, MEF2D-BCL9 fusion protein. Therefore, it can be used for the inspection method of the present invention.

The antibody of the present invention can be prepared using an immunological technique, a phage display method, a ribosome display method, or the like. Preparation of a polyclonal antibody by an immunological technique can be performed by the following procedure. An antigen (MEF2D-BCL9 fusion protein or a part thereof (including a fusion site)) is prepared and used to immunize animals such as rabbits. An antigen can be obtained by purifying a biological sample. A recombinant antigen can also be used. The recombinant antigen can be expressed, for example, by introducing a gene encoding the MEF2D-BCL9 fusion protein (ie, MEF2D-BCL9 fusion gene) into a suitable host using a vector, and expressing it in the resulting recombinant cell. Can be prepared.

In order to enhance the immunity-inducing action, an antigen to which a carrier protein is bound may be used. As the carrier protein, KLH (KeyholeHLimpet） Hemocyanin), BSA (Bovine Serum Albumin), OVA (Ovalbumin) and the like are used. The carbodiimide method, the glutaraldehyde method, the diazo condensation method, the MBS (maleimidobenzoyloxysuccinimide) method, etc. can be used for the coupling | bonding of carrier protein. On the other hand, an antigen in which MEF2D-BCL9 fusion protein (or part thereof) is expressed as a fusion protein with GST, β-galactosidase, maltose-binding protein, histidine (His) tag or the like can also be used. Such a fusion protein can be easily purified by a general method.

Immunization is repeated as necessary, and blood is collected when the antibody titer has sufficiently increased, and serum is obtained by centrifugation or the like. The obtained antiserum is affinity purified to obtain a polyclonal antibody.

On the other hand, monoclonal antibodies can be prepared by the following procedure. First, an immunization operation is performed in the same procedure as described above. Immunization is repeated as necessary, and antibody-producing cells are removed from the immunized animal when the antibody titer sufficiently increases. Next, the obtained antibody-producing cells and myeloma cells are fused to obtain a hybridoma. Subsequently, after this hybridoma is monoclonalized, a clone that produces an antibody having high specificity for the target protein (ie, MEF2D-BCL9 fusion protein) is selected. The target antibody can be obtained by purifying the culture medium of the selected clone. On the other hand, the desired antibody can be obtained by growing the hybridoma to a desired number or more, then transplanting it into the abdominal cavity of an animal (for example, a mouse), growing it in ascites, and purifying the ascites. For purification of the culture medium or ascites, affinity chromatography using protein G, protein A or the like is preferably used. Alternatively, affinity chromatography in which an antigen is immobilized may be used. Furthermore, methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods can be used alone or in any combination.

Various modifications can be made to the obtained antibody on condition that the specific binding property to the MEF2D-BCL9 fusion protein is maintained. It may also be labeled. Examples of labeling substances that can be used for labeling include fluorescent dyes such as fluorescein, rhodamine, Texas red, oregon green, enzymes such as horseradish peroxidase, microperoxidase, alkaline phosphatase, β-D-galactosidase, luminol, acridine Chemical or bioluminescent compounds such as dyes, various radioisotopes, biotin.

The antibodies of the present invention include antibodies from non-human animals such as mice and rats, chimeric antibodies, humanized antibodies, and human antibodies in which a part of the region has been replaced with those from other animals (including humans). The class of the antibody is not particularly limited. For example, antibodies of the IgG class (for example, those belonging to human antibody subclasses IgG1, IgG2, IgG3, and IgG4).

Note that matters not specifically mentioned in the present specification (conditions, operation methods, etc.) may follow conventional methods, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York), Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987), Current protocols in Immunology, John Wiley & Sons Inc.

1. Discovery of MEF2D-BCL9 fusion gene in pediatric ALL (1) Method 59 patients with recurrent pediatric ALL (including 3 infant ALL, 6 Ph1-positive ALL, 4 T-cell ALL, 3 mature B-cell ALL) RNA was extracted from bone marrow containing leukemia cells using RNeasy (registered trademark) Mini Kit (Qiagen). After confirming the degree of RNA degradation using Agilent 2200 TapeStation and RNA ScreenTape (Agilent), a library for RNA sequencing analysis using NEBNext (registered trademark) Ultra RNA Prep Kit for Illumina (New England Biolabs) Created. Next generation sequencing was performed using HiSeq ^™ 2500 (Illumina), and the obtained sequence data was analyzed using Tophat-fusion software to detect the fusion gene.

(2) Results MEF2D-BCL9 fusion gene was detected in 4 cases. The detected fusion gene was fused in-frame, suggesting the possibility of producing a functional protein. MEF2D and BCL9 are both located on the long arm of chromosome 1, and the distance between these two genes is about 9 Mb (FIG. 1A). A partial inversion of this site forms a MEF2D-BCL9 fusion gene. This chromosomal abnormality was not detected by G-band staining.

(3) Discussion In all 4 cases, genomic breakpoints were identified by direct sequencing. A breakpoint existed in

intron

6 or 7 on the MEF2D side and in exon 8 or intron 9 on the BCL9 side (an example is shown in FIG. 1B). The MEF2D-BCL9 fusion gene has been observed in 4 patients, suggesting that it is involved in the recurrence of ALL.

2. Detection of fusion gene mRNA by RT-PCR (1) Method For patients with MEF2D-BCL9 detected, cDNA extracted from bone marrow containing leukemia cells as a template using ThermoScript ^™ RT-PCR system (Thermo) Was synthesized. RT-PCR was performed using the following primer set corresponding to the sequences of MEF2D and BCL9 and PrimeSTAR (registered trademark) GXL DNA polymerase. The PCR product was subjected to direct sequence analysis using BigDye (registered trademark) Terminater 3.1 (Life Technologies).

<Primer set 1>
Forward primer (region spanning exons 3 to 4 of MEF2D): 5'-CATCATCGAGACCCTGAGGAAG-3 '(SEQ ID NO: 1)
Reverse primer (exon 10 region of BCL9): 5'-TGTGGGGGAGACTGTACTGG-3 '(SEQ ID NO: 2)
<Primer set 2>
Forward primer (region spanning exons 5-6 of MEF2D): 5'-GGCGCTATGGGTCAACTGTC-3 '(SEQ ID NO: 3)
Reverse primer (exon 10 region of BCL9): 5'-CGTCCTTGAGGTACCATCGG-3 '(SEQ ID NO: 4)
<Primer set 3>
Forward primer (region of exon 6 of MEF2D): 5'-GCCCGTGTCCAATCAGAGC-3 '(SEQ ID NO: 5)
Reverse primer (exon 10 region of BCL9): 5'-CCGGGCATTGTAGATTGTGC-3 '(SEQ ID NO: 6)

(2) Results The results for case 1 are shown in the upper part of FIG. A PCR product of 918 bp was confirmed for both the specimen at the time of diagnosis and the specimen at the time of recurrence (using primer set 3). No PCR product was confirmed for cDNA from normal bone marrow specimens. It was confirmed by direct sequencing that MEF2D exon 7 and BCL9 exon 10 were joined. The results for case 4 are shown in the lower part of FIG. A PCR product of 1,353 bp was confirmed in the specimen at the time of recurrence (using primer set 3), but no specimen in remission was observed. The PCR product was directly sequenced and confirmed to be a product of exon 7 of MEF2D and exon 9 of BCL9.

(3) Discussion The presence of the MEF2D-BCL9 fusion gene detected by RNA sequencing was also confirmed by RT-PCR. The specificity of the RT-PCR method could be confirmed by using healthy subjects as controls. In addition, MEF2D-BCL9 in 115 cases of first acute leukemia (100 cases of B progenitor ALL, 4 cases of T cell ALL, 10 cases of acute myeloid leukemia, 1 case of mixed plasma acute leukemia, 1 case of acute mixed leukemia) Although the fusion gene was screened, no new cases of MEF2D-BCL9 fusion gene were detected.

3. Detection of breakpoints in genomic DNA (1) Method For patients with MEF2D-BCL9 detected, using the DNA extracted from bone marrow containing leukemia cells as a template, the following primer set corresponding to the sequences of MEF2D and BCL9 and PrimeSTAR (registered) (Trademark) PCR was performed using GXL DNA polymerase. The PCR product was subjected to direct sequence analysis using BigDye (registered trademark) Terminater 3.1 (Life Technologies).

<Primer set 4>
Forward primer (region of exon 2 of MEF2D): 5'-AGGCTGTGCAGAAGGTATCC-3 '(SEQ ID NO: 7)
Reverse primer (exon 10 region of BCL9): 5'-GTGCAACACATGACCGATGG-3 '(SEQ ID NO: 8)
<Primer set 5>
Forward primer (region of exon 5 of MEF2D): 5'-TTCTGTGGGCCAGAAATGGA-3 '(SEQ ID NO: 9)
Reverse primer (exon 10 region of BCL9): 5'-GGGACCCCATGAGGAGGTAT-3 '(SEQ ID NO: 10)

(2) Results The results of PCR using primer set 4 are shown in the upper left of FIG. Positive bands were confirmed for

cases

1 and 3. The results of PCR using primer set 5 are shown in the upper right of FIG. Positive bands were confirmed for

cases

2, 3, and 4. The results of direct sequencing each PCR product and determining the genomic breakpoint in each case are shown in the lower part of FIG. Case 1 was MEF2D intron 7 and BCL9 intron 9, and Case 2 was MEF2D intron 6 and BCL9 intron 9, confirming that the chromosomes were joined. For

cases

3 and 4, the insertion sequence of unknown origin is the junction site (in case 3, MEF2D intron 7 and BCL9 intron 9 are joined via the insertion sequence, and in case 4 MEF2D intron 7 and BCL9 exon 8 are located via the insertion sequence. It was confirmed that this occurred in the bonding).

(3) Discussion The presence of the MEF2D-BCL9 fusion gene detected by RNA sequencing was supported by the presence of a breakpoint in the genome. It was also confirmed that this fusion gene could be detected by PCR using the genome as a template. In addition, MEF2D-BCL9 in 115 cases of first acute leukemia (100 cases of B progenitor ALL, 4 cases of T cell ALL, 10 cases of acute myeloid leukemia, 1 case of mixed plasma acute leukemia, 1 case of acute mixed leukemia) Although the fusion gene was screened, no new cases of MEF2D-BCL9 fusion gene were detected.

4). Expression profile analysis (1) Method Using the Tophat software and Cufflinks software, FPKM (Fragments per kilobase of exon per million mapped sequence reads) value is calculated from the sequence data obtained by RNA sequence analysis. did. Samples were clustered based on Spearman's correlation coefficient.

(2) Results It was confirmed that samples derived from cases having the MEF2D-BCL9 fusion gene accumulate in small clusters (FIG. 4).

(3) Discussion It is thought that the MEFD-BCL9 fusion gene has a major effect on the expression profile of leukemia cells and a similar expression profile has been constructed in 4 cases. The fact that similar expression profiles were observed suggests that the leukemia cells in these cases have similar biological properties. That is, it is suggested that similar characteristics such as drug sensitivity exist for these cases. In addition, 4 cases of MEF2D-BCL9 positive were B precursor progenitor immunophenotype (CD19 positive, CD20 negative, CD3 negative, CD13 negative, CD33 negative), relatively older age of 10 years or older, during treatment They also had common clinical features in terms of recurrence and poor prognosis (all deaths). Four cases of MEF2D-BCL9 positive leukemia cells are typical in terms of (i) medium to large blasts, (ii) strong basophil cytoplasm, and (iii) marked vacuolation. It had morphological characteristics different from those of B precursor cells. These findings are similar to those of mature B-cell leukemia, but are differentiated based on the presence of cell surface markers such as CD20 and immunoglobulin, and the presence or absence of an IGH-MYC fusion gene.

5). Functional analysis of MEF2D-BCL9 fusion gene (1) Method The expression level of HDAC9 in MEF2D-BCL9 positive and negative cases was determined using Taqman (registered trademark) Gene Expression Assay (Cat no # 4331182, Applied Biosystem) and ABI Prism (registered) Measurement was performed by quantitative RT-PCR using a trademark 7000 (Life Technologies). In addition, NALM-6, which is an ALL cell line, and NALM-6 in which MEF2D-BCL9 fusion gene was forcibly expressed using a lentivirus were cultured, and the expression level of HDAC9 was similarly measured. For measurement, BD FACSCalibur ^™ (BD) and CountBright ^™ Absolute Counting Beads (Life Technologies) were used.

(2) Results In the MEF2D-BCL9 positive case, the HDAC9 expression level was higher than the negative case with a statistically significant difference (FIG. 5A). In addition, when the MEF2D-BCL9 fusion gene was introduced into NALM-6, the expression level of HDAC9 increased (FIG. 5B), and the cell growth rate also increased (FIG. 5C).

(3) Discussion It was shown that high expression of HDAC9 in MEF2D-BCL9 positive ALL was the result of expression of the MEF2D-BCL9 fusion gene. It was also shown that the MEF2D-BCL9 fusion gene promotes cell proliferation. These results suggest that the MEF2D-BCL9 fusion gene functions as a driver gene in leukemia cells.

6). Effect of molecular target drug (1) Method In vitro drug susceptibility test against vorinostat, quisinostat, and bortezomib using primary cultured cells established from MEF2D-BCL9 positive patient leukemia cells went. Place 2.5 × 10 ⁴ leukemia cells in 200 μL of culture solution set to each drug concentration, incubate for 24 or 48 hours, stain with Annevin V-FITC and 7-AAD, and measure the number of viable cells with a flow cytometer did. The results are shown as a percentage (%) relative to DMSO control.

(2) Results (Fig. 6)
All of vorinostat (A), xinostat (B), and bortezomib (C) showed anti-leukemic cell action at a concentration considered to be a therapeutic concentration range. The 50% growth inhibitory concentration (IC50) for each drug was 1.91 μM for vorinostat, 43.3 nM for xinostat, and 27.1 nM for bortezomib.

(3) Discussion It was suggested that vorinostat and xinostat, which are HDAC inhibitors, may be effective in treating MEF2D-BCL9 positive ALL. In recent years, bortezomib, a proteasome inhibitor that is expected to be effective in combination with other drugs against relapsed and refractory ALL, also showed cell growth inhibitory activity in vitro. This was consistent with clinical experience that one of four MEF2D-BCL9 positive patients had relapse treatment and a third response was obtained with bortezomib combination chemotherapy.

Â € ¢ We succeeded in identifying a new fusion gene (MEF2D-BCL9 fusion gene) useful for predicting the prognosis of ALL and deciding treatment policy. In treating ALL, cases are stratified by predicting prognosis using various indicators, and the optimal treatment strategy is determined for each case. The present invention provides a new index useful for stratification of cases, and contributes to optimization of ALL treatment policy and maximization of therapeutic effect. The MEF2D-BCL9 fusion gene is also useful as a target for molecular targeted therapy. Therefore, the present invention is also expected to contribute to the establishment of a new treatment strategy for ALL.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

SEQ ID NO: 1: description of artificial sequence: forward primer SEQ ID NO: 2: description of artificial sequence: reverse primer SEQ ID NO: 3: description of artificial sequence: forward primer SEQ ID NO: 4: description of artificial sequence: reverse primer SEQ ID NO: 5: artificial sequence Description: forward primer SEQ ID NO: 6: description of artificial sequence: reverse primer SEQ ID NO: 7: description of artificial sequence: forward primer SEQ ID NO: 8: description of artificial sequence: reverse primer SEQ ID NO: 9: description of artificial sequence: forward primer sequence Number 10: description of artificial sequence: reverse primer SEQ ID NO: 21: description of artificial sequence: cDNA of

exons

1, 2, 3, 4, 5, 6 of MEF2D gene
SEQ ID NO: 22: Description of artificial sequence: cDNA, exon 10 of BCL9 gene, exon 10 cDNA
SEQ ID NO: 23: Description of artificial sequence: cDNA encoding MEF2D-BCL9 fusion protein
SEQ ID NO: 24: Description of artificial sequence: cDNA encoding MEF2D-BCL9 fusion protein
SEQ ID NO: 25 Description of artificial sequence: cDNA encoding MEF2D-BCL9 fusion protein
SEQ ID NO: 26: Description of artificial sequence: cDNA encoding MEF2D-BCL9 fusion protein
SEQ ID NO: 27 Description of artificial sequence: cDNA encoding MEF2D-BCL9 fusion protein
SEQ ID NO: 28: Description of artificial sequence: cDNA encoding MEF2D-BCL9 fusion protein

Claims

Method for testing acute lymphocytic leukemia, including the following steps (1) to (3):
(1) preparing a specimen containing leukemia cells isolated from a patient with acute lymphoblastic leukemia;
(2) detecting the presence or absence of a fusion gene of the MEF2D gene and the BCL9 gene or a fusion protein encoded by the fusion gene in the specimen;
(3) A step of determining that the prognosis is poor or difficult to treat when the fusion gene or the fusion protein is detected.
The test method according to claim 1, wherein the fusion gene is formed by chromosomal inversion in which a breakpoint is present in intron 6 or 7 in the MEF2D gene and a breakpoint is present in exon 8 or intron 9 in the BCL9 gene.
The detection of the fusion gene in step (2) is selected from the group consisting of RT-PCR method, PCR method, PCR-RFLP, PCR-SSCP method, RNA sequence analysis, target sequence analysis, FISH method and whole genome analysis The test method according to claim 1 or 2, wherein the detection is performed by any detection method, and the detection of the fusion protein in step (2) is performed by an immunological measurement method.
The test method according to any one of claims 1 to 3, wherein the patient with acute lymphoblastic leukemia is a child.
The inspection method according to any one of claims 1 to 4, further comprising the following step (4) or (4 '):
(4) identifying a risk group to which the acute lymphoblastic leukemia patient belongs based on the determination in the step (3), and determining or changing a treatment policy;
(4 ′) A step of identifying a risk group to which the patient with acute lymphocytic leukemia belongs and determining or changing a treatment policy based on the determination in step (3) and the results of other tests.
The inspection method according to claim 5, wherein the risk group is a high risk group.
The inspection method according to claim 5 or 6, wherein the treatment policy after the change in step (4) or (4 ') includes a therapy that is strengthened than before the change.
The therapeutic policy determined or changed in the step (4) or (4 ') includes treatment by administration of a histone deacetylase inhibitor and / or a proteasome inhibitor. Inspection method.
The test method according to claim 8, wherein the histone deacetylase inhibitor is a histone deacetylase 9 inhibitor.
The test method according to claim 5 or 6, wherein the treatment policy determined or changed in step (4) or (4 ') includes indication of hematopoietic stem cell transplantation.
The test method according to claim 10, wherein the hematopoietic stem cell transplantation is performed in a first remission period.
A method for treating acute lymphoblastic leukemia, comprising treating the patient with acute lymphoblastic leukemia according to a treatment policy determined or changed by the testing method according to any one of claims 5 to 11.
A pharmaceutical for treating patients with acute lymphocytic leukemia characterized by the formation of a fusion gene between the MEF2D gene and the BCL9 gene, including a histone deacetylase inhibitor and / or a proteasome inhibitor.
The medicament according to claim 13, wherein the histone deacetylase inhibitor is a histone deacetylase 9 inhibitor.
Including therapeutically effective doses of drugs containing histone deacetylase inhibitors and / or proteasome inhibitors for patients with acute lymphocytic leukemia characterized by the formation of a fusion gene between the MEF2D gene and the BCL9 gene How to treat acute lymphoblastic leukemia.
The treatment method according to claim 15, wherein the histone deacetylase inhibitor is a histone deacetylase 9 inhibitor.
The transcription product of the fusion gene of MEF2D gene and BCL9 gene formed by chromosomal inversion in which breakpoint exists in intron 6 or 7 in MEF2D gene and breakpoint exists in exon 8 or intron 9 in BCL9 gene A primer set consisting of forward and reverse primers designed to specifically amplify DNA complementary to mRNA.
In the MEF2D gene, a breakpoint exists in intron 6 or 7; in the BCL9 gene, a fusion gene between the MEF2D gene and the BCL9 gene, which is formed by a chromosomal inversion with a breakpoint in exon 8 or intron 9, is specifically amplified. Primer set consisting of forward primer and reverse primer designed to be able to.
A kit for detecting a genetic abnormality characterized by formation of a fusion gene of MEF2D gene and BCL9 gene, comprising the primer set according to claim 17 or 18.
The detection kit according to claim 19, comprising the following primer set as the primer set:
A forward primer consisting of a sequence complementary to a part of exon 1 to 6 of the MEF2D gene and consisting of 13 bases or more, and a part of the exon 10 region of the BCL9 gene consisting of 13 bases or more Primer set consisting of reverse primers consisting of complementary sequences
The detection kit according to claim 19, comprising one or more of the following (a1) to (a3) as the primer set:
(a1) Forward primer consisting of a sequence complementary to a part of the exon 3 to 4 region of the MEF2D gene and 13 bases or more, and a part of the exon 10 region of the BCL9 gene from 13 bases or more A primer set consisting of a reverse primer consisting of a sequence complementary to
(a2) Forward primer consisting of a sequence complementary to a part of exon 5 to 6 of the MEF2D gene and consisting of 13 bases or more, and part of the exon 10 region of the BCL9 gene from 13 bases or more A primer set consisting of a reverse primer consisting of a sequence complementary to
(A3) A part of the exon 6 region of the MEF2D gene that is complementary to a portion consisting of 13 bases or more and a part of the exon 10 region of the BCL9 gene that is a portion of 13 bases or more A primer set consisting of a reverse primer consisting of a sequence complementary to.
The detection kit according to claim 19, comprising the following (b1) and / or (b2) as the primer set:
(b1) A part of the exon 2 region of the MEF2D gene that is complementary to a portion consisting of 13 bases or more and a part of the exon 10 region of the BCL9 gene that is a portion of 13 bases or more A primer set consisting of a reverse primer consisting of a sequence complementary to
(b2) A part of the exon 5 region of the MEF2D gene that is complementary to the portion consisting of 13 bases or more and a part of the exon 10 region of the BCL9 gene that is a portion of 13 bases or more A primer set consisting of a reverse primer consisting of a sequence complementary to.
A method for screening a substance effective for the treatment of patients with acute lymphocytic leukemia characterized by formation of a fusion gene of MEF2D gene and BCL9 gene, comprising the following steps (i) to (iii):
(i) providing a cell expressing a fusion gene of MEF2D gene and BCL9 gene;
(ii) culturing the cell in the presence of a test substance;
(iii) measuring the number of viable cells and determining the effectiveness of the test substance.
Fusion gene of MEF2D gene and BCL9 gene.
The fusion gene according to claim 24, which is generated by a chromosomal inversion in which a breakpoint is present in intron 6 or 7 in the MEF2D gene and a breakpoint is present in exon 8 or intron 9 in the BCL9 gene.
The fusion gene according to claim 25, comprising exons 1 to 6 of the MEF2D gene and exon 10 of the BCL9 gene or a part thereof.
The fusion gene according to claim 26, comprising the sequence of SEQ ID NO: 11 and the sequence of SEQ ID NO: 12.
A fusion protein encoded by the fusion gene according to any one of claims 24 to 27.
The fusion protein according to claim 28, comprising the sequence of SEQ ID NO: 13 and the sequence of SEQ ID NO: 14.
A DNA complementary to mRNA which is a transcription product of the fusion gene according to any one of claims 24 to 27.
The DNA according to claim 30, comprising the sequence of SEQ ID NO: 15 and the sequence of SEQ ID NO: 16.
An antibody that recognizes the fusion protein encoded by the fusion gene of MEF2D gene and BCL9 gene.
The antibody according to claim 32, wherein the fusion protein is a fusion protein as defined in claim 28 or 29.