WO2021149752A1

WO2021149752A1 - Method for determining prognosis of glioma

Info

Publication number: WO2021149752A1
Application number: PCT/JP2021/001970
Authority: WO
Inventors: 邦夫三宅; 岳史犬飼; 渡邊　敦; 博之木内; 智之川瀧; 光人埴原; 有理子根本
Original assignee: 国立大学法人山梨大学; 積水メディカル株式会社
Priority date: 2020-01-21
Filing date: 2021-01-21
Publication date: 2021-07-29
Also published as: JPWO2021149752A1

Abstract

Provided is a method for determining the prognosis of a glioma. In the present invention, the level of DNA methylation of a target DNA region included in the promoter region of the MGMT gene in the genome DNA derived from glioma cells or a tissue including the cells is measured. When the level of DNA methylation is a predetermined reference level or more, the prognosis of the glioma is determined as good. When the level is less than the reference level, the prognosis of the glioma is determined as poor.

Description

Prognosis determination method for glioma

The present invention relates to a method for determining the prognosis of glioma.

Glioma is a brain tumor that develops from glial cells. Gliomas are broadly divided into astrocytomas and oligodendrocytes, and about 85% of gliomas are astrocytomas. Brain tumors are classified into grades 1 to 4 according to their malignancy. Grade 1 is a benign tumor such as pilocytic astrocytoma that occurs frequently in children. Grade 2 is diffuse astrocytoma and oligodendroglioma, and when they become more malignant, they become grade 3 anaplastic astrocytoma or anaplastic oligodendroglioma. Grade 4 is glioblastoma, the most malignant. Grades 3 and 4 are sometimes collectively referred to as malignant glioma. Glioblastoma accounts for about 10% of intracranial tumors, most of which occur in adults and have a very poor prognosis.

Previously, postoperative radiotherapy was used to treat glioblastoma, but in recent years, it has been found to be effective in combination with postoperative drug therapy (temozolomide) radiotherapy, which is now in Japan. It is used as standard treatment. On the other hand, it has been reported that the effect of temozolomide depends on the expression of the DNA repair enzyme O6-methyl-guanine-DNA-methyltransphase (MGMT) in tumor cells. Expression of MGMT in tumor cells is epigenetically regulated, and methylation of the MGMT gene promoter region reduces MGMT expression and increases susceptibility to temozolomide. On the other hand, patients without methylation of the MGMT gene promoter region are resistant to alkylating agents such as temozolomide because DNA methylation is rapidly repaired by MGMT expression. Methylation of the MGMT gene promoter region has been suggested to be a prognostic factor for glioma. Non-Patent Document 1 reports that methylation of the MGMT gene promoter region is associated with tumor shrinkage and prolongation of survival after chemotherapy. Non-Patent Document 2 states that in patients with methylation of the MGMT gene promoter region, temozolomide combination therapy statistically significantly prolonged survival compared to radiation monotherapy, but methylation of the MGMT gene promoter region was observed. It has been reported that there was no significant difference in survival between radiation monotherapy and temozolomide combination therapy in patients who did not. On the other hand, it has also been reported that a relationship between methylation of the MGMT gene promoter region and prognosis was observed even in the case of radiation monotherapy (Non-Patent Document 3).

As a method for analyzing methylated DNA, a method using the bisulfite method has already been established and is widely used. Examples of the methylated DNA analysis method based on the bisulfite method include a methylation-Special PCR (MSP) method, a Combined Bisulfite Restoration Analysis (COBRA) method, a BAC array-based methylated CpG island amplification method (BAMCA method), and the following. Sequencing by a generation sequencer (MethylCap-seq), a method of detecting a single-base extension reaction with a methylated / unmethylated DNA-specific probe (for example, Infinium® assay), methylation-sensitivity restoration enzyme measurement (MRE). -Seq) method is used. Patent Document 1 discloses a method for detecting DNA methylation levels based on the difference in retention time by ion exchange chromatography.

International Publication No. 2014/136930

The present invention provides a method for evaluating the prognosis of a tumor based on the methylation level of DNA in glioma.

By measuring the DNA methylation level of a specific region in the promoter region of the gene encoding O6-methyl-guanine-DNA-methyltransphase (MGMT) of glioma (MGMT gene), the present inventors have glioma. We found that the prognosis of tumors could be evaluated.

Therefore, the present invention provides the following.
[1] A method for determining cells or tissues derived from glioma with a good or poor prognosis.
1) To measure the DNA methylation level of the target DNA region contained in the promoter region of the gene encoding O6-methyl-guanine-DNA-methyltransferase in DNA derived from glioma cells or tissues containing the same.
Here, the target DNA region is at least one region selected from the group consisting of the following regions 1 and 2:
Region 1) A region consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity thereof,
Region 2) A region consisting of the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 90% identity thereof,
2) When the measured DNA methylation level is equal to or higher than the following criteria, the cell or tissue containing it is judged to be a glioma-derived cell or tissue with a good prognosis, or the measured DNA methylation level is When it is less than the following criteria, the cells or tissues containing them are judged to be glioma-derived cells or tissues having a poor prognosis:
Criteria for region 1 A value selected from the range of 10-40%,
Criteria for region 2 Value selected from the range of 5-30%,
Including methods.
[2] A method for determining the prognosis of glioma.
1) To measure the DNA methylation level of the target DNA region contained in the promoter region of the gene encoding O6-methyl-guanine-DNA-methyltransphase in DNA derived from the subject's glioma cells or tissues containing the same. ,
Here, the target DNA region is at least one region selected from the group consisting of the following regions 1 and 2:
Region 1) A region consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity thereof,
Region 2) A region consisting of the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 90% identity thereof,
2) When the measured DNA methylation level is equal to or higher than the following criteria, the subject's glioma is judged to have a good prognosis, or when the measured DNA methylation level is less than the following criteria, the subject is judged to have a good prognosis. Judging glioma as poor prognosis:
Criteria for region 1 A value selected from the range of 10-40%,
Criteria for region 2 Value selected from the range of 5-30%,
Including methods.
[3] A method for acquiring data to be used for determining cells or tissues derived from glioma having a good or poor prognosis, or for determining the prognosis of glioma.
1) To measure the DNA methylation level of the target DNA region contained in the promoter region of the gene encoding O6-methyl-guanine-DNA-methyltransphase in DNA derived from the subject's glioma cells or tissues containing the same. ,
Here, the target DNA region is at least one region selected from the group consisting of the following regions 1 and 2:
Region 1) A region consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity thereof,
Region 2) A region consisting of the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 90% identity thereof,
2) Obtaining data on whether or not the measured DNA methylation level is above the following criteria:
Criteria for region 1 A value selected from the range of 10-40%,
Criteria for region 2 Value selected from the range of 5-30%,
Including methods.
[4] The method according to any one of [1] to [3], wherein the promoter region consists of the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having at least 90% identity thereof.
[5] The item according to any one of [1] to [4], wherein the region 1 is a region consisting of the nucleotide sequence of SEQ ID NO: 1 and the region 2 is a region consisting of the nucleotide sequence of SEQ ID NO: 2. the method of.
[6] The measurement of the DNA methylation level comprises measuring the DNA methylation level of at least one region selected from the group consisting of the bisulfite-treated regions 1 and 2. [1] to [ 5] The method according to any one of paragraphs 1.
[7] The bisulfite-treated region 1 is a region sandwiched between a primer consisting of the nucleotide sequence of SEQ ID NO: 4 and a primer consisting of the nucleotide sequence of SEQ ID NO: 5 (however, the primer binding sequence is not included).
The bisulfite-treated region 2 is a region sandwiched between a primer consisting of the nucleotide sequence of SEQ ID NO: 6 and a primer consisting of the nucleotide sequence of SEQ ID NO: 7 (however, the primer binding sequence is not included).
[6] The method according to the above.
[8] The method according to [6] or [7], wherein the measurement of the DNA methylation level is performed using ion exchange chromatography.
[9] The method according to any one of [1] to [8], wherein the glioma is glioblastoma.
[10] A kit for carrying out the method according to any one of [1] to [9].
It comprises a set of PCR primers for PCR amplification of a target DNA region contained in the promoter region of a bisulfite-treated gene encoding O6-methyl-guanine-DNA-methyltransphase, wherein the bisulfite-treated prior to the bisulfite treatment. The target DNA region is at least one region selected from the group consisting of regions 1 and 2 below:
Region 1) A region consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity thereof,
Region 2) A region consisting of the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 90% identity thereof,
And,
A guide for determining whether or not the DNA methylation level of the target DNA region is equal to or higher than the following criteria is included.
Criteria for region 1 A value selected from the range of 10-40%,
Criteria for region 2 Value selected from the range of 5-30%,
kit.
[11] The PCR primer set is a primer set consisting of a primer consisting of the nucleotide sequence of SEQ ID NO: 4 and a primer consisting of the nucleotide sequence of SEQ ID NO: 5, or a primer consisting of the nucleotide sequence of SEQ ID NO: 6 and the nucleotide of SEQ ID NO: 7. The kit according to [10], which is a primer set with a primer consisting of a sequence.
[12] A system for carrying out the method according to any one of [1] to [9].
[13] The system according to [12], comprising an ion exchange chromatography apparatus.

The present invention provides a criterion for determining the prognosis of glioma, and a method for determining the prognosis of glioma using the criterion. According to the present invention, more reliable prognosis of glioma can be determined.

Correlation of glioma patients' hateless survival (PFS) and overall survival (OS) to DNA methylation levels. PFS and OS in the hypermethylated group (dotted line) and the hypomethylated group (solid line) of P1 (A) and P2 (B). Changes in survival rate (Probability) depending on the number of postoperative days (left: PFS, right: OS). The reference values for methylation are P1: 30% and P2: 7.5%. PFS and OS in the hypermethylated group (dotted line) and the hypomethylated group (solid line) of P1 (A) and P2 (B). Changes in survival rate (Probability) depending on the number of postoperative days (left: PFS, right: OS). The reference values for methylation are P1: 34.15% and P2: 8.84%. Changes in PFS (Probability) in the hypermethylated and hypomethylated groups of P1 depending on the number of postoperative days. Data based on the methylation level of DNA derived from FFPE specimens.

In the present specification, the identity of amino acid sequences and nucleotide sequences can be determined using the algorithm BLAST (Pro. Natl. Acad. Sci. USA, 1993, 90: 5873-5877) by Carlin and Arthur. Based on this BLAST algorithm, programs called BLASTN, BLASTX, BLASTP, BLASTN and TBLASTX have been developed (J. Mol. Biol., 1990, 215: 403-410). When using these programs, the default parameters of each program can be used. Specific methods for these analysis methods are known (see [www.ncbi.nlm.nih.gov]).

As used herein, "at least 90% identity" with respect to amino acid sequences and nucleotide sequences means 90% or more identity, preferably 95% or more identity, more preferably 97% or more identity, even more preferably. Means 98% or more identity, more preferably 99% or more identity.

In the present specification, the "corresponding position" on the amino acid sequence and the nucleotide sequence refers to the target sequence and the reference sequence (for example, the nucleotide sequence of SEQ ID NO: 1) as a conserved amino acid residue existing in each amino acid sequence or nucleotide sequence. It can be determined by aligning the groups or nucleotides to give them maximum homology. Alignment can be performed using known algorithms and the procedure is known to those of skill in the art. For example, alignment can be performed by using the Clustal W multiple alignment program (Thompson, J. D. et al, 1994, Nucleic Acids Res., 22: 4673-4680) with default settings. Clustal W is, for example, the European Bioinformatics Institute (EBI [www.ebi.ac.uk/index.html]) and the DNA Data Bank of Japan (DDBJ [www. It can be used on the website of ddbj.nig.ac.jp/index.html]).

In the present specification, "glioma" refers to a brain tumor that develops from glial cells (glioma cells). Glioblastomas are classified according to the World Health Organization (WHO) classification, including pilocytic astrocytoma (grade 1), diffuse astrocytoma, and oligodendroglioma (grade 2), and anaplastic astrocytoma. , And anaplastic oligodendroglioma (grade 3), and glioblastoma (grade 4). The glioma to be determined for prognosis in the present invention is preferably a grade 2 to 4 glioma, more preferably a grade 3 to 4 malignant glioma, and even more preferably a grade 4 glioblastoma. It is a tumor.

As used herein, the "prognosis" of a tumor refers to the future condition of the tumor. For example, a "poor prognosis" of a tumor means a low survival rate (after treatment) of a patient having the tumor, while a "good prognosis" of a tumor means a prognosis of a patient having the tumor (after treatment). Indicates that the survival rate (after treatment) is not low. For example, in a glioma patient, if the progression-free survival (PFS) is 50% or less 500 days after surgery, or if the overall survival (OS) is 1000 days after surgery, the overall survival rate (OS) is If less than 20%, the glioma or patient has a "poor prognosis".

In the present specification, "CpG" means a site in DNA where a phosphodiester bond (p) is formed between cytosine (C) and guanine (G). The region where CpG frequently appears is called a CpG island and is often present in the promoter region of a gene. As used herein, the term "CpG of a gene (is)" means CpG present in the promoter region of the gene, unless otherwise defined.

In the present specification, "DNA methylation" means a state in which cytosine (5-carbon in cytosine) of DNA is methylated. Further, in the present specification, the "DNA methylation level" (or simply referred to as "methylation level") of a DNA region means the ratio of cytosine contained in the DNA region being methylated. For example, the methylation level of a DNA region can be expressed as the ratio of the number of methylated cytosines to the total number of cytosines (methylated cytosine and unmethylated cytosine) contained in the DNA region.

One aspect of the present invention relates to a method for determining the prognosis of glioma. One embodiment of this embodiment is a method for determining the prognosis of glioma, wherein the method is: O6-methyl-guanine-DNA-methyltransferase (MGMT) in DNA derived from a subject's glioma cells or a tissue containing the same. ) Is measured in the DNA methylation level of the target DNA region contained in the promoter region of the gene encoding; and the glioma is based on whether or not the measured DNA methylation level is equal to or higher than a predetermined standard. Includes determining whether a tumor has a poor prognosis.

Another embodiment of this embodiment is a method for determining cells or tissues derived from glioma having a good or poor prognosis, which method: encodes MGMT in DNA derived from glioma cells or tissues containing the same. The cell or tissue has a poor prognosis based on measuring the DNA methylation level of the target DNA region contained in the promoter region of the gene to be used; and whether the measured DNA methylation level is above a predetermined standard. Includes determining whether a cell or tissue is derived from a promoter.

Another embodiment of this embodiment is a method of obtaining data for use in determining cells or tissues derived from glioma with good or poor prognosis, or in determining the prognosis of glioma, wherein the method is: test. To measure the DNA methylation level of the target DNA region contained in the promoter region of the gene encoding MGMT in DNA derived from glioma cells of the body or tissues containing the same; and the measured DNA methylation level is predetermined. Includes obtaining data on whether or not it is above the criteria of. The method for acquiring data according to the present invention is a method that does not include prognostic diagnosis of glioma by a doctor or the like. For example, the method is a method for acquiring data necessary for prognosis diagnosis by a doctor or the like, which is carried out in a research institute or a clinical laboratory.

In the following specification, the method for determining the prognosis of glioma according to the present invention, the method for determining cells or tissues derived from glioma having a good or poor prognosis, and the method for acquiring data are collectively referred to as the method of the present invention. It is called.

In the present invention, examples of the subject include those who need to determine the prognosis of glioma, for example, patients suffering from glioma, preferably patients who have been treated for glioma by surgery. Can be mentioned. Preferably, these patients are receiving radiation therapy, or a combination of radiation therapy and chemotherapy (eg, administration of an alkylating agent such as temozolomid), or radiation therapy or a combination of the same. Patients who are being considered for receiving.

Examples of the DNA derived from the glioma cell or the tissue containing the glioma cell used in the method of the present invention include genomic DNA prepared from the glioma cell of the subject or the tissue containing the glioma cell. Examples of the glioma cells or tissues containing them include fresh glioma cells collected in surgery or tissues containing them; glioma cells frozen after collection or frozen tissues containing them; formalin after collection. Examples include fixed and paraffin-embedded glioma cells or tissues containing them. Of these, frozen glioma cells or frozen tissues containing them are preferable from the viewpoint of suppressing the decomposition of DNA in cells or tissues and detecting the DNA methylation level more efficiently.

The method for preparing sample DNA from tissues or cells is not particularly limited, and a known method can be appropriately selected and used. Known methods for preparing DNA include the phenol chloroform method, a commercially available DNA extraction kit, for example, DNeasy Blood & Tissue Kit (manufactured by Qiagen), QIAamp DNA Mini kit (manufactured by Qiagen), Clean Colors (manufactured by NexTech), and the like. Examples thereof include a DNA extraction method using AquaPure (manufactured by Bio-Rad), ZR Plant / Seed DNA Kit (manufactured by Zymo Research), prepGEM (manufactured by ZyGEM), BuccalQuick (manufactured by TrimGen), and the like.

Preferably, the prepared sample DNA is bisulfite-treated. The method for bisulfite treatment of DNA is not particularly limited, and a known method can be appropriately selected and used. Known methods for bisulfite treatment include, for example, EZ DNA methylation-Gold kit, EZ DNA methylation-Lightning Kit (all manufactured by Zymo Research), EpiTect Bisulfite Kit (48) (manufactured by Qagen), and Qagen. Human Genetic Signatures Pty), Cells-to-CpG Bisulfite Conversion Kit (Applied Biosystems), CpGenome Turbo Bisulfite Models, CpGenome Turbo Bisulfite Mods, etc.

Further, it is preferable to amplify the bisulfite-treated sample DNA. The amplification method is not particularly limited, but PCR is preferably used. As the amplification method and conditions, known methods and conditions can be appropriately selected and used according to the sequence, length, amount and the like of the DNA to be amplified.

In the method of the present invention, the promoter region of the gene encoding O6-methyl-guanine-DNA-methyltransphase (MGMT) in the sample DNA derived from the glioma-derived DNA prepared as described above (hereinafter, also referred to as MGMT promoter region). ), The DNA methylation level of the target DNA region is measured. The MGMT promoter region can be identified as a region on genomic DNA consisting of the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having at least 90% identity thereof. Alternatively, the MGMT promoter region on genomic DNA can be identified with reference to the NCBI database ([www.ncbi.nlm.nih.gov/]).

The target DNA region in which the DNA methylation level is measured is at least one region selected from the group consisting of the following regions 1 and 2 in the MGMT promoter region:
Region 1) A region consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity thereof, wherein the nucleotide sequence of SEQ ID NO: 1 is the nucleotide sequence Nos. 2319 to 2470 of SEQ ID NO: 3. Equivalent to;
Region 2) A region consisting of the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 90% identity thereof, wherein the nucleotide sequence of SEQ ID NO: 2 is the nucleotide sequence of SEQ ID NO: 3 from No. 2534 to No. 2777. Equivalent to.

Alternatively, the regions 1 and 2 are represented as regions within the MGMT promoter region as defined below:
Region 1) After bisulfite treatment, a region sandwiched between a primer consisting of the nucleotide sequence of SEQ ID NO: 4 and a primer consisting of the nucleotide sequence of SEQ ID NO: 5 (not including the primer binding sequence);
Region 2) A region (not including the primer binding sequence) sandwiched between a primer consisting of the nucleotide sequence of SEQ ID NO: 6 and a primer consisting of the nucleotide sequence of SEQ ID NO: 7 after bisulfite treatment.

The present inventor has found that the DNA methylation levels in the regions 1 and 2 in the MGMT promoter region are effective as markers for determining the prognosis of glioma. More specifically, the present inventor has found that the prognosis of glioma can be determined based on whether or not the DNA methylation level in any of the above regions 1 and 2 meets a predetermined criterion. In the method of the present invention, the prognosis of glioma is determined based on whether or not the DNA methylation level of at least one region selected from the group consisting of the regions 1 and 2 meets a predetermined criterion. Conventionally, it has been suggested that DNA methylation of the MGMT promoter region may affect the prognosis of glioma (Non-Patent Documents 1 to 3), but the prognosis of glioma based on DNA methylation of the MGMT promoter region. No clear criteria have been provided so far to determine.

In one embodiment of the method of the present invention, data on whether or not the DNA methylation level in any one or more of the regions 1 and 2 in the sample DNA is equal to or higher than a predetermined reference is acquired. At this time, the criterion for the region 1 is a value selected from the range of 10 to 40%, for example, but not limited to, 10%, 20%, 23%, 25%, 28%, 30%, 33%. It may be set to 35%, 38%, or 40%. Preferably, the criteria for region 1 is a value selected from the range of 25-35%, or 25-36%, eg, but not limited to 25%, 28%, 30%, 33%, 35. It may be set to% or 36%. More preferably, the criteria for region 1 is a value selected from the range 29-36% or 29-31%, eg, but not limited to 29%, 30%, 31%, 32%, 33. It may be set to%, 34%, 34.2%, 34.5%, 35%, or 36%. More preferably, the standard for region 1 may be set in the range of 30% or 30 to 36%, more preferably in the range of 30 to 34.2%. Criteria for region 2 are values selected from the range of 5-30%, such as, but not limited to, 5%, 7.5%, 9%, 11%, 13%, 15%, 20%, 25. It may be set to% or 30%. Preferably, the criterion for region 2 may be set to a value selected from the range of 5-11%, for example, but not limited to, 5%, 7.5%, 9%, or 11%. More preferably, the criterion for region 2 is a value selected from the range of 6.5 to 8.5%, or 6.5 to 9.5%, eg, but not limited to 6.5%. , 7.5%, 8.5%, 8.8%, 8.9%, 9.0%, or 9.5%. More preferably, the standard for region 2 may be set in the range of 7.5%, 7.5 to 9.5%, and more preferably 7.5 to 8.9%. For each of Regions 1 and 2, DNA methylation levels above the criteria indicate a good (or not poor) prognosis for glioma, while DNA methylation levels below the criteria are neurological. Indicates a poor prognosis for glioma.

In another embodiment, when the DNA methylation level of the region 1 in the sample DNA is equal to or higher than the above criteria, the prognosis of the subject's glioma may be determined to be good (or not bad). On the other hand, when the DNA methylation level of the region 1 is less than the above criteria, the prognosis of the subject's glioma can be determined to be poor.
In another embodiment, when the DNA methylation level of the region 1 in the sample DNA is equal to or higher than the above criteria, the cell or tissue from which the sample DNA is derived has a good (or not bad) prognosis. It can be determined that the cell or tissue is of origin, while the cell or tissue from which the sample DNA is derived is derived from glioma with a poor prognosis when the DNA methylation level of the region 1 is below the above criteria. Can be determined to be.

In another embodiment, when the DNA methylation level of the region 2 in the sample DNA is equal to or higher than the above criteria, the prognosis of the subject's glioma may be determined to be good (or not bad). On the other hand, when the DNA methylation level of the region 2 is less than the above criteria, the prognosis of the subject's glioma can be determined to be poor.
In another embodiment, when the DNA methylation level of the region 2 in the sample DNA is equal to or higher than the above criteria, the cell or tissue from which the sample DNA is derived has a good (or not bad) prognosis. It can be determined that the cell or tissue is of origin, while the cell or tissue from which the sample DNA is derived is derived from glioma with a poor prognosis when the DNA methylation level of the region 2 is below the above criteria. Can be determined to be.

In yet another embodiment, the DNA methylation levels of regions 1 and 2 can be combined to determine the prognosis of glioma. For example, when the DNA methylation levels of both regions 1 and 2 are both below the criteria, the prognosis of the subject's glioma is poor, or the cell or tissue from which the sample DNA is derived has a prognosis. It can be determined to be of poor glioma origin. Alternatively, when the DNA methylation levels in both regions 1 and 2 are both above the criteria, the prognosis for glioma in the subject is good (or not bad), or the sample DNA is derived. Cells or tissues that produce can be determined to be derived from glioma with a good (or not poor) prognosis.

Therefore, when the bisulfite-treated genomic DNA is amplified by PCR or the like in the method of the present invention, any one or more of the regions 1 and 2 may be amplified, or the regions 1 and 2 may be amplified. Both may be amplified.

In the method of the present invention, the method for measuring the DNA methylation level of the regions 1 and 2 is not particularly limited, and a known DNA methylation level quantification method can be appropriately used. Examples of such known methods include the first to eighth methods shown below.

The first method is DNA methylation in the target DNA region using a 1-base extension reaction using a probe constructed to have a base complementary to methylated or unmethylated cytosine at the 3'end. Is a method of quantifying. For example, in the first method, a probe that hybridizes to bisulfite-treated genomic DNA has a base complementary to methylated or unmethylated cytosine of CpG in the target DNA region at the 3'end. Use a probe. Bisulfite treatment converts unmethylated cytosine to uracil, but not methylated cytosine. That is, the base at the 3'end of the probe for methylated cytosine is guanine, and the base at the 3'end of the probe for unmethylated cytosine is adenine. The two types of probes are hybridized with the single-stranded fragmented bisulfite-treated DNA, and a single-base extension reaction is carried out in the presence of a fluorescently labeled base. Depending on the methylation or unmethylation of the target cytosine, a one-base extension reaction occurs with either probe to incorporate the fluorescently labeled base. The DNA methylation level can be calculated from the intensity of fluorescence emitted by the probe for methylated cytosine and / or the probe for unmethylated cytosine.

Alternatively, in another aspect of the first method, a probe that hybridizes to bisulfite-treated genomic DNA and has a base complementary to CpG guanine in the target DNA region at its 3'end is used. You may. Then, the probe is hybridized with the single-stranded fragmented DNA, and a one-base extension reaction is carried out in the presence of guanine labeled with a fluorescent substance and / or adenine labeled with a fluorescent dye different from the fluorescent substance. I do. Fluorescently labeled guanine or adenine is incorporated into the probe depending on the methylation or unmethylation of the target cytosine. The DNA methylation level can be calculated from the fluorescence emitted by each fluorescent substance incorporated into the probe.

A preferred example of such a first method is, for example, the bead array method (eg, Infinium® assay).

The second method is a method of quantifying methylated DNA by mass spectrometry. Preferred examples of the second method include, for example, MassARRAY® (Jurinke C et al., Mutat Res, 2005, 573, pp. 83-95). In MassARRAY®, the target DNA region after bisulfite treatment is amplified, transcribed into RNA, and then specifically cleaved at the uracil site by RNAase. As a result, RNA fragments having different lengths are generated depending on the presence or absence of DNA methylation (conversion of cytosine to uracil). By subjecting the obtained RNA fragment to mass analysis, RNA fragments derived from methylated DNA and RNA fragments derived from unmethylated DNA were separated according to the difference in molecular weight, and from the mass ratio of these fragments detected. , The DNA methylation level of the target DNA region is calculated. For mass spectrometry of RNA fragments, MALDI-TOF MAS (for example, MassARRAY Analyzer 4 manufactured by SEQUENOM), which can detect a difference in mass of one base, can be used.

A third method includes a pyrosequencing method (registered trademark, Pyrosequencing) (see Anal. Biochem. (2000) 10: 103-110). First, the target DNA region treated with bisulfite is amplified, and the amplified DNA is dissociated into a single strand. An extension reaction (sequencing) is performed one base at a time using one strand of the obtained single-stranded DNA. In the extension reaction, unmethylated cytosine (converted to uracil) is shown as thymine. The pyrophosphate produced during the reaction is enzymatically emitted to emit light, and the intensity of the emission is measured. The luminescence intensity derived from methylated cytosine (cytosine) is compared with the luminescence intensity derived from unmethylated cytosine (thymine), and for example, the DNA methylation level (%) of the target DNA region is calculated by the following formula.
DNA methylation level (%) = cytosine luminescence intensity x 100 / (cytosine luminescence intensity + thymine luminescence intensity).

As a fourth method, methylation-sensitive high resolution melting (MS-HRM, Wojidacz TK et al., Nat Protocol., 2008, Vol. 3, pp. 1903-8) is used. Can be mentioned. In the fourth method, the melting curve of the amplification product of the bisulfite-treated target DNA region is measured in a reaction system containing an intercalator that fluoresces when inserted between DNA double strands. The DNA methylation level of the target DNA region is calculated by comparing the melting curve with methylated / unmethylated controls.

As a fifth method, for example, methylation-specific polymerase chain reaction (MS-PCR) using real-time quantative PCR such as the Methylation method using a TaqMan probe (registered trademark) is mentioned. Be done. In the fifth method, a bisulfite-treated target DNA region is amplified by using a primer set that specifically amplifies methylated DNA and a primer set that specifically amplifies unmethylated DNA. DNA methylation levels are calculated by comparing the amounts of each amplification product.

Examples of the sixth method include bisulfite direct sequencing and bisulfite cloning sequencing (Kristensen LS et al., Clin Chem, Vol. 14, 2009, Vol. 55). See page 83). As a seventh method, for example, COBRA (analysis using a combination of bisulfite and a restriction enzyme) can be mentioned.

The eighth method is a method using ion exchange chromatography. For example, in the eighth method, first, the bisulfite-treated genomic DNA is fragmented, and the target DNA region is amplified by PCR using the obtained DNA fragment as a template. The amplified DNA fragment is then subjected to ion exchange chromatography to calculate the DNA methylation level of the target DNA region based on the peak retention time.

In the eighth method, unmethylated cytosine in the target DNA region is converted to uracil by bisulfite treatment and then further converted to thymine by PCR. On the other hand, methylated cytosine remains cytosine after bisulfite treatment and PCR. Due to this base difference, the methylated cytosine-containing fragment (methylated fragment) and the unmethylated fragment are detected as separate peaks with different retention times in ion exchange chromatography. Preferably, the peak retention time of the methylated fragment is shorter than that of the unmethylated fragment, and the higher the methylation level, the earlier the retention time peak is detected. Therefore, the DNA methylation level of the target DNA region can be calculated based on the retention time of the peak in ion exchange chromatography.

In the eighth method, the DNA methylation level of the target DNA region can be calculated based on a calibration curve prepared in advance using DNA derived from the same DNA region whose methylation level is known. Alternatively, it is investigated whether or not the DNA methylation level of the target DNA region is equal to or higher than the reference value by comparing the retention time of the peak with the DNA (Reference) derived from the same DNA region whose methylation level is the reference value. Can be done. The retention time of the reference peak may be measured together with the sample DNA, or may be determined in advance. Sample DNA detected at the same or earlier retention time as Reference is determined to have a methylation level above the reference, while sample DNA detected at a retention time below the reference has a methylation level of said. It is judged to be below the standard.

The ion exchange chromatography performed by the eighth method is preferably anion exchange chromatography. The column packing material is not particularly limited as long as it is a base particle having a strong cationic group on the surface, but is shown in International Publication No. 2012/108516, which has a strong cationic group and a weak cationic property on the packing surface. Substrate particles having both groups are preferred. More preferably, the substrate particles are coated polymer particles in which a layer of a hydrophilic polymer having a strongly cationic group (preferably a quaternary ammonium salt) is copolymerized on the surface of the hydrophobic crosslinked polymer particles. , A base material particle containing a weak cationic group (preferably a tertiary amino group) introduced into the surface of the coated polymer particles. The column temperature during the chromatographic analysis is preferably 30 ° C. or higher and lower than 90 ° C.

As described above, the method that can be suitably used as the "method for measuring the DNA methylation level" in the present invention has been exemplified, but the method is not limited to this. In the first to eighth methods, as described above, genomic DNA prepared from glioma cells or tissues containing the glioma cells is further subjected to bisulfite treatment. Therefore, the DNA used to measure DNA methylation levels in the methods of the invention is preferably bisulfite-treated genomic DNA from glioma cells or tissues containing them.

For patients who are determined to have a good prognosis for glioma by the method of the present invention, it is possible to actively administer chemotherapy (for example, administration of an alkylating agent such as temozolomide) in combination with radiotherapy for the survival period of the patient. It is effective for extension of. On the other hand, for patients who are determined to have a poor prognosis of glioma by the method of the present invention, the dose of the alkylating agent may be increased, or a therapy other than chemotherapy with the alkylating agent may be applied, or the condition may be more cautious. It is possible to take appropriate measures for the patient, such as monitoring the patient. The present invention can contribute to improving the quality of life and prognosis of an individual patient by providing the individual patient with information for selecting an appropriate treatment.

Therefore, the present invention also provides a method for treating glioma, which comprises treating a subject who is determined to have a good or poor prognosis for glioma by the method for determining prognosis of the present invention. As a means of treatment, it is preferable to use radiation therapy in combination with chemotherapy (for example, administration of an alkylating agent such as temozolomid) for subjects with a good prognosis, and only radiation therapy for subjects with a poor prognosis. , The combination of radiation therapy and administration of a higher dose of alkylating agent than usual, or the combination of radiation therapy and administration of an alkylating agent other than the administration of an alkylating agent is preferable, but is not particularly limited.

Furthermore, the present invention provides primers or probes for use in the methods of the present invention. Preferably, the primer or probe is used for determining the prognosis of glioma, for determining cells or tissues derived from glioma with good or poor prognosis, or for determining cells or tissues derived from glioma by any of the first to eighth methods. It is used to acquire the data used for those determinations. Preferably, the primer or probe is a primer or probe for measuring the DNA methylation level of said region 1 or 2 in the MGMT promoter region. More preferably, the primer or probe is a primer or probe that hybridizes to the bisulfite-treated MGMT promoter region.

The chain length of the primer or probe of the present invention may be at least 12 bases, but is preferably at least 15 bases, and more preferably 15 to 200 bases. In the case of a probe, the preferred chain length is 100-200 bases, more preferably 100-150 bases. In the case of PCR primers, the preferred chain length is 12-60 bases, more preferably 15-40 bases. Further, the primer or probe of the present invention may be labeled (for example, fluorescently labeled). Further, the primer or probe of the present invention is preferably a primer or probe that can be used in any of the first to eighth methods.

As one embodiment of the primer or probe, a primer set for PCR amplification of the region 1 or 2 in the bisulfite-treated MGMT promoter region (for example, a primer set that can be used in the above-mentioned eighth method). Can be mentioned. An example of such a primer set is a primer set consisting of a primer consisting of the nucleotide sequence of SEQ ID NO: 4 and a primer consisting of the nucleotide sequence of SEQ ID NO: 5, which PCR-amplifies the bisulfite-treated region 1, or a primer set complementary thereto. Primer set consisting of primers having a specific sequence; and a primer set consisting of a primer consisting of the nucleotide sequence of SEQ ID NO: 6 and a primer consisting of the nucleotide sequence of SEQ ID NO: 7, which PCR-amplifies the bisulfite-treated region 2. A primer set consisting of primers having a sequence complementary to the above can be mentioned.

As another example of the primer set, a primer having a sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 4 and at least 90% of the nucleotide sequence of SEQ ID NO: 5 that PCR-amplifies the bisulfite-treated region 1. A primer set with primers consisting of the same sequence, or a primer set consisting of primers having a sequence complementary thereto; and at least 90% identical to the nucleotide sequence of SEQ ID NO: 6 that PCR-amplifies the bisulfite-treated region 2. Examples thereof include a primer set consisting of a primer having a similar sequence and a primer having a sequence consisting of at least 90% identical to the nucleotide sequence of SEQ ID NO: 7, or a primer set consisting of a primer having a sequence complementary thereto.

In another embodiment of the primer or probe of the present invention, it hybridizes to either of the bisulfite-treated regions 1 and 2 and is complementary to methylated or unmethylated cytosine at its 3'end. Primers or probes constructed to have a specific base (for example, primers or probes that can be used in the first method described above) can be mentioned.

As another embodiment of the primer or probe of the present invention, a primer capable of hybridizing to any of the bisulfite-treated regions 1 and 2 and carrying out an extension reaction one base at a time (for example, the above-mentioned third primer). Primer for pyrosequencing) that can be used in the above method.

Another embodiment of the primer or probe of the present invention is a primer set capable of specifically amplifying bisulfite-treated DNA derived from the methylated region 1 or 2, and the unmethylated region. Examples thereof include a primer set capable of specifically amplifying bisulfite-treated DNA derived from 1 or 2 (for example, a primer set that can be used in the above-mentioned fifth method).

Furthermore, the present invention provides a kit for performing the method of the present invention, which comprises the primer or probe of the present invention. Preferably, the kit of the present invention is used for determining the prognosis of glioma, for determining cells or tissues derived from glioma having a good or poor prognosis, or for determining cells or tissues derived from glioma by any of the first to eighth methods. It is used to acquire the data used for those determinations.

The kit of the present invention can contain components other than the primer or probe of the present invention. Examples of such components include reagents required for bisulfite treatment (eg, sodium hydrogen sulfite solution, etc.), reagents required for PCR reactions (eg, deoxyribonucleotides, heat-resistant DNA polymerase, etc.), and infinium (registered trademark). ) Reagents required for the assay (eg, fluorescently labeled nucleotides), Reagents required for MassARRAY® (eg, RNase for performing base-specific cleavage reactions), required for pyrosequencing Reagents (eg, ATP sulfylase for detection of pyrophosphate, adenosine-5'-phosphosulfate, luciferase, luciferin, streptavidin for separating single-stranded DNA, etc.), reagents required for detection of labels (eg, substrate) And enzymes), buffers used for diluting and washing samples (tissue-derived DNA, etc.), and the like, but are not limited thereto. In addition, the kit may include its instruction manual.

Preferably, the kit of the present invention comprises a guide for determining whether or not the measured DNA methylation level is above the above-mentioned criteria for determining the prognosis of glioma. In one embodiment, the guide is an indication indicating the criteria for determining the prognosis of the aforementioned glioma. In one embodiment, the guide is the DNA of either region 1 or 2 whose methylation level is at the reference value, and the user of the kit is one of regions 1 and 2 of the sample DNA. By comparing the DNA methylation level with the reference, it can be determined whether or not the methylation level of any of regions 1 and 2 of the sample DNA is equal to or higher than the reference.

Preferably, the kit of the present invention utilizes the eighth method and the DNA methylation level of any one or more of the regions 1 and 2 is equal to or higher than the above-mentioned criteria for determining the prognosis of glioma. It is used to obtain data about the presence or absence. The kit includes a primer set that can be used in the eighth technique and utilizes the eighth technique to measure the DNA methylation level of any one or more of the regions 1 and 2. The kit can further include reagents other than the primer set required for the eighth procedure, such as bisulfite treatment reagents, PCR reagents, chromatographic reagents, and the like. Preferably, the kit comprises a display indicating a criterion for determining the prognosis of the glioma, or DNA (reference) of any one or more of regions 1 and 2 whose methylation level is at the reference value. By comparing the DNA methylation level of either region 1 or 2 of the sample DNA with the methylation level of the indicated reference or the reference, the DNA methylation level of either region 1 or 2 of the sample DNA Can be determined whether or not is greater than or equal to the standard.

Furthermore, the present invention provides a DNA methylation level measurement system for carrying out the method of the present invention. Preferably, the system utilizes the eighth technique to determine if the DNA methylation level of any one or more of the regions 1 and 2 is above the criteria for determining the prognosis of the glioma described above. It is a system that makes it possible to acquire data on whether or not. For example, the system comprises an ion exchange chromatography (preferably anion exchange chromatography) apparatus used in the eighth technique described above. If desired, the system may be equipped with a sample DNA preparation device (including, for example, a device for bisulfite processing of DNA and a PCR device). The bisulfite-treated and PCR-treated sample DNA is sent to a chromatographic device and separated according to the methylation level. The DNA methylation level of any of the regions 1 and 2 can be measured based on the retention time of the chromatographic peak. Further, the system determines whether or not the DNA methylation level of any one or more of the regions 1 and 2 calculated from the chromatography result is equal to or higher than the above-mentioned criteria for determining the prognosis of glioma. It is equipped with a methylation level detection device for detection. For example, the detection device is a computer. The detection device detects whether or not the measured DNA methylation level of any one or more of the regions 1 and 2 is equal to or higher than the reference level, and outputs the result. If necessary, the detector measures DNA methylation levels from the retention time of the chromatographic peak.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the following Examples.

Example 1
1) Patient and tissue specimens Frozen glioma specimens (n = 107) were prepared. These specimens included 2 patients with pilocytic astrocytoma (grade 1), 9 patients with diffuse astrocytoma and 4 patients with oligodendroglioma (grade 2), and 8 anaplastic stars. 54 patients (9-83 years; average age 47 years, including astrocytoma and 8 anaplastic oligodendroglioma (grade 3) patients, and 23 glioblastoma (grade 4) patients, It was prepared by rapidly cryopreserving the tissue containing glioma, which was surgically removed from 28 males and 26 females) at -80 ° C. These specimens also included specimens from the same patient taken from different sites of tumor tissue at different times. Twenty-one of the 23 patients with glioblastoma (GBM) received radiation chemotherapy (radiation + temozolomide) after surgery (mean treatment period 453 days).

2) DNA preparation DNA was extracted from the prepared frozen tumor sample using the DNeasy Blood & Tissue Kit (Qiagen, Cat. No. 69504) according to the attached protocol. The extracted DNA was bisulfite-treated according to the attached protocol using EZ DNA Methylation-Lightning Kits (ZymoResearch).

3) PCR amplification Using the bisulfite-treated DNA obtained in 2) as a template, 2 regions (P1 and P2; SEQ ID NOs: 1 and 2) in the MGMT promoter region (SEQ ID NO: 3) shown in Table 1 are amplified by PCR. did. In PCR, ^{50 μL of reaction solution (containing 20 ng of template) using TaKaRa EpiTaq TM} HS was prepared, and the reaction was carried out according to the attached protocol. Table 2 shows the primers used for PCR amplification of the two regions.

4) Preparation of anion exchange column 200 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) and triethylene glycol dimethate in 2000 mL of a 3 wt% polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd.) aqueous solution in a reactor with a stirrer. A mixture of 100 g of acrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.), 100 g of glycidyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) and 1.0 g of benzoyl peroxide (manufactured by Kishida Chemical Co., Ltd.) was added. The mixture was heated with stirring and polymerized at 80 ° C. for 1 hour in a nitrogen atmosphere. Next, as a hydrophilic monomer having a strong cationic group, 100 g of ethyltrimethylammonium chloride methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in ion-exchanged water. This was added to the same reactor and polymerized in the same manner at 80 ° C. for 2 hours under a nitrogen atmosphere with stirring. The obtained polymerization composition was washed with water and acetone to obtain coated polymer particles having a layer of a hydrophilic polymer having a quaternary ammonium group on the surface. The obtained coated polymer particles were measured using a particle size distribution measuring device (AccuSizer780 / Particle Sigmaning Systems), and the average particle size was 10 μm. 10 g of the obtained coated polymer particles were dispersed in 100 mL of ion-exchanged water to prepare a pre-reaction slurry. Next, while stirring this slurry, 10 mL of N, N-dimethylaminopropylamine (manufactured by Wako Pure Chemical Industries, Ltd.), which is a reagent having a weak cationic group, was added and reacted at 70 ° C. for 4 hours. After completion of the reaction, the supernatant was removed using a centrifuge (“Himac CR20G” manufactured by Hitachi, Ltd.) and washed with ion-exchanged water. After washing, the supernatant was removed using a centrifuge. This washing with ion-exchanged water was repeated four more times to obtain a filler for ion-exchange chromatography having a quaternary ammonium group and a tertiary amino group on the surface of the substrate particles. The obtained filler for ion exchange chromatography was packed in a stainless steel column (column size: inner diameter 4.6 mm × length 20 mm) of a liquid chromatography system.

5) HPLC analysis The PCR reaction product obtained in 3) was purified and analyzed by HPLC under the following conditions to detect the retention time of the peak.
System: LC-20A series (manufactured by Shimadzu Corporation)
Column: Anion exchange column (prepared in 4 above)
Eluent: Eluent A 25 mM MES-NaOH (pH 6.0)
Eluent B 25 mM MES-NaOH (pH 6.0)
+ 2M guanidine sulfate Analysis time: 15 minutes Elution method: The mixing ratio of eluent B was linearly increased under the following gradient conditions.
0 minutes (eluent B 30%) → 10 minutes (eluent B 50%)
Specimen: PCR amplification product obtained in 3) Flow rate: 1.0 mL / min
Detection wavelength: 260 nm
Sample injection volume: 5 μL
Column temperature: 70 ° C

6) Calculation of DNA methylation level 0% methylated DNA and 100% methylated DNA consisting of the same sequence were analyzed by HPLC in the same manner, and a calibration curve was prepared from the retention time of the detected peak. The DNA methylation levels of P1 and P2 in the sample DNA were calculated according to the calibration curve. If the chromatograms of P1 and P2 did not show a single peak (having shoulders or bipeaks), they were converted to a single peak by fitting and then the DNA methylation level was calculated based on the retention time of the peak.

7) Effect of DNA methylation level on glioma prognosis Figure 1 shows the progression-free survival (Days of) of glioma patients from which the sample DNA is derived, relative to the DNA methylation level of the sample DNA (P1 and P2). A plot of progression free survival (PFS days) and days of overall survival (OS days) is shown. For both P1 and P2, the number of PFS days and the number of OS days tended to increase as the DNA methylation level increased, suggesting a correlation between the DNA methylation levels of P1 and P2 and the prognosis of glioma.

Patients were divided into two groups according to the DNA methylation level of P1 and P2, and the difference in PFS or OS between the groups was statistically tested. When multiple samples were obtained from the same patient, the methylation level of the central tumor sample was adopted, and when there were multiple central samples, the median of those methylation levels was adopted. Boundary values of DNA methylation levels that have a significant effect on PFS or OS were determined. FIG. 2 shows changes in PFS or OS (Probability) with respect to postoperative days for 21 GBM patients who received preoperative radiochemotherapy. FIG. 2A shows the data of P1 divided into two groups having a methylation level of 30% or more (dotted line) and less than that (solid line). FIG. 2B shows the data of P2 divided into two groups, that is, the methylation level is 7.5% or more (dotted line) and less than that (solid line). Regarding P1, the PFS was statistically significantly different between the hypermethylated group and the hypomethylated group (Log rank test, P <0.05), and the OS tended to be different. Regarding P2, PFS and OS were statistically significantly different between the hypermethylated group and the hypomethylated group (Log rank test, P <0.01). Therefore, it was shown that about 30% for P1 and about 7.5% for P2 can be used as the reference value of the DNA methylation level that distinguishes between good prognosis and poor prognosis.

FIG. 3 shows changes in PFS or OS (Probability) with respect to postoperative days for 21 GBM patients (20 patients common to FIG. 2 and another GBM patient) who received preoperative radiochemotherapy. For P1 (Fig. 3A), the methylation level is 34.15% or more (dotted line) and less than that (solid line). For P2 (Fig. 3B), the methylation level is 8.84% or more (dotted line). ) And less than that (solid line). Regarding P1, the PFS was statistically significantly different between the hypermethylated group and the hypomethylated group (Log rank test, P <0.05), and the OS tended to be different. Regarding P2, PFS and OS were statistically significantly different between the hypermethylated group and the hypomethylated group (Log rank test, P <0.01). Therefore, it was shown that about 34.2% for P1 and about 8.8 to 8.9% for P2 can be used as the reference value of the DNA methylation level that distinguishes between good prognosis and poor prognosis.

From the above results, as a reference value for the DNA methylation level that distinguishes good prognosis from poor prognosis, P1 is in the range of 29 to 31%, or 29 to 36%, or 25 to 36%, or 25 to 25. A value selected from the range of 35% or 10-40%, preferably in the range of 30-36%, more preferably in the range of 30-34.2%, can be adopted, and for P2 6.5- A value selected from the range of 8.5%, or 6.5 to 9.5%, or 5 to 11%, or 5 to 30%, preferably 7.5 to 9.5%. It was expected that the range of 7.5 to 8.9% could be adopted, more preferably the range of 7.5 to 8.9%. A sample having a DNA methylation level equal to or higher than the reference value can be determined to have a good prognosis, and a sample having a DNA methylation level lower than the reference value can be determined to have a poor prognosis.

Example 2
DNA methylation levels of P1 and P2 were measured using a formalin-fixed paraffin-embedded (FFPE) GBM sample (n = 32) in the same procedure as in Example 1. The FFPE specimen was prepared from a tissue containing glioma surgically removed from 30 patients (17-83 years old; average age 55.2 years old, 17 males, 13 females). These specimens included specimens from the same patient taken from different times. All of the patients were GBM patients, and 28 of the 30 patients received radiation chemotherapy (radiation + temozolomide) after surgery (mean treatment period 389 days). As a result, DNA methylation levels were measured from 29 samples for P1 and 8 samples for P2. It was considered that the reason why the number of acquired data was small in P2 was that the DNA strand length was longer than that in P1 and therefore the DNA damage caused by the FFPE treatment was more affected. The following analysis shows 26 of the 28 first-onset GBMs receiving radiochemotherapy, excluding those with a genetic disorder (17-83 years; average 56.7 years, 15 males, 11 females, Only for P1 during the average treatment period (342 days).

FIG. 4 shows changes in PFS (Probability) with respect to postoperative days for GBM patients (26 cases above) from which FFPE samples were derived, divided into a hypermethylated group and a hypomethylated group in the same manner as in FIG. The reference value of the DNA methylation level was set to 30%, which is the same as in FIG. Even when the FFPE sample was used, it was possible to determine a patient with a good prognosis and a patient with a poor prognosis based on the same standard value of DNA methylation level as that of the frozen sample.

Example 3
Twenty-six GMS patients used in the analysis of FIG. 4 were subjected to multivariate analysis between PFS or OS and other information. As variables, performance status before surgery, presence / absence of IDH mutation, gender, age at surgery, presence / absence of irradiation (total 66 Gy), average methylation rate of P1 region, average methylation rate of P1 region is 30% or more. Or, whether or not the average methylation rate of the P2 region and the average methylation rate of the P2 region is 7.5% or more was used. As a result, for PFS, whether or not the average methylation rate of the P1 region was 30% or more and the age at the time of surgery showed a particularly high correlation, and for OS, the average methylation rate of the P1 region and the average methylation of the P1 region were shown. Whether or not the conversion rate was 30% or more and the age at the time of surgery showed a particularly high correlation (Table 3). The average methylation rate of the P1 region <30% group was 17% worse in PFS and 14.6% worse in OS than in the> = 30% group. Also, with each increase in age, PFS deteriorated by 4% and OS deteriorated by 3.5%.

Example 4
Twenty-six GMS patients used in the analysis of FIG. 4 were subjected to multivariate analysis between PFS or OS and other information. As variables, performance status before surgery, presence / absence of IDH mutation, gender, age at surgery, presence / absence of irradiation (total 66 Gy), average methylation rate of P1 region, average methylation rate of P1 region is 34.15% or more. Whether or not the average methylation rate of the P2 region and whether or not the average methylation rate of the P2 region was 8.84% or more were used. As a result, for PFS, the age at surgery showed a particularly high correlation, and for OS, the average methylation rate of the P1 region, whether or not the average methylation rate of the P1 region was 34.15% or more, and the age at surgery. Showed a particularly high correlation (Table 4). The mean methylation rate of the P1 region in the <34.15% group was 2.6% worse than that in the> = 34.15% group. Also, with each increase in age, PFS deteriorated by 2.5% and OS deteriorated by 2.8%.

Claims

A method for determining cells or tissues derived from glioma with a good or poor prognosis.
1) To measure the DNA methylation level of the target DNA region contained in the promoter region of the gene encoding O6-methyl-guanine-DNA-methyltransferase in DNA derived from glioma cells or tissues containing the same.
Here, the target DNA region is at least one region selected from the group consisting of the following regions 1 and 2:
Region 1) A region consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity thereof,
Region 2) A region consisting of the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 90% identity thereof,
2) When the measured DNA methylation level is equal to or higher than the following criteria, the cell or tissue containing it is judged to be a glioma-derived cell or tissue with a good prognosis, or the measured DNA methylation level is When it is less than the following criteria, the cells or tissues containing them are judged to be glioma-derived cells or tissues having a poor prognosis:
Criteria for region 1 A value selected from the range of 10-40%,
Criteria for region 2 Value selected from the range of 5-30%,
Including methods.
A method for determining the prognosis of glioma,
1) To measure the DNA methylation level of the target DNA region contained in the promoter region of the gene encoding O6-methyl-guanine-DNA-methyltransphase in DNA derived from the subject's glioma cells or tissues containing the same. ,
Here, the target DNA region is at least one region selected from the group consisting of the following regions 1 and 2:
Region 1) A region consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity thereof,
Region 2) A region consisting of the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 90% identity thereof,
2) When the measured DNA methylation level is equal to or higher than the following criteria, the subject's glioma is judged to have a good prognosis, or when the measured DNA methylation level is less than the following criteria, the subject is judged to have a good prognosis. Judging glioma as poor prognosis:
Criteria for region 1 A value selected from the range of 10-40%,
Criteria for region 2 Value selected from the range of 5-30%,
Including methods.
A method for acquiring data to be used for determining cells or tissues derived from glioma having a good or poor prognosis, or for determining the prognosis of glioma.
1) To measure the DNA methylation level of the target DNA region contained in the promoter region of the gene encoding O6-methyl-guanine-DNA-methyltransphase in DNA derived from the subject's glioma cells or tissues containing the same. ,
Here, the target DNA region is at least one region selected from the group consisting of the following regions 1 and 2:
Region 1) A region consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity thereof,
Region 2) A region consisting of the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 90% identity thereof,
2) Obtaining data on whether or not the measured DNA methylation level is above the following criteria:
Criteria for region 1 A value selected from the range of 10-40%,
Criteria for region 2 Value selected from the range of 5-30%,
Including methods.
The method according to any one of claims 1 to 3, wherein the promoter region comprises the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having at least 90% identity thereof.
The method according to any one of claims 1 to 4, wherein the region 1 is a region consisting of the nucleotide sequence of SEQ ID NO: 1 and the region 2 is a region consisting of the nucleotide sequence of SEQ ID NO: 2.
Any of claims 1-5, wherein the measurement of the DNA methylation level comprises measuring the DNA methylation level of at least one region selected from the group consisting of the bisulfite-treated regions 1 and 2. The method described in item 1.
The bisulfite-treated region 1 is a region sandwiched between a primer consisting of the nucleotide sequence of SEQ ID NO: 4 and a primer consisting of the nucleotide sequence of SEQ ID NO: 5 (however, it does not include the primer binding sequence).
The bisulfite-treated region 2 is a region sandwiched between a primer consisting of the nucleotide sequence of SEQ ID NO: 6 and a primer consisting of the nucleotide sequence of SEQ ID NO: 7 (however, the primer binding sequence is not included).
The method according to claim 6.
The method according to claim 6 or 7, wherein the measurement of the DNA methylation level is performed using ion exchange chromatography.
The method according to any one of claims 1 to 8, wherein the glioma is glioblastoma.
A kit for carrying out the method according to any one of claims 1 to 9.
It comprises a set of PCR primers for PCR amplification of a target DNA region contained in the promoter region of a bisulfite-treated gene encoding O6-methyl-guanine-DNA-methyltransphase, wherein the bisulfite-treated prior to the bisulfite treatment. The target DNA region is at least one region selected from the group consisting of regions 1 and 2 below:
Region 1) A region consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity thereof,
Region 2) A region consisting of the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 90% identity thereof,
And,
A guide for determining whether or not the DNA methylation level of the target DNA region is equal to or higher than the following criteria is included.
Criteria for region 1 A value selected from the range of 10-40%,
Criteria for region 2 Value selected from the range of 5-30%,
kit.
The PCR primer set consists of a primer set consisting of a primer consisting of the nucleotide sequence of SEQ ID NO: 4 and a primer consisting of the nucleotide sequence of SEQ ID NO: 5, or a primer consisting of the nucleotide sequence of SEQ ID NO: 6 and the nucleotide sequence of SEQ ID NO: 7. The kit according to claim 10, which is a primer set with a primer.
A system for implementing the method according to any one of claims 1 to 9.
The system according to claim 12, further comprising an ion exchange chromatography apparatus.