JP7105460B2 - Methods for detecting adult T-cell leukemia/lymphoma - Google Patents

Description

The present invention relates to methods for detecting adult T-cell leukemia/lymphoma (ATL), and the like.

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell tumor resulting from infection of T cells with the retrovirus Human T-cell Lymphotropic Virus type-1 (HTLV-1). HTLV-1 spreads mainly through breast-feeding, sexual intercourse, and blood transfusions. 0 million people (Non-Patent Document 1). The number of HTLV-1 infected people in Japan is estimated to be 1.1 million (Non-Patent Document 1), 40-50% of which are unevenly distributed in the Kyushu and Okinawa regions. HTLV-1 infected people in Japan are increasing.

HTLV-1-infected T cells undergo a multistep oncogenic process in which they acquire clonal proliferation ability while accumulating genetic mutations and epigenetic abnormalities. Therefore, the period from infection to the onset of ATL is very long, and as a result, many elderly carriers are characterized by the onset of ATL (Non-Patent Document 1). On the other hand, many of the HTLV-1 infected people end their lives as asymptomatic patients, but 3-5% of the infected people develop ATL, and from the clinical pathology, smoldering type, chronic type, acute type, and lymphoma type. are categorized. The acute type, lymphoma type, and some chronic types with poor prognostic factors are highly malignant and have an extremely poor prognosis. On the other hand, the smoldering type and the chronic type without poor prognostic factors are regarded as low-grade ATL that follows a relatively indolent course, and direct therapeutic intervention is basically not performed. However, about half of patients with low-grade ATL undergo blast crisis during the course, and their long-term prognosis is poor (Non-Patent Document 2). Therefore, it is desired to develop an effective method for preventing the onset of ATL, a method for treating the disease, and the like.

Ishitsuka et al. , Lancet Oncol (2014), 15(11), p. e517-26 Takasaki et al. , Blood (2010), 115(22), p. 4337-43

Under the above circumstances, a new method for detecting adult T-cell leukemia/lymphoma (ATL) has been desired.

As a result of extensive research, the present inventors have found that SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, The DNA methylation status of the LZTFL1, KLHL34 and BCL9 genes reflects the pathology of ATL, the subject develops ATL by using the methylation of at least one of these genes as a marker, or ATL The present invention has been completed based on the knowledge that it is possible to detect that there is a risk of developing.
The following are provided here:

[1-1] SERPINB6, NELL2, THEEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1 in the subject biological sample , KLHL34 and BCL9 genes, comprising measuring the presence or absence of DNA methylation of at least one gene selected from the group consisting of genes, wherein the subject is an adult T A method for detecting ATL, indicating that the patient has developed or is likely to develop cellular leukemia/lymphoma (ATL).
[1-2] The method of [1-1] above, wherein the DNA methylation of the at least one gene is measured by a bisulfite sequencing method.
[1-3] the at least one gene is
(1) a combination containing SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL34 and BCL9 genes ,
(2) a combination comprising the LYPD3, THEEMIS, NELL2, and C2orf40 genes;
(3) combinations comprising LYPD3, THEMIS, NELL2, C2orf40, HOOK1, FHIT, CD7, MDS2, and LAIR1 genes;
(4) a combination comprising the LYPD3, THEMIS, NELL2, C2orf40, HOOK1, FHIT, CD7, MDS2, LAIR1, RNF130, POMC, and BCL9 genes, or (5) LYPD3, THEMIS, NELL2, C2orf40, HOOK1, FHIT, CD7, The method of [1-1] or [1-2] above, which is a combination containing MDS2, LAIR1, RNF130, POMC, BCL9, LZTFL1, ZIK1, and TMEM45B genes.

[2-1] from SERPINB6, NELL2, THEEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL34 and BCL9 genes A kit for detecting adult T-cell leukemia/lymphoma (ATL), comprising reagents for detecting the presence or absence of DNA methylation of the promoter CpG of at least one gene selected from the group consisting of:
[2-2] The kit according to [2-1] above, which is used to measure DNA methylation of the at least one gene by a bisulfite sequencing method.
[2-3] the at least one gene is
(1) a combination containing SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL34 and BCL9 genes ,
(2) a combination comprising the LYPD3, THEEMIS, NELL2, and C2orf40 genes;
(3) combinations comprising LYPD3, THEMIS, NELL2, C2orf40, HOOK1, FHIT, CD7, MDS2, and LAIR1 genes;
(4) a combination comprising the LYPD3, THEMIS, NELL2, C2orf40, HOOK1, FHIT, CD7, MDS2, LAIR1, RNF130, POMC, and BCL9 genes, or (5) LYPD3, THEMIS, NELL2, C2orf40, HOOK1, FHIT, CD7, The method of [2-1] or [2-2] above, which is a combination containing MDS2, LAIR1, RNF130, POMC, BCL9, LZTFL1, ZIK1, and TMEM45B genes.

[3-1] A subject who has been shown to be developing or likely to develop ATL by the method for detecting ATL according to any one of [1-1] to [1-3] above, A method for preventing or treating ATL, comprising administering a drug for preventing or treating ATL.

In some aspects of the present invention, new methods for detecting ATL are provided.

FIG. 2 shows the onset/progression of ATL and changes in the expression of cell surface markers (CADM1 and CD7). 1 is a flow chart for extracting a DNA methylation-abnormal region that reflects the pathology of ATL. FIG. 3 shows cluster formation reflecting the progression of ATL pathology by the methylation profile of the HTLV-1-specific DNA hypermethylation region. FIG. 10 is a diagram showing the results of extracting a group of abnormally expressed genes associated with abnormal DNA methylation using a gene expression database; FIG. 2 shows examples of CpG sites targeted for methylation detection in each gene. FIG. 2 shows examples of CpG sites targeted for methylation detection in each gene. FIG. 2 shows examples of CpG sites targeted for methylation detection in each gene.

The present invention will be described in detail below.
All documents and publications mentioned herein are hereby incorporated by reference in their entirety regardless of their purpose.

1. Overview In the Examples described later, by comparing normal T cells and HTLV-1 infected cells in the same patient, a DNA methylation-abnormal region specific to HTLV-1 infected cells was extracted with high accuracy (Figs. 1 to 3). . The extracted DNA methylation aberrant regions contained approximately 900 genes. Next, we extracted approximately 400 genes that are expressed in normal T cells and whose expression is suppressed in HTLV-1 infected cells. Compared with about 900 genes contained in the above-mentioned HTLV-1-specific DNA hypermethylation region, 22 common genes (SERPINB6, NELL2, THEEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC , FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL34, BCL9) were identified (Fig. 4). Hierarchical clustering analysis using the expression profiles of the selected 22 genes revealed that the pathology was reflected in the same way as the DNA methylation status (Fig. 4). It was found to be suppressed, thereby contributing to the proliferation of HTLV-1-infected cells, that is, to the onset and progression of ATL.
Here, in a biological sample of interest, we show the and measuring the presence or absence of DNA methylation of at least one gene selected from the group consisting of BCL9 gene, and the subject is adult T-cell leukemia that said at least one gene is DNA methylated / To provide a method for detecting ATL, etc., indicating that lymphoma (ATL) has developed or is likely to develop. The ATL detection method and the like will be described in detail below.

2. Methods for Detection of Adult T-Cell Leukemia/Lymphoma (ATL) Methods for the detection of ATL provided herein include SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, A method comprising detecting the presence or absence of DNA methylation in at least one gene selected from the group consisting of FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL34 and BCL9 genes. . In some embodiments, DNA methylation of said at least one gene is used as an ATL detection marker.

"Adult T-cell leukemia/lymphoma" or "ATL" is a peripheral T-cell neoplasm resulting from infection of T cells with the retrovirus Human T-cell Lymphotropic Virus type-1 (HTLV-1). ATL is classified according to its clinical pathology into smoldering, chronic, acute, and lymphoma types.

A "subject" is a mammal, including humans, such as humans and monkeys, preferably humans.

The "biological sample" is not limited as long as the presence or absence of DNA methylation of genes in T cells can be detected. Biological samples include, for example, (i) peripheral blood (whole blood), (ii) peripheral blood mononuclear cells separated from peripheral blood (mixture containing CD4-positive T cells and CD8-positive T cells), (iii) peripheral blood or T cells isolated from peripheral blood mononuclear cells, or isolated from peripheral blood or samples other than peripheral blood mononuclear cells; (iv) cell-free DNA in plasma or serum;

"T cell", also called T lymphocyte, means a thymus-derived cell among lymphocytes involved in an immune response. T cells include CD8 positive T cells (cytotoxic T cells: CTL), CD4 positive T cells (helper T cells), suppressor T cells, regulatory T cells such as regulatory T cells, effector cells, naive T cells, Included are either memory T cells, αβ T cells expressing α and β chain TCRs, or γδ T cells expressing γ and δ chain TCRs. T-cells include T-cell progenitor cells committed to differentiate into T-cells.

"Samples other than peripheral blood or peripheral blood mononuclear cells" are, for example, skin lesions, lymph node lesions, bone marrow cells, and the like.

"DNA methylation" refers to hypermethylation of the 5-position carbon atom of cytosine (C) in the untranslated region of the gene on the genome of interest, particularly in the CpG island (also referred to as "promoter CpG island") sequence of the promoter region. refers to the phenomenon of CpG islands are cytosine (C) guanine (G)-rich regions that occur in untranslated regions in the genomic sequence of interest and often contain repeated sequences of dinucleotides consisting of CG.

In each gene, the region where DNA methylation is detected is determined, for example, as follows. Quantified CpG site information is obtained from oligonucleotide probe information used for DNA methylation quantification of each gene in the Infinium HumanMethylation 450K BeadChip manufactured by Illumina used in the comprehensive DNA methylation analysis. DNA methylation at that site or at CpG sites in the vicinity thereof is quantified by the bisulfite-pyrosequencing method or the like. A standard DNA with known methylation rate is used to validate the CpG site measurement and to determine the region where DNA methylation is to be detected. In some aspects, when comparing normal T cells and HTLV1-infected T cells, CpG sites with a β value change of 0.2 or more are determined as DMPs (Differential Methylation Positions), and the CpG sites are measured. It is judged to be a valid position (Cancer Lett. (2013) 330(1), p.33-40). Preferably, at this time, normal T cells and HTLV1-infected T cells in the same patient are compared to determine changes in the β value.

Genes targeted by the detection method are SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL34 and at least one gene selected from the group consisting of the BCL9 gene.

Below are examples of human genes. The sequences of relevant genes of mammals other than humans can be obtained from, for example, the GenBank (NCBI) database.

The human base sequence of SERPINB6 is registered in GenBank (NCBI, USA) under the accession number of "5269". Examples of peripheral sequences containing the promoter CpG of the human SERPINB6 gene are shown in FIG. 5 (SEQ ID NOS: 1-3). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human base sequence of NELL2 is registered in GenBank (NCBI) under the accession number of "4753". FIG. 5 shows an example of the peripheral sequence containing promoter CpG of the human NELL2 gene (SEQ ID NO: 4). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human base sequence of THEEMIS is registered in GenBank (NCBI) under the accession number "387357". An example of the surrounding sequence containing promoter CpG of the human THEEMIS gene is shown in FIG. 5 (SEQ ID NOS:5-6). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human nucleotide sequence of ZSCAN18 is registered in GenBank (NCBI) under the accession number of "65982". Examples of surrounding sequences containing promoter CpG islands of the human ZSCAN18 gene are shown in FIG. 5 (SEQ ID NOs:7-11). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human base sequence of LAIR1 is registered in GenBank (NCBI) under the accession number of "3903". FIG. 5 shows examples of peripheral sequences containing the promoter CpG of the human LAIR1 gene (SEQ ID NOs: 12-15). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human nucleotide sequence of CD7 is registered in GenBank (NCBI) under the accession number of "924". FIG. 5 shows an example of a surrounding sequence containing the promoter CpG of the human CD7 gene (SEQ ID NOS: 16-17). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human base sequence of RNF130 is registered in GenBank (NCBI) under the accession number of "55819". An example of the surrounding sequence containing the promoter CpG island of the human RNF130 gene is shown in FIG. 5 (SEQ ID NO: 18). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human nucleotide sequence of ZIK1 is registered in GenBank (NCBI) under the accession number of "284307". Examples of surrounding sequences containing promoter CpG islands of the human ZIK1 gene are shown in FIG. 5 (SEQ ID NOS: 19-21). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human base sequence of LYPD3 is registered in GenBank (NCBI) under the accession number of "27076". FIG. 5 shows an example of the peripheral sequence containing promoter CpG of the human LYPD3 gene (SEQ ID NO: 22). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human base sequence of TMEM45B is registered in GenBank (NCBI) under the accession number of "120224". An example of the flanking sequence containing the promoter CpG of the human TMEM45B gene is shown in FIG. 5 (SEQ ID NOS:23-24). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human base sequence of POMC is deposited in GenBank (NCBI) under the accession number "5443". Examples of flanking sequences containing the promoter CpG island of the human POMC gene are shown in FIG. 5 (SEQ ID NOs:25-27). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human base sequence of FHIT has been deposited in GenBank (NCBI) under the accession number "2272". An example of the flanking sequence containing the promoter CpG island of the human FHIT gene is shown in Figure 5 (SEQ ID NO: 28). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human base sequence of MDS2 is registered in GenBank (NCBI) under the accession number of "259283". FIG. 5 shows an example of the peripheral sequence containing promoter CpG of the human MDS2 gene (SEQ ID NO: 29). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human nucleotide sequence of HOOK1 is registered in GenBank (NCBI) under the accession number of "51361". FIG. 5 shows an example of the peripheral sequence containing promoter CpG of the human HOOK1 gene (SEQ ID NO: 30). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human base sequence of SORCS3 is registered in GenBank (NCBI) under the accession number of "22986". An example of the flanking sequence containing the promoter CpG island of the human SORCS3 gene is shown in FIG. 5 (SEQ ID NOS:31-32). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human nucleotide sequence of C2orf40 is registered in GenBank (NCBI) under the accession number of "84417". Examples of flanking sequences containing promoter CpG islands of the human C2orf40 gene are shown in FIG. 5 (SEQ ID NOS:33-36). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human base sequence of ZNF662 is registered in GenBank (NCBI) under the accession number of "389114". An example of the flanking sequence containing the promoter CpG island of the human ZNF662 gene is shown in Figure 5 (SEQ ID NO: 37). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human nucleotide sequence of SPG20 is registered in GenBank (NCBI) under the accession number of "23111". An example of the surrounding sequence containing the promoter CpG island of the human SPG20 gene is shown in Figure 5 (SEQ ID NO: 38). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human base sequence of ZNF717 is registered in GenBank (NCBI) under the accession number of "100131827". Examples of surrounding sequences containing promoter CpG islands of the human ZNF717 gene are shown in FIG. 5 (SEQ ID NOS:39-40). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human base sequence of LZTFL1 is registered in GenBank (NCBI) under the accession number of "54585". An example of the flanking sequence containing the promoter CpG island of the human LZTFL1 gene is shown in FIG. 5 (SEQ ID NOs:41-42). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human base sequence of KLHL34 is registered in GenBank (NCBI) under the accession number of "257240". An example of the flanking sequence containing the promoter CpG island of the human KLHL34 gene is shown in FIG. 5 (SEQ ID NOs:43-44). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

The human base sequence of BCL9 is registered in GenBank (NCBI) under the accession number of "607". FIG. 5 shows an example of the surrounding sequence containing promoter CpG of human BCL9 gene (SEQ ID NO: 45). Examples of target CpG sequences for detecting DNA methylation are underlined in the sequences of FIG. Examples of target CpG sequences for detecting DNA methylation in the preferred embodiment are further bracketed.

In some aspects, the SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL34 and BCL9 genes The target CpG sequences for detecting DNA methylation in at least one gene selected from the group consisting of are, for example, all or part of the underlined CpG sequences in FIG. In the at least one gene, the target CpG sequences for detecting DNA methylation are, for example, all or part of the CpG sequences further bracketed in FIG.

Measurement of DNA methylation can be performed, for example, as follows.
Genomic DNA is extracted from a biological sample by a known method. When the biological sample is a bodily fluid, the solid content is removed by separation means such as filter filtration and centrifugation, and then genomic DNA is extracted from the supernatant by a known technique such as the phenol/chloroform method or using a commercially available kit. . When the biological sample is a tissue, it is homogenized with purified water, subjected to the same separation means to remove solids, and the supernatant is subjected to a known technique such as the phenol/chloroform method or using a commercially available kit to extract genomic DNA. Extract. The extracted DNA is then used to detect methylation.

A method for detecting DNA methylation is not particularly limited, and a known method can be used.

Methods for analyzing DNA methylation using extracted genomic DNA include, for example, bisulfite sequencing, pyrosequencing, methylation specific PCR (MSP), quantitative MSP, COBRA (Combined Bisulfite Restriction Analysis) method, or single base typing by Tm analysis method using Quenching probe. These methods differ greatly in analysis range, sensitivity, precision, and required equipment. All of these utilize the principle that only unmethylated cytosine is converted to uracil by bisulfite treatment.

The method for measuring DNA methylation will be described.
In the "bisulfite sequencing method", unmethylated cytosine in DNA (unmethylated cytosine) is converted to uracil by treating DNA with bisulfite. On the other hand, methylated cytosines are not converted by bisulfite treatment. Therefore, such treatment can distinguish between methylated and unmethylated cytosines. Then, DNA methylation can be analyzed by a technique such as determining the base sequence of converted DNA.

The "pyrosequencing method" is a method of decoding base sequences by detecting the luciferase luminescence reaction caused by the polymerase base extension reaction. Highly accurate quantitative analysis can be performed. Based on this principle, the T%/C% of the CpG site of the bisulfite-treated DNA is decoded to determine the DNA methylation ratio.

"Methylation-specific PCR (MSP) method" converts DNA to bisulfite and uses this DNA as a template to specifically amplify methylated DNA and primers to specifically amplify unmethylated DNA. PCR is performed separately using , and the presence or absence of DNA methylation is determined by the presence or absence of PCR products.

The "quantitative MSP method" is a methylation-specific PCR technique in which DNA treated with Na bisulfite is PCR-amplified using primers of predicted sequences specific to the methylated and unmethylated regions inside the target gene. is based on. Real-time quantitative MSP can be performed by using multiple fluorophores, where methylated and unmethylated region-specific primers are used.

In the "COBRA method", after PCR amplification of the target DNA using primers common to methylated DNA and unmethylated DNA after bisulfite treatment, restriction to recognize sites where the sequences differ between methylated DNA and unmethylated DNA. After enzymatic treatment, the fragments are concentrated by ethanol precipitation and electrophoresis can distinguish between methylated and unmethylated DNA. PCR amplification can be performed similarly to the bisulfite sequencing method.

In the "Tm analysis method using a quenching probe", a DNA fragment containing a target CpG site is amplified by PCR from a DNA sample after bisulfite conversion, and a QProbe with a complementary sequence (fluorescently labeled cytosine base At the end of the QProbe, the fluorescence decreases when it binds to the DNA fragment, and emits light when it dissociates. Single base typing can be performed by detecting the resulting fluorescence.

Here, in each of the above methods, a primer set for PCR can be designed and obtained by a known method or a method based thereon. A primer set can be designed, for example, by inputting sequences into a program MethPrimer (http://www.urogene.org/methprimer/index1.html) on the Internet. Moreover, in each of the above methods, the probe can be designed and obtained by a known method or a method based thereon.

In some aspects, DNA methylation measurements are performed by bisulfite sequencing. A preferred example thereof is a method using a bead array (Infinium (registered trademark) Human Methylation 450 BeadChip) manufactured by Illumina. In this method, methylated cytosine is distinguished from unmethylated cytosine by converting unmethylated cytosine (unmethylated cytosine) in DNA to uracil by bisulfite treatment. Then, after hybridization of probes immobilized on two beads, a probe for methylation (M type) and a probe for unmethylation (U type), which are specific for each site, 1 base using labeled ddNTP An extension reaction is performed, and the ratio of methylation and non-methylation is calculated from this fluorescence intensity signal. Thereby, comprehensive DNA methylation analysis can be easily performed.

In some embodiments, the frequency of DNA methylation (e.g., methylation of CpG sequences) of each gene or the frequency of DNA methylation (e.g., methylation of CpG sequences) of a combination of genes detected as described above is , when high (e.g., 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30 % or more, 35% or more, 40% or more, 45% or more, or 50% or more, or a β value of 0.05 or more, 0.10 or more, 0.15 or more, 0.20 or more, 0.25 or more, 0.30 or higher, 0.35 or higher, 0.40 or higher, 0.45 or higher, or 0.50 or higher), it can be determined that each gene is DNA methylated. More specifically, the frequency of DNA methylation (e.g., CpG sequence methylation) for each gene is compared to the frequency of corresponding genomic DNA methylation (e.g., CpG sequence methylation) in normal cells. When high, it can be determined that the gene is DNA methylated.

In some other embodiments, the frequency of DNA methylation (e.g., CpG sequence methylation) of each gene detected as described above or the frequency of DNA methylation (e.g., CpG sequence methylation) of a combination of genes When the frequency is, for example, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, or the β value is 0.5 or more, 0.6 or more, 0.7 or more, 0 When the gene is 0.8 or more or 0.9 or more, it can be judged that the DNA is methylated.
In some further embodiments, the frequency of DNA methylation (e.g., CpG sequence methylation) of each gene detected as described above or DNA methylation (e.g., CpG sequence methylation) of a combination of genes is high (e.g., 5% or more, 10% or more, 15% or more, 20% or more, 25% or more , 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more higher, or the β value is 0.05 or more, 0.10 or more, 0.15 or more, 0.20 or more, or 0.05 or more. 25 or more, 0.30 or more, 0.35 or more, 0.40 or more, 0.45 or more, or 0.50 or more higher), and the methylation frequency is 50% or more, 60% or more, 70% or more, When the gene is 80% or more, or 90% or more, or when the β value is 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, or 0.9 or more, the gene is DNA methyl It can be determined that the

A "normal cell" is a T cell that is not infected with HTLV-1.

Among the above marker genes, at least one gene (i.e., one gene, or a combination of genes, i.e., two genes, three genes, four genes, five genes, six genes, 7 genes, 8 genes, 9 genes, 10 genes, 11 genes, 12 genes, 13 genes, 14 genes, 15 genes, 16 genes, By analyzing the DNA methylation of 17 genes, 18 genes, 19 genes, 20 genes, 21 genes, or 22 genes), the presence or absence of ATL onset in the subject, or The presence or absence of the possibility of developing ATL in the future is determined. The at least one gene is, eg, 1 or at least 2 genes, eg, 4-9 genes, 10-15 genes, or 16-22 genes. In some aspects, the at least one gene is all 22 genes.

In some aspects, the subject has adult T-cell leukemia/lymphoma ( It can be determined that ATL) has developed or is likely to develop.

In some other embodiments, the genes analyzed for DNA methylation are a combination of genes, i.e., at least two genes (e.g., two genes, three genes, four genes, five genes, six genes). species genes 7 genes 8 genes 9 genes 10 genes 11 genes 12 genes 13 genes 14 genes 15 genes 16 species genes, 17 genes, 18 genes, 19 genes, 20 genes, 21 genes, or 22 genes), and of the at least 2 genes, for example, 40 Adult T-cell leukemia/lymphoma (ATL) if the subject could be determined to be DNA methylated for %, 50%, 60%, 70%, 80%, 90%, or 100% of the genes It can be determined that it has developed or is likely to develop.

Here, examples of combinations of two or more genes are as follows.

(1) a combination containing SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL34 and BCL9 genes (all 22 genes).

(2) a combination (4 genes) containing the LYPD3, THEEMIS, NELL2, and C2orf40 genes.

(3) Combinations containing LYPD3, THEMIS, NELL2, C2orf40, HOOK1, FHIT, CD7, MDS2, and LAIR1 genes (9 genes).

(4) Combinations containing LYPD3, THEMIS, NELL2, C2orf40, HOOK1, FHIT, CD7, MDS2, LAIR1, RNF130, POMC, and BCL9 genes (12 genes).

(5) Combinations containing LYPD3, THEMIS, NELL2, C2orf40, HOOK1, FHIT, CD7, MDS2, LAIR1, RNF130, POMC, BCL9, LZTFL1, ZIK1, and TMEM45B genes (15 genes).

In some embodiments, in combinations (1) to (5) above, the target CpG sequences for detecting DNA methylation are, for example, all or part of the underlined CpG sequences in FIG. . In the combinations (1) to (5) above, the target CpG sequences for detecting DNA methylation are, for example, all or part of the CpG sequences shown in brackets ([]) in FIG.

In some aspects, the frequency of DNA methylation in each gene or combination of genes is 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more (or a β value of 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, or 0.9 or more), the probability that the subject has developed ATL is, for example, 40% or more, 50% or more, 60% or more, It can be determined to be 70% or more, 80% or more, 90% or more, or 100%.
Alternatively, the frequency of DNA methylation in each gene or combination of genes is 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more (or a β value of 0.5 or more, 0.6 or more) , 0.7 or more, 0.8 or more, or 0.9 or more), the subject is likely to develop ATL, and the probability of developing ATL in the future is, for example, 40% or more, 50% or more, 60% or more , 70% or more, 80% or more, 90% or more, or 100%.

The reference value F% of the methylation frequency at which the probability of developing ATL or the probability of developing ATL in the future is P% may be adjusted by accumulating cases. Statistical processing for adjusting the reference value F% of the methylation frequency is not particularly limited, and can be performed by a method known in the art.

As shown in the Examples below, the DNA methylation status is well correlated with the onset and progression of ATL. can also be determined as follows. In some aspects, the degree of ATL progression is determined based on the frequency of DNA methylation in each gene or combination of genes, as follows. That is, the higher the frequency of DNA methylation of each gene or the frequency of DNA methylation of a combination of genes, the higher the malignancy of ATL. can be judged to be low.

In addition, as shown in the examples below, the DNA methylation state is well correlated with the onset and progression of ATL. Based on the frequency of transformation, it can be an indication of the extent to which aggressive HTLV-1 infected cells are present in a biological sample (eg, in collected peripheral blood). In some aspects, the frequency of DNA methylation in each gene or gene combination is used to determine the extent to which aggressive HTLV-1 infected cells are present in a biological sample, as follows. That is, it can be determined that the higher the frequency of DNA methylation of each gene or the frequency of DNA methylation of a combination of genes, the more malignant HTLV-1 infected cells are present in the biological sample. On the other hand, the lower it is, the less or no highly malignant HTLV-1 infected cells are present in the biological sample. Furthermore, subjects with many highly malignant HTLV-1 infected cells in the biological sample can be judged to have a high risk of developing ATL, a high risk of progression of ATL, or a poor prognosis. On the other hand, subjects with few or no high-grade HTLV-1-infected cells in the biological sample are considered to have a low risk of developing ATL, a low risk of developing ATL, or a good prognosis. can judge.
In some embodiments, prognostication, ie prediction of onset and progression of ATL, is performed as follows. If the frequency of DNA methylation in each gene or combination of genes in the HTLV-1 carrier state is X% (the β value is X _β ), the probability of developing ATL is determined to be A%. In addition, if the frequency of DNA methylation in each gene or combination of genes in the state of low-grade ATL is Y% (Y _β in the β value), the probability of transition to high-grade ATL is determined to be B%. . At this time, the specific numerical values of the frequency of methylation and the probability of onset or progression can be set and adjusted as a methylation score by accumulating cases.

Here, "frequency of DNA methylation" refers to the proportion (%) of methylated cytosine, guanine, adenine, and thymine in DNA in the genome. The term "methylation frequency of CpG sequences" refers to the ratio (%) of methylated cytosines among the cytosines in the CpG sequences.

In some aspects, these frequencies can be expressed as a number from 0-100%. For example, if cytosine is methylated in one CpG sequence, the frequency of methylation of the CpG sequence is 100%, whereas if cytosine is demethylated in one CpG sequence, the CpG sequence The frequency of methylation of is 0%. The methylation frequency of a CpG sequence is obtained by summing the methylation frequencies of each CpG sequence and dividing the sum by the number of CpG sequences when the methylation frequency is determined for two or more CpG sequences. It may be an average value. That is, if Y (Y ≥ 1) CpG sequences out of X (X ≥ 2) CpG sequences in a certain region are methylated, the methylation frequency Z% of the CpG sequences in this region is given by the formula Z (%) = [100 (%) x Y (pieces)]/X (pieces).

Further, the methylation frequency in one CpG sequence or the methylation frequency in two or more CpG sequences may be an average value of methylation frequencies obtained by two or more measurements. That is, when Zi% is the methylation frequency in the i-th (i=1, 2, . Z(%)=[Z1(%)+Z2(%)+...+Zi(%)+...+Zn(%)]/n (times).

In some other aspects, the frequency of methylation is indicated by the β value. Note that the β value is calculated by the following formula.
β value = (maximum fluorescence value of methyl detection probe)/(maximum fluorescence value of non-methyl detection probe + maximum fluorescence value of methyl detection probe + 100)

DNA methylation detected as described above is used as an ATL detection marker, but in making a diagnosis that a subject has or is at risk of developing adult T-cell leukemia/lymphoma (ATL). In addition to the detection results of DNA methylation, the results of detection of other markers, quantification of infectious virus amount, blood morphology test, soluble IL-2 receptor level, etc. are combined to comprehensively It is also possible to make a diagnosis by making a judgment. In this way, multiple detection results such as DNA methylation and ATL detection results with other markers, antibody tests, infectious viral load quantification, blood morphology tests, soluble IL-2 receptor levels, etc. By combining test results, it is possible to make a more accurate diagnosis of developing or likely to develop ATL.

Moreover, the detection results obtained by the method for detecting ATL are also useful for predicting the therapeutic effect of administering a preventive or therapeutic agent for ATL. Therefore, the following methods for preventing or treating ATL are also provided here.

A method for preventing or treating ATL, comprising administering a preventive or therapeutic agent for ATL to a subject indicated to be developing or likely to develop ATL by a method for detecting ATL.

Prophylactic or therapeutic agents for ATL include, for example, DNA demethylating agents; immunomodulators such as lenalidomide; anti-CCR-4 antibody drugs such as mogamulizumab; immune checkpoint inhibitors such as anti-PD-1/PD-L1 antibody drugs drugs can be mentioned. In some aspects, the agent for preventing or treating ATL is a DNA demethylating agent. Examples of DNA demethylating agents include azacytidine, decitabine, guadecitabine (SGI-110), and ASTX727.

3. Adult T-Cell Leukemia/Lymphoma (ATL) Testing Kit Also provided herein is an adult T-cell leukemia/lymphoma (ATL) detection kit. The kit may be used in the methods for detecting ATL described above.

The kit comprises genes selected from SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL34 and BCL9. and reagents for detecting the presence or absence of DNA methylation of at least one gene. DNA methylation of genes is as described above.

In some embodiments, reagents can include probes that include sequences complementary to DNA methylation regions of the at least one gene and can detect the presence or absence of DNA methylation.

In some other embodiments, the reagents can include primers for amplifying DNA methylated regions of said at least one gene.

The reagents may further include at least one selected from bisulfite reagents, instructions for use, and the like.

The kit contains at least one set, or at least two sets for each gene.

Bisulfite reagents are reagents for converting unmethylated cytosines to uracil, and may include Na bisulfite, scavengers such as hydroquinone, and the like.

The instructions for use may include protocols for testing DNA methylation, and the like.

Kits may further include phenol/chloroform or phenol/chloroform/isoamyl alcohol solutions, restriction enzymes, etc. for DNA extraction and restriction digestion.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

In order to extract DNA methylation abnormal regions that are particularly important in the pathogenesis of ATL, healthy subjects (3 subjects), HTLV-1 infected subjects (3 subjects), smoldering ATL patients (3 subjects), chronic ATL patients ( 4 persons), peripheral blood mononuclear cells were separated from the peripheral blood of acute ATL patients (3 persons) using a lymphocyte separation medium (MP Biomedical) according to the instruction manual, and the cell surface proteins CADM1 and CD7 were isolated. A normal T cell fraction (P fraction) and an HTLV-1-infected T cell fraction (D, N fractions) were sorted using a cell sorter (BD Biosciences). This cell sorting method is described in detail in a paper by Kobayashi et al. in 2014 (Kobayashi et al., Clin Cancer Res (2014), 20(11), p.2851-61). This cell sorting method takes advantage of changes in the expression of CADM1 and CD7 on the surface of T cells that correlate well with the progression of ATL (Fig. 1).

Here, as shown in FIG. 1, the period from the infection of T cells with HTLV-1 to the onset of ATL is several decades, which is extremely long. More than 95% of those infected with HTLV-1 end their lives as asymptomatic carriers (AC), but 3-5% of those infected develop ATL. ATL is classified into Smoldering (S), Chronic (C), Acute (A), and Lymphoma (L) according to its clinical pathology. Acute, lymphoma, and some chronic forms with poor prognostic factors are highly malignant. On the other hand, the smoldering type and the chronic type without poor prognostic factors are regarded as low-grade ATL that follows a relatively indolent course. Among these, asymptomatic patients with high proviral load (AC), smoldering ATL patients (S), chronic ATL patients (C), acute ATL patients (A), and lymphoma ATL patients (L) peripheral Analysis of cell surface proteins of blood mononuclear cells has shown that the aforementioned HTLV-1-infected T cell fractions (D, N fractions) increase as the disease progresses. On the other hand, HTLV-1 non-infected cells are sorted into the normal T cell fraction (P fraction) regardless of HTLV-1 non-infected or infected individuals.

Next, DNA was extracted from each cell, and comprehensive DNA methylation analysis was performed using Infinium Human Methylation 450K BeadChip and MethylationEPIC BeadChip (Illumina). Using the obtained methylation profile, hierarchical cluster analysis was performed on the abnormal DNA methylation specifically observed in HTLV-1-infected cells, and regions with hypermethylated DNA that well reflected the progression of the disease were extracted. (Figs. 2 and 3). In 2015, Kataoka et al. reported on DNA methylation abnormal regions in ATL from comprehensive analysis using peripheral blood mononuclear cell DNA derived from healthy subjects and ATL patients (Kataoka et al., Nat Genet (2015) , 47(11), p1304-15), in this analysis, by comparing normal T cells and HTLV-1-infected cells in the same patient, the DNA methylation abnormal region specific to HTLV-1-infected cells was accurately identified. I was able to extract it.

Here, the flowchart of FIG. 2 will be described. A flow chart shows a method for extracting 22 genes of interest in this example. First, using the Infinium HumanMethylation 450K BeadChip manufactured by Illumina Inc., about 480,000 DNA methylation nucleotide sites were obtained. The β value of the N fraction was compared between 3 patients with smoldering ATL and 4 patients with chronic ATL. 12,025 probes with increased DNA methylation and 33,581 probes with decreased DNA methylation were extracted in common in the N fraction of 7 low-grade ATL patients. Hierarchical cluster analysis using β values of hypermethylated probes formed clusters that better reflected pathology. Therefore, probe 1,207 contained within 200 bp upstream (TSS200) from the transcription initiation site, which is particularly important for gene expression control, was further extracted. Hierarchical cluster analysis using the β values of those probes formed clusters that particularly reflected the progression of pathology.

Also, cluster formation in FIG. 3 will be described. In a hierarchical cluster analysis using the β value of the probe contained in TSS200, which was commonly hypermethylated in seven low-grade ATL patients, healthy subjects, HTLV-1 infected subjects, ATL The P fraction was included in all patients. On the other hand, the acute N-fraction and some low-grade ATL N-fractions formed clusters with particularly enhanced methylation. Moderately methylated clusters included HTLV-1-infected individuals, smoldering ATL, and D and N fractions of chronic ATL. In addition, it is understood that the average β value of each fraction increases stepwise with the progression of pathology, and that there is a strong correlation between the progression of pathology and hypermethylation of DNA in a specific region.

HTLV-1 infected cells were isolated by the same cell fractionation method, gene expression analysis data (GSE55851) was downloaded from the NCBI GEO database, expressed in normal T cells, and expressed in HTLV-1 infected cells. Approximately 400 repressed genes were extracted. Compared with the approximately 900 genes included in the aforementioned HTLV-1-specific DNA hypermethylation region, 22 common genes (SERPINB6, NELL2, THEEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL34, BCL9) were identified (Fig. 4).

Here, the hierarchical clustering analysis of FIG. 4 will be described. For the 22 genes described above, gene expression data was downloaded from the Gene Expression Omnibus database of NCBI (GSE55851), and hierarchical cluster analysis was performed based on gene expression levels.

Hierarchical clustering analysis using the expression profiles of the selected 22 genes revealed that the pathology was reflected in the same way as the DNA methylation status (Fig. 4). It is thought to be suppressed, thereby contributing to the proliferation of HTLV-1-infected cells, that is, to the onset and progression of ATL. The 22 genes actually include the following factors that suppress the T cell receptor signaling pathway (TCR pathway) that promotes proliferation of T cells, and tumor suppressors.

Factors that suppress the TCR pathway (i) THEEMIS (Paster et al., EMBO J (2015), 34(3), p.393-409; Kinosada et al., PLoS Pathog (2017), 13(1), p. .e1006120)
(ii) LAIR1 (Kinosada et al., PLoS Pathog (2017), 13(1), p.e1006120)
(iii) RNF130 (Guais et al., Gene (2006), 374, p.112-20; Nurieva et al., Immunity (2010), 32(5), p.670-80.)),

Tumor suppressor (iv) C2orf40 (Li et al., Int J Cancer (2009), 125(7), p.1505-13)
(v) FHIT (Pichiorri et al., Clin Cancer Res (2006), 12(11Pt 1), p.3494-501)

Since the DNA methylation status of the 22 gene clusters correlates well with the onset and progression of ATL, it is possible to develop ATL among HTLV-1 infected individuals by measuring the DNA methylation status of these gene clusters. It is effective in identifying 3-5% of infected people. It is also useful for predicting the therapeutic effect of administering a DNA demethylating agent that is expected to be effective in preventing and treating the onset of ATL.

As described above, measuring the methylation status of the 22-gene group is useful for identifying HTLV-1-infected cells, and increases in correlation with the progression of disease. For this reason, for example, measuring the methylation state of all peripheral blood mononuclear cells is considered to be an indication of the extent to which HTLV-1-infected cell clones with particularly high malignant potential are present in peripheral blood. Therefore, among HTLV-1-infected individuals, those infected with high methylation status of 22 gene clusters have many high-grade cell clones and are expected to have a high risk of developing ATL.

Claims (4)

SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL934 and BCL9 genes in a subject biological sample comprising measuring the presence or absence of DNA methylation of at least one gene selected from the group consisting of: DNA methylation of the at least one gene indicates that the subject has adult T-cell leukemia/lymphoma ( A method for detecting ATL, which indicates that ATL has developed or is likely to develop. 2. The method of claim 1, wherein DNA methylation of said at least one gene is measured by bisulfite sequencing. the at least one gene is
(1) a combination containing SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL34 and BCL9 genes ,
(2) a combination comprising the LYPD3, THEEMIS, NELL2, and C2orf40 genes;
(3) combinations comprising LYPD3, THEMIS, NELL2, C2orf40, HOOK1, FHIT, CD7, MDS2, and LAIR1 genes;
(4) a combination comprising the LYPD3, THEMIS, NELL2, C2orf40, HOOK1, FHIT, CD7, MDS2, LAIR1, RNF130, POMC, and BCL9 genes, or (5) LYPD3, THEMIS, NELL2, C2orf40, HOOK1, FHIT, CD7, 3. The method of claim 1 or 2, which is a combination comprising the MDS2, LAIR1, RNF130, POMC, BCL9, LZTFL1, ZIK1, and TMEM45B genes. SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL34 and BCL9 genes selected from the group consisting of Adult T-cell leukemia/lymphoma (ATL) detection kit comprising reagents for detecting the presence or absence of DNA methylation of the promoter CpG of at least one gene.

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