CN113498436A

CN113498436A - Detection method of adult T cell leukemia/lymphoma

Info

Publication number: CN113498436A
Application number: CN202080015975.5A
Authority: CN
Inventors: 渡边达郎; 末冈荣三朗; 木村晋也; 嬉野博志
Original assignee: Saga University NUC; Ohara Pharmaceutical Co Ltd
Current assignee: Saga University NUC; Ohara Pharmaceutical Co Ltd
Priority date: 2019-03-01
Filing date: 2020-02-28
Publication date: 2021-10-12
Also published as: WO2020179646A1; JP7105460B2; JPWO2020179646A1

Abstract

The present invention provides a method for detecting ATL comprising detecting the presence or absence of DNA methylation of at least one gene 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 genes in a biological sample of a subject, said DNA methylation of at least one gene being indicative of a subject having or at risk of having adult T cell leukemia/lymphoma (ATL). Thus, a novel method for detecting adult T cell leukemia/lymphoma (ATL) is provided.

Description

Detection method of adult T cell leukemia/lymphoma

Technical Field

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

Background

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell tumor caused by T-cell infection with the retrovirus Human Lymphotropic Virus type I (HTLV-1). The main infection routes of HTLV-1 include lactation, sexual intercourse, blood transfusion, etc., while those infected with HTLV-1 are distributed in japan, the caribbean coastal region, central africa, south america, etc., and the number of infected persons is estimated to be 500 to 2000 thousands (non-patent document 1). It is estimated that there are 11O thousands of japanese HT LV-1 infected persons (non-patent document 1), of which 40 to 50% live in kyushu and okinawa areas, but in recent years, there is a tendency that there are more HTLV-1 infected persons who live in large cities such as osaka, tokyo, and erichia.

T cells infected with HTLV-1 undergo a multi-step oncogenic process that acquires clonal proliferation potential while accumulating genetic mutations and epigenetic abnormalities. Therefore, the period from infection to onset of ATL is long, and therefore, the elderly carriers have a characteristic that many ATL-affected patients are present (non-patent document 1). On the other hand, most HTLV-1 infected patients belong to asymptomatic patients for the whole life, but 3-5% of infected patients suffer from ATL, which is classified into stasis type, chronic type, acute type and lymphoma type according to clinical pathology. Acute and lymphoma types have poor prognosis factors, and some chronic types have high malignancy and poor prognosis. In contrast, the stasis and chronic forms without prognostic badness are relatively slow-progressing, low-malignancy ATL, and generally do not undergo direct therapeutic intervention. However, about half of patients with ATL of low malignancy have acute transformation during the course of disease, and their long-term prognosis is poor (non-patent document 2). Therefore, development of effective methods for preventing and treating the onset of ATL is desired.

Documents of the prior art

Non-patent document

Non-patent document 1: lshitsuka et al, Lancet Oncol (2014), 15(11), p.e517-26

Non-patent document 2: takasaki et al, Blood (2010), 115(22), p.4337-43

Disclosure of Invention

Problems to be solved by the invention

In the above-mentioned background, development of a new method for detecting adult T-cell leukemia/lymphoma (ATL) and the like is required.

Problem solving scheme

After many intensive studies, the inventors of the present invention have completed the present invention based on the findings that the DNA methylation status of SERPINB6, NELL2, THEMIS, ZSC AN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, lhl kl 34, and BCL9 genes reflect the pathology of ATL, and that methylation of at least 1 of the above genes is a marker, it can be detected that a subject has ATL or is at risk of ATL.

Here, the following is provided.

[1-1] an ATL detection method comprising detecting the presence or absence of DNA methylation of at least one gene selected from the group consisting of SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, TFL1, KLHL34 and BCL9 genes, which indicates that a subject is suffering from or at risk of suffering from adult T cell leukemia/lymphoma (ATL), in a biological sample of the subject.

[1-2] the method according to the above [1-1], wherein the DNA methylation of the at least one gene is detected by bisulfite sequencing.

[1-3] the method according to the above [1-1] or [1-2], wherein

Said at least one gene is

(1) Including 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) comprising a combination of LYPD3, THEMIS, NELL2 and C2orf40 genes,

(3) comprises a combination of LYPD3, THEMIS, NELL2, C2orf40, HOOK1, FHIT, CD7, MDS2 and LAIR1 genes,

(4) comprises a combination of LYPD3, THEMIS, NELL2, C2orf40, HOOK1, FHIT, CD7, MDS2, LAIR1, RNF130, POMC and BCL9 genes, or

(5) Including combinations of LYPD3, THEMIS, NELL2, C2orf40, HOOK1, FHIT, CD7, MDS2, LAIR1, RNF130, POMC, BCL9, LZTFL1, ZIK1, and TMEM45B genes.

[2-1] A kit for detecting adult T-cell leukemia/lymphoma (ATL), comprising an agent for detecting the presence or absence of DNA methylation of CpG that is a promoter of at least one gene selected from the group consisting of SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FH IT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL34 and BCL9 genes.

[2-2] the kit according to the above [2-1], for detecting DNA methylation of the at least one gene by bisulfite sequencing.

[2-3] the method according to the above [2-1] or [2-2], wherein the at least one gene is

(2) comprising a combination of LYPD3, THEMIS, NELL2 and C2orf40 genes,

[3-1] A method for preventing or treating ATL, which comprises administering an ATL preventive or therapeutic agent to a subject who is indicated to have or be at risk of having ATL by the method for detecting ATL according to any one of [1-1] to [1-3 ].

Advantageous effects of the invention

In several embodiments of the present invention, novel methods of detecting ATL are provided.

Drawings

FIG. 1 is a graph showing the onset, progression and changes in expression of cell surface markers (CADM1 and CD7) of ATL.

FIG. 2 is a flow chart for extracting DNA methylation abnormality regions reflecting ATL pathology.

FIG. 3 is a graph showing cluster formation reflecting the pathological development of ATL by methylation profiles of HTLV-1-specific DNA methylation-aggravated regions.

FIG. 4 is a view showing the results of extracting an expression abnormality gene group having DNA methylation abnormality using a gene expression database.

FIG. 5-1 shows examples of CpG sites in each gene as a subject for detecting methylation.

FIG. 5-2 shows examples of CpG sites in each gene as a subject for detecting methylation.

FIGS. 5 to 3 show examples of CpG sites in each gene as a subject for detecting methylation.

Detailed Description

The present invention will be described in detail below.

All documents and publications mentioned in the present specification are incorporated herein by reference in their entirety for all purposes.

1. Summary of the invention

In the examples described later, the regions of abnormal DNA methylation specific to cells infected with HTLV-1 were extracted with high accuracy by comparing normal T cells and cells infected with HTLV-1 in the same patient (FIGS. 1 to 3). The extracted DNA methylation abnormality region includes about 900 genes. Next, about 400 genes, whose expression was suppressed in HTLV-1-infected cells, were extracted. In comparison with about 900 genes contained in the HTLV-1-specific DNA methylation abnormality aggravated region described above, 22 identical genes were identified (SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, LZNF 717, ZNL 1, KLHL34, BCL9) (FIG. 4). Since hierarchical clustering analysis was performed using the expression profiles of the selected 22 genes, pathology was reflected as well as DNA methylation status (fig. 4), and it was found that expression of these gene groups was suppressed due to increased DNA methylation, thereby promoting proliferation of cells infected with HTLV-1, i.e., onset and progression of pathology.

Herein, provided are methods of detecting ATL, and the like, comprising detecting in a biological sample of a subject the presence or absence of DNA methylation of at least one gene selected from SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, Z1K1, LYPD3, TMEM45B, POMG, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL34, and BCL9 genes, the DNA methylation of the at least one gene being indicative of a subject having or at risk of having adult T cell leukemia/lymphoma (ATL). The detection method of ATL and the like are described in detail below.

2. Method for detecting adult T cell leukemia/lymphoma (ATL)

The detection methods for ATL provided herein include methods for detecting the presence or absence of DNA methylation of at least one gene selected from SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, H00K1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL34, and BCL9 genes in a biological sample of a subject. In several embodiments described below, DNA methylation of the at least one gene is used as an ATL detection marker.

An "adult T cell leukemia/lymphoma" or "ATL" is a peripheral T cell tumor caused by T cell infection with retroviral Human T Lymphotropic Virus type I (Human T-cell Lymphotropic Virus type-1, HTLV-1). ATL is classified into stasis type, chronic type, acute type and lymphoma type according to clinical pathology.

A "subject" is a mammal including a human, such as a human and a monkey, preferably a human.

The "biological sample" is not particularly limited, and it is sufficient if DNA methylation of a gene in a T cell can be detected. Examples of the biological sample include (i) peripheral blood (whole blood), (ii) peripheral blood mononuclear cells (a mixture containing CD 4-positive T cells and CD 8-positive T cells) isolated from peripheral blood, (iii) T cells isolated from peripheral blood or peripheral blood mononuclear cells, or isolated from a sample other than peripheral blood or peripheral blood mononuclear cells, and (iv) free DNA in plasma or serum.

"T cell" also known as T lymphocyte refers to a cell derived from the thymus among lymphocytes involved in an immune response. T cells include any of regulatory T cells such as CD8 positive T cells (cytotoxic T cells: CTL), CD4 positive T cells (helper T cells), suppressive T cells, and regulatory T cells, effector cells, naive T cells, memory T cells, a β T cells expressing α chain and β chain TCRs, or γ δ T cells expressing γ chain and δ chain TCRs. T cells include progenitor cells of T cells that have been committed to differentiate into T cells.

The "peripheral blood or a sample other than peripheral blood mononuclear cells" is, for example, a skin lesion, a lymph node lesion, a bone marrow cell, or the like.

"DNA methylation" refers to the phenomenon that the carbon atom at position 5 of cytosine (C) is hypermethylated in the sequence of a CpG island (also referred to as "promoter CpG island") in an untranslated region, particularly a promoter region, of a gene on a target genome. CpG islands are regions of the subject's genomic sequence that are rich in cytosine (C) guanine (G) present in untranslated regions, and typically include repeats of dinucleotides composed of CG.

In each gene, the region in which DNA methylation is detected is determined, for example, as follows. In a human methylation450K Chip (Infinium methylation450K Bead Chip) manufactured by illumina used for whole genome DNA methylation analysis, quantitative CpG site information was obtained from oligonucleotide probe information for quantifying DNA methylation of each gene. DNA methylation at the site or at a CpG site in the vicinity thereof is quantified by bisulfite pyrosequencing or the like. The effectiveness of determining their CpG sites is confirmed using standard DNA of known methylation rate and the region to detect DNA methylation is determined. In some embodiments, comparing normal T cells to those infected with HTLV1, CpG sites with a change in β value above 0.2 are identified as Differential Methylation sites (DMP), the effective Positions when judged to determine CpG sites (Cancer Lett. (2013)330(1), p.33-40). Preferably, normal T cells and HTLV infected T cells in the same patient at this time are compared to determine changes in the beta values.

The gene as a detection target in the detection method is at least one gene selected from the group consisting of SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL34 and BCL9 genes.

Examples of human genes are shown below. In addition, the corresponding gene sequences of mammals other than humans are available, for example, from GenBank (NCBI) database and the like.

The SERPINB6 human base sequence was registered with GenBank (NCBI, usa) under the registration number "5269". FIG. 5 shows an example of the vicinity of promoter CpG including the human SERPINB6 gene (SEQ ID NO: 1. about.3). In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

NELL2 human nucleotide sequence was registered in GenBank (NCBI) under the accession number "4753". FIG. 5 shows an example of the vicinity of CpG of a promoter including the human NELL2 gene (SEQ ID NO: 4). In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

The THEMIS human base sequence was registered in GenBank (NCBI) under the accession number "387357". FIG. 5 shows an example of the vicinity of CpG including the promoter of the human THEMIS gene (SEQ ID Nos. 5 to 6). In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

ZSCAN18 human base sequence is registered in GenBank (NCBI) with the registration number of "65982". FIG. 5 shows an example of the sequences (SEQ ID NO: 7-11) including the vicinity of the promoter CpG island of the human ZSCAN18 gene. In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

The LAIR1 human base sequence was registered in GenBank (NCBI) under the accession number "3903". FIG. 5 shows an example of the sequences including the vicinities of promoter CpG of the human LAIR1 gene (SEQ ID NOS: 12 to 15). In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

The CD7 human base sequence was registered in GenBank (NCBI) under the registration No. "924". FIG. 5 shows an example of the vicinity of promoter CpG including the human CD7 gene (SEQ ID NO: 16-17). In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

The RNF130 human base sequence was registered in GenBank (NCBI) under the accession number "55819". FIG. 5 shows an example of the vicinity of a promoter CpG island including the human RNF130 gene (SEQ ID NO: 18). In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

ZIK1 human nucleotide sequence is registered in GenBank (NCBI) under the accession number "284307". FIG. 5 shows an example of the vicinity of a promoter CpG island including the human ZIK1 gene (SEQ ID NO: 19 to 21). In the sequence of fig. 5, an example of a target cpg sequence for detecting DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

LYPD3 human base sequence is registered in GenBank (NCBI) under the accession number "27076". FIG. 5 shows an example of the vicinity of CpG of a promoter including the human LYPD3 gene (SEQ ID NO: 22). In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

TMEM45B human base sequence was registered in GenBank (NCBI) under the accession number "120224". FIG. 5 shows an example of the vicinity of promoter CpG including the human TMEM45B gene (SEQ ID NO: 23-24). In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

The POMC human base sequence is registered in GenBank (NCBI) under the registration number "5443". FIG. 5 shows an example of the sequences (SEQ ID NOS: 25 to 27) including the vicinities of promoter CpG islands of the human POMC gene. In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

FHIT human base sequence was registered in GenBank (NCBI) under the accession number "2272". FIG. 5 shows an example of the nearby sequence of the promoter CpG island including the human FHIT gene (SEQ ID NO: 28). In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

MDS2 human nucleotide sequence is registered in GenBank (NCBI) under the registration number of "259283". Fig. 5 shows an example of the vicinity of the promoter CpG including the human MDS2 gene (sequence No. 29). In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

The base sequence of HOOK1 is registered in GenBank (NCBI) under the registration number "51361". FIG. 5 shows an example of the vicinity of CpG of a promoter including the human HOOK1 gene (SEQ ID NO: 30). In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

The S0RCS3 human base sequence was registered in GenBank (NCBI) under the accession number "22986". FIG. 5 shows an example of the vicinity of a promoter CpG island including the human SORCS3 gene (SEQ ID NO: 31 to 32). In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

The C2orf40 human base sequence was registered in GenBank (NCBI) under the accession number "84417". FIG. 5 shows an example of the vicinity of a promoter CpG island including the human C2orf40 gene (SEQ ID NO: 33 ~ 36). In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

ZNF662 human base sequence is registered in GenBank (NCBI) under the registration number "389114". FIG. 5 shows an example of the sequences (SEQ ID NO: 37) including the vicinity of the promoter CpG island of the human ZNF662 gene. In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

The SPG20 human base sequence was registered in GenBank (NCBI) under the accession number "23111". Fig. 5 shows an example of the vicinity of a promoter CpG island (sequence No. 38) including the human SPG20 gene. In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

ZNF717 human nucleotide sequence is registered in GenBank (NCBI) under the registration number "100131827". FIG. 5 shows an example of the sequences (SEQ ID NO: 39-40) including the vicinity of the promoter CpG island of the human ZNF717 gene. In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of target CpG sequences for detection of DN a methylation in preferred embodiments are further indicated in parentheses.

The LZTFL1 human base sequence was registered in GenBank (NCBI) under the accession number "54585". FIG. 5 shows an example of the sequence around the CpG island of the promoter including the human LZTFL1 gene (SEQ ID NO: 41-42). In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

The KLHL34 human nucleotide sequence was registered in GenBank (NCBI) under the accession number "257240". FIG. 5 shows an example of the vicinity of a promoter CpG island including the human KLHL34 gene (SEQ ID NO: 43-44). In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

The BCL9 human base sequence was registered in genbank (ncbi) under the accession number "607". FIG. 5 shows an example (SEQ ID NO: 45) of a sequence including the vicinity of promoter CpG of the human BCL9 gene. In the sequence of FIG. 5, an example of a target CpG sequence for detection of DNA methylation is underlined. Examples of the CpG sequences of interest for detection of DNA methylation in the preferred embodiment are further indicated in parentheses.

In several embodiments, in at least one gene selected from the group consisting of SERPINB6, NELL2, THEMIS, ZSCAN18, LAlR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, hl kl 34, and BCL9 genes, the CpG sequence of interest for detecting DNA methylation is, for example, all or a portion of the CpG sequence underlined in fig. 5.

The measurement of DNA methylation can be carried out, for example, as follows.

Genomic DNA is extracted from a biological sample using known methods. When the biological sample is a body fluid, after removing solids by a separation method such as filtration through a filter or centrifugation, the supernatant is extracted by a known method such as a phenol/chloroform method, or a genomic DNA is extracted using a commercially available kit. When the biological sample is a tissue, purified water is added to homogenize the mixture, and after removing solids by the same separation method, the supernatant is extracted by a known method such as phenol/chloroform method, or a commercially available kit is used to extract genomic DNA. The extracted DNA is then used for methylation detection.

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

Examples of a method for analyzing DNA Methylation using the extracted genomic DNA include Bisulfite sequencing, pyrosequencing, Methylation Specific PCR (MSP), quantitative MSP, COBRA (Combined Bisulfite Restriction Analysis), or single-base typing by Tm Analysis using a Quenching probe (Quenching probe). In the above methods, the respective analysis ranges, sensitivities, accuracies, and necessary instruments are greatly different. The above methods all use the principle of converting only unmethylated cytosines to uracil by bisulfite treatment.

The above DNA methylation assay is described.

"bisulfite sequencing" is a process by which unmethylated cytosines (unmethylated cytosines) in DNA are converted to uracil by bisulfite treatment of the DNA. In contrast, methylated cytosines are not converted by bisulfite treatment. Thus, methylated cytosines can be distinguished from unmethylated cytosines by this treatment. Then, DNA methylation can be analyzed by means of, for example, determining the nucleotide sequence of the DNA after transformation.

The "pyrosequencing method" is a method of decoding a base sequence by detecting a luciferase luminescence reaction generated by a polymerase base extension reaction, and can perform quantitative analysis with high accuracy because pyrophosphate generated when the extension reaction is performed is in a completely proportional relationship with the luminescence intensity. By this principle, the T%/C% of CpG sites of DNA subjected to bisulfite treatment was decoded, and the ratio of DNA methylation was determined.

The "methylation-specific PCR (MSP) method" is a method in which DNA is converted into bisulfite, PCR is performed using a primer that specifically amplifies methylated DNA and a primer that specifically amplifies unmethylated DNA using the DNA as a template, and the presence or absence of DNA methylation is determined based on the presence or absence of a PCR product.

The "quantitative MSP method" is a methylation-Specific PCR (methylation Specific PCR) technique, and basically performs PCR amplification of DNA treated with sodium bisulfite using primers having desired sequences Specific to a methylated region and an unmethylated region in the target gene. MSP can be quantified in real time by using multiple fluorophores (fluorophores), when using methylation region specific primers and non-methylation region specific primers.

The "COBRA method" is a method in which after bisulfite treatment, a target DNA is PCR-amplified using a primer common to methylated DNA and unmethylated DNA, then treated with a restriction enzyme that recognizes the position of a sequence difference between methylated DNA and unmethylated DNA, and fragments are concentrated by ethanol precipitation, and then the methylated DNA and unmethylated DNA are distinguished by electrophoresis. PCR amplification can be performed in the same manner as bisulfite sequencing.

The "Tm analysis method using a quenching probe" is a method in which a DNA fragment including a target CpG site is PCR-amplified from a bisulfite-converted DNA sample, and bound to a QProbe (a probe having a fluorescently labeled cytosine base at the end, which is reduced in fluorescence by binding to the DNA fragment and emits light upon dissociation) having a complementary sequence, and the dissociation temperature by QProbe differs depending on the degree of matching of the complementary sequence, and single-base typing can be performed by detecting fluorescence obtained by dissociation.

In each of the above methods, a primer set for PCR can be designed and obtained by a known method or a method in accordance therewith. For example, the primer set can be designed by entering the sequence in the item MethPrimer (http:// www.urogene.org/MethPrimer/index1.htmI) on the network. In addition, in the above-mentioned methods, a probe can be designed and obtained by a known method or a method according to the known method.

In some embodiments, the DNA methylation assay is performed using bisulfite sequencing. As a preferred example, a method using BeadArray (Infinium (registered trademark) human methylation450 chips, Humanmethylation450 Beadchips) available from illumina may be used. In this method, unmethylated cytosines (unmethylated cytosines) in DNA are converted to uracils by bisulfite treatment, thereby distinguishing methylated cytosines from unmethylated cytosines. Then, after probes immobilized on two types of magnetic beads, i.e., a methylation probe (M-type) and a non-methylation probe (U-type), which are site-specific, were hybridized, a single-base extension reaction using a labeled ddNTP was performed, and the ratio of methylation and non-methylation was calculated from the fluorescence intensity signal. Thus, the whole DNA methylation analysis can be easily performed.

In some embodiments, when 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 gene combination detected as described above is higher than the methylation frequency of DNA methylation (e.g., methylation of CpG sequences) corresponding to normal cells (e.g., higher by 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 higher by β value by 0.05 or more, 0.10 or more, 0.15 or more, 0.20 or more, 0.25 or more, 0.30 or more, 0.35 or more, 0.40 or more, 0.45 or more, or 0.50 or more), each gene can be judged to be methylated. More specifically, when the frequency of DNA methylation (e.g., methylation of CpG sequences) of each gene is higher than the frequency of genomic DNA methylation (e.g., methylation of CpG sequences) corresponding to normal cells, it can be judged that the gene is DNA-methylated.

In other embodiments, the frequency of DNA methylation (e.g., methylation of CpG sequence) of each gene or the frequency of DNA methylation (e.g., methylation of CpG sequence) of a combination of genes detected as described above can be determined to be DNA methylation when the frequency is, for example, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, or when the frequency is O.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, or 0.9 or more.

In other 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 higher than the frequency of DNA methylation (e.g., methylation of CpG sequences) corresponding to normal cells (e.g., higher by 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 β value is higher by 0.05 or more, 0.10 or more, 0.15 or more, 0.20 or more, 0.25 or more, 0.30 or more, 0.35 or more, 0.40 or more, 0.45 or more, or 0.50 or more), and when the frequency of methylation is 50% or more, 60% or more, 70% or more, 80% or more, or 90% 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, methylation by DNA can be judged.

"Normal cells" are T cells that are not infected with HTLV-1.

Of the above marker genes, the presence or absence of the onset of ATL in a subject or the possibility of the onset of ATL in the future of the subject is determined by analyzing DNA methylation of at least 1 gene (i.e., 1 gene, or a combination of genes, i.e., 2 genes, 3 genes, 4 genes, 5 genes, 6 genes, 7 genes, 8 genes, 9 genes, 10 genes, 11 genes, 12 genes, 13 genes, 14 genes, 15 genes, 16 genes, 17 genes, 18 genes, 19 genes, 20 genes, 21 genes, or 22 genes). At least one gene is, for example, 1 or at least 2 genes, for example, 4 to 9 genes, 10 to 15 genes, or 16 to 22 genes. In some embodiments, at least 1 gene is a full 22 genes.

In some embodiments, where it can be determined that at least one gene (i.e., 1 gene, or a combination of genes) is methylated by DNA, the subject can be determined to have, or be at risk of having, adult T-cell leukemia/lymphoma (ATL).

In other embodiments, the genes that resolve DNA methylation are a combination of genes, i.e., at least 2 genes (e.g., 2 genes, 3 genes, 4 genes, 5 genes, 6 genes, 7 genes, 8 genes, 9 genes, 10 genes, 11 genes, 12 genes, 13 genes, 14 genes, 15 genes, 16 genes, 17 genes, 18 genes, 19 genes, 20 genes, 21 genes, or 22 genes), and the subject can be determined to have adult T cell leukemia/lymphoma (ATL) or be at risk of developing ATL if, for example, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the at least 2 genes are methylated.

Here, examples of combinations of 2or more genes are as follows.

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

(2) Including combinations of LYPD3, THEMIS, NELL2 and C2orf40 genes (4 genes).

(3) Including LYPD3, THEMIS, NELL2, C2orf40, HOOK1, FHIT, CD7, MDS2 and LAIR1 gene combinations (9 genes).

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

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

In some embodiments, in the combinations (1) to (5), the CpG sequence of interest for detecting DNA methylation is all or a part of the CpG sequence underlined in fig. 5, for example. In the above combinations (1) to (5), the CpG sequences of interest for detecting DNA methylation are, for example, all or a part of the CpG sequences further indicated by parentheses ([ ]) in FIG. 5.

In some embodiments, the subject is judged to have a probability of, for example, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more (or β value is 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, or 0.9 or more) of ATL when 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, 90% or more, or 100%.

Or 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 the beta value is 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, or 0.9 or more), it can be judged that the subject is at risk of developing ATL, and the future incidence of the disease is, for example, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%.

The baseline value of methylation frequency, F%, for the incidence of ATL or the incidence of P% in the future can be adjusted by accumulating cases. The statistical process for adjusting the methylation frequency reference value, e.g., F%, is not limited, and any method known in the art may be used.

As shown in the examples described later, since the DNA methylation status is highly correlated with the onset and progression of ATL, the progression of ATL can be judged based on the DNA methylation frequency in each gene or combination of genes as follows. In several embodiments, the progression of ATL is judged as follows based on the frequency of DNA methylation in each gene or combination of genes. 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 degree of malignancy of ATL can be judged, and conversely, the lower the degree of malignancy of ATL can be judged.

In addition, as shown in the examples described later, since the DNA methylation status is highly correlated with the onset and progression of ATL, the DNA methylation frequency based on the whole biological sample (for example, the whole mononuclear cells in the collected peripheral blood) can be used as an index for measuring the extent to which cells infected with HTLV-1 having a high degree of malignancy are present in the biological sample (for example, in the collected peripheral blood). In some embodiments, the degree to which HTLV-1-infected cells with high malignancy are present in a biological sample is determined based on the frequency of DNA methylation in each gene or combination of genes as follows. That is, the higher the DNA methylation frequency of each gene or the DNA methylation frequency of the combination of genes, the more highly malignant cells infected with HTLV-1 are present in the biological sample, while the lower the DNA methylation frequency, the less or none of the highly malignant cells infected with HTLV-1 are present in the biological sample. Further, it is judged that the subject having a large number of cells with high malignancy infected with HTLV-1 in the biological sample is at high risk of developing ATL, or the risk of developing ATL is high, or the prognosis is poor, and on the other hand, it is judged that the subject having a small number of cells with high malignancy infected with HTLV-1 in the biological sample or having no cells with high malignancy infected with HTLV-1 is at low risk of developing ATL, or the prognosis is good.

In some embodiments, prognosis is predicted in a manner that predicts ATL onset and progression. The frequency of DNA methylation in each gene or combination of genes was X% in the state of carrying HTLV-1 (beta value is X)_β) Then, the incidence of ATL is judged to be A%. In addition, in the state of low ATL malignancy, the frequency of DNA methylation in each gene or gene combination is Y% (the value of. beta. is Y)_β) In this case, the probability of conversion to ATL with high malignancy was judged to be B%. In this case, the specific values of methylation frequency and incidence or progression can be collectively set and adjusted by accumulating cases as methylation scores (score).

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

In some embodiments, the frequency can be represented by a number from 0 to 100%. For example, in the case of methylation of cytosine in 1 CpG sequence, the methylation frequency of CpG sequence is 100%, whereas in the case of demethylation of cytosine in 1 CpG sequence, the methylation frequency of CpG sequence is 0%. When the methylation frequency of 2or more CpG sequences is determined, the methylation frequency of CpG sequences may be an average value obtained by summing up the methylation frequencies of CpG sequences and dividing the sum by the number of CpG sequences. That is, when Y (Y.gtoreq.1) CpG sequences have been methylated among X (X.gtoreq.2) CpG sequences in a certain region, the methylation frequency Z% of the CpG sequences in the region can be determined by the formula Z (%) - (100 (%) xY/X (s)).

The methylation frequency of the 1 CpG sequence or the methylation frequency of the 2or more CpG sequences may be an average value of the methylation frequencies obtained by measuring 2or more times. That is, when the methylation frequency in the i-th measurement (i ═ 1, 2, …, n) is Zi% in n measurements (n.gtoreq.2), the methylation frequency Z% can be determined by the formula Z (%) - (Z1 (%) + Z2 (%) + … + Zi (%) + … + Zn (%)/n (th).

In several other embodiments, the frequency of methylation is represented by the beta value. The β value is calculated by the following equation.

The value of β is (maximum value of fluorescence value of probe for methyl group detection)/(maximum value of fluorescence value of probe for non-methyl group detection + maximum value of fluorescence value of probe for methyl group detection +100)

As described above, the detected DNA methylation is used as an ATL detection marker, but when a subject is diagnosed as having or likely to have adult T-cell leukemia/lymphoma (ATL), the diagnosis can be made by performing comprehensive judgment in addition to the detection result of DNA methylation, in combination with the detection results using other markers, such as the quantification of the amount of infectious virus, the blood morphology test, and the amount of soluble IL-2 receptor. In this manner, possible diagnoses of patients with ATL or ATL can be more accurately performed by combining a plurality of test results such as the result of detection of ATL using DNA methylation and other markers, antibody test, quantification of infectious virus amount, blood morphology test, and test result such as the amount of soluble lL-2 receptor.

In addition, the detection result obtained by the detection method of ATL can be used for prediction of the therapeutic effect in prevention of ATL or administration of a therapeutic agent. Accordingly, also provided herein are methods of preventing or treating the following ATL.

The method for preventing or treating ATL comprises administering a prophylactic or therapeutic agent for ATL to a subject who is indicated to have ATL or is at risk of developing it by the ATL detection method.

Examples of the preventive or therapeutic agent for ATL include DNA demethylating agents, immunomodulators such as lenalidomide, anti-CCR-4 antibody agents such as Mogamulizumab, and immune checkpoint inhibitors such as anti-PD-1/PD-L1 antibody agents. In some embodiments, the ATL prophylactic or therapeutic agent is a DNA demethylating agent. Examples of the DNA demethylating agent include azacytidine, decitabine, Guadecitabine (SGI-110), and ASTX 727.

3. Adult T cell leukemia/lymphoma (ATL) examination kit

In addition, provided herein is an adult T-cell leukemia/lymphoma (ATL) test kit. The kit can be used for the detection method of the ATL.

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

In some embodiments, the reagent comprises a sequence complementary to a DNA methylation region of the at least one gene, and may comprise a probe capable of detecting the presence or absence of DNA methylation.

In other embodiments, the reagents may include primers for amplifying the DNA methylation region of the at least one gene.

The reagent may further comprise a bisulfite reagent, at least 1 selected from instructions for use, and the like.

At least one group, or at least two groups, of each gene is included in the kit.

The bisulfite reagent is a reagent for converting unmethylated cytosine to uracil, and may include sodium bisulfite (Na bisulfate), a scavenger (hydroquinone, etc.), and the like.

The instructions may describe protocols for detecting DNA methylation, etc.

The kit may further comprise a phenol/chloroform solution or a phenol/chloroform/isoamyl alcohol solution, a restriction enzyme, etc. for extracting DNA and restriction digestion.

Examples

The present invention is further specifically illustrated by the following examples. However, the present invention is not limited to the following examples.

In order to extract a DNA methylation abnormality region important for the pathological formation of ATL, peripheral blood mononuclear cells were isolated from peripheral blood of healthy persons (3), HTLV-1 infected persons (3), pooled ATL patients (3), chronic ATL patients (4), acute ATL patients (3) using lymphocyte separation media (MP biological) according to the instructions for use, and a normal T cell fraction (P fraction) and a T cell fraction (D, N fraction) infecting HTLV-1 were further isolated from the expression patterns of cell surface proteins CADM1 and CD7 using a cell sorter (BD Biosciences). This cell isolation method is described in detail in a paper published by Kobayashi et al in 2014 (Kobayashi et al, Clin Cancer Res (2014), 20(11), p.2851-61). This cell isolation method utilizes the changes in the expression of CADM1 and CD7 on the surface of T cells, which are highly correlated with the development of ATL pathology (fig. 1).

Here, as shown in FIG. 1, the period from T cell infection with HTLV-1 to the onset of ATL is several decades long. More than 95% of HTLV-1 infected persons are Asymptomatic (AC) infected persons for the whole life, but 3-5% of infected persons have ATL. ATL is clinically classified into a stasis type (Smoldering; S), a Chronic type (Chronic; C), an Acute type (Acute; A), and a Lymphoma type (Lymphoma; L). Acute type and lymphoma type, and some chronic types with poor prognostic factors. In contrast, the stasis and chronic forms without poor prognostic factors are relatively slow-progressing, less malignant ATLs. Among them, the cell surface proteins of peripheral blood mononuclear cells of asymptomatic infected patients (AC), patients with pooled ATL (S), patients with chronic ATL (C), patients with acute ATL (A) and patients with lymphoma ATL (L) with high provirus were analyzed, and it was shown that the T cell fraction (D, N fraction) of HTLV-1 infection increased with the progress of the pathology. On the other hand, cells not infected with HTLV-1 were isolated into the normal T cell fraction (P fraction) regardless of whether they were non-infected or infected.

Next, DNA was extracted from each cell, and total DNA methylation analysis was performed using an Infinium Human methylation450 KBeadChip and a methylation EPIC BeadChip (Illumina). The obtained methylation map is used to perform hierarchical clustering analysis on the DNA methylation abnormality areas specifically recognized in cells infected with HTLV-1, and DNA methylation abnormality aggravation areas which sufficiently reflect pathological development are extracted (FIG. 2 and FIG. 3). In 2015, Kataoka et al reported that the DNA methylation abnormality region in ATL was completely analyzed using peripheral blood mononuclear cell DNA from healthy persons and ATL patients (Kataoka et al, Nat Genet (2015), 47(11), p1304-15), but in this analysis, the DNA methylation abnormality region specific to cells infected with HTLV-1 could be extracted with high accuracy by comparing normal T cells and cells infected with HTLV-1 in the same patient.

The flowchart of fig. 2 will be explained here. The flow chart shows the extraction method of 22 genes of interest in this example. First, the beta values of the P fraction of normal T cells which were not infected with HTLV-1 and the N fraction of the cell fraction which was infected with HTLV-1 in 3 patients of stasis-type ATL and 4 samples of patients of slow-type ATL were compared with each other at about 48 ten thousand DNA methylated nucleotide sites obtained using an Infinium Humanmethylation450K BeadChip manufactured by ill uma. 12, 025 probes common to the N-fraction of 7 patients with low-malignancy ATL and with increased DNA methylation and 33, 581 probes with reduced DNA methylation were extracted. When hierarchical clustering analysis using the beta values of methylation-enhancing probes was performed, clusters more reflecting pathology were formed. Therefore, 1, 207 probes contained within 200bp upstream (TSS200) from the transcription initiation point important for gene expression control were further extracted. Hierarchical clustering analysis using the beta values of these probes forms clusters that reflect in particular the development of pathology.

The clustering in fig. 3 will be described. In hierarchical clustering analysis using the probe β values contained in TSS200, which is common to 7 patients with low-malignancy ATL but whose methylation is exacerbated, P-moieties were included as non-methylated clusters in healthy persons, HTLV-1 infected persons, and ATL patients. On the other hand, the acute N-part and a part of the N-part of the low-malignancy ATL form clusters in which methylation is particularly exacerbated. In the moderately methylated cluster, HTLV-1 infected patients, pooled ATL, and the D, N portion of chronic ATL were mixed. In addition, the average value of β values of each fraction increases stepwise with the development of the pathology, which is strongly correlated with the progress of DNA methylation 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 GEO database at NCBI, and about 400 genes whose expression was suppressed in normal T cells and in HTLV-1-infected cells were extracted. The same 22 genes were identified (SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, LZF 717, ZNL 1, KLHL34 and BCL9) in comparison with about 900 genes contained in the HTLV-1 specific DNA methylation abnormality aggravated region (FIG. 4).

The hierarchical cluster analysis of fig. 4 will be explained here. Gene expression data (GSE55851) were downloaded from the Gene expression Omnibus database of NCB I, and the 22 genes were subjected to hierarchical clustering analysis based on the Gene expression levels.

Hierarchical clustering analysis was performed using expression profiles of 22 selected genes, and pathology was reflected in the same manner as the DNA methylation status (fig. 4), and it was considered that expression of these gene groups was suppressed due to increased DNA methylation, thereby promoting proliferation of cells infected with HTLV-1, i.e., onset and progression of pathology of ATL. In fact, among the 22 genes, the following factors that inhibit the T Cell Receptor (TCR) signaling pathway that promotes T cell proliferation, and tumor suppressor factors are included.

Agents that inhibit TCR pathways

(i)THEMIS(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 factor

(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 22 gene groups is highly correlated with the onset and progression of ATL, it is effective to identify 3 to 5% of infected individuals with ATL from HTLV-1 infected individuals by measuring the DNA methylation status of these gene groups. In addition, it can be used for predicting the therapeutic effect of a DNA demethylating agent which is effective for the prevention and treatment of the onset of ATL.

As described above, determination of the methylation status of 22 gene populations can be used to identify cells infected with HTLV-1, and is also exacerbated in association with the development of pathology. Therefore, for example, by measuring the methylation state of the whole peripheral blood mononuclear cell, it is possible to obtain an index of the degree of infection of HTLV-1, particularly a cell clone with high malignancy, in peripheral blood. Therefore, it is presumed that, among HTLV-1 infected patients, those with high methylation status of 22 gene groups had many highly malignant cell clones and the risk of developing AT L was high.

Sequence listing

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<400> 26

cggggagacg cgctggaaag gggctggaat tagcactgtc ctgccgaagg cgcacctggc 60

cgctcgccct caggaagaac ttaattatgg atggcgttgg gtctccgctt agaacgggcg 120

gg 122

<210> 27

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 27

gaaggcgcac ctggccgctc gccctcagga agaacttaat tatggatggc gttgggtctc 60

cgcttagaac gggcgggagg cttagcaccc ccgtgtgggg caggccgggc agcctctgag 120

gt 122

<210> 28

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 28

acggtagggg cgggacccaa gatggccgct tgtctgggga caggagcgga ggccaatacg 60

cgcagagcat gcgcctacgc cgggccaatt gaaagccata gtgacagtaa ccctgattca 120

gg 122

<210> 29

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 29

tcaaatggtt tttatgtggt gtggccccag atacaagcca accatttcac ctgtggtgag 60

cggaaggcgc agctatctat aacacctgag aaaaaaagaa cctgctgtac tagctgtcag 120

ct 122

<210> 30

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 30

gactggtgcg cggccaggtc actcagggcg gcggggcgag gtgtgcggga gtgggcagga 60

cgggtgggag gaggggaggg gtgggagaag gcggggctca ggtgcctggc agcgcggccg 120

gg 122

<210> 31

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 31

atttctggag gagctcagtg gggaggattg gggggaatct gattttaaga aagaaagcac 60

cgaaggggca attattaatt ttcctcgggt ttggaatcac cctctggaca agagaacggg 120

cg 122

<210> 32

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 32

ttggaggctg gatttcggag agactcggga ttggggtcta ttgccggccc ctcctggatc 60

cggcttgatt ttcatatttt aagaagattt tcttattacc tttgattact cctttttacc 120

aa 122

<210> 33

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 33

gcccccgccg ccggcggttc tccgtggcca agcatccttg gccttggagc ccaggggctg 60

cgttcccctt ggggccgggg cgggagagag gacctcggtg gtactcgccc gtgcgctggg 120

cg 122

<210> 34

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 34

ggttctccgt ggccaagcat ccttggcctt ggagcccagg ggctgcgttc cccttggggc 60

cggggcggga gagaggacct cggtggtact cgcccgtgcg ctgggcgcag ccgcttggcc 120

ct 122

<210> 35

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 35

ccagcttgtc ctaaccgctt tcgctgcggg cagcgctggc cacgcggccc ccgccgccgg 60

cggttctccg tggccaagca tccttggcct tggagcccag gggctgcgtt ccccttgggg 120

cc 122

<210> 36

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 36

tcctaaccgc tttcgctgcg ggcagcgctg gccacgcggc ccccgccgcc ggcggttctc 60

cgtggccaag catccttggc cttggagccc aggggctgcg ttccccttgg ggccggggcg 120

gg 122

<210> 37

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 37

gtgcagcgag ctggagagga aggggtggcc tagagcgcag gggaaggatt cctccccagc 60

cgcctgcacc cctaccccgg tagcggtccc tgggatcgtc cgtgtctcca ggagaaccgg 120

ac 122

<210> 38

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 38

actctgacct tctaaatggt acgtgggagg acgtccgtcc ccttcggacc caagagtcac 60

cgtaacactc tagaagggga gaaaaggagc gagggcggca ggcgacagag aacctcgcga 120

gt 122

<210> 39

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 39

acttcacttg ctaaagagac accgggaaac ccaactaata caaacgccca gtttaagact 60

cgaggcgcac gcgtttcgca ctactcctct gggaatgggg aacgtctccc gagaactgtg 120

tg 122

<210> 40

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 40

gcgcacgcgt ttcgcactac tcctctggga atggggaacg tctcccgaga actgtgtgtt 60

cgcactggga cagatgggca aactgagcgt catgcgggtt ggtaaccggc tccctcagcg 120

gc 122

<210> 41

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 41

cacgtggact gcaattatgc attttcattg gtcctcagga tcacgcgaca ggaagtattg 60

cgtaaccggt tgactgccac atgcgcattg gcttccaggg ccggaagtcc cacctttttg 120

tg 122

<210> 42

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 42

gaagtcccac ctttttgtgc cagttcactt ttgggagctg actgcagttg cagaaggtag 60

cgggagggtt gggctgcctg gagttgcgtg tggccaccaa cgcgcttcgc gcctcgtggc 120

gt 122

<210> 43

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 43

tcagagcggc gggtcagagg cgtgattggg cgccgcacat aacatgccgg tagctcacag 60

cgacaggaat agcgcgccgc ctcggcctcc cgggcgcgcc ccgccccgtg ccctgactgc 120

tg 122

<210> 44

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 44

gtcagaggcg tgattgggcg ccgcacataa catgccggta gctcacagcg acaggaatag 60

cgcgccgcct cggcctcccg ggcgcgcccc gccccgtgcc ctgactgctg gccgcgaacc 120

ga 122

<210> 45

<211> 122

<212> DNA

<213> human (Homo Sapiens)

<400> 45

gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgcctgcgt gcgcagctag gccaagcctg 60

cgagcctgcc tgtcactgga gagttttagt ttgcattcag aagaaaggag gaggggagac 120

aa 122

Claims

1. A method of detecting ATL comprising detecting the presence or absence of DNA methylation of at least one gene 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 genes in a biological sample of a subject, said methylation of at least one gene being indicative of the subject having or at risk of having adult T cell leukemia/lymphoma (ATL).

2. The method of claim 1, wherein the DNA methylation of the at least one gene is detected by bisulfite sequencing.

3. The method of claim 1 or 2, wherein

Said at least one gene is

(1) Including SERPlNB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, LZTFL1, KLHL34 and BCL9 genes,

(2) comprising a combination of LYPD3, THEMIS, NELL2 and C2orf40 genes,

(3) including LYPD3, THEMLS, NELL2, C2orf40, HOOK1, FHIT, CD7, MDS2 and LAIR1 genes,

(5) Including combinations of LYPD3, THEMIS, NELL2, C2orf40, HOOK1, FHIT, CD7, MDS2, LAIR1, RNFl30, POMC, BCL9, LZTFL1, ZIK1, and TMEM45B genes.

4. An adult T-cell leukemia/lymphoma (ATL) detection kit comprising reagents for detecting the presence or absence of DNA methylation of CpG of a promoter of at least one gene selected from SERPINB6, NELL2, THEMIS, ZSCAN18, LAIR1, CD7, RNF130, ZIK1, LYPD3, TMEM45B, POMC, FHIT, MDS2, HOOK1, SORCS3, C2orf40, ZNF662, SPG20, ZNF717, ltfl 1, lzhl 34 and BCL9 genes.