JP5522348B2 - Prognostic method and kit for adult T-cell leukemia / lymphoma - Google Patents

Description

  The present invention provides data for predicting the prognosis of adult T-cell leukemia / lymphoma (ATLL), preferably the onset of the carrier of adult T-cell leukemia virus (HTLV-I) or the malignant transformation of indolent ATLL that is the cause thereof. The present invention relates to a kit used for prognostic estimation testing, a system for prognostic estimation testing, a method for providing data for prognosis determination, and the like.

  In recent years, research on the development of genetic diagnosis techniques for hematopoietic tumors including adult T-cell leukemia / lymphoma (ATLL) has been progressing rapidly (see Non-Patent Document 1). This is because the medical understanding of hematopoietic tumors and related genes is deepened, and coupled with the development of genomic research and gene handling technology, research is being promoted in a form that combines the two. It is. The target and contents of genetic diagnosis vary widely, but (i) the presence or absence of cancer cells using genetic abnormalities as an index, (ii) the malignancy or drugs of cancer, the nature of cancer cells sensitive to radiation, and (iii) the onset of cancer It is roughly classified into previous diagnosis and risk estimation.

  In an attempt to identify hematopoietic cell proliferation abnormality by detecting methylation of CpG island (CpG island) present in the promoter region that controls gene expression for genetic diagnosis targeting hematopoietic tumors. A search for methylation was carried out for about 80 genes, which are assumed to be involved in the abnormalities of these genes (see Patent Document 1).

  The present inventors have so far proposed a highly specific method for confirming the presence or absence of hematopoietic tumor cells for one piece of genetic information in a maximum of 4 steps (see Patent Document 2). The method quantifies a protein tyrosine phosphatase SHP1 protein or mRNA specific for hematopoietic cells contained in a specimen containing hematopoietic cells, and a CpG island contained in the base sequence of the SHP1 gene obtained from the specimen. In which DNA methylation is identified and further allelic loss is detected.

  Furthermore, the present inventor detects a methylated DNA from a small amount of a cell sample by omitting the step of extracting nucleic acid from cells in the sample and including a step of amplifying the DNA by polymerase chain reaction (PCR). The method which can do was developed (refer patent document 3).

  The data provided for genetic diagnosis is expected from the clinical site side to present an index that links the expression change of the gene involved in the target hematopoietic tumor and the identified disease state based on statistical support based on the data. Is done. Furthermore, data that can contribute to prediction of cancer progression, prognosis determination, and risk estimation leading to onset are also desired. However, the development of testing methods that provide such data has not been found for hematopoietic tumors such as adult T-cell leukemia / lymphoma.

Special table 2004-528837 gazette JP 2004-128 A JP 2005-58217 A

Harris NL, et al., Hematology. 2001; 1: 194-220., Staudt LM, Dave S. Adv Immunol. 2005; 87: 163-208

  In view of the above circumstances, the present inventors have so far developed to establish a prognosis estimation for predicting the onset and malignancy of adult T-cell leukemia / lymphoma (ATLL) and a test for providing data for the estimation. The DNA methylation detection method was applied to clinical specimens for further research. As a result, it is used for prognostic estimation tests that provide data for appropriately predicting the prognosis of ATLL pathology, including the onset of adult T cell leukemia virus (HTLV-I) carriers (virus-infected persons) or malignant indolent ATLL. The present invention related to a kit, a prognostic estimation system, a method for providing prognostic estimation data, and the like has been completed.

  The present invention provides a prognostic estimation test method that provides prognostic estimation data that can be used for estimation of prognosis of adult T-cell leukemia / lymphoma, preferably for predicting the onset of HTLV-I carrier and progression to aggressive disease type, and It aims at providing the inspection kit for that. Therefore, another object of the present invention is to provide a method for presenting data for prognostic estimation, the types of genes to be investigated and combinations thereof.

  By examining the expression control level of a specific gene based on the present invention, it is possible to predict the onset and progression of ATLL that follows a multistage carcinogenic process and to evaluate the prognosis. The present invention is specified as an invention having the following configuration.

The prognostic test kit of the present invention is
Prognosis used in prognostic tests to provide data for predicting the onset of adult T cell leukemia virus (HTLV-I) carriers or indolent ATLL causing adult T cell leukemia / lymphoma (ATLL) An estimation test kit,
Selecting two or more genes from the gene group consisting of SHP1 gene, p15 gene, p16 gene, p73 gene, hMLH1 gene, MGMT gene, DAPK gene and HCAD gene in a sample from the carrier or indolent ATLL patient, Methylation profile of CpG islands in the promoter region of the above-mentioned gene prepared by measuring the methylation status of the selected tumor suppressor gene or cancer-related gene promoter region, or adult T cell leukemia / lymphoma obtained therefrom It is a prognostic test kit used in a prognostic test that provides data for estimating the prognosis of those carriers or patients, including data of prognostic markers indicating the onset or malignancy of the disease.

  The selected gene is preferably a gene set consisting of SHP1, p15, p16, p73, hMLH1, MGMT, DAPK and HCAD genes.

  The kit comprises a lysis solution for dissolving a specimen, a bisulfite-containing reagent, and methyl for measuring methylation of CpG islands in the promoter region of two or more selected tumor suppressor genes or cancer-related genes. It is desirable to include a chemical detection amplification reagent.

The predictive marker data is preferably CIMP or the methylation status of the SHP1, p16, MGMT, DAPK, and HCAD genes.
The malignant transformation of indolent ATLL is a transition to aggressive ATLL.

  The specimen is a cell collected from an organ or tissue selected from the group consisting of tonsils, bone marrow, lymph nodes, digestive organs, respiratory organs, spleen, liver, sensory organs, central nervous system, motor organs, skin and peripheral blood. A contained sample is preferred.

  An adult T-cell leukemia virus (HTLV-) comprising the above-described prognostic test kit, a tool for processing and analyzing data obtained in the prognostic test, and an output device for data processing results The prognostic test system for predicting the onset of carrier of I) or malignant transformation of indolent ATLL is also included in the present invention. Such tools may include data processing programs and / or programs using prognostic statistical analysis algorithms.

  The method of the present invention is used in the collection of data for estimating the prognosis of adult T-cell leukemia / lymphoma (ATLL) in the SHP1 gene, p15 gene, p16 gene, p73 of isolated peripheral blood mononuclear cells (PBMC). CIMP is calculated as a prognostic factor by measuring the methylation state of CpG islands in the promoter region of the gene group consisting of the gene, hMLH1 gene, MGMT gene, DAPK gene and HCAD gene, and the methylated gene, methyl By displaying the stratification of risk based on the ATLL onset / progression risk score calculated from the number of activated genes and the CIMP value and / or the methylation status of the SHP1, p16, MGMT, DAPK and HCAD genes, Adult T-cell leukemia virus (HTLV-I It is a method of creating a ATLL prognostic data that makes it possible to predict the onset of the carrier or progression to aggressive type ATLL of indolent type ATLL.

  Furthermore, there is provided a method for generating data used for screening of a drug administered in the prognostic course of adult T-cell leukemia / lymphoma (ATLL) and / or for determining a therapeutic effect of administration, and comprising isolated peripheral blood mononuclear cells (PBMC) By measuring the methylation state of CpG islands in the promoter region of the gene group consisting of SHP1 gene, p15 gene, p16 gene, p73 gene, hMLH1 gene, MGMT gene, DAPK gene and HCAD gene of A methylation profile of CpG island in the promoter region of the above gene is prepared, and methylation status of CIMP and SHP1, p16, MGMT, DAPK, and HCAD genes as prognostic factors is determined. And a method for creating data used for screening of administered drugs and / or determination of therapeutic effects of administration, wherein ATLL onset / progression risk score is calculated from the prognostic factors and the ATLL onset / progression risk score is used as the data Are also included in the present invention.

  Furthermore, the method of the present invention is for predicting the development of a carrier of adult T cell leukemia virus (HTLV-I) causing adult T cell leukemia / lymphoma (ATLL) or progress of indolent ATLL to aggressive ATLL. A method for preparing prognostic test data, wherein the promoter regions of the SHP1, p15, p16, p73, hMLH1, MGMT, DAPK, and HCAD genes in a sample from the carrier or indolent ATLL patient The methylation status of CpG islands was measured to obtain methylation profiles of the eight genes, CIMP was obtained from the methylation profiles, and ATLL onset / progression risk score was calculated from the methylation profiles and the CIMP. The ATLL onset / progression risk score is Comprising a, it is also the creation method for estimating prognosis inspection data.

  The method for providing data for estimating the prognosis of adult T-cell leukemia / lymphoma of the present invention relates to changes in expression control levels of a plurality of gene groups related to or closely related to carcinogenesis and cancer progression. Is a test method that provides data used for risk assessment of ATLL onset and prediction of prognosis. The present invention is also a prognostic test kit used in a method for providing such data.

Therefore, the present invention is useful as a test method and test kit that provide data for genetic diagnosis of adult T-cell leukemia / lymphoma that takes a multistage carcinogenic process.
Adult T-cell leukemia / lymphoma may develop from early prediction of HTLV-I carrier onset based on the data obtained by the method of the present invention, relatively favorable indolent ATLL to poor aggressive ATLL Can be predicted early. Since these predictions are made with high accuracy, prognosis management is possible in which high-risk patients are treated intensively at an early stage to prevent tumor progression.

  The method of the present invention and the kit therefor detect changes in gene status based on an epigenetic mechanism closely related to carcinogenesis and its progress, and therefore accurately predict the onset and prognosis. Can do.

  Establish medication and treatment protocol early by estimating prognosis of adult T-cell leukemia / lymphoma, and through or in the process of its implementation and treatment management, ie, screening of administered drugs and / or determination of medication / treatment effect Based on the accumulated treatment results, it is also possible to develop new onset and recurrence prevention treatment methods. Furthermore, the present invention is useful for establishing tumor markers, anticancer drug sensitivity indicators, and developing novel methylation inhibitors.

FIG. 1 shows the ATLL evolution scheme and disease type. FIG. 2 represents the clinical criteria for each disease type in ATLL. FIG. 3 shows the relationship between the intracellular functions of eight genes measured in the Examples and specific gene methylation. FIG. 4 shows the relationship between the ATLL evolution scheme and the methylation of 8 genes. FIG. 5 shows the relationship between methylation of 8 genes and clinical data. FIG. 6 shows the correlation between the number of methylated genes, the number of WBCs, and lymphocytes. FIG. 7 shows the correlation between the number of methylated genes and abnormal lymphocytes and lactate dehydrogenase (LDH). FIG. 8 shows the correlation between the number of methylated genes, soluble interleukin 2 receptor (sIL2R), and HTLV1 viral load. FIG. 9 shows the correlation between the number of methylated genes and the serum Ca value. FIG. 10 shows the survival curve of ATLL by clinical disease type. FIG. 11 is a survival curve showing the presence / absence and prognosis of p16 gene methylation. FIG. 12 is a survival curve showing the presence / absence and prognosis of p73 gene methylation. FIG. 13 is a survival curve showing the presence or absence and prognosis of DAPK gene methylation. FIG. 14 is a survival curve showing the presence or absence and prognosis of HCAD gene methylation. FIG. 15 is a survival curve showing the presence or absence and prognosis of MGMT gene methylation. FIG. 16 is a survival curve showing the presence or absence and prognosis of SHP1 gene methylation. FIG. 17 is a survival curve showing the presence and prognosis of hMLH1 gene methylation. FIG. 18 is a survival curve showing the presence / absence and prognosis of p15 gene methylation. FIG. 19 is a survival curve showing the presence or absence of CIMP and prognosis. FIG. 20 shows the results of ATLL onset / progression risk scoring (sum of MSP and CIMP). FIG. 21 shows the results of ATLL onset / progression risk scoring (sum of MSP and CIMP). FIG. 22 shows the results of ATLL onset / progression risk scoring (sum of MSP and CIMP). FIG. 23 shows the results of ATLL onset / progression risk scoring (the sum of MSP, number of methylated genes and CIMP). FIG. 24 shows the results of ATLL onset / progression risk scoring (the sum of MSP, number of methylated genes and CIMP). FIG. 25 shows the results of ATLL onset / progression risk scoring (the sum of MSP, number of methylated genes and CIMP).

  In the process of searching for the correlation between the pathological state of ATLL and its progress and the methylation of a series of genes including a tumor suppressor gene or a cancer-related gene, the present inventors investigated the methylation and ATLL of a specific gene therein. It was found that there was a strong correlation with prognosis. The present invention pays attention to such correlation and provides a genetic diagnosis tool that is extremely useful for HTLV-I carriers and prognosis management of ATLL patients. Hereinafter, the present invention will be described in the order of a prognostic test kit, a test method, and a test system.

As used herein, “cancer” refers to a malignant tumor, and is sometimes simply referred to as “tumor”.
“Gene” refers to genomic DNA that carries genetic information that expresses some function, but it may also simply be referred to as DNA that is a chemical entity.

  “Tumor suppressor gene” means a gene that suppresses the onset of cancer, and “cancer-related gene” means a gene involved in the onset of cancer. In this specification, the time point when a specific disease is diagnosed by comprehensive judgment based on disease-specific clinical symptoms, test data, etc. is referred to as “onset”.

The “preclinical phase” refers to a pre-onset state before a disease-specific clinical symptom appears, and an early state in which a minute amount of malignant tumor cells are already present.
DNA methylation means that the carbon at the 5-position of cytosine on the CpG island in the DNA base sequence is methylated.

  "Prognosis estimation" predicts the transition of the pathological condition from the current point of view from the viewpoint of patient treatment, and predicts not only the determination of good / bad prognosis but also the prediction of progression of the disease type and the estimation of malignancy. .

Prognostic Test Kit The prognostic test kit of the present invention is a carrier of adult T-cell leukemia virus (HTLV-I) causing adult T-cell leukemia / lymphoma (ATLL), or malignant ATLL pathology, In particular, it is a kit used for prognostic estimation that provides data for predicting malignant transformation of indolent ATLL,
From a gene group consisting of SHP1, p15, p16, p73, hMLH1, MGMT, DAPK, and HCAD genes that are tumor suppressor genes or cancer-related genes in specimens from the carrier or indolent ATLL patients Selecting two or more genes, measuring the methylation state of the CpG island in the promoter region of the selected tumor suppressor gene or cancer-related gene, and the methylation profile of the CpG island in the promoter region of the prepared gene, Or prognostic estimation used in prognostic estimation tests that provide data for estimating the prognosis of those carriers or patients, including data of prognostic markers indicating the onset or malignancy of adult T-cell leukemia / lymphoma obtained therefrom This is a test kit. The data preferably includes an ATLL onset / progression risk score. The ATLL onset / progression risk score will be described later.

  Specifically, the kit provides a prognostic test that provides data for estimating the prognosis of a carrier or patient based on the data of a prognostic marker indicating the onset or malignancy of adult T-cell leukemia / lymphoma. A kit comprising a lysate for dissolving a specimen, a bisulfite-containing reagent, and a methylation detection amplification reagent for performing, and two or more selected tumor suppressor genes or cancer-related by a test using this kit A methylation profile of CpG islands in the promoter region of the gene is created.

  “Tumor suppressor gene or cancer-related gene” is a gene involved in canceration of cells and involved in cancer development. In particular, abnormal expression of tumor suppressor genes is thought to be directly linked to carcinogenesis and cancer progression. Such a tumor suppressor gene is not particularly limited. For example, protein tyrosine phosphatase SHP1 gene, p16Ink4a gene, p15Ink4b gene, CDH1 gene, HDAC gene, p14ARF gene, DAPK gene, p73 gene, APC gene, GSTP1 gene, anthrogen receptor gene , Estrogen receptor gene, TGF-β1 gene, TGF-β2 gene, p130 gene, BRCA gene, NF1 gene, NF2 gene, TSG101 gene, MDG1 gene, GST-pi gene, caltonin gene, HIC-1 gene, endothelin B receptor Gene such as body gene, TIMP-2 gene, TIMP-3 gene, O6-MGMT gene, hMLH1 gene, MSH2 gene and GFAP gene It is.

  The reason for selecting two or more tumor suppressor genes or cancer-related genes is as follows. The methylation of tumor suppressor genes or cancer-related genes also generally tends to progress and expand with aging. Variations in the methylation of one gene are also assumed to be associated with one or more diseases. Furthermore, the level of expression control in each tumor classified as a hematopoietic tumor changes in a differential manner, but there are several genes that change simultaneously, and it is led by the type of individual tumor. The genes involved are also assumed to be different. It is possible to detect the simultaneous methylation of a plurality of genes by measuring a sample, and improve the accuracy in genetic diagnosis from the obtained data. That is, in the present invention, it is preferable to measure DNA methylation in the above-described tumor suppressor gene in addition to the eight genes that measure the methylation state of CpG islands in the promoter region.

-Eight genes In the prognostic test kit used for the prognostic test of the present invention, a gene consisting of SHP1, p15, p16, p73, hMLH1, MGMT, DAPK, and HCAD genes in the sample It is desirable that the gene selected from the group is a gene set consisting of all genes, that is, SHP1 gene, p15 gene, p16 gene, p73 gene, hMLH1 gene, MGMT gene, DAPK gene and HCAD gene.

  These eight genes are markers that have been found to have characteristics that predetermine at the gene level the future development of ATLL or the malignancy of the previous ATLL pathology (ie, “predictive markers”), each of which is a DNA repair (repair). One of the biological functions such as DNA), apoptosis, cell adherence, tumor suppression, and signal transduction regulation is exerted in the cell (FIG. 3).

  These are related to the preclinical status and development of adult T-cell leukemia / lymphoma through the regulation of gene expression, and are specific to the progression of adult T-cell leukemia / lymphoma. The inventor has found that the property is high. The degree of involvement and mode vary, but comprehensive data for evaluating the data for diagnosis of adult T-cell leukemia / lymphoma, the preclinical status of the tumor, or the likelihood of developing it It is an extremely useful gene group for obtaining accurate data. That is, a combination that should be called a specific gene set selected by gene screening.

  By selecting two or more genes from these eight gene groups and measuring the methylation status of CpG islands in the promoter region of the selected genes, the future development of ATLL or the malignancy of the previous ATLL pathology Data useful for prognosis estimation can be obtained. That is, all the eight genes may be examined, or two or more of the eight genes may be appropriately selected in any combination depending on the disease type.

  In addition, the above eight gene groups are designated as “core target genes”, and two or more genes are selected from them, and the expression control of those selected genes including genes selected separately other than the core gene group is also performed. It may be an aspect in which the level is detected. In that case, selection of candidate genes other than the core target gene group is preferably a gene that provides reinforcing or supplementary data for measurement of the core target gene. Examples of such a gene include the above-described cancer suppressor genes.

  Among the above 8 genes, the following prognostic analysis regarding the methylation status of the SHP1, p16, MGMT, DAPK and HCAD genes is particularly useful for the prognosis management of ATLL patients. Therefore, it is desirable to use the methylation status of these five genes (that is, the data of predictive marker) as a parameter for determining the ATLL onset / progression risk score, which will be described later, together with the number of CIMP and methylated genes.

  In gene diagnosis that examines methylation of genes and malignant tumor formation (canceration) of cells, it is closely and specifically related to the target malignant tumor rather than a gene set that does not consider biological functions. A combination that tends to occur frequently and that can be expected to be methylated simultaneously is desirable in terms of search efficiency. From these meanings, the present inventors have recognized that the above eight genes are useful in adult T-cell leukemia / lymphoma, and that these gene groups are particularly suitable as a gene set for preparing a gene expression profile. In other words, the intracellular activity of each of the above eight genes is known as follows.

  The SHP1 gene was included in genes whose expression was greatly reduced in adult T-cell leukemia / lymphoma from DNA microarray analysis. Other genes are expected to be methylated from a group of genes whose expression is decreased in adult T-cell leukemia / lymphoma.

  In human hematopoietic tumors such as malignant lymphoma and leukemia, strong expression suppression of SHP1 protein was observed at a high frequency of 90% or more in many types (for example, American Journal of Pathology, Vol. 159, No. 4, October 2001: 1495-1505 etc.). Thus, in malignant adult T-cell leukemia / lymphoma cells, the suppression of the expression of the SHP1 protein is seen very frequently, whereas this phenomenon is not seen in normal blood cells. In patients with adult T-cell leukemia / lymphoma, especially malignant lymphoma and leukemia patients, the CpG region present in the promoter region of the SHP1 gene is methylated among the above genes, and thus is negative in the cell signaling mechanism. A mechanism is known in which the suppression of the expression of protein tyrosine phosphatase SHP1 protein, which regulates the above, is observed at a very high frequency, and the inhibitory control of cell proliferation becomes ineffective. Therefore, suppression of SHP1 protein expression is due to methylation of the SHP1 gene. Furthermore, one allele of the SHP1 gene is lost before and after the transcriptional repression of the SHP1 gene by DNA methylation.

  Based on the above, selected in a test method that provides data for diagnosing adult T-cell leukemia / lymphoma, or data for evaluating the possibility that the tumor is in a preclinical state or developing the tumor. It is desirable that one of these genes includes the SHP1 gene that is deeply involved in the development of malignant lymphoma and leukemia.

The p15, p16 and p73 genes are known as tumor suppressor genes.
The hMLH1 and MGMT genes are DNA repair enzyme-related genes.
The HCAD gene is a cell adhesion (cadherin) -related gene. Cadherin is a glycoprotein related to cell-cell adhesion with a molecular weight of 120 kDa, and plays an important role in embryonic tissue organization and organogenesis. It is known that E cadherin (derived from epithelium) (CDH1) is also responsible for adhesion between cancer cells in cancer cells, and the process of cancer metastasis in which cancer cells infiltrating into blood vessels dissociate and drift to the target organ Is believed to be involved.

E-cadherin is known to lose its expression in tumor cells of acute myeloid leukemia or Hodgkin lymphoma, and an association with onset has been suggested.
In addition, it is known that the HCAD (or CDH13 or H-cadherin) gene lacks an intracellular domain and is related not only to intercellular adhesion but also to intracellular signaling.

In addition, it has been reported that the expression of H-cadherin disappears in various cancers such as ovarian cancer, breast cancer, lung cancer, and colon cancer due to abnormal DNA methylation or gene deletion. Recently, several studies, including those of our group, have observed strong methylation of the HCAD promoter in early chronic myelogenous leukemia or interferon-treated hyporesponsive chronic myelogenous leukemia, and It is known that in diffuse large B-cell lymphoma, a decrease or disappearance of gene expression is observed due to abnormal methylation of HCAD gene DNA and allelic deletion.
The DAPK (Death-associated protein kinase) gene is an apoptosis-related gene, and is assumed to be associated with in vivo apoptosis associated with various pathological conditions.

Adult T-cell leukemia / lymphoma The subject of the method of the present invention is an adult T-cell leukemia / lymphoma included in the category of hematopoietic tumor, and the method of the present invention and the prognostic test kit detect the disease type. In addition, it is possible to provide data for predicting the likelihood of onset and evaluating prognosis.

  Adult T-cell leukemia / lymphoma (ATLL) is mostly human T-lymphotropic virus type I (HTLV) that is vertically infected from mothers through breast milk in most cases. -I) Hematopoietic tumor arising from a carrier. The other main routes of transmission of HTLV-I are sexual intercourse and blood transfusion. ATLL occurs every year at a rate of 0.5 to 1.0 per 1000 people from HTLV-I carriers, with a lifetime risk of developing ATLL of 5-10%. It usually develops after an incubation period of 40 to 50 years. In the case of acute ATLL, about 70% die within one year from the onset, and it is an intractable leukemia with a very poor prognosis. The number of carriers nationwide is estimated to be about 1 million to 2 million, and the number of ATLL cases is said to be about 700 a year.

  Such ATLL disease types are classified into four disease types: smoldering type, chronic type, lymphoma type, and acute (acute transformation) type. In the present specification, the smoldering type and the chronic type are referred to as indolent types, and the lymphoma type and the acute (acute transformation) type are referred to as aggressive types. The malignant transformation of indolent ATLL is the transition to aggressive type. All of these disease types clinically develop ATLL. The ATLL progress scheme and disease type are shown in FIG.

  The clinical criteria for ATLL disease type are shown in FIG. In FIG. 2, “*” indicates that there is no restriction on the condition, that is, the disease type cannot be determined by examining the item, and “+” indicates that the disease type must be observed. , “-” Indicates that the disease type is not found. The “lymphocyte count” represents the lymphocyte count per 1 μl of blood.

  The smoldering type is a state in which monoclonal proliferation of ATLL cells is demonstrated in peripheral blood, lungs or skin, and is considered as an early stage of onset. Although atypical cells are found in peripheral blood, they are asymptomatic, and spontaneous remission is often observed without progression of lung or skin lesions.

  The chronic type has 10% or more atypical cells in peripheral blood, but the clinical course is stable, and there are cases where it survives for almost 10 years without treatment. Although the lymph nodes, liver, and spleen are swollen, other organs are not infiltrated and do not have hypercalcemia.

  The disease types that require treatment are acute and lymphoma types. For chronic and smoldering types that are understood as precursor lesions, the condition is maintained for a long period of time, and clinical follow-up is the main focus, but it is often converted to an acute type. Seen. In addition, HTLV-I carrier is present as a pre-leukemia state (state that does not show clinically significant findings, ie, does not develop ATLL) in front of them.

  In the acute type, atypical lymphocytes are confirmed in the blood, but in the lymphoma type, there are differences such as the absence of atypical lymphocytes in the blood, but in most cases, lactate dehydrogenase (LDH) increases, Symptoms such as decreased cellular immunity and hypercalcemia occur. In addition, hyperuricemia, hyperbilirubinemia (liver infiltration), alkaline phosphatase elevation (bone destruction), parathyroid hormone-related protein (PTHrP) elevation (bone destruction) are observed.

  The lymphoma type is particularly resistant to treatment compared to other malignant lymphomas, and infiltrates the lymph nodes and organs throughout the body to form lymphoma. In the acute type, a large number of ATLL tumor cells that are usually atypical are found in the peripheral blood and exhibit typical leukemia symptoms. Furthermore, lymphadenopathy, liver dysfunction, and edema are observed, and ATLL cells infiltrate many organs such as bone marrow and central nervous system (FIG. 2).

  Here, “detecting an ATLL disease type” means detecting a smoldering type, chronic type, lymphoma type, or acute (acute inversion) type, which is a state of developing ATLL. In the smoldering type, the white blood cell count is normal, but there is a characteristic that atypical ATLL tumor cells are present in the blood in an amount of 3% or less in the blood, and symptoms are hardly noticed. In the chronic type, the number of white blood cells in the blood increases, and 10% or more of atypical ATLL tumor cells appear in the peripheral blood, but there is no rapid increase in the number of atypical ATLL tumor cells, and the clinical course is stable. Therefore, in clinical diagnosis, diagnosis of acute type and lymphoma type that require urgent treatment is natural, and even if it is chronic type or smoldering type that does not require treatment for the time being, detecting them is an estimation of prognosis And especially for follow-up.

  HTLV-I carrier, smoldering type, chronic type, lymphoma type, and acute type ATLL are accurately identified and detected, and appropriate management of the prognosis of the disease is early in the onset of lymphoma type and acute type ATLL with poor prognosis It is very important for starting treatment and for effective treatment such as suppressing the growth of tumor cells before drug resistance is acquired, or for developing prophylactic treatment to prevent blast crisis It is.

  From this viewpoint, it is desirable to obtain data specifying lymphoma type or acute type ATLL by selecting two or more types of tumor suppressor genes or cancer-related genes in a specimen and examining their expression control levels. In general, the gene product is quantified, and the expression profile of the gene group is created based on the quantification result, or the regulation of expression is examined by detecting the methylation status. Perform early detection and diagnosis.

  Thus, data for diagnosing ATLL disease type, for evaluating the possibility of progressing to a disease type requiring treatment, and further data for early detection of progress to a disease type with poor prognosis can be obtained ( (See FIG. 1).

  In particular, by selecting two or more genes from the gene group consisting of the above eight genes (particularly preferably all eight genes are selected) and detecting the expression control level of the selected genes, lymphoma type or acute The type ATLL can be detected and specified. For examining the expression control level of genes, it is preferable to examine methylation of CpG islands in the promoter region of those genes as described above. Based on this, the patient will be provided with the necessary information regarding the disease type, and will be used for self-management by providing appropriate guidance during follow-up.

  The data provided by the method of the present invention and the prognostic test kit that allow for ATLL disease type identification and / or prognosis estimation are smoldering or chronic, especially when developing and progressing from HTLV-I carriers to smoldering Detects and diagnoses such changes at an early stage when the patient becomes acutely converted from erythrocytes (ie, falls under “indolent ATLL malignancy”), and when lymphoma type or acute type ATLL develops from an HTLV-I carrier Very useful for the purpose.

  “The methylation profile of the CpG island in the promoter region of the gene created above, or the data of prognostic markers indicating the onset or malignant development of adult T-cell leukemia / lymphoma obtained therefrom” refers to the methylation of individual genes selected This refers to the aging state, the methylation profile obtained for the selected gene set or the survival curve and the ATLL onset / progression risk score based on the classification criteria of gene methylation.

The predictive marker refers to the above eight types of cancer suppressor genes or cancer-related genes or genes specifically selected from them.
The methylation profile refers to the methylation state measured for a selected gene group, or processing data created through necessary processing based on the methylation state. For example, the average number of MSP (+) genes.

  Furthermore, the “methylation state” means the state of methylation of CpG islands in the promoter region of an individual gene, that is, no (−), presence / absence (+/−), existence (+, ++, +++), It may refer to the methylation rate (methylation frequency) of each gene, the average number of methylated genes, the total of data regarding the methylation status, and the like.

  The prognostic test kit according to the present invention includes various equipment (for example, a gene amplification device, an analysis device, etc.) or materials (for example, a blood collection device, a blood separation device, etc.), a reagent, which are necessary for performing the method of the present invention And / or primers for performing gene amplification. These reagents include various enzymes, buffers, washing solutions, lysis solutions, and the like.

  Specifically, it contains a lysis solution for dissolving a specimen, a bisulfite-containing reagent, a methylation detection amplification reagent, and a PCR primer for detecting a promoter region variant of a tumor suppressor gene or a cancer-related gene. Including. The kit element may further include a necessary set of equipment such as a microtiter plate capable of simultaneously processing a large number of specimens and a DNA amplification tool.

  The high-throughput mode of the inspection method according to the present invention may include a microreactor type device, specifically a chip-shaped device, in the kit. As will be described in more detail later, the improved mode of such a configuration is a mode in which a digitized version of a signal obtained from a chip is taken, a file is created through computer processing, and stored in a predetermined directory on the computer. The system employed is preferred.

  The numerical data is statistically processed to examine the ability to regulate the expression (methylation profile) of two or more, preferably the above eight types of tumor suppressor genes or cancer-related genes. Can be included in the system up to the ability to estimate. Data processing is carried out using suitable software that allows statistical analysis after necessary corrections and normalization. A person skilled in the art can construct a system for such data processing using existing techniques, methods, and procedures.

  The “specimen” may be any organ, tissue, cell or cell extract that can be detected from adult T-cell leukemia / lymphoma isolated from a patient or the like. Such specimens may be isolated from a human or mammalian animal that is in preclinical state or developing adult T-cell leukemia / lymphoma or from a human or mammalian animal that does not have adult T-cell leukemia / lymphoma or tumor The specimen to be separated.

  The specimen is not particularly limited, for example, a tissue obtained from a patient (human or mammal), a test patient or a laboratory animal, specifically bone marrow tissue (for example, from a biopsy or autopsy), tonsils, bone marrow, lymph nodes Digestion, respiratory organs, spleen, liver, sensory organs, central nervous system, motor organs, skin, peripheral blood, whole blood, cell lysate, mammalian cell culture, or any other cell-containing sample, extraction from it Such as things.

  Preferably, the specimen is taken from an organ or tissue selected from the group consisting of tonsils, bone marrow, lymph nodes, digestive organs, respiratory organs, spleen, liver, sensory organs, central nervous system, motor organs, skin and peripheral blood. Cell-containing sample.

Test Method by Detection of Gene Methylation One aspect of the present invention is a genetic test that provides data for prognosis estimation of adult T-cell leukemia / lymphoma. That is, the method of the present invention
In collecting data for estimating the prognosis of adult T-cell leukemia / lymphoma (ATLL), isolated peripheral blood mononuclear cells (PBMC) SHP1, p15, p16, p73, hMLH1, MGMT CIMP was calculated as a prognostic factor by measuring the methylation state of CpG islands in the promoter region of genes, DAPK genes and HCAD genes, and the methylated genes, the number of methylated genes and the CIMP value By displaying the stratification of the risk by the ATLL onset / progression risk score calculated from the above, the change trend of the CIMP value and / or the methylation state of the SHP1, p16, MGMT, DAPK and HCAD genes , Adult T cell leukemia virus (HT This is a method of creating data for estimating ATLL prognosis that makes it possible to predict the onset of carriers of LV-I) or the malignancy of ATLL pathological conditions, particularly the malignancy of indolent ATLL.

  In this test method, two or more types of tumor suppressor genes or cancer-related genes contained in a specimen are selected as predictive markers, and their gene expression is examined in terms of regulation. In terms of regulation, the methylation status of the CpG island in the promoter region of the selected gene is measured, and the state of chromatin in which the gene is present and the expression of the gene are determined from changes in prognostic factors expressed as data. Based on judging the degree.

  Specimens to be used for the above-mentioned examination are HTLV-I carriers as described above or suspected to be, or tonsils, bone marrow, lymph nodes, digestive organs, respiratory organs, spleen isolated from ATLL patients It is preferably a cell-containing sample collected from an organ or tissue selected from the group consisting of liver, sensory organs, central nervous system, motor organs, skin and peripheral blood.

・ Methylation of CpG island
When there is a region rich in CpG sequences in the promoter region of a gene, that is, a CpG island, cytosine methylation in the CpG island affects the transcriptional control of the gene. Therefore, by detecting cytosine methylation in the promoter region of a gene, abnormal expression of the gene (for example, whether transcription is activated or suppressed) can be detected. Further, when the gene is a tumor suppressor gene, whether or not the cell contained in the cell-containing sample may be a cancer cell by detecting methylation of cytosine of CpG island in the promoter region of the tumor suppressor gene. Can be detected.

In order to know the actual state of cytosine methylation in CpG islands of gene promoter regions involved in gene transcription control, for example, the methylation state may be measured. Therefore, a method (Patent Document 3) that can easily detect methylated DNA from a small amount of cell sample without using the step of extracting nucleic acid from cells of the cell sample is suitable. That is,
A step of preparing a cell-containing sample lysate by dissolving the cell-containing sample with a lysate,
The cell-containing sample lysate obtained in this step is directly treated with a bisulphite-containing reagent to convert unmethylated cytosine in the base sequence of the CpG-containing DNA contained in the cell-containing sample lysate into uracil. Process,
Amplifying the obtained CpG-containing DNA by polymerase chain reaction using a predetermined methylation-specific oligonucleotide primer and an unmethylation-specific oligonucleotide primer;
Next, based on a method including a step of detecting whether or not the CpG-containing DNA is amplified, DNA methylation can be detected easily and rapidly.

  In these treatments, unmethylated cytosine in the sample DNA is converted to thymine via uracil, while 5-methylated cytosine is finally converted into cytosine. Therefore, methylated cytosine and unmethylated cytosine Discrimination can be made.

  “Select two or more tumor suppressor genes or cancer-related genes in a specimen” is as described above, and is to select a target gene for measuring methylation. By measuring the methylation status of two or more target genes, by comparing them with each other, it is possible to obtain knowledge such as the degree of methylation, slow methylation, and simultaneous methylation. Many useful information can be obtained by associating with. As described above, it is preferable to select all eight genes.

  As one of the preferred embodiments of the above-described test method, in the collection of data for estimating the prognosis of adult T-cell leukemia / lymphoma (ATLL), the SHP1 gene and p15 gene of isolated peripheral blood mononuclear cells (PBMC) CIMP is calculated as a prognostic factor by measuring the methylation state of CpG islands in the promoter region of the gene group consisting of the p16 gene, p73 gene, hMLH1 gene, MGMT gene, DAPK gene and HCAD gene, Onset of carriers of adult T cell leukemia virus (HTLV-I) by displaying the CIMP value and / or the risk stratification by methylation status of the SHP1, p16, MGMT, DAPK and HCAD genes Or predicting malignant transformation of indolent ATLL Creating a data ATLL estimating prognosis that enables Rukoto the like.

  The significance and specificity of these gene groups are as described above, and FIG. 3 shows the relationship between the intracellular functions of the above eight genes and methylation. As will be described in detail later, from the research results of the present inventors, CpG island methylator phenotype (CIMP: CpG islands of promoter regions of various specific target gene groups are frequently methylated by P-value statistical analysis, It was found that the target genes related to the phenotype in which the expression of the target gene group disappears one after another are SHP1, p15, p16, p73, DAPK, and MGMT. It was concluded that these genes should be noted from the CIMP aspect.

  In the method of the present invention, which provides data for diagnosing adult T-cell leukemia / lymphoma, or data for evaluating the possibility that the tumor is in a preclinical state or the possibility of developing it, etc. It is desirable that at least the SHP1 gene is included in one of the generated genes (FIG. 4).

Measurement of methylation state The method of the present invention is a method for preparing data for estimating the prognosis of HTLV-1 carriers or ATLL patients by measuring the methylation state of CpG islands in the promoter region of a selected gene. The detection of the methylation frequency can be performed after treating the cell lysate obtained by dissolving the specimen with bisulfite.

Specifically, the method for detecting the methylation state includes the following steps.
(1) a cell lysis step of preparing a cell-containing sample lysate by lysing a specimen (cell-containing sample) containing cells with a lysate;
(2) The cell-containing sample lysate obtained by the cell lysis step is directly treated with a bisulfite-containing reagent, and unmethylated cytosine in the base sequence of the CpG-containing DNA contained in the cell-containing sample lysate is converted into uracil. A DNA conversion step of converting to
(3) a DNA amplification step for amplifying the CpG-containing DNA obtained by the DNA conversion step by polymerase chain reaction (PCR) using a predetermined methylation-specific oligonucleotide primer and an unmethylation-specific oligonucleotide primer; ,
(4) A methylation detection step for detecting whether or not the CpG-containing DNA is amplified by the DNA amplification step.

Treatment of DNA with bisulfite converts unmethylated cytosine to uracil.
Specifically, unmethylated cytosine is sulfonated with bisulfite (Sulphonation), hydrolyzed and deaminated (Alkaline desulphonation) in the presence of alkali, Converted to uracil. In contrast, methylated cytosine is not converted to uracil even when treated with bisulfite.

  In non-methylated DNA, all cytosine is converted to uracil by bisulfite treatment as described above. In contrast, in methylated DNA, methylated cytosine is not converted to uracil by bisulfite treatment, and only other unmethylated cytosine is converted to uracil. That is, even in the case of DNA having the same base sequence, there is a difference in the base sequence after bisulfite treatment depending on whether it is methylated or not. By detecting this difference in base sequence, the presence or absence of methylation of the CpG-containing DNA can be detected.

  The “CpG-containing DNA” is not particularly limited as long as it is DNA contained in the cell-containing sample and is a DNA sample having a base sequence containing a CpG sequence. The DNA specimen here is usually genomic DNA, that is, a gene. Furthermore, the gene is more preferably a tumor suppressor gene or a cancer-related gene. This “CpG-containing DNA” is preferably present in the promoter region of the gene. In order to specifically detect methylation of the promoter region, a primer designed for a base sequence including the CpG sequence of the promoter region of the gene is used.

  As a DNA amplification method, a PCR amplification method or a modification thereof is used, but an amplification method such as recently developed ICAN (Isothermal chimera primer initiated nucleic acid amplification) may be used.

  Furthermore, the method for detecting a methylation state described above is characterized in that a cell lysate obtained by dissolving a specimen is directly treated with bisulfite, and the methylation frequency is measured without extracting a gene from the specimen. Yes. DNA may be extracted from the cell lysate and separated to detect methylation. However, the present inventor has already proposed that the cell lysate can be directly treated with bisulfite without extracting the gene DNA from the specimen (Patent Document 3). The operation of extracting DNA from a sample is complicated, and the above detection method can be avoided from the situation that a sample containing only a small amount of gene cannot be examined.

  A “specimen” may be any organ, tissue, cell or cell extract isolated from a patient that can detect adult T-cell leukemia / lymphoma. Such specimens include, but are not limited to, organs selected from the group consisting of tonsils, bone marrow, lymph nodes, digestive organs, respiratory organs, spleen, liver, sensory organs, central nervous system, motor organs, skin and peripheral blood A cell-containing sample collected from a tissue is desirable.

  The “dissolution solution” is not particularly limited as long as it can dissolve the sample and cleave the membrane, but a reagent that causes protein denaturation is preferable. Specific examples of the solution include a solution containing a conventionally known protein denaturant such as guanidine thiocyanate, sodium iodide, urea, or SDS. Further, these may contain a conventionally known crosslinking agent such as β-mercaptoethanol.

The “bisulfite-containing reagent” is not particularly limited as long as it is a conventionally known reagent containing bisulfite, and examples thereof include sodium bisulfite (Na ₂ S ₂ O ₅ , metabisulfite). Sodium sulfite, sodium disulfite, or sodium pyrosulfite) can be preferably used. Further, a bisulfite compound and urea may be used in combination.

  For the detection of the methylation state, a methylation sensitive restriction enzyme may be used. As a basic method, Patent Document 2 discloses a method using a methylation-sensitive restriction enzyme including a gene cleavage step, a gene amplification step, and a gene amplification confirmation step. A methylation-sensitive restriction enzyme includes cytosine in the base sequence to be recognized in double-stranded DNA, and when cytosine in this base sequence is methylated, the double strand of the base sequence The restriction enzyme is not particularly limited as long as it is a restriction enzyme that cannot cleave DNA. Specific examples include HpaII, EagI, and NaeI.

-Correlation between progression of ATLL disease type and methylation In prognostic test of adult T cell leukemia / lymphoma in which the prognostic test kit of the present invention is used, in order to provide data for estimating ATLL prognosis, Two or more genes are selected from the gene group consisting of SHP1 gene, p15 gene, p16 gene, p73 gene, hMLH1 gene, MGMT gene, DAPK gene and HCAD gene, and the methyl of CpG island in the promoter region of the selected gene The crystallization state is measured. Next, identification of HTLV-I carrier and discrimination of smoldering type, chronic type, lymphoma type, and acute type in ATLL disease type are required.

  The above 8 gene groups are particularly useful for adult T-cell leukemia / lymphoma as a gene set specific for disease type detection, diagnosis, and prognosis estimation, and are therefore preferably used as core target genes (FIG. 4). ). Details of the methylation status of each gene and ATLL disease type discrimination are disclosed in Japanese Patent Application Laid-Open No. 2007-244377, but only the main points are described below.

  The SHP1 gene or the p73 gene is effective as a target gene (discriminating marker) for healthy individuals and HTLV-I carriers. Furthermore, DAPK gene and HCAD gene are useful as markers for the development of smoldering ATLL. Between healthy individuals and chronic ATLL, the SHP1 gene, DAPK gene, and HCAD gene can be good discrimination markers. In addition, examples of markers for the development of acute ATLL from healthy individuals include the SHP1 gene, DAPK gene and HCAD gene, and markers for the development of lymphoma ATLL include the SHP1 gene, p16 gene, DAPK gene, MGMT gene and HCAD gene.

  In the discrimination between HTLV-I carrier and smoldering type, the presence or absence of methylation of HCAD gene and SHP1 gene is an effective index, and in the discrimination between smoldering type and acute type, methylation of SHP1 gene is an effective index. In distinguishing between HTLV-I carrier and acute type, methylation of HCAD gene and SHP1 gene, and in distinguishing between HTLV-I carrier and lymphoma type, methylation of SHP1 gene, DAPK gene, and HCAD gene are extremely effective indicators, and p16 Detection accuracy is improved by adding the gene to the marker.

  Since methylation of the SHP1 gene, p16 gene, DAPK gene, and HCAD gene is significantly different between lymphoma type ATLL and HTLV-I carrier, these genes are markers for the onset of lymphoma type ATLL from HTLV-I carrier. Can be. In distinguishing between smoldering and lymphoma types, it is effective to select at least two types of target genes from the SHP1 gene, MGMT gene, and DAPK gene. If both of the two selected genes show high methylation frequency, the disease type is likely to have progressed from smoldering type to lymphoma type, and the target gene is the p73 gene that is hardly methylated in the smoldering type The detection accuracy or reliability is improved by increasing the number of p16 genes to 4 or more.

  In distinguishing between smoldering type and acute type, SHP1 gene is used as a target gene. For discrimination between smoldering and chronic types, it is desirable to select the SHP1 gene as the target gene. In the discrimination between acute type and lymphoma type, if the p16 gene and MGMT gene are selected as target genes and the p16 gene shows a high methylation frequency, the disease type is likely to be lymphoma type. If the MGMT gene shows a high methylation frequency, it is highly likely that it is an acute type.

Prognostic estimation test system and prognostic estimation Through the previous researches, the present inventors have developed (i) an increase in the number of genes exhibiting methylation, (ii) methylation of a specific gene, (iii) CIMP with the progress of ATLL. It was clarified that methylation of a specific gene is involved from the HTLV-I infection to the onset of ATLL. The present inventors also demonstrated the presence of CIMP in ATLL for the first time because of the high proportion of acute and lymphoma types showing CIMP, suggesting that CIMP could be a biomarker of ATLL progression. Acute and lymphoma types both have a poor prognosis, but the genes that are methylated are different, which is thought to reflect differences in the pathogenesis of the disease type. Based on these findings, research was also conducted on the application of ATLL onset and prognosis estimation.

  The test system of the present invention is a prognostic test system for predicting the onset of a carrier of adult T cell leukemia virus (HTLV-I), or malignancy of ATLL pathology, particularly malignancy of indolent ATLL. And a kit for processing and analyzing the data obtained by the prognostic estimation test, and a data processing / analysis result output device. A tool for processing and analyzing data obtained by prognostic estimation inspection includes a data processing program, the above statistical processing program, a program using a clustering algorithm, and a program using an algorithm including a judgment / estimation judgment procedure. Or a procedural scheme, etc., which may take the form of computer software. Alternatively, the prognosis determination / estimation determination procedure may be in the form of a flowchart.

  The data processing is, for example, checking a methylated gene item, summing the number of methylated genes, and determining CIMP positive / negative, and analyzing the data is the risk of developing or developing ATLL. This refers to calculating the degree score.

  The statistical processing program is a program that gives a computer an instruction to select a higher one than the ATLL onset / progression threshold or to display the degree of risk based on the ATLL onset / progression risk score.

  The clustering algorithm analyzes the methylation positive / negative data for each gene and stratifies the high risk group and the low risk group with respect to the onset or progression based on the combination pattern or profile of the methylation positive gene group Refers to the processing procedure.

  The algorithm including the judgment / estimation judgment procedure is to analyze the data of methylation positive / negative for each gene, and from the methylation profile of the gene group, the HTLV-I carrier develops an ATLL risk level score "Or a procedure for calculating a" risk score "for patients with smoldering or chronic indolent ATLL to develop into acute or lymphoma ATLL. In this algorithm, parameters of blood test data usually performed, such as white blood cell count, lymphocyte count, presence / absence of 5% or more abnormal lymphocytes, LDH value, HTLV-I DNA copy number, sIL2R value, etc. In addition to the parameters for calculating the risk score, higher accuracy may be achieved.

  The output device of the data processing result is a data display device such as a display or a printer used in a normal computer. If necessary, the output device may include a communication protocol or the like.

・ Prognostic factors and data formats The differential expression profile of two or more selected tumor suppressor genes or cancer-related genes can be used for epigenetics-related disease monitoring and diagnosis, as well as predicting the likelihood of onset and prognosis. The estimation can be evaluated early with high sensitivity and high accuracy. One such useful profile is the methylation profile of CpG islands in the promoter region of tumor suppressor genes or cancer-related genes. A method for providing data for estimating ATLL prognosis based on such a methylation profile is presented as one aspect of the present invention.

That is,
By using the above prognostic test kit or prognostic test system,
Regarding a group of genes consisting of SHP1, p15, p16, p73, hMLH1, MGMT, DAPK and HCAD genes of isolated peripheral blood mononuclear cells (PBMC), CpG in the promoter region of those genes By measuring the methylation state of the island,
While creating a methylation profile of CpG island in the promoter region of the above gene, CIMP, which is one of prognostic factors, is calculated based on this profile,
“Risk score” regarding the onset and progression of ATLL is calculated from methylated genes, number of methylated genes and CIMP value, and risk stratification and / or SHP1, p16, MGMT, DAPK and HCAD genes By displaying the methylation status of HTLV, it is possible to predict the onset of carriers of adult T cell leukemia virus (HTLV-I) or the malignancy of ATLL pathology, in particular, the malignancy of indolent ATLL. Estimation data can be provided.

  Here, the “prognostic factor” is a substance or event that provides a clue to foreseeing the pathology of the disease, future development of the disease type, or patient outcome. Malignant transformation of ATLL pathology includes transition from smoldering type to chronic or aggressive type, transition from chronic type to aggressive type, and exacerbation from acute type to lymphoma type in aggressive type. In particular, the fact that the transition from the indolent ATLL to the aggressive disease type can be predicted from the change in the gene level has great clinical significance as well as the onset prediction of the carrier of HTLV-I.

  As a method of providing data, a change in the expression of a gene and the identified pathology is an indicator linked to the support of statistical data, that is, a change in prognostic factors, more preferably a change over time (or at multiple time points). Is more preferable. Examples include the average number of MSP (+) genes, CIMP (CpG Island Methylator Phenotype), and ATLL onset / progression risk score. Statistical analysis can be performed by selecting a suitable method from various statistical methods and test methods used in the industry.

(Average number of MSP (+) genes)
The “average number of MSP (+) genes” is the average number of genes that gave a positive result in MSP, that is, in which methylation was recognized by methylation specific PCR (MSP). In the healthy person DNA and the ATLL patient DNA, the average number of methylated genes in the above eight gene groups, that is, the average number of MSP (+) genes, was 0.5 for healthy person DNA and HTLV-1 carrier DNA: 1. 6 genes, smoldering DNA: 1.9, chronic DNA: 3.2, acute DNA: 2.5, lymphoma DNA: 3.4, number of genes showing methylation in ATLL patients Tend to increase as the stage progresses.

  There is a significant difference between the average number of MSP (+) genes in healthy subjects and the average number of MSP (+) genes in each disease type of HTLV-I carrier, smoldering, chronic, acute, and lymphoma ATLL Therefore, “average number of MSP (+) genes” is useful as a marker for discriminating HTLV-I carriers from healthy subjects and ATLL of each disease type from healthy subjects.

  Furthermore, between “average MSP (+) gene number” of HTLV-I carrier and “average MSP (+) gene number” of chronic type, acute type or lymphoma type ATLL, or “average MSP (+) gene type of smoldering ATLL” There is also a significant difference between the “number” and the “average MSP (+) gene number” of chronic or lymphoma ATLL.

  Therefore, the “average number of MSP (+) genes” is a prognostic factor that can predict the onset of chronic, acute, or lymphoma ATLL from an HTLV-I carrier, or malignant transformation from smoldering ATLL to chronic or lymphoma ATLL. Can be used as a prognostic factor.

(Analysis based on index CIMP (+))
CIMP is useful as an index for associating each ATLL disease type with DNA methylation. In general, CIMP (CpG Island Methylator Phenotype) is an expression in which CpG islands in the promoter region of various specific target gene groups are frequently methylated, thereby causing the expression of the target gene groups to disappear one after another. Refers to the type.

That is, in CIMP, DNA is frequently methylated (hypermethylation) and "gene
This parameter indicates that silencing has occurred. By increasing the cut-off level (if there are genes that are methylated in excess of this number, it is determined that high-frequency DNA methylation expression is positive). In addition, prediction of the possibility of onset of HTLV-I carriers, or the criteria for indolent prognosis estimation, which is a concern for malignant transformation in ATLL patients, will become stricter.

  In the present invention, when three or more genes are methylated in 8 gene groups (SHP1 gene, p15 gene, p16 gene, p73 gene, hMLH1 gene, MGMH gene, DAPK gene, HCAD gene), CIMP (+): Defined as CIMP positive. CIMP is one of the indicators for scoring risk of HTLV-I carrier, lymphoma type, smoldering type and lymphoma type, smoldering type and chronic type, and acute type and lymphoma type. The effectiveness of the analysis is shown in the examples below.

  There is a significant difference in the number of genes in which methylation of the target gene occurs between HTLV-I carriers and ATLL chronic, acute and lymphoma types. Further, the number of CIMP (+) cases increases as the disease type progresses. Therefore, the average MSP (+) gene number of target genes and CIMP are also used as prognostic factors for the progression of ATLL disease type. Can be adopted as

  Furthermore, it has become clear from the survival curve study of these 5-gene methylations by the present inventors that the methylation status of the SHP1, p16, MGMT, DAPK, and HCAD genes is extremely useful for prognosis of ATLL patients. It was. Therefore, it is reasonable to position the SHP1 gene, p16 gene, MGMT gene, DAPK gene, and HCAD gene as predictive markers for predicting the likelihood of developing ATLL and estimating prognosis. These 5 genes may be displayed as prognostic factors together with the methylation aspect, the average number of MSP (+) genes, and CIMP.

(ATLL onset / progression risk score)
The above “ATLL onset / progression risk score” gave a positive result in MSP, that is, a methylation-specific PCR method (methylation specific PCR (MSP)) in which methylation was observed, the number of methylated genes and This is a value obtained as the sum of values weighted by the Hazard Ratio (HR) obtained by univariate analysis or multivariate analysis in the CIMP term. The calculation formula for calculating the ATLL onset / progression risk score includes clinical data (eg, white blood cell count, lymphocyte count, presence or absence of abnormal lymphocytes of 5% or more, LDH value, HTLV-I DNA copy number, sIL2R value, etc.) Data) etc. may be added. This will be described in more detail below.

  The ATLL onset / progression risk score is (1) a gene that gave a positive result in MSP, that is, a gene that was methylated by methylation specific PCR (MSP), and (2) methylation The number of genes and (3) CIMP value, and the above three risk factor terms weighted by the Hazard Ratio (HR) (see Table 1 below) determined by univariate analysis or multivariate analysis, Required for each patient.

  The HR determined by the univariate analysis or multivariate analysis is the statistical calculation (univariate analysis or multivariate analysis) of the methylation status of eight genes of ATLL carriers and patients. Is a numerical value indicating the relationship (risk) of each gene, the number of methylated genes, and CIMP to ATLL, and the higher the numerical value, the more the gene is methylated, and ATLL develops or ATLL There is a high risk of becoming malignant. This HR is expected to be a more accurate numerical value by increasing the population parameter of the group performing statistical calculation.

Second, HR weighting measures the gene methylation status for each patient,
(1) The gene that gave a positive result with MSP is set to plus 1, the gene that gave a negative result with MSP is set to 0, and each value is multiplied by the value of Hazard Ratio (HR) corresponding to each gene. And sum the numbers obtained by multiplication,
(2) Multiply the number of methylated genes detected in the patient by the Hazard Ratio (HR) value of the number of methylated genes,
(3) When the patient is CIMP positive, that is, when 3 or more of 8 genes are methylated, plus 1 is set, and when CIMP is negative, 0 is set. Multiply by the value of Hazard Ratio (HR).

Sum of (1) to (3), (1) and (2), (2) and (3), or (1) and (3) And
It is arbitrary whether to use a value obtained by univariate analysis or a value obtained by multivariate analysis as HR.

  Regarding (1), it is not always necessary to calculate the values for all eight genes. For example, among the eight genes, genes particularly deeply involved in ATLL (for example, SHP1 gene, p16 gene, MGMT gene, Only the values for DAPK gene and HCAD gene) may be taken into account.

  Regarding (2), the number of methylated genes can take a numerical value of 0-8, and when this is multiplied by the HR of the number of methylated genes, the numerical value may be too large for (1) and (3). Therefore, as the value of (2), for example, the value obtained by multiplying the number of methylated genes by HR is divided by the total number of analyzed genes (8 if 8 genes are analyzed) ((1) and It is preferable to adopt the numerical value of (3) and the numerical value standardized so that the contribution to the ATLL onset / progression risk score is equivalent.

  Since the ATLL onset / progression risk score obtained by such calculation increases as the malignancy of ATLL increases (aggressive type is higher than indolent type), the ATLL onset / progression risk score also increases in patients with the same disease type. The higher the ATLL, the more likely it is to become malignant, so that patients with one disease type can be divided into groups that are more likely to become malignant (this group can be further divided into multiple groups) and groups that are less likely to become malignant (more It can be divided into different groups.

・ Detection and diagnosis of epigenetic abnormalities, prognosis management (prognosis judgment or estimation)
DNA methylation is deeply involved in cell differentiation and aging as a long-term memory device of cells. It is known that the abnormality is deeply involved as a cause in the development and progression of cancer, and even if it is not the cause, it may be closely correlated with the characteristics of individual cancers. Indeed, epigenetic mechanisms such as DNA methylation and histone modification abnormalities are key to abnormal gene expression in cancer. In particular, silencing by methylation of CpG islands in the tumor suppressor gene promoter region has been widely recognized as an inactivation mechanism of tumor suppressor genes.

  Previous studies have revealed that methylation abnormalities occur in genes involved in all processes necessary for carcinogenesis such as cell cycle, apoptosis, signal transduction, metastasis and invasion. The present inventors not only detect cancer cell-specific DNA methylation abnormalities, but also detect DNA methylation abnormalities closely related to cancer characteristics. Therefore, research has been conducted under the concept that clinically useful information (data and indicators that contribute to the formulation of appropriate treatment policies and prognosis management) can be obtained.

  As described in the Examples below, the correlation between the number of methylated genes and each item of clinical data is examined, and the survival function related to the survival rate over a certain (morbid) period is determined for each target gene, or The base of prognosis estimation was established by performing the analysis by Kaplan-Meier method for every gene methylation event (for example, CIMP). That is, the Kaplan-Meier method was used to assess survival as a function of time.

  The data obtained from the prognostic test is the methylation profile of CpG islands in the promoter region of two or more selected tumor suppressor genes or cancer-related genes, but their processing and analysis are mainly required for statistical analysis Data processing and subsequent statistical analysis. Specific determination / estimation methods and procedures vary depending on the index to be applied, the purpose of processing, the contents of data, the statistical method to be used, and the like. For example, the significance of differences or differences in prognostic factors is tested using log-rank statistics. The risk (hazard ratio) of a potential prognostic factor is calculated for that particular prognostic factor in its unit of change at each level relative to the basal level. That is, it is a prognostic analysis in which multi-factor analysis based on Kaplan-Meier analysis and Cox proportional hazard model is performed by SPSS using the above DNA methylation profile and ATLL clinical information.

  The study of the present inventors has revealed that patients with methylation and CIMP (+) of the SHP1, p16, MGMT, DAPK, and HCAD genes have a significantly poor prognosis. That is, the number of DNA methylation genes of the SHP1, p16, MGMT, DAPK and HCAD genes correlates with clinical disease progression data, and CIMP is independent of age, sex, disease type, etc. It was found to be a prognostic factor. Therefore, it is desirable to estimate the prognosis based on the methylation status of these 5 genes and the above-mentioned CIMP, clinical data, and ATLL test data.

  Diagnosis including prognosis estimation for individual patients, including prediction of the likelihood of ATLL onset of carriers of HTLV-I, or malignancy of ATLL pathology, particularly indolent ATLL, is actually performed by the physician In addition to the data provided by this method, other clinical data and the circumstances of each subject (age, sex, medical history, lifestyle, etc.) are comprehensively taken into consideration. Such prognostic predictions include those in which data on gene methylation detects epigenetic abnormalities through changes in biomarkers that reflect the level of expression control of two or more tumor suppressor genes or cancer-related genes in the sample. Is directly linked to the carcinogenic process and is an independent prognostic factor as well as a clinicopathological index, further increasing the reliability.

In determining whether the prognosis is good or not, a procedure is established for determining whether the prognosis is good or bad by comparing the reference value with the measured value by quantifying the standard of the methylation state.
Not only such simple determinations related to the prognosis of prognosis, but also survival curves related to the presence or absence of methylation of SHP1 gene, p16 gene, MGMT gene, DAPK gene and HCAD gene, and survival curves related to the presence or absence of CIMP (FIGS. 11 to 19) By comparing the gene methylation data obtained from specimens derived from individual patients, a highly reliable prognostic judgment including the prediction of the progress of the pathological condition is derived. Preferably, a more appropriate prognosis can be estimated by stratifying the onset / progress risk based on the above-described numerical value of the ATLL onset / progress risk score.

Specifically, the risk of potential prognostic factors and the range of associated 95% confidence intervals are estimated with Cox's “proportional-hazards model”, and the P-value is based on a two-sided test.
As a result, useful data that contributes to the formulation of treatment and administration plans can be obtained. The prognosis management of the patient is followed up based on the prognostic factor CIMP, the number of methylated genes and the ATLL onset / progression risk score calculated from the indices of the above five gene groups. If CIMP is positive and the number of methylated genes in the five gene groups shows a tendency to increase and the ATLL onset / progression risk score rises, suspected the progression of the disease type and detailed examination It is important to do further.

  Further, in connection with the above prognostic estimation, the following data providing method that is promising in view of the usefulness of data for detecting epigenetic abnormality and the reliability of estimation based thereon is also proposed as one aspect of the present invention. The

A method for generating data used for screening of administered drugs and / or determining the effects of treatment in the prognosis of adult T-cell leukemia / lymphoma,
Regarding a group of genes consisting of SHP1, p15, p16, p73, hMLH1, MGMT, DAPK and HCAD genes of isolated peripheral blood mononuclear cells (PBMC), CpG in the promoter region of those genes By measuring the methylation state of the island,
A methylation profile of CpG island in the promoter region of the above gene is prepared, and as a prognostic factor, the methylation status of CIMP and SHP1, p16, MGMT, DAPK and HCAD genes is determined, and the methylation profile and A method for preparing data used for screening of a drug to be administered and / or for determining a therapeutic effect of administration, wherein an ATLL onset / progress risk score is calculated from the prognostic factors, and the ATLL onset / progress risk score is used as the data.

The method of the present invention provides data useful for screening of administered drugs and / or determining the effects of administered treatment, which are required in actual treatment and prognosis management of ATLL.
Based on the methylation profile of the CpG island in the promoter region of the created gene, using indicators such as the presence or absence of gene methylation, CIMP based on statistical analysis of those data, ATLL onset / progression risk score, The present invention, which makes it possible to determine the disease type of adult T-cell leukemia / lymphoma disease, as well as to catch slight signs of the progression of the disease type, and to predict the progression of the disease type from the detected signs, This is an extremely useful technique for individually confirming treatment effects (determination of administration treatment effects) and establishing appropriate treatment / medication plans (screening for administered drugs).

  For example, if the prognosis is followed in the same patient and the data before treatment is compared with the data at the time of recurrence and exacerbation, if the type and number of genes methylated in both data are the same, treatment It is suspected that the same clones with leukemia cells that had decreased due to the increase again and relapsed. Complete remission (CR) usually eliminates any methylation of the target gene. Although it recurred after transplantation treatment, if only SHP1 was methylated during partial remission (PR), it is possible that methylation of SHP1 is involved at the boundary between PR and CR, which is specified in the prognosis It shows the significance of exploring the methylation of genes.

  Although the method of the present invention measures DNA methylation, such a measurement is much more sensitive than a normal blood test. Therefore, even in patients who are judged to have no abnormalities in HTLV-I carriers that are in the previous ATLL state due to the appearance of ATLL tumor cells in blood tests, they are HTLV-I carriers with a high risk of onset without missing them. There is a possibility that diagnosis can be performed with high sensitivity and high accuracy.

  The apparatus name, the concentration of the material used, the amount used, the processing time, the processing conditions, the numerical conditions such as the processing method, and the like used in the following examples are merely preferred examples within the scope of the present invention. The following description is made with reference to several figures, which may be shown schematically to the extent that the invention can be understood.

[Example 1]
Detection of gene methylation and discrimination of ATLL disease type In adult T cell leukemia / lymphoma (ATLL), which is one of the hematopoietic tumor diseases, methylation in two or more gene promoter regions selected from 8 gene groups The presence or absence of methylation and the frequency of methylation were confirmed using methylation specific PCR (MSP), and their clinical significance was examined.

・ Subjects and specimens Clinical specimens for measurement were collected from a total of 111 people. The breakdown consists of 13 healthy volunteers and 98 carriers and patients, ie 18 HTLV-I virus carriers (carriers), 11 chronic T-cell leukemia / lymphoma patients, and 15 smoldering patients. There are 29 patients with lymphoma type and 25 patients with acute type. Peripheral blood mononuclear cells (PBMC) or lymph node tissue cells were collected from these healthy subjects, carriers and patients. Peripheral blood as a sample was collected from the earlobe, brachial vein, etc., and mononuclear cells (PBMC) were prepared by a conventional method.

・ Search genes (adult T-cell leukemia / lymphoma gene set)
As a gene set for adult T-cell leukemia / lymphoma, the following eight types of tumor suppressor genes or cancer-related genes were targeted, and methylation of CpG islands in the promoter region was examined:
SHP1 gene, p15 gene, p16 gene, p73 gene, hMLH1 gene, MGMT gene, DAPK gene and HCAD gene.

-Detection of methylation Subsequently, in order to obtain genomic DNA from the specimen, the specimen and the cell lysate were mixed, and then the mixed solution was heated for a certain time. By this treatment, the cells in the specimen were destroyed with the lysis solution, and the genomic DNA in the cells was extracted. The reaction conditions such as the composition, concentration, reaction temperature, and reaction time of the lysate in the cell lysis step were those described in Patent Document 3.

As previously reported, DNA extracted from clinical specimens was treated with bisulfite (bisulfite, disulfite).
Using the modified DNA solution after the DNA conversion step, the presence or absence of methylation of the CpG island in the promoter region of the gene group (SHP1, p16, p15, p73, hMLH1, MGMH, DAPK, HCAD), the degree of methylation Was subjected to CpG island assay (methylation assay) using MSP (methylation specific PCR) method. According to this method, methylated DNA is detected with higher accuracy in order to PCR-amplify CpG-containing DNA after the DNA conversion step using a methylation-specific primer and an unmethylation-specific primer. Can do.

  The methylation-specific primer MSP and unmethylation-specific primer UMSP used in the assay are primers designed for the nucleotide sequences including the CpG sequence of the promoter region of each of the eight genes searched above. The base sequences are shown in Table 1 of JP 2007-244377 A. Further, details of primer preparation and MSP are described in Patent Document 3.

  In addition, in order to eliminate measurement errors due to false positive and false negative in MSP, a positive control and a negative control were processed simultaneously with the specimen. As a positive control, DNA obtained from peripheral blood mononuclear cells of healthy subjects treated with SssI methylase (New England BioLabs Inc., Beverly, Mass.) (PBMC (SssI)) was used. As a negative control, MSP was performed using a DNA sample of a healthy person.

The reaction was carried out for each DNA specimen using the following assay conditions.
Reaction solution: (0.4 μM primer: 1 μl bisulfite modified DNA: 1 × PCR buffer: 200 μM dNTP: 1.5 mM magnesium chloride: AmpliTaq Gold DNA
Polymerase 0.5 units; in a final reaction volume of 20 μl)
Reaction conditions: (95 ° C for 10 minutes): [(94 ° C for 15 seconds): (AT (annealing temperature) for 1 minute): (72 ° C for 1 minute)] 35 to 40 cycles: (72 ° C for 7 minutes) ).

  Detection of the PCR product after amplification was electrophoresed on a 3% agarose gel to detect the presence or absence of methylation. The presence or absence of a band corresponding to the size of the PCR product and the product size were detected with a marker (50 base pair ladder).

・ Relationship between gene methylation and clinical data DNA methylation of 8 genes measured and clinical data of ATLL (white blood cell count (WBC) in 1 μl of blood, lymphocyte count, abnormal lymphocyte count, calcium level, LDH , SIL2R and HTLV1 viral load). The result is shown in FIG. Correlation analysis was based on Spearman's rank correlation test. In FIG. 5, “number” is an item indicating a P value as to whether or not there is a correlation between the absolute number of the item and methylation of each gene. For example, between age and methylation of the SHP1 gene, This means that there is no correlation with a probability of 26.2%. Similarly, “≧ 60 / ≦ 59” in the age item indicates whether there is a significant difference in the methylation status of each gene between a person over 60 years old and a person under 59 years old. . CIMP3 represents a phenotype in which 3 or more of 8 genes are methylated.

Statistical analysis was performed using SPSS for Windows (registered trademark), Release 11.5, and significant difference was found when the p value was 0,05 or less. Items that have significant differences are colored. Regarding the relationship between the number of methylated genes and clinical laboratory data, among the eight genes, methylation of the SHP1 gene was the most correlated with clinical laboratory data items. When the SHP1 gene had methylation, the WBC (x103 / μl) number increased, the LDH (IU / L) increased, the HTLV1 viral load increased, and the sIL2R (U / mL) increased.
Further, FIGS. 6 to 9 show whether or not there is a correlation between the number of methylated genes and clinical laboratory data. As the number of methylated genes increased, the number of WBC (x103 / μl) increased, the number of lymphocytes (%) increased, the number of abnormal lymphocytes (%) increased, the LDH (IU / L) increased, the sIL2R (U / mL) increased A correlation was observed between the number of methylated genes and these laboratory data. Moreover, the tendency which shows the high amount of HTLV-1 virus amount was recognized as the number of methylation genes increased (P = 0.06). There was no correlation between the number of methylated genes and serum Ca (mg / dl).

[Example 2]
The relationship between gene methylation and prognosis Whether or not gene methylation affects prognosis was analyzed using three statistical methods. Statistical analysis was performed using SPSS for Windows (registered trademark), Release 11.5, and a significant difference was found when the p-value was 0.05 or less. The presence or absence of a correlation between the 8 genes and the prognosis of the 111 persons was examined. The results (survival curves) are shown in FIGS. 11 to 19 and Table 1 below. In Table 1, CI indicates Confidence Interval: 95% confidence interval. FIG. 10 is a survival curve for each disease type of ATLL shown in “Brit J Haematol. 79: 428-437, 1991”. In FIG. 10, “smoldering type (45)” means 45 cases. It represents the survival curve of smoldering patients.

(1) In creating the survival curve, the Kaplan-Meier method was used for analysis, and the log rank test was used for the difference test. The censored example indicates that no outcome (death) has occurred at the time of analysis. When methylation of the SHP1 gene, p16 gene, MGMT gene, DAPK gene, and HCAD gene was carried out, the survival days were significantly reduced in the case of having CIMP.
(2) Univariate analysis shows that methylation of SHP1, p16, MGMT, DAPK, and HCAD genes, an increase in the number of methylated genes, and the presence of CIMP are significant prognostic factors. It was done.
(3) Multivariate analysis showed that methylation of SHP1, p16, MGMT, and DAPK genes, the presence of CIMP, and an increase in the number of methylated genes were independent prognostic factors. The SHP1 gene and the p16 gene have the highest hazard ratio (HR) (risk ratio), indicating that they are important among prognostic factors.

From the above examples, it was shown that methylation of a specific gene and the number of methylated genes correlate with ATLL clinical laboratory data and can be independent prognostic factors for ATLL.

Next, the ATLL onset / progression risk scoring was performed by the following two methods based on the data obtained for each carrier and patient.
Specifically, as the first method, in each patient,
(1) For 5 genes of SHP1 gene, p16 gene, MGMT gene, DAPK gene and HCAD gene, plus 1 for genes that gave a positive result in MSP, 0 for genes that gave a negative result in MSP, Each numerical value was multiplied by a Hazard Ratio (HR) value (HR value obtained by univariate analysis shown in Gene Table 1) corresponding to each gene and totaled.
(3) For the above 5 genes, the positive value was 1 when CIMP was positive and 0 when CIMP was negative, and the value was multiplied by the Hazard Ratio (HR) value of CIMP determined by univariate analysis.

The sum of (1) and (3) above, rounded to the first decimal place for convenience, was used as the ATLL onset / progression risk score.
The results of the above ATLL onset / progression risk scoring (total score distribution) are shown in Table 2 below and FIGS. As a result of analyzing this ATLL onset / progression risk score by the Kruscal Wallis test, it became clear that the ATLL onset / progression risk score shows a significant difference between each disease type, and this is a book for calculating the ATLL onset / progression risk score It was shown that each disease type can be separated by the method (p = 0.000).

  In addition, there is a group (score = 2 to 6) having a high ATLL onset / progression risk score among carriers, and it is suspected that it is a high risk group for developing ATLL. Further, among smoldering and chronic patients, patients with an ATLL onset / progression risk score of 8 or more are suspected to be a high-risk group in which ATLL may further develop (malignant). It was shown that the high risk group can be stratified in each disease type.

In Table 2, “Frequency” represents the number of carriers or patients. “Average value” represents the average value of the ATLL onset / progression risk score shown in FIGS. Similarly, “standard deviation”, “standard error”, “95% confidence interval of average value”, “minimum value”, and “maximum value” indicate values of respective items related to the ATLL onset / progression risk score.

Specifically, as the second method, in each patient,
(1) For eight genes, plus 1 is given for genes that gave a positive result with MSP, and 0 is given for a gene that gave a negative result with MSP, and each numerical value has a Hazard Ratio ( HR) values (HR values obtained by univariate analysis shown in Gene Table 1) were multiplied and totaled.
(2) The number of methylated genes was multiplied by the Hazard Ratio (HR) value of the number of methylated genes determined by univariate analysis, and divided by 8 (divided by the total number of analyzed genes and normalized).
(3) The value was positive 1 when CIMP was positive and 0 when CIMP was negative, and the value was multiplied by the Hazard Ratio (HR) value of CIMP determined by univariate analysis.

The sum of (1) to (3) above, rounded to the first decimal place for convenience, was used as the ATLL onset / progression risk score.
The results of the above ATLL onset / progression risk scoring (total score distribution) are shown in Table 3 below and FIGS. As a result of analyzing this ATLL onset / progression risk score by the Kruscal Wallis test, it became clear that the ATLL onset / progression risk score shows a significant difference between each disease type, and this is a book for calculating the ATLL onset / progression risk score It was shown that each disease type can be separated by the method (p = 0.000).

  In addition, there is a group (score = 2 to 6) having a high ATLL onset / progression risk score among carriers, and it is suspected that it is a high risk group for developing ATLL. Further, among smoldering and chronic patients, patients with an ATLL onset / progression risk score of 8 or more are suspected to be a high-risk group in which ATLL may further develop (malignant). It was shown that the high risk group can be stratified in each disease type.

In Table 3, “Frequency” represents the number of carriers or patients. “Average value” represents the average value of the ATLL onset / progression risk score shown in FIGS. Similarly, “standard deviation”, “standard error”, “95% confidence interval of average value”, “minimum value”, and “maximum value” indicate values of respective items related to the ATLL onset / progression risk score.

Claims (9)

In collecting data to estimate the prognosis of adult T-cell leukemia / lymphoma (ATLL),
Regarding a group of genes consisting of SHP1, p15, p16, p73, hMLH1, MGMT, DAPK and HCAD genes of isolated peripheral blood mononuclear cells (PBMC), CpG in the promoter region of those genes By measuring the methylation state of the island,
While creating a methylation profile of CpG islands in the promoter region of the above gene, the CIMP value was calculated as a prognostic factor by the following (A) ,
By displaying the stratification of the risk by the ATLL onset / progression risk score calculated by the following (B) from the methylated gene, the number of methylated genes and the CIMP value,
Carrier adult T-cell leukemia virus (HTLV-I), i ndolent type ATLL and / or aggressive type ATLL method of creating a prognosis estimation data for.
(A): Among the genes in the gene group, when 3 or more genes of the patient are methylated, plus 1, when the patient has 2 or less methylated genes 0.
(B):
(1) SPSS for Windows (registered trademark) Release in univariate analysis or multivariate analysis on the correlation between the methylation of genes of the above gene group (methylation of CpG islands in the promoter region) and the survival rate of the population Among the genes whose P value by 11.5 (hereinafter referred to as P value) is 0.05 or less and whose Hazard Ratio (HR) (hereinafter referred to as Hazard Ratio (HR)) by SPSS for Windows (registered trademark) Release 11.5 is calculated About a gene group containing at least one of (hereinafter referred to as a selection gene group)
The gene that gave a positive result in the patient's MSP (methylation specific PCR method) is plus 1, and the gene that gave a negative result in the patient's MSP is 0, and each of those values is assigned to each gene. Multiply the corresponding population Hazard Ratio (HR) values, and sum the numbers obtained by multiplication,
(2) The number of methylated genes detected in the patient in the selected gene group is multiplied by the Hazard Ratio (HR) value of the number of methylated genes in the population, and the number of genes in the selected gene group Split
(3) Multiply the numerical value calculated by (A) by the value of Hazard Ratio (HR) of the CIMP of the population,
The sum of (1) to (3), the sum of (1) and (2), the sum of (2) and (3),
Or, the sum of (1) and (3) is used as the ATLL onset / progression risk score.
However, as the Hazard Ratio (HR), it is arbitrary whether to use a value obtained by univariate analysis or a value obtained by multivariate analysis. The method for creating prognostic estimation data according to claim 1, further comprising displaying the methylation status of the SHP1 gene, p16 gene, MGMT gene, DAPK gene and HCAD gene. A method for generating data used for screening of administered drugs and / or for determining the therapeutic effect of treatment in the prognosis of adult T-cell leukemia / lymphoma (ATLL),
Regarding a group of genes consisting of SHP1, p15, p16, p73, hMLH1, MGMT, DAPK and HCAD genes of isolated peripheral blood mononuclear cells (PBMC), CpG in the promoter region of those genes By measuring the methylation state of the island,
A methylation profile of CpG island in the promoter region of the gene is prepared, and as a prognostic factor, the CIMP value according to (A) according to claim 1 and the methylation of SHP1, p16, MGMT, DAPK and HCAD genes Seeking the state of
The ATLL onset / progression risk score according to (B) according to claim 1 is calculated from the methylation profile and the prognostic factor, and the administered drug screening and / or the ATLL onset / progression risk score is used as the data, and / or Alternatively, a method of creating data used to determine the treatment effect. Preparation of prognostic test data for predicting the onset of adult T cell leukemia virus (HTLV-I) carrier causing adult T cell leukemia / lymphoma (ATLL) or progression of indolent ATLL to aggressive ATLL A method,
Measuring the methylation status of CpG islands in the promoter regions of SHP1, p15, p16, p73, hMLH1, MGMT, DAPK and HCAD genes in specimens from the carrier or indolent ATLL patients; Obtain methylation profiles of 8 genes,
The CIMP value according to (A) of claim 1 is determined from the methylation profile,
A prognosis estimation test comprising calculating an ATLL onset / progression risk score according to (B) according to claim 1 from the methylation profile and the CIMP value , and using the ATLL onset / progression risk score as the data To create data. 8 types of reagents including a lysate for dissolving a specimen, a bisulfite-containing reagent, and methylation detection amplification reagents for SHP1, p15, p16, p73, hMLH1, MGMT, DAPK, and HCAD genes A kit for measuring the methylation status of CpG islands in the promoter region of the gene of
A tool for processing and analyzing the data obtained by the kit and the data obtained by prognostic estimation; and
A data creation system for creating data according to any one of claims 1 to 4, comprising a data processing / analysis result output device,
(1) methylation profiles of the eight genes,
(2) the CIMP value according to (A) of claim 1, and
(3) ATLL onset / progression risk score according to (B) of claim 1
A data creation system that includes tools for processing and analyzing data to create data. further,
  (4) Methylation status of SHP1 gene, p16 gene, MGMT gene, DAPK gene and HCAD gene
The data creation system according to claim 5, comprising a tool for displaying the data. The data creation system according to claim 5 or 6, wherein the tool for processing and analyzing the data includes a data processing program and / or a program using a prognostic statistical analysis algorithm. The selected gene group is a gene group consisting of SHP1 gene, p15 gene, p16 gene, p73 gene, hMLH1 gene, MGMT gene, DAPK gene and HCAD gene,
In the univariate analysis or multivariate analysis of the correlation between the gene methylation (methylation of CpG islands in the promoter region) and the survival rate for the population, the P value is 0.05 or less, and the Hazard Ratio The method for creating data according to any one of claims 1 to 4, wherein the gene group includes all genes for which (HR) is calculated. The selected gene group is a gene group consisting of SHP1 gene, p15 gene, p16 gene, p73 gene, hMLH1 gene, MGMT gene, DAPK gene and HCAD gene,
In the univariate analysis or multivariate analysis of the correlation between the gene methylation (methylation of CpG islands in the promoter region) and the survival rate for the population, the P value is 0.05 or less, and the Hazard Ratio The data creation system for data according to any one of claims 5 to 7, which is a gene group including all genes for which (HR) is calculated.