JP2018163019A - Method of supporting prediction of cancer recurrence - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of supporting prediction of cancer recurrence applicable at a point of discontinuation of tyrosine kinase inhibitor or at an early stage within several months from the discontinuation.SOLUTION: The method of supporting prediction of cancer recurrence includes the steps of: measuring the number of eTreg cells in blood collected from a cancer patient; acquiring the value relevant to the number of eTreg cells based on the measurement result; classifying the patient into a recurrence low/high risk group on the basis of the value relevant to the obtained number of eTreg cells, in which the cancer patient has tyrosine kinase inhibitor administration history, and the blood is collected 20 to 50 days after stopping the administration of the tyrosine kinase inhibitor.SELECTED DRAWING: None

Description

  The present invention relates to a method for assisting in predicting recurrence of cancer.

  Chronic myeloid leukemia (CML) results from the uncontrollable activation of BCR-ABL1 tyrosine kinase by the Philadelphia chromosome, an abnormal chromosome. Imatinib is known as a therapeutic agent for CML. Imatinib is a kind of tyrosine kinase inhibitor (TKI). According to the prospective observational study “TARGET” in Japan, the overall survival rate at 7.5 years for 639 CML patients was 95.1%. Although CML was once considered an incurable disease, the advent of imatinib has made it possible to dramatically improve the prognosis of CML patients.

  In recent years, cases have been reported that do not recur even after CML treatment is discontinued. In the prospective STIM study in France (STop IMatinib), 39% of patients with chronic phase CML who had maintained complete molecular genetic remission for more than 2 years had 39% of imatinib without molecular genetic recurrence. It has been reported that administration could be stopped for a long time. This result has been supplemented by the TWISTER trial in Australia, and it has been shown that some patients with CML can cease imatinib treatment.

  Some patients want to discontinue TKI, including imatinib, because of the financial burden, the presence of side effects, and the desire for pregnancy in young women.

Imagawa J. et al., “Discontinuation of dasatinib in patients with chronic myeloid leukemia who have maintained deep molecular response for longer than 1 year (DADI trial): a multicenter phase 2 trial” The Lancet Haematology, Volume 2, Issue 12, December 2015, Pages e528-e535

  Against this background, it is important to identify patients who can and should not be treated with imatinib. Non-Patent Document 1 reports the results of a dasatinib (a kind of TKI) administration discontinuation test in the treatment of chronic myelogenous leukemia. This document describes that the prognosis was good in a patient group with a small number of Treg cells (regulatory T cells) one year after discontinuation of dasatinib administration. However, this document does not describe a method that can provide information about the prognosis of CML patients when a physician actually decides whether or not to discontinue imatinib, or at an early stage within several months after discontinuation. Absent.

  Therefore, an object of the present invention is to provide a method for assisting prediction of cancer recurrence, which can be applied at an early stage within several months from the discontinuation of administration of a tyrosine kinase inhibitor.

  The present inventors have found that there is a correlation between the number of blood eTreg cells (effector regulatory T cells) approximately 1 month after imatinib administration is discontinued and the prognosis of cancer patients. It came to complete.

  That is, according to the present invention, a step of measuring the number of eTreg cells in blood collected from a cancer patient, a step of obtaining a value related to the number of eTreg cells based on the measurement result, and the number of obtained eTreg cells Categorizing the patient into a low risk group for recurrence based on the value for, wherein the cancer patient has a history of administration of a tyrosine kinase inhibitor, and the blood is 20 to 20 A method is provided to help predict cancer recurrence, which is blood collected after 50 days.

  ADVANTAGE OF THE INVENTION According to this invention, the prediction assistance method of the recurrence of cancer applicable to the early stage of about one month from the discontinuation of administration of a tyrosine kinase inhibitor can be provided.

It is an example of the external appearance of the reagent kit of this embodiment. It is an example of the external appearance of the reagent kit of this embodiment. It is the schematic which showed an example of the prediction assistance apparatus. It is a block diagram which shows the function structure of the prediction assistance apparatus of FIG. It is a block diagram which shows the hardware constitutions of the prediction auxiliary | assistance apparatus shown by FIG. It is an example of the flowchart of the determination for acquiring the data which assists the prediction of the recurrence of cancer using the prediction assistance apparatus shown by FIG. It is a figure which shows the classification result based on the increase rate of the number of eTreg cells. It is a figure which shows the classification result based on the number of eTreg cells / CD8 ⁺ T cells. It is a figure which shows the classification result based on the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells. It is a figure which shows the classification result based on the number of PD-1 ⁺ eTreg cells / CD8 ⁺ T cells. It is a figure which shows the classification result based on the number of PD-1 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells. It is a figure which shows the classification result based on the CTLA-4 ⁺ eTreg cell number / CD8 ⁺ T cell number. It is a figure which shows the classification result based on the number of CTLA-4 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells. It is a figure which shows the classification result based on the number of LAG-3 ⁺ eTreg cells / CD8 ⁺ T cells. It is a figure which shows the classification result based on the number of LAG-3 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells. It is a figure which shows the classification result based on the number of Tim-3 ⁺ eTreg cells / CD8 ⁺ T cells. It is a figure which shows the classification result based on the number of Tim-3 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells. It is a figure which shows the classification result based on the number of TIGIT ⁺ eTreg cells / CD8 ⁺ T cells. It is a figure which shows the classification result based on the number of TIGIT ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells. It is a figure which shows the classification result based on the number of ICOS ⁺ eTreg cells / CD8 ⁺ T cells. It is a figure which shows the classification result based on the number of ICOS ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells.

  The method for assisting in predicting the recurrence of cancer according to this embodiment (hereinafter also simply referred to as “method”) includes a step of measuring the number of eTreg cells in blood collected from a cancer patient. A cancer patient is a patient who has been diagnosed as suffering from cancer and has a history of administration of a tyrosine kinase inhibitor, and preferably has been administered a tyrosine kinase inhibitor for more than 3 years. A patient who has had a complete and complete remission for more than 2 years.

  “Has a tyrosine kinase inhibitor dosing history” means that the patient has been administered a tyrosine kinase inhibitor more than once. “Molecular genetic complete remission” means that a high-sensitivity RQ-PCR (Real-time Quantitative PCR) method or nested PCR method does not detect a cancer causative gene such as the BCR-ABL gene twice consecutively. Means that.

  The cancer is not particularly limited as long as it is a cancer caused by an abnormality of tyrosine kinase. Leukemia, specifically, chronic myeloid leukemia (CML), Philadelphia chromosome positive acute lymphoblastic leukemia (Ph + ALL) and chronic lymph Leukemia, gastrointestinal stromal tumor (GIST), non-small cell lung cancer, renal cell carcinoma, pancreatic neuroendocrine tumor, malignant soft tissue tumor (soft tissue sarcoma), malignant melanoma, HER2-overexpressing breast cancer, idiopathic pulmonary fibrosis, Although small lymphocytic lymphoma etc. are mentioned, Preferably it is chronic myeloid leukemia.

Blood drawn from cancer patients includes whole blood, serum or plasma. The blood is particularly preferably one that can be easily collected, and examples thereof include peripheral blood and serum and plasma obtained therefrom.
The blood is blood collected 20 to 50 days after cessation of administration of the tyrosine kinase inhibitor, preferably 25 to 42 days after cessation of administration of the tyrosine kinase inhibitor.

  The tyrosine kinase inhibitor is not particularly limited, and imatinib, dasatinib, gefitinib, erlotinib, osmeltinib, afatinib, bosutinib, vandetanib, sunitinib, axitinib, pazopanib, lenvatinib, lapatinib, nintedinib, nilotinib, etc. It is.

eTreg cells are regulatory T cells that are in the final differentiation stage and exhibit strong immunosuppressive activity. eTreg cells are cells characterized by expressing CD3, CD4 and FOXP3 and not CD45RA (CD3 ⁺ CD4 ⁺ FOXP3 ⁺ CD45RA ^- cells). Here, in the present specification, expressions such as “express” and “+” for a cell surface marker indicate that the signal is equal to or higher than a predetermined threshold when the cell surface marker is labeled with a labeled antibody described later. Means that. For example, a CD3 ⁺ cell is a cell that exhibits a fluorescence intensity equal to or higher than a predetermined threshold when labeled with an anti-CD3 labeled antibody. In the present specification, expressions such as “not expressed” and “−” for a cell surface marker mean that the signal becomes less than a predetermined threshold when the cell surface marker is labeled with a labeled antibody described below. To do. For example, a CD45RA ^- cell is a cell that exhibits a fluorescence intensity below a predetermined threshold when labeled with an anti-CD45RA labeled antibody.

CD3 (Cluster of Differentiation 3) is a transmembrane protein known to complex with T cell receptors. CD4 (Cluster of Differentiation 4) is a co-receptor expressed on the surface of helper T cells. FOXP3 (Forkhead box P3) is a gene encoding a Treg master transcription factor. CD45RA (Cluster of Differentiation 45 RA) is one of the naïve T cell markers. The number of eTreg cells can be measured by counting the number of CD3 ⁺ CD4 ⁺ FOXP3 ⁺ CD45RA ⁻ cells by a method known to those skilled in the art. The number of eTreg cells can be measured, for example, as follows: a cell collected from blood is mixed with a labeled anti-CD3 antibody, a labeled anti-CD4 antibody and a labeled anti-CD45RA antibody, and the cell surface is mixed. The antibody is bound to the antigen to be expressed, the cells are washed, stained with a labeled anti-FoxP3 antibody, and the cells are washed, and then using flow cytometry, the labeled anti-CD3 antibody and the labeled anti-CD4 All the signals derived from the antibody and the labeled anti-FoxP3 antibody are emitted, and the number of cells not emitting the signal derived from the labeled anti-CD45RA antibody is counted. The number of eTreg cells can be measured, for example, according to the description of Nishikawa et al. (Current Opinion in Immunology, 2014, 27: 1-7).

  The method of this embodiment further comprises a step of measuring the number of eTreg cells in blood collected 10 to 50 days before cessation of administration of the tyrosine kinase inhibitor, preferably 15 to 44 days before cessation of administration of the tyrosine kinase inhibitor. May be included.

The method of this embodiment is at least one selected from the group consisting of the number of CD8 ⁺ T cells and the number of PD-1 ⁺ CD8 ⁺ T cells in blood collected 20 to 50 days after the administration of the tyrosine kinase inhibitor is stopped. It may further include a step of measuring one. As shown in the following examples, the number of eTreg cells further expressing PD-1, CTLA-4, LAG-3, Tim-3, TIGIT, or ICOS is the number of CD8 ⁺ T cells or PD-1 ⁺ CD8 ⁺ Good classification results were obtained when parameters obtained by dividing by the number of T cells were used.

CD8 (Cluster of Differentiation 8) is a co-receptor expressed on the surface of cytotoxic T cells. CD8 ⁺ T cells are cells (CD3 ⁺ CD8 ⁺ T cells or CD3 ⁺ CD8a ⁺ T cells) that are characterized by expressing CD3 and CD8 (CD8a). Thus, CD8 ⁺ T cell number, by methods known to those skilled in the art, can be determined by measuring the number of such CD3 ⁺ CD8 ⁺ T cells (or CD3 ⁺ CD8a ⁺ T cells). The number of CD8 ⁺ T cells can be determined by, for example, labeling anti-CD3 antibody, labeled anti-CD4 antibody, labeled anti-FoxP3 antibody and labeled anti-CD45RA antibody instead of labeled anti-CD3 antibody and labeled anti-CD8 antibody (or labeled The measurement can be performed in the same manner as described with respect to the measurement of the number of eTreg cells, except that an anti-CD8a antibody) is used. CD8 forms the CD8 family including CD8a and the like, but it is CD8a that is measured in the present invention. Hereinafter, CD8a is sometimes simply referred to as “CD8”.

PD-1 ⁺ CD8 ⁺ T cell count is a cell characterized by expressing CD3, CD8 (CD8a) and PD-1 (PD-1 ⁺ CD3 ⁺ CD8 ⁺ T cell or PD-1 ⁺ CD3 ⁺ CD8a ⁺ T cells). PD-1 (Programmed cell death 1) is an immune checkpoint protein known to suppress T cell activation through interaction with PD-L1 or PD-L2 ligand. PD-1 ⁺ CD8 ⁺ T cell count is determined by counting the number of PD-1 ⁺ CD3 ⁺ CD8 ⁺ T cells (or PD-1 ⁺ CD3 ⁺ CD8a ⁺ T cells) by methods known to those skilled in the art can do. The number of PD-1 ⁺ CD8 ⁺ T cells is, for example, a labeled anti-CD3 antibody, a labeled anti-CD8 antibody instead of a labeled anti-CD3 antibody, a labeled anti-CD4 antibody, a labeled anti-FoxP3 antibody and a labeled anti-CD45RA antibody. Except for using an antibody (or labeled anti-CD8a antibody) and a labeled anti-PD-1 antibody, the measurement can be performed in the same manner as described for counting the number of eTreg cells.

The method of the present embodiment comprises the number of PD-1 ⁺ eTreg cells, CTLA-4 ⁺ eTreg cells, LAG-3 ⁺ eTreg cells in blood collected 20 to 50 days after cessation of tyrosine kinase inhibitor administration, The method may further include a step of measuring at least one selected from the group consisting of the number of Tim-3 ⁺ eTreg cells, the number of TIGIT ⁺ eTreg cells, and the number of ICOS ⁺ eTreg cells. As shown in the following examples, good classification results are obtained when the number of eTreg cells further expressing PD-1, CTLA-4, LAG-3, Tim-3, TIGIT, or ICOS is used. It was.

PD-1 ⁺ eTreg cells are cells characterized by expressing PD-1, CD3, CD4 and FOXP3 but not CD45RA (PD-1 ⁺ CD3 ⁺ CD4 ⁺ FOXP3 ⁺ CD45RA ⁻ cells). The number of PD-1 ⁺ eTreg cells can be measured by counting the number of PD-1 ⁺ CD3 ⁺ CD4 ⁺ FOXP3 ⁺ CD45RA ⁻ cells by a method known to those skilled in the art. The number of PD-1 ⁺ eTreg cells can be measured, for example, as follows: cells collected from blood, labeled anti-PD-1 antibody, labeled anti-CD3 antibody, labeled anti-CD4 antibody and label Mixed with anti-CD45RA antibody, bind the antibody to the antigen expressed on the cell surface, wash the cell, stain with the labeled anti-FoxP3 antibody, wash the cell, then use flow cytometry Of cells that emit all signals derived from labeled anti-PD-1 antibody, labeled anti-CD3 antibody, labeled anti-CD4 antibody and labeled anti-FoxP3 antibody, but not signals derived from labeled anti-CD45RA antibody. Measure the number.

CTLA-4 ⁺ eTreg cells are cells characterized by expressing CTLA-4, CD3, CD4 and FOXP3 and not CD45RA (CTLA-4 ⁺ CD3 ⁺ CD4 ⁺ FOXP3 ⁺ CD45RA ⁻ cells). CTLA-4 (Cytotoxic T-lymphocyte-associated antigen 4) is a gene encoding an immune checkpoint protein known to be involved in suppression of T cell immune response. The number of CTLA-4 ⁺ eTreg cells can be measured by counting the number of CTLA-4 ⁺ CD3 ⁺ CD4 ⁺ FOXP3 ⁺ CD45RA ⁻ cells by a method known to those skilled in the art. The number of CTLA-4 ⁺ eTreg cells can be measured, for example, as follows: cells collected from blood, labeled anti-CTLA-4 antibody, labeled anti-CD3 antibody, labeled anti-CD4 antibody and label Mixed with anti-CD45RA antibody, bind the antibody to the antigen expressed on the cell surface, wash the cell, stain with the labeled anti-FoxP3 antibody, wash the cell, then use flow cytometry Of cells that emit all signals derived from labeled anti-CTLA-4 antibody, labeled anti-CD3 antibody, labeled anti-CD4 antibody and labeled anti-FoxP3 antibody, but not signals derived from labeled anti-CD45RA antibody. Measure the number.

LAG-3 ⁺ eTreg cells are cells characterized by expressing LAG-3, CD3, CD4 and FOXP3 and not CD45RA (LAG-3 ⁺ CD3 ⁺ CD4 ⁺ FOXP3 ⁺ CD45RA ⁻ cells). LAG-3 (lymphocyte activation gene-3) is a gene encoding a membrane protein belonging to the immunoglobulin superfamily. The number of LAG-3 ⁺ eTreg cells can be measured by counting the number of LAG-3 ⁺ CD3 ⁺ CD4 ⁺ FOXP3 ⁺ CD45RA ⁻ cells by a method known to those skilled in the art. The number of LAG-3 ⁺ eTreg cells can be measured, for example, as follows: cells collected from blood, labeled anti-LAG-3 antibody, labeled anti-CD3 antibody, labeled anti-CD4 antibody and label Mixed with anti-CD45RA antibody, bind the antibody to the antigen expressed on the cell surface, wash the cell, stain with the labeled anti-FoxP3 antibody, wash the cell, then use flow cytometry Of cells that emit all signals derived from labeled anti-LAG-3 antibody, labeled anti-CD3 antibody, labeled anti-CD4 antibody and labeled anti-FoxP3 antibody, but not signals derived from labeled anti-CD45RA antibody. Measure the number.

Tim-3 ⁺ eTreg cells are cells characterized by expressing Tim-3, CD3, CD4 and FOXP3 but not CD45RA (Tim-3 ⁺ CD3 ⁺ CD4 ⁺ FOXP3 ⁺ CD45RA ⁻ cells). Tim-3 (T-cell immunoglobulin and mucin containing protein-3) is a gene encoding a membrane protein belonging to the immunoglobulin superfamily. The number of Tim-3 ⁺ eTreg cells can be measured by counting the number of Tim-3 ⁺ CD3 ⁺ CD4 ⁺ FOXP3 ⁺ CD45RA ⁻ cells by a method known to those skilled in the art. The number of Tim-3 ⁺ eTreg cells can be measured, for example, as follows: cells collected from blood, labeled anti-Tim-3 antibody, labeled anti-CD3 antibody, labeled anti-CD4 antibody and label Mixed with anti-CD45RA antibody, bind the antibody to the antigen expressed on the cell surface, wash the cell, stain with the labeled anti-FoxP3 antibody, wash the cell, then use flow cytometry Of cells that emit all signals derived from labeled anti-Tim-3 antibody, labeled anti-CD3 antibody, labeled anti-CD4 antibody and labeled anti-FoxP3 antibody, but not signals derived from labeled anti-CD45RA antibody. Measure the number.

TIGIT ⁺ eTreg cells are cells characterized by expressing TIGIT, CD3, CD4 and FOXP3 and not CD45RA (TIGIT ⁺ CD3 ⁺ CD4 ⁺ FOXP3 ⁺ CD45RA ⁻ cells). TIGIT (T cell immunoreceptor with Ig and ITIM domain) is a gene encoding a membrane protein belonging to the immunoglobulin superfamily. The number of TIGIT ⁺ eTreg cells can be measured by counting the number of TIGIT ⁺ CD3 ⁺ CD4 ⁺ FOXP3 ⁺ CD45RA ⁻ cells by a method known to those skilled in the art. TIGIT ⁺ ETreg cell numbers, for example, can be measured as follows: the cells taken from the blood, labeled anti-TIGIT antibodies, labeled anti-CD3 antibody, labeled anti-CD4 antibody and labeled anti-CD45RA antibody The antibody is bound to the antigen expressed on the cell surface, the cell is washed, stained with a labeled anti-FoxP3 antibody, washed with the cell, and then labeled with flow cytometry. All the signals derived from the anti-TIGIT antibody, labeled anti-CD3 antibody, labeled anti-CD4 antibody and labeled anti-FoxP3 antibody are emitted, and the number of cells not emitting the signal derived from the labeled anti-CD45RA antibody is counted.

ICOS ⁺ eTreg cells are cells characterized by expressing ICOS, CD3, CD4 and FOXP3 and not CD45RA (ICOS ⁺ CD3 ⁺ CD4 ⁺ FOXP3 ⁺ CD45RA ⁻ cells). ICOS (Inducible T cell costimulator) is a gene encoding a costimulatory molecule involved in inducing differentiation of Treg cells. The number of ICOS ⁺ eTreg cells can be measured by counting the number of ICOS ⁺ CD3 ⁺ CD4 ⁺ FOXP3 ⁺ CD45RA ⁻ cells by a method known to those skilled in the art. The number of ICOS ⁺ eTreg cells can be measured, for example, as follows: cells collected from blood, labeled anti-ICOS antibody, labeled anti-CD3 antibody, labeled anti-CD4 antibody and labeled anti-CD45RA antibody The antibody is bound to the antigen expressed on the cell surface, the cell is washed, stained with a labeled anti-FoxP3 antibody, washed with the cell, and then labeled with flow cytometry. All the signals derived from the anti-ICOS antibody, the labeled anti-CD3 antibody, the labeled anti-CD4 antibody and the labeled anti-FoxP3 antibody are emitted, and the number of cells not emitting the signal derived from the labeled anti-CD45RA antibody is counted.

The labeled antibody means an antibody labeled with a labeling substance. The labeling substance is not particularly limited as long as a detectable signal is generated. For example, it may be a substance that itself generates a signal (hereinafter also referred to as a “signal generating substance”) or a substance that generates a signal by catalyzing the reaction of another substance. Preferably it is a generating substance. Examples of such signal generating substances include fluorescent substances and radioisotopes. Examples of the fluorescent substance include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, Alexa Fluor (registered trademark), and fluorescent proteins such as green fluorescent protein (GFP). Examples of the radioisotope include ¹²⁵ I, ¹⁴ C, and ³² P. Examples of the substance that generates a detectable signal by catalyzing the reaction of another substance include an enzyme. Examples of the enzyme include alkaline phosphatase, peroxidase, β-galactosidase, luciferase and the like. The enzyme substrate can be appropriately determined by those skilled in the art depending on the type of enzyme selected. The labeling substance is particularly preferably a fluorescent substance.

  In the present specification, “detecting a signal” includes qualitatively detecting the presence or absence of a signal, quantifying the signal intensity, and semi-quantitatively detecting the intensity of the signal. Semi-quantitative detection refers to showing the intensity of a signal in stages, such as “no signal generated”, “weak”, “medium”, “strong”, and the like. Methods for detecting signals themselves are known in the art. In the present embodiment, a measurement method corresponding to the type of signal derived from the labeling substance may be selected as appropriate. For example, when the labeling substance is a fluorescent substance, the fluorescent signal emitted therefrom can be measured by using a known device such as a flow cytometer. In addition, an excitation wavelength and a fluorescence wavelength can be suitably determined according to the kind of used fluorescent substance.

  In the present embodiment, the number of cells such as eTreg cells is measured from the signal detection result. For example, the value of the number of signal detections can be used as the number of cells. Examples of the number of cells include a value obtained by subtracting the value of the signal detection number of the negative control sample or the background value from the value of the signal detection number. A negative control sample can be selected as appropriate, and examples thereof include blood obtained from a healthy person.

The method of the present embodiment includes a step of obtaining a value related to the number of eTreg cells based on the measurement result.
Values related to the number of eTreg cells are parameters such as the degree of increase in the number of eTreg cells in the blood collected from cancer patients and the ratio to the number of other cells present in the blood, such as CD8 ⁺ T cells, or Or a combination thereof.

In one embodiment, the value for the number of eTreg cells may be the rate of increase in the number of eTreg cells or the reciprocal of the rate of increase in the number of eTreg cells.
Here, the rate of increase in the number of eTreg cells is expressed by the following formula:
(Number of eTreg cells in blood collected 20 to 50 days after cessation of administration of tyrosine kinase inhibitor) / (Number of eTreg cells in blood collected 10 to 50 days before cessation of administration of tyrosine kinase inhibitor)
Represented by

The reciprocal of the increase in the number of eTreg cells is given by the following formula:
(Number of eTreg cells in blood collected 10 to 50 days before cessation of administration of tyrosine kinase inhibitor) / (Number of eTreg cells in blood collected 20 to 50 days after cessation of administration of tyrosine kinase inhibitor)
Represented by

  Since Treg cells originally have an effect of suppressing immune function, when eTreg cells increase, immune function is lowered, and the prognosis of cancer patients is likely to deteriorate. However, contrary to such common technical knowledge, the present inventors have found that the prognosis of cancer patients is good when the number of blood eTreg cells is increased about one month after discontinuation of imatinib. I found it.

In another embodiment, one or more parameters selected from the following group:
eTreg cells / CD8 ⁺ T cells, eTreg cells / PD-1 ⁺ CD8 ⁺ T cells, PD-1 ⁺ eTreg cells / CD8 ⁺ T cells, PD-1 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells, CTLA-4 ⁺ eTreg cells / CD8 ⁺ T cells, CTLA-4 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells, LAG-3 ⁺ eTreg cells / CD8 ⁺ T cells Number, LAG-3 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells, Tim-3 ⁺ eTreg cells / CD8 ⁺ T cells, Tim-3 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells , TIGIT ⁺ eTreg cells / CD8 ⁺ T cells, TIGIT ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells, ICOS ⁺ eTreg cells / CD8 ⁺ T cells, ICOS ⁺ eTreg cells / PD- 1 ⁺ CD8 ⁺ T cell count; or one or more reciprocal parameters selected from the following groups:
CD8 ⁺ T cell count / eTreg cell count, PD-1 ⁺ CD8 ⁺ T cell count / eTreg cell count, CD8 ⁺ T cell count / PD-1 ⁺ eTreg cell count, PD-1 ⁺ CD8 ⁺ T cell count / PD- 1 ⁺ eTreg cells, CD8 ⁺ T cells / CTLA-4 ⁺ eTreg cells, PD-1 ⁺ CD8 ⁺ T cells / CTLA-4 ⁺ eTreg cells, CD8 ⁺ T cells / LAG-3 ⁺ eTreg cells Count, PD-1 ⁺ CD8 ⁺ T cell count / LAG-3 ⁺ eTreg cell count, CD8 ⁺ T cell count / Tim-3 ⁺ eTreg cell count, PD-1 ⁺ CD8 ⁺ T cell count / Tim-3 ⁺ eTreg cell count Number, CD8 ⁺ T cell count / TIGIT ⁺ eTreg cell count, PD-1 ⁺ CD8 ⁺ T cell count / TIGIT ⁺ eTreg cell count, CD8 ⁺ T cell count / ICOS ⁺ eTreg cell count and PD-1 ⁺ CD8 ⁺ T cell count Number / ICOS ⁺ eTreg cell number;
It may be.

Usually, continuous and continuous increase in the number of eTreg cells in cancer-bearing organisms is considered to work in the direction of suppressing cancer immunity. In the present invention, the administration of imatinib over a long period of time results in an immune state in which eTreg cells are reduced in the patient's body and the suppression is released. By stopping imatinib administration, eTreg cells were captured in a transient state (one month after discontinuation). Activation of eTreg cells first occurs at a month after imatinib withdrawal, after which eTreg cell rebound settles. On the other hand, the activation of CD8 ⁺ T cells captures whether sufficient activation was induced during the decline of eTreg cells. That is, when there is a sufficient decrease in eTreg cells during imatinib administration, and the immune suppression via eTreg cells by the tumor is released, CD8 ⁺ T cells in the body are sufficiently activated. An index that captures both the sufficient decrease of eTreg cells by imatinib and activation of CD8 ⁺ T cells is “eTreg cell number / CD8 ⁺ T cell number”. In this embodiment, the higher the value of the number of eTreg cells / CD8 ⁺ T cells one month after discontinuation of administration, the later the immune state is recovered and normalized, and as a result, it is considered to be a non-relapse index. eTreg cells suppress the function of effector cells including CD8 ⁺ T cells, while CD8 ⁺ T cells are effector T cells having cytotoxic effects. By taking the ratio of the number of these cells, it is considered that an indicator of whether the immune state is relatively suppressive or activated can be obtained.

  In another embodiment, the value related to the number of eTreg cells may be a combination of the above-described increase rate of the number of eTreg cells, a parameter, and an inverse parameter. By using a plurality of parameters in combination for analysis, the accuracy of the classification process can be improved.

More specifically, the value relating to the number of eTreg cells is, for example, two parameters selected from the above group, namely a combination of the following first parameter and the following second parameter or two reciprocal parameters, ie the following first parameter: It may be a combination of the reciprocal parameter and the following second reciprocal parameter:
The first parameter is the rate of increase in the number of eTreg cells;
The first reciprocal parameter is the reciprocal of the rate of increase in eTreg cell number;
The second parameter is the number of eTreg cells / CD8 ⁺ T cells, eTreg cells / PD-1 ⁺ CD8 ⁺ T cells, PD-1 ⁺ eTreg cells / CD8 ⁺ T cells, PD-1 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cell count, CTLA-4 ⁺ eTreg cell count / CD8 ⁺ T cell count, CTLA-4 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, LAG-3 ⁺ eTreg cell count / CD8 ⁺ T cell count, LAG-3 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, Tim-3 ⁺ eTreg cell count / CD8 ⁺ T cell count, Tim-3 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cells, TIGIT ⁺ eTreg cells / CD8 ⁺ T cells, TIGIT ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells, ICOS ⁺ eTreg cells / CD8 ⁺ T cells, ICOS ⁺ eTreg A value of 1 selected from the group consisting of cell number / PD-1 ⁺ CD8 ⁺ T cell number;
The second reciprocal parameter is the number of CD8 ⁺ T cells / eTreg cells, PD-1 ⁺ CD8 ⁺ T cells / eTreg cells, CD8 ⁺ T cells / PD-1 ⁺ eTreg cells, PD-1 ⁺ CD8 ⁺ T cell count / PD-1 ⁺ eTreg cell count, CD8 ⁺ T cell count / CTLA-4 ⁺ eTreg cell count, PD-1 ⁺ CD8 ⁺ T cell count / CTLA-4 ⁺ eTreg cell count, CD8 ⁺ T cell count / LAG-3 ⁺ eTreg cell count, PD-1 ⁺ CD8 ⁺ T cell count / LAG-3 ⁺ eTreg cell count, CD8 ⁺ T cell count / Tim-3 ⁺ eTreg cell count, PD-1 ⁺ CD8 ⁺ T cell count / Tim-3 ⁺ eTreg cells, CD8 ⁺ T cells / TIGIT ⁺ eTreg cells, PD-1 ⁺ CD8 ⁺ T cells / TIGIT ⁺ eTreg cells, CD8 ⁺ T cells / ICOS ⁺ eTreg cells and PD- The value is 1 selected from the group consisting of 1 ⁺ CD8 ⁺ T cell number / ICOS ⁺ eTreg cell number.

Alternatively, the value for the number of eTreg cells is a combination of three parameters selected from the above group, namely the above first parameter, the above second parameter and the following third parameter, or three reciprocal parameters, ie, the above first parameter. It may be a combination of the reciprocal parameter, the above second reciprocal parameter and the following third reciprocal parameter:
The third parameter is the number of eTreg cells / CD8 ⁺ T cells, eTreg cells / PD-1 ⁺ CD8 ⁺ T cells, PD-1 ⁺ eTreg cells / CD8 ⁺ T cells, PD-1 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cell count, CTLA-4 ⁺ eTreg cell count / CD8 ⁺ T cell count, CTLA-4 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, LAG-3 ⁺ eTreg cell count / CD8 ⁺ T cell count, LAG-3 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, Tim-3 ⁺ eTreg cell count / CD8 ⁺ T cell count, Tim-3 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cells, TIGIT ⁺ eTreg cells / CD8 ⁺ T cells, TIGIT ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells, ICOS ⁺ eTreg cells / CD8 ⁺ T cells, ICOS ⁺ eTreg A parameter different from the second parameter selected from the group consisting of cell number / PD-1 ⁺ CD8 ⁺ T cell number;
The third reciprocal parameter is the number of eTreg cells / CD8 ⁺ T cells, eTreg cells / PD-1 ⁺ CD8 ⁺ T cells, PD-1 ⁺ eTreg cells / CD8 ⁺ T cells, PD-1 ⁺ eTreg cells Number / PD-1 ⁺ CD8 ⁺ T cell count, CTLA-4 ⁺ eTreg cell count / CD8 ⁺ T cell count, CTLA-4 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, LAG-3 ⁺ eTreg cell count Number / CD8 ⁺ T cell count, LAG-3 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, Tim-3 ⁺ eTreg cell count / CD8 ⁺ T cell count, Tim-3 ⁺ eTreg cell count / PD- 1 ⁺ CD8 ⁺ T cell count, TIGIT ⁺ eTreg cell count / CD8 ⁺ T cell count, TIGIT ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, ICOS ⁺ eTreg cell count / CD8 ⁺ T cell count, ICOS ⁺ The reciprocal parameter is different from the second reciprocal parameter selected from the group consisting of the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells.

In a preferred embodiment, the value for eTreg cell number is a combination of the following first parameter, the following second parameter and the following third parameter:
The first parameter is the rate of increase in the number of eTreg cells;
The second parameter is the number of eTreg cells / CD8 ⁺ T cells;
The third parameter is the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells.

The method of this embodiment includes a step of classifying patients into a low risk group for recurrence based on the value regarding the number of eTreg cells obtained as described above.
The classification may be performed by, for example, comparing a parameter with a predetermined threshold value.
The predetermined threshold is not particularly limited and can be set as appropriate. For example, a predetermined threshold value may be set as follows. First, the number of eTreg cells in blood collected from multiple cancer patients is measured. In addition, the number of eTreg cells in blood collected from those cancer patients 10 to 50 days before cessation of administration of the tyrosine kinase inhibitor is measured. Thereafter, the increase rate of the number of eTreg cells is calculated from the measured data of the number of eTreg cells. Then, the obtained increase rate of the number of eTreg cells is classified into data of a patient group having a recurrence history and data of a patient group having no recurrence history. “There is a recurrence history” means that the cancer patient has relapsed in the past. And the most accurate distinction between the rate of increase in the number of eTreg cells in blood collected from patients with a history of recurrence and the rate of increase in the number of eTreg cells in blood collected from patients without a history of recurrence A value is obtained and the value is set as a predetermined threshold value. In setting the predetermined threshold, it is preferable to consider sensitivity, specificity, positive predictive value, negative predictive value, and the like.

In a more specific embodiment, for example, when the rate of increase in the number of eTreg cells is greater than or equal to a predetermined threshold, the patient is classified into a low-risk group in the classification step. Further, when the reciprocal of the increase rate of the number of eTreg cells is less than a predetermined threshold, the patient is classified into a low risk group for recurrence in the classification process. Such a classification is supported, for example, in Table 1.
In another embodiment, for example, if one parameter is greater than or equal to a predetermined threshold, or if one reciprocal parameter is less than a predetermined threshold, the classification step classifies the patient into a low-risk group. Such classification is supported, for example, in Tables 2-15.

  If the value for the number of eTreg cells includes multiple parameters, the classification can be performed by, for example, individually comparing each parameter or reciprocal parameter with a predetermined threshold corresponding to each parameter or reciprocal parameter, and all parameters or reciprocal parameters are It may be performed according to criteria such as whether or not a predetermined threshold value or more, and whether or not any one parameter or reciprocal parameter is a predetermined threshold value or more.

  In a more specific embodiment, for example, if the first parameter is greater than or equal to a predetermined threshold or the second parameter is greater than or equal to a predetermined threshold, the cancer patient is classified into a low risk group for recurrence in the classification step. When the first reciprocal parameter is less than a predetermined threshold value or the second reciprocal parameter is less than a predetermined threshold value, the cancer patient is classified into a low recurrence risk group in the classification step. Such classification is supported by (16-1), (16-3), and (16-5) of Example 16, for example.

  In another embodiment, for example, if the first parameter is greater than or equal to a predetermined threshold, the second parameter is greater than or equal to a predetermined threshold, or the third parameter is greater than or equal to a predetermined threshold, the cancer patient is classified into the low risk group for recurrence Is done. In addition, when the first reciprocal parameter is less than the predetermined threshold, the second reciprocal parameter is less than the predetermined threshold, or the third reciprocal parameter is less than the predetermined threshold, the cancer patient is classified into a low recurrence risk group in the classification step. Such classification is supported by (16-7) of Example 16, for example.

  In another embodiment, for example, the first parameter is a predetermined threshold or more and the second parameter is a predetermined threshold or more, the first parameter is a predetermined threshold or more and the third parameter is a predetermined threshold or more, or the second parameter is predetermined If the third parameter is equal to or greater than the predetermined threshold and the third parameter is equal to or greater than the predetermined threshold, the patient is classified as a low-risk group. Such classification is supported by (16-2), (16-4), and (16-6) of Example 16, for example.

  In another embodiment, for example, if the first parameter is greater than or equal to a predetermined threshold, the second parameter is greater than or equal to a predetermined threshold, and the third parameter is greater than or equal to a predetermined threshold, the patient is classified into the low recurrence risk group. Such a classification is supported by (16-8) of Example 16, for example.

  The scope of the present invention includes a method for treating cancer. According to one embodiment, a method for treating cancer includes the measurement step, the acquisition step, and the determination step described above, and further includes a treatment step. In the treatment method according to the present embodiment, in the determination step, patients are classified into a low risk group for recurrence and another group. In the treatment process, TKI administration is resumed for patients classified into other groups. For patients classified into the low-risk relapse group, drug administration is not resumed (drug re-administration), and measures such as follow-up can be taken.

The scope of the present invention also includes a reagent kit used in the method for assisting in predicting the recurrence of cancer.
In the first embodiment, the reagent kit is an eTreg cell detection reagent kit. The reagent kit of the first embodiment includes an anti-CD3 antibody (first antibody), an anti-CD4 antibody (second antibody), an anti-FoxP3 antibody (third antibody), an anti-CD45RA antibody (fourth antibody), and four types of reagents. Contains labeling substance. When the labeling substance is an enzyme, the reagent kit of this embodiment may further include a substrate for the enzyme.

In the second embodiment, the reagent kit is a CD8 ⁺ T cell detection reagent kit. The reagent kit of the second embodiment includes an anti-CD3 antibody (first antibody), an anti-CD8 antibody (fifth antibody), and two types of labeling substances. When the labeling substance is an enzyme, the reagent kit of this embodiment may further include a substrate for the enzyme.

  The forms of the first antibody, the second antibody, the third antibody, the fourth antibody, the fifth antibody, the labeling substance and the substrate are not particularly limited, and may be solid (eg, powder, crystal, lyophilized product, etc.). However, it may be a liquid (eg, solution, suspension, emulsion, etc.). In the present embodiment, the first antibody, the second antibody, the third antibody, the fourth antibody, the fifth antibody, the labeling substance and the substrate are preferably stored in separate containers or individually packaged. . The details of the blood sample, the first antibody, the second antibody, the third antibody, the fourth antibody, the fifth antibody, the labeling substance and the substrate are the same as described in the explanation of the prediction assisting method.

  The reagent kit further includes one or more antibodies selected from the group consisting of anti-PD-1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-Tim-3 antibody, anti-TIGIT antibody and anti-ICOS antibody. May be. In this case, the reagent kit further includes a labeling substance for labeling the selected antibody. The labeling substance for labeling the selected antibody is of a different type from the labeling substance used for labeling the first to fifth antibodies. When the labeling substance for labeling the selected antibody is an enzyme, the reagent kit may further contain a substrate for the enzyme.

The reagent kit may be provided to the user by packing a container containing the above various reagents in a box. This box may contain a package insert of the reagent kit. The package insert preferably describes, for example, the configuration of the reagent kit, the measurement protocol of the biomarker concentration in the blood sample, and the like.
In addition to the above-described various reagents, the reagent kit may appropriately include a cleaning solution, a calibrator, and the like.

  FIG. 1A shows an example of the appearance of the reagent kit of the first embodiment. In the figure, 510 represents a reagent kit, 511 represents a first container containing a first antibody labeled with a first labeling substance, and 512 represents a second antibody labeled with a second labeling substance. 513 shows a third container containing a third antibody labeled with a third labeling substance, and 514 contains a fourth antibody labeled with a fourth labeling substance , 515 indicates a package insert, and 516 indicates a packaging box.

  FIG. 1B shows an example of the appearance of the reagent kit of the second embodiment. In the figure, 520 represents a reagent kit, 521 represents a first container containing a first antibody labeled with a first labeling substance, and 522 represents a fifth antibody labeled with a fifth labeling substance. 523 indicates a package insert, and 524 indicates a packing box.

  The scope of the present invention includes the use of the above reagent for the production of a reagent kit for predicting recurrence of cancer. That is, the present invention also relates to the use of an antibody and a labeling substance for producing a reagent kit for predicting recurrence of cancer.

  The present invention includes an apparatus suitable for acquiring data that assists in predicting cancer recurrence. The present invention also includes a computer program product that causes a computer to acquire data that assists in predicting the recurrence of cancer. Note that the medium included in the computer program product may be a medium in which the computer program is recorded non-temporarily and is readable by the computer.

  Hereinafter, an example of an apparatus suitable for carrying out the method of the present embodiment will be described with reference to the drawings. However, the present invention is not limited to the form shown in this example. FIG. 2 is a schematic diagram of a prediction assisting device for obtaining data that assists in predicting the recurrence of cancer. The prediction auxiliary device 11 shown in FIG. 2 includes a measurement device 22 and a determination device 33 connected to the measurement device 22.

  In this embodiment, the kind of measuring apparatus is not specifically limited, It can select suitably according to the measuring method of a cell number. In the example shown in FIG. 2, the measuring device 22 is a flow cytometer. The flow cytometer is not particularly limited as long as the signal based on the labeling substance can be detected, and can be appropriately selected according to the type of the labeling substance. In the example described below, the signal is optical information such as a chemiluminescent signal or a fluorescent signal.

  When the cell sample subjected to the antigen-antibody reaction is set in the measuring device 22, the measuring device 22 acquires optical information based on the labeled antibody and transmits the obtained optical information to the determination device 33.

  The determination device 33 includes a computer main body 33a, an input unit 33b, and a display unit 33c that displays sample information, determination results, and the like. The determination device 33 receives optical information from the measurement device 22. Then, the processor of the determination device 33 executes a program that acquires data that assists in predicting recurrence of cancer based on the optical information.

  FIG. 3 is a block diagram showing software of the computer main body 33a of the prediction assisting apparatus 11 as functional blocks. As illustrated in FIG. 3, the determination device 33 includes a reception unit 301, a storage unit 302, a calculation unit 303, a determination unit 304, and an output unit 305. The receiving unit 301 is connected to the measurement device 22 via a network so that communication is possible.

  The receiving unit 301 receives information transmitted from the measurement device 22. The storage unit 302 stores formulas, processing programs, and the like for calculating a predetermined threshold necessary for determination and the number of cells. The calculation unit 303 uses the information acquired by the reception unit 301 to calculate the number of cells according to the formula stored in the storage unit 302. The determination unit 304 determines whether the number of cells calculated by the calculation unit 303 is greater than or equal to a predetermined threshold stored in the storage unit 302. The output unit 305 outputs the determination result by the determination unit 304 as data that assists in predicting the recurrence of cancer.

  FIG. 4 is a block diagram showing a hardware configuration of the computer main body 33a shown in FIG. As shown in FIG. 4, the computer main body 33a includes a CPU (Central Processing Unit) 330, a ROM (Read Only Memory) 331, a RAM (Random Access Memory) 332, a hard disk 333, an input / output interface 334, A reading device 335, a communication interface 336, and an image output interface 337 are provided. The CPU 330, the ROM 331, the RAM 332, the hard disk 333, the input / output interface 334, the reading device 335, the communication interface 336, and the image output interface 337 are connected by a bus 338 so that data communication is possible.

  The CPU 330 can execute computer programs stored in the ROM 331 and computer programs loaded in the RAM 332. Each function shown in FIG. 3 is executed by the CPU 330 executing the application program. Thereby, the determination apparatus 33 functions as an apparatus for acquiring data that assists in predicting recurrence of cancer.

  The ROM 331 is configured by a mask ROM, PROM, EPROM, EEPROM, or the like. The ROM 331 stores computer programs executed by the CPU 330 and data used for the computer programs.

  The RAM 332 is configured by SRAM, DRAM, and the like. The RAM 332 is used for reading out computer programs recorded in the ROM 331 and the hard disk 333. The RAM 332 is also used as a work area for the CPU 330 when executing these computer programs.

  The hard disk 333 is installed with an operating system to be executed by the CPU 330, a computer program such as an application program (a computer program for obtaining data for assisting prediction of cancer recurrence), and data used for executing the computer program. ing.

  The reading device 335 includes a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, and the like. The reading device 335 can read the computer program or data recorded on the portable recording medium 340.

  The input / output interface 334 includes, for example, a serial interface such as USB, IEEE 1394, and RS-232C, a parallel interface such as SCSI, IDE, and IEEE1284, and an analog interface including a D / A converter and an A / D converter. It is configured. An input unit 33b such as a keyboard and a mouse is connected to the input / output interface 334. The operator can input various commands and data to the computer main body 33a through the input unit 33b.

  The communication interface 336 is, for example, an Ethernet (registered trademark) interface. The computer main body 33a can transmit print data to a printer or the like via the communication interface 336. The measurement device 22 can transmit measurement data (optical information) to the determination device 33 through the communication interface 336.

  The image output interface 337 is connected to a display unit 33c configured with an LCD, a CRT, or the like. Thereby, the display part 33c can output the video signal according to the image data given from CPU330. The display unit 33c displays an image (screen) according to the input video signal.

  Next, a data acquisition processing procedure for assisting prediction of cancer recurrence by the prediction assisting device 11 will be described. FIG. 5 is an example of a flowchart for obtaining prediction data of cancer recurrence. Here, a case will be described as an example where the increase rate of the number of eTreg cells is calculated from optical information based on the labeled antibody and the determination is made based on the calculated rate. However, the present embodiment is not limited to this example.

  First, in step S <b> 1-1, the receiving unit 301 of the prediction assisting device 11 receives optical information from the measuring device 22. Next, in step S1-2, the calculation unit 303 is collected from the optical information received by the reception unit 301 20 to 50 days after the administration of the tyrosine kinase inhibitor is stopped according to the formula stored in the storage unit 302. The number of eTreg cells in blood and the number of eTreg cells in blood collected 10 to 50 days before cessation of administration of tyrosine kinase inhibitor are measured. In step S <b> 1-3, the calculation unit 303 calculates the increase rate of the number of eTreg cells according to the formula stored in the storage unit 302.

  Thereafter, in step S1-4, the determination unit 304 determines whether the increase rate calculated in step S1-3 is greater than or less than a predetermined threshold stored in the storage unit 302. Here, when the increase rate is equal to or greater than the predetermined threshold, the routine proceeds to step S1-5, and the determination unit 304 outputs a determination result indicating that the cancer patient is classified into the low recurrence risk group. To 305. On the other hand, when the increase rate is less than the predetermined threshold, the routine proceeds to step S1-6, and the determination unit 304 outputs a determination result indicating that the cancer patient is not classified into the low recurrence risk group to the output unit 305. Send.

  In step S1-7, the output unit 305 outputs the determination result and displays it on the display unit 33c. Thereby, the prediction assistance apparatus 11 can provide a doctor or the like with data that assists in the prediction of cancer recurrence.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

Example 1: Classification based on the rate of increase in the number of eTreg cells Peripheral blood collected from chronic myeloid leukemia patients (55; including 19 relapsed cases and 36 non-relapsed cases) 25 to 42 days after discontinuation of imatinib administration Dilute with RPMI1640 (Nipro) and collect PBMC (peripheral blood mononuclear cells) layer by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare Japan) It was suspended in the liquid and stored frozen under liquid nitrogen until measurement analysis. Liquid nitrogen frozen cell vials were warmed in a 37 ° C. constant temperature layer, suspended in PBS or RPMI1640 solution containing 2% FCS, and cells were collected by centrifugation. The collected cells were suspended in PBS containing 100 μL of 2% FCS, Fc block (manufactured by eBioscience) was added to block non-specific binding, and the reaction was allowed to proceed for 15 minutes. Thereafter, FVD (Fixable Viability Dye; manufactured by eBioscience) for dead cell removal and anti-human CD3 antibody, anti-human CD4 antibody and anti-human CD45RA antibody (each manufactured by eBioscience) for cell surface antigen staining were added, The antibody reaction was performed for 20-30 minutes. After washing, for intracellular staining, cell fixation and membrane permeabilization were performed using FoxP3 / Transcription Factor Staining Buffer Set (manufactured by eBioscience), and nuclear staining was performed using anti-FoxP3 antibody (manufactured by eBioscience). After washing, measurement analysis was performed with a flow cytometer (manufactured by BD), and the number of eTreg cells was counted.
In addition, PBMCs were collected from peripheral blood collected from patients with chronic myelogenous leukemia 15 to 44 days before discontinuation of imatinib administration, and the number of eTreg cells was counted in the same manner as described above.
Based on the above results, the rate of increase in the number of eTreg cells was determined, and the recurrence risk group was classified.

  The results are shown in FIG. As shown in FIG. 6, in the non-recurrent group (Non-Ret), a large number of specimens whose parameters exceeded a predetermined threshold appeared. On the other hand, in the relapse group (Ret), there were almost no specimens whose parameters exceeded a predetermined threshold. From this, it became clear that patients whose parameters are greater than or equal to a predetermined threshold can be classified into a low-risk group. In addition, as shown in Table 1 below, the accuracy rate of the relapse low risk group classification was very high. Here, the correct answer rate means the probability that a patient classified into the low-risk relapse group did not recur as classified. In the table, false positive means that the patient was relapsed but was classified as low-risk group. True positive means that the patient who did not relapse was classified as low-risk group. Means. In the case of Table 1 below, there is one false-positive patient classified as a low-risk group among 19 recurrent cases, and true-positive patients classified as a low-risk group among 36 non-recurrent cases There were 10 people.

Example 2: Classification based on the number of eTreg cells / CD8 ⁺ T cells 25 to 42 days after discontinuation of imatinib, collected from 55 patients with chronic myelogenous leukemia (of which 19 were relapsed and 36 were non-relapsed) Peripheral blood is diluted with RPMI1640 (manufactured by Nipro) and the PBMC (peripheral blood mononuclear cells) layer is recovered by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare Japan) The suspension was suspended in a cell cryopreservation solution and stored frozen under liquid nitrogen until measurement analysis. Liquid nitrogen frozen cell vials were warmed in a 37 ° C. constant temperature layer, suspended in PBS or RPMI1640 solution containing 2% FCS, and cells were collected by centrifugation. The collected cells were suspended in PBS containing 100 μL of 2% FCS, Fc block (manufactured by eBioscience) was added to block non-specific binding, and the reaction was allowed to proceed for 15 minutes. Thereafter, FVD (Fixable Viability Dye; manufactured by eBioscience) for removing dead cells, and anti-human CD3 antibody, anti-human CD4 antibody, anti-human CD8a antibody and anti-human CD45RA antibody (respectively eBioscience) for cell surface antigen staining The antibody reaction was carried out for 20-30 minutes. After washing, for intracellular staining, cell fixation and membrane permeabilization were performed using FoxP3 / Transcription Factor Staining Buffer Set (manufactured by eBioscience), and nuclear staining was performed using anti-FoxP3 antibody (manufactured by eBioscience). After washing, measurement analysis was performed with a flow cytometer (manufactured by BD), and the number of eTreg cells and the number of CD8a ⁺ T cells were counted. Then, the number of eTreg cells / the number of CD8a ⁺ T cells was obtained, and the recurrence risk group classification was performed.

  The results are shown in FIG. As shown in FIG. 7, in the non-recurrent group (Non-Ret), a large number of specimens whose parameters exceeded a predetermined threshold appeared. On the other hand, in the relapse group (Ret), there were almost no specimens whose parameters exceeded a predetermined threshold. From this, it became clear that patients whose parameters are greater than or equal to a predetermined threshold can be classified into a low-risk group. In addition, as shown in Table 2 below, the accuracy rate of the relapse low risk group classification was very high.

Example 3: Classification based on the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells Using peripheral blood collected from patients with chronic myelogenous leukemia (55; including 19 relapse cases and 36 non-relapse cases) Anti-human PD-1 antibody (manufactured by BioLegend) was further used for antigen staining on the cell surface, and the number of eTreg cells and PD-1 ⁺ CD8a ⁺ T cells were counted to determine the number of eTreg cells / PD-1 ⁺ CD8a ⁺ The recurrence risk group classification was performed in the same manner as in Example 2 except that the number of T cells was determined.

  The results are shown in FIG. As shown in FIG. 8, in the non-relapse group (Non-Ret), a large number of specimens whose parameters exceeded a predetermined threshold appeared. On the other hand, in the relapse group (Ret), there were almost no specimens whose parameters exceeded a predetermined threshold. From this, it became clear that patients whose parameters are greater than or equal to a predetermined threshold can be classified into a low-risk group. In addition, as shown in Table 3 below, the accuracy rate of the relapse low-risk group classification was very high.

Example 4: Classification based on the number of PD-1 ⁺ eTreg cells / CD8 ⁺ T cells Using peripheral blood collected from chronic myeloid leukemia patients (57; of which 20 were relapsed and 37 were non-relapsed) Anti-human PD-1 antibody (manufactured by BioLegend) was further used for antigen staining on the cell surface, and the number of PD-1 ⁺ eTreg cells and CD8a ⁺ T cells was counted to obtain PD-1 ⁺ eTreg cells / CD8a ⁺ The recurrence risk group classification was performed in the same manner as in Example 2 except that the number of T cells was determined.

  The results are shown in FIG. As shown in FIG. 9, in the non-recurrent group (Non-Ret), a large number of specimens whose parameters exceeded a predetermined threshold appeared. On the other hand, in the relapse group (Ret), there were almost no specimens whose parameters exceeded a predetermined threshold. From this, it became clear that patients whose parameters are greater than or equal to a predetermined threshold can be classified into a low-risk group. In addition, as shown in Table 4 below, the accuracy rate of the relapse low risk group classification was very high.

Example 5: Classification based on PD-1 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count Peripheral samples collected from patients with chronic myelogenous leukemia (57; of which 20 were relapsed and 37 were non-relapsed) Using blood, anti-human PD-1 antibody (manufactured by BioLegend) was further used for antigen staining on the cell surface, and the number of PD-1 ⁺ eTreg cells and PD-1 ⁺ CD8a ⁺ T cells were counted to obtain PD- The recurrence risk group classification was performed in the same manner as in Example 2 except that the number of 1 ⁺ eTreg cells / PD-1 ⁺ CD8a ⁺ T cells was determined.

  The results are shown in FIG. As shown in FIG. 10, in the non-relapse group (Non-Ret), a large number of specimens whose parameters exceeded a predetermined threshold appeared. On the other hand, in the relapse group (Ret), there were almost no specimens whose parameters exceeded a predetermined threshold. From this, it became clear that patients whose parameters are greater than or equal to a predetermined threshold can be classified into a low-risk group. In addition, as shown in Table 5 below, the accuracy rate of the relapse low risk group classification was very high.

Example 6: Classification based on CTLA-4 ⁺ eTreg cell count / CD8 ⁺ T cell count Using peripheral blood collected from patients with chronic myelogenous leukemia (57; including 20 relapse cases and 37 non-relapse cases) Anti-human CTLA-4 antibody (manufactured by BioLegend) was further used for antigen staining on the cell surface, and the number of CTLA-4 ⁺ eTreg cells and CD8a ⁺ T cells were counted to obtain CTLA-4 ⁺ eTreg cells / CD8a ⁺ The recurrence risk group classification was performed in the same manner as in Example 2 except that the number of T cells was determined.

  The results are shown in FIG. As shown in FIG. 11, in the non-recurrent group (Non-Ret), many specimens whose parameters exceeded a predetermined threshold appeared. On the other hand, in the relapse group (Ret), there were almost no specimens whose parameters exceeded a predetermined threshold. From this, it became clear that patients whose parameters are greater than or equal to a predetermined threshold can be classified into a low-risk group. In addition, as shown in Table 6 below, the accuracy rate of the relapse low risk group classification was very high.

Example 7: CTLA-4 ⁺ eTreg cell number / PD-1 ⁺ CD8 ⁺ T cell numbers based on the classification of chronic myelogenous leukemia patients; peripheral taken from (57 of whom recurrent 20 people and non-recurrence 37 persons) Using blood, anti-human CTLA-4 antibody (manufactured by BioLegend) was further used for antigen staining on the cell surface, and the number of CTLA-4 ⁺ eTreg cells and PD-1 ⁺ CD8a ⁺ T cells were counted. The recurrence risk group classification was performed in the same manner as in Example 6 except that the number of 4 ⁺ eTreg cells / PD-1 ⁺ CD8a ⁺ T cells was determined.

  The results are shown in FIG. As shown in FIG. 12, in the non-recurrent group (Non-Ret), a large number of specimens whose parameters exceeded a predetermined threshold appeared. On the other hand, in the relapse group (Ret), there were almost no specimens whose parameters exceeded a predetermined threshold. From this, it became clear that patients whose parameters are greater than or equal to a predetermined threshold can be classified into a low-risk group. In addition, as shown in Table 7 below, the accuracy rate of the relapse low risk group classification was very high.

Example 8: Classification based on the number of LAG-3 ⁺ eTreg cells / CD8 ⁺ T cells Using peripheral blood collected from patients with chronic myelogenous leukemia (20; of which 6 were relapsed and 14 were non-relapsed) An anti-human LAG-3 antibody (manufactured by ENZO) was further used for antigen staining on the cell surface, and the number of LAG-3 ⁺ eTreg cells and CD8a ⁺ T cells was counted to determine the number of LAG-3 ⁺ eTreg cells / CD8a ⁺ The recurrence risk group classification was performed in the same manner as in Example 2 except that the number of T cells was determined.

  The results are shown in FIG. As shown in FIG. 13, in the non-relapse group (Non-Ret), a large number of specimens whose parameters exceeded a predetermined threshold appeared. On the other hand, in the relapse group (Ret), there were almost no specimens whose parameters exceeded a predetermined threshold. From this, it became clear that patients whose parameters are greater than or equal to a predetermined threshold can be classified into a low-risk group. In addition, as shown in Table 8 below, the accuracy rate of the relapse low risk group classification was very high.

Example 9: Classification based on the number of LAG-3 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells Peripheries collected from patients with chronic myelogenous leukemia (20 patients, including 6 recurrent cases and 14 non-recurrent cases) Using blood, anti-human LAG-3 antibody (manufactured by ENZO) was further used for antigen staining on the cell surface, and the number of LAG-3 ⁺ eTreg cells and PD-1 ⁺ CD8a ⁺ T cells were counted to determine LAG- The recurrence risk group classification was performed in the same manner as in Example 8 except that the number of 3 ⁺ eTreg cells / PD-1 ⁺ CD8a ⁺ T cells was determined.

  The results are shown in FIG. As shown in FIG. 14, in the non-recurrent group (Non-Ret), a large number of specimens whose parameters exceeded a predetermined threshold appeared. On the other hand, in the relapse group (Ret), there were almost no specimens whose parameters exceeded a predetermined threshold. From this, it became clear that patients whose parameters are greater than or equal to a predetermined threshold can be classified into a low-risk group. In addition, as shown in Table 9 below, the accuracy rate of the relapse low risk group classification was very high.

Example 10: Classification based on the number of Tim-3 ⁺ eTreg cells / CD8 ⁺ T cells Using peripheral blood collected from patients with chronic myelogenous leukemia (20; of which 6 were relapsed and 14 were non-relapsed) Anti-human Tim-3 antibody (manufactured by BioLegend) was further used for antigen staining on the cell surface, and the number of Tim-3 ⁺ eTreg cells and CD8a ⁺ T cells were counted to determine the number of Tim-3 ⁺ eTreg cells / CD8a ⁺ The recurrence risk group classification was performed in the same manner as in Example 2 except that the number of T cells was determined.

  The results are shown in FIG. As shown in FIG. 15, in the non-recurrent group (Non-Ret), a large number of specimens whose parameters exceeded a predetermined threshold appeared. On the other hand, in the relapse group (Ret), there were almost no specimens whose parameters exceeded a predetermined threshold. From this, it became clear that patients whose parameters are greater than or equal to a predetermined threshold can be classified into a low-risk group. In addition, as shown in Table 10 below, the accuracy rate of the relapse low risk group classification was very high.

Example 11: Classification based on the number of Tim-3 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells Peripheries collected from patients with chronic myelogenous leukemia (20 patients, including 6 recurrent cases and 14 non-recurrent cases) Using blood, anti-human Tim-3 antibody (manufactured by BioLegend) was further used for antigen staining on the cell surface, and the number of Tim-3 ⁺ eTreg cells and PD-1 ⁺ CD8a ⁺ T cells were counted. The recurrence risk group classification was performed in the same manner as in Example 10 except that the number of 3 ⁺ eTreg cells / PD-1 ⁺ CD8a ⁺ T cells was determined.

  The results are shown in FIG. As shown in FIG. 16, in the non-recurrent group (Non-Ret), a large number of specimens whose parameters exceeded a predetermined threshold appeared. On the other hand, in the relapse group (Ret), there were almost no specimens whose parameters exceeded a predetermined threshold. From this, it became clear that patients whose parameters are greater than or equal to a predetermined threshold can be classified into a low-risk group. In addition, as shown in Table 11 below, the accuracy rate of the relapse low risk group classification was very high.

Example 12: Classification based on the number of TIGIT ⁺ eTreg cells / CD8 ⁺ T cells The cell surface using peripheral blood collected from chronic myeloid leukemia patients (22; of which 8 were relapsed and 14 were non-recurrent) Except that anti-human TIGIT antibody (manufactured by eBioscience) was further used for antigen staining of TIGIT ⁺ eTreg cells / CD8a ⁺ T cells by counting TIGIT ⁺ eTreg cells and CD8a ⁺ T cells In the same manner as in Example 2, the recurrence risk group classification was performed.

  The results are shown in FIG. As shown in FIG. 17, in the non-relapse group (Non-Ret), a large number of specimens whose parameters exceeded a predetermined threshold appeared. On the other hand, in the relapse group (Ret), there were almost no specimens whose parameters exceeded a predetermined threshold. From this, it became clear that patients whose parameters are greater than or equal to a predetermined threshold can be classified into a low-risk group. In addition, as shown in Table 12 below, the accuracy rate of the relapse low risk group classification was very high.

Example 13: Classification based on the number of TIGIT ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells Peripheral blood collected from patients with chronic myelogenous leukemia (22; of which 8 were relapsed and 14 were non-relapsed) In addition, anti-human TIGIT antibody (manufactured by eBioscience) was further used for antigen staining on the cell surface, and the number of TIGIT ⁺ eTreg cells and PD-1 ⁺ CD8a ⁺ T cells were counted, and the number of TIGIT ⁺ eTreg cells / PD- The recurrence risk group classification was performed in the same manner as in Example 12 except that the number of 1 ⁺ CD8a ⁺ T cells was determined.

  The results are shown in FIG. As shown in FIG. 18, in the non-recurrent group (Non-Ret), a large number of specimens whose parameters exceeded a predetermined threshold appeared. On the other hand, in the relapse group (Ret), there were almost no specimens whose parameters exceeded a predetermined threshold. From this, it became clear that patients whose parameters are greater than or equal to a predetermined threshold can be classified into a low-risk group. In addition, as shown in Table 13 below, the accuracy rate of the relapse low risk group classification was very high.

Example 14: Classification based on the number of ICOS ⁺ eTreg cells / CD8 ⁺ T cells The cell surface using peripheral blood collected from patients with chronic myelogenous leukemia (32; of which 12 were relapsed and 20 were non-recurrent) Except that anti-human ICOS antibody (manufactured by eBioscience) was further used for antigen staining, and the number of ICOS ⁺ eTreg cells and CD8a ⁺ T cells were counted to obtain the number of ICOS ⁺ eTreg cells / CD8a ⁺ T cells In the same manner as in Example 2, the recurrence risk group classification was performed.

  The results are shown in FIG. As shown in FIG. 19, in the non-recurrent group (Non-Ret), a large number of specimens whose parameters exceeded a predetermined threshold appeared. On the other hand, in the relapse group (Ret), there were almost no specimens whose parameters exceeded a predetermined threshold. From this, it became clear that patients whose parameters are greater than or equal to a predetermined threshold can be classified into a low-risk group. In addition, as shown in Table 14 below, the accuracy rate of the relapse low risk group classification was very high.

Example 15: Classification based on the number of ICOS ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells Peripheral blood collected from patients with chronic myelogenous leukemia (32; of which 12 were relapsed and 20 were non-relapsed) In addition, anti-human ICOS antibody (manufactured by eBioscience) was further used for antigen staining on the cell surface, and the number of ICOS ⁺ eTreg cells and PD-1 ⁺ CD8a ⁺ T cells were counted to determine the number of ICOS ⁺ eTreg cells / PD- The recurrence risk group classification was performed in the same manner as in Example 14 except that the number of 1 ⁺ CD8a ⁺ T cells was determined.

  The results are shown in FIG. As shown in FIG. 20, in the non-recurrent group (Non-Ret), a large number of specimens whose parameters exceeded a predetermined threshold appeared. On the other hand, in the relapse group (Ret), there were almost no specimens whose parameters exceeded a predetermined threshold. From this, it became clear that patients whose parameters are greater than or equal to a predetermined threshold can be classified into a low-risk group. In addition, as shown in Table 15 below, the accuracy rate of the relapse low risk group classification was very high.

Examples 16 to 22: Combination analysis of multiple parameters Using the results of the one-parameter measurement obtained in Examples 1 to 15, in Examples 16 to 22, a plurality of parameters are combined and used for analysis. Went.

Example 16: Combined analysis of the rate of increase in the number of eTreg cells, the number of eTreg cells / CD8 ⁺ T cells and the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells The parameters obtained in Examples 1 to 3 (target patients) Based on a comparison with a predetermined threshold value corresponding to each parameter, a recurrence risk group classification was performed using a total of 55 people, including 19 relapse cases and 36 non-relapse cases. More specifically, classification was performed as follows.

(16-1) When the rate of increase in the number of eTreg cells was equal to or higher than a predetermined threshold value, or the number of eTreg cells / CD8 ⁺ T cells was equal to or higher than a predetermined threshold value, the cells were classified into a low-risk group.
(16-2) When the rate of increase in the number of eTreg cells was equal to or higher than a predetermined threshold and the number of eTreg cells / CD8 ⁺ T cells was equal to or higher than a predetermined threshold, the cells were classified into a low risk group for recurrence.
(16-3) When the rate of increase in the number of eTreg cells was equal to or higher than a predetermined threshold value, or the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells was equal to or higher than a predetermined threshold value, the cells were classified into a low risk group for recurrence.
(16-4) When the rate of increase in the number of eTreg cells was equal to or higher than a predetermined threshold value, and the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells was equal to or higher than a predetermined threshold value, the cells were classified into a low recurrence risk group.

(16-5) When the number of eTreg cells / CD8 ⁺ T cells was a predetermined threshold or more, or the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells was a predetermined threshold or more, the cells were classified into a low risk group for recurrence.
(16-6) If the number of eTreg cells / CD8 ⁺ T cells is equal to or greater than the predetermined threshold and the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells is equal to or greater than the predetermined threshold, the cells were classified as low-risk relapse groups. .
(16-7) The rate of increase in the number of eTreg cells is greater than or equal to a predetermined threshold, the number of eTreg cells / CD8 ⁺ T cells is greater than or equal to a predetermined threshold, or the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells is a predetermined threshold In these cases, we classified it as a low-risk group.
(16-8) The rate of increase in the number of eTreg cells is greater than or equal to a predetermined threshold, the number of eTreg cells / CD8 ⁺ T cells is greater than or equal to a predetermined threshold, and the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells is a predetermined threshold. If it was above the threshold, it was classified as a low-risk group.

(16-9) The rate of increase in the number of eTreg cells is greater than or equal to a predetermined threshold and the number of eTreg cells / CD8 ⁺ T cells is greater than or equal to a predetermined threshold, or the rate of increase in the number of eTreg cells is greater than or equal to a predetermined threshold and the number of eTreg cells / PD When the number of -1 ⁺ CD8 ⁺ T cells was equal to or higher than a predetermined threshold, the cell was classified as a low-risk group.
(16-10) The number of eTreg cells / CD8 ⁺ T cells is equal to or greater than a predetermined threshold and the rate of increase in the number of eTreg cells is equal to or greater than a predetermined threshold, or the number of eTreg cells / CD8 ⁺ T cells is equal to or greater than a predetermined threshold and eTreg cells If the number / number of PD-1 ⁺ CD8 ⁺ T cells was equal to or higher than a predetermined threshold, the cell was classified into the low-risk group.
(16-11) eTreg cell number / PD-1 ⁺ CD8 ⁺ T cell count more than a predetermined threshold value and ETreg cell count increase rate of not less than a predetermined threshold value, or ETreg cell number / PD-1 ⁺ CD8 ⁺ T cell numbers If the number of eTreg cells / CD8 ⁺ T cells is equal to or higher than a predetermined threshold, the cell was classified into the low risk group for recurrence.

  The results are shown in Table 16 below. As shown in Table 16, the accuracy rate of the relapse low-risk group classification was very high in any combination pattern of a plurality of parameters.

Example 17: Increase rate of the number of eTreg cells, number of eTreg cells / CD8 ⁺ T cells, PD-1 ⁺ eTreg cells / CD8 ⁺ T cells and PD-1 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T Combination analysis of cell number Using the parameters obtained in Examples 1, 2, 4 and 5 (number of target patients: 55; including 19 relapse cases and 36 non-relapse cases), a predetermined threshold corresponding to each parameter Based on the comparison, risk group classification was performed. More specifically, classification was performed as follows.

(17-1) When the rate of increase in the number of eTreg cells was equal to or higher than a predetermined threshold, or the number of PD-1 ⁺ eTreg cells / CD8 ⁺ T cells was equal to or higher than a predetermined threshold, the cells were classified into a low risk group for recurrence.
(17-2) If the rate of increase in the number of eTreg cells is equal to or higher than the predetermined threshold value, or the number of PD-1 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells is equal to or higher than the predetermined threshold value, it is classified into the low risk group for recurrence .
(17-3) When the rate of increase in the number of eTreg cells was equal to or higher than a predetermined threshold and the number of PD-1 ⁺ eTreg cells / CD8 ⁺ T cells was higher than a predetermined threshold, the cells were classified into a low risk group for recurrence.
(17-4) If the rate of increase in the number of eTreg cells is greater than or equal to a predetermined threshold and the number of PD-1 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells is greater than or equal to a predetermined threshold, it is classified as a low-risk group did.

(17-5) When the number of eTreg cells / CD8 ⁺ T cells was a predetermined threshold or more, or the number of PD-1 ⁺ eTreg cells / CD8 ⁺ T cells was a predetermined threshold or more, the cells were classified into a low risk group for recurrence.
(17-6) If the number of eTreg cells / CD8 ⁺ T cells is greater than or equal to a predetermined threshold or the number of PD-1 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells is greater than or equal to a predetermined threshold, the risk of recurrence is low Classified.
(17-7) If the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells is greater than or equal to a predetermined threshold, or the number of PD-1 ⁺ eTreg cells / CD8 ⁺ T cells is greater than or equal to a predetermined threshold, the risk of recurrence is low Classified.
(17-8) When the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells is a predetermined threshold or more, or the number of PD-1 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells is a predetermined threshold or more, Classified as low-risk group.

The results are shown in Table 17 below. As shown in Table 17, the correct rate of relapse low risk group classification was very high in any combination pattern of a plurality of parameters.

Example 18: Increase rate of eTreg cell number, eTreg cell number / CD8 ⁺ T cell number, CTLA-4 ⁺ eTreg cell number / CD8 ⁺ T cell number and CTLA-4 ⁺ eTreg cell number / PD-1 ⁺ CD8 ⁺ T Combination analysis of cell number Using the parameters obtained in Examples 1, 2, 6 and 7 (number of target patients: 55; including 19 relapse cases and 36 non-relapse cases), a predetermined threshold corresponding to each parameter Based on the comparison, risk group classification was performed. More specifically, except that replaced the "PD-1 ⁺ eTreg cell number" and "CTLA-4 ⁺ eTreg cell number" in Example 17 (17-1) - (17-8) of Example 17 Classification was performed in the same manner.

The results are shown in Table 18 below. As shown in Table 18, the correct rate of relapse low-risk group classification was very high in any combination pattern of a plurality of parameters.

Example 19: Increase rate of the number of eTreg cells, number of eTreg cells / CD8 ⁺ T cells, number of LAG-3 ⁺ eTreg cells / CD8 ⁺ T cells and number of LAG-3 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T Combination analysis of cell number Using the parameters obtained in Examples 1, 2, 8 and 9 (number of target patients: 19; of which 6 were relapsed and 13 were non-relapsed), a predetermined threshold corresponding to each parameter Based on the comparison, risk group classification was performed. More specifically, except that replaced the "PD-1 ⁺ eTreg cell number" and "LAG-3 ⁺ eTreg cell number" in Example 17 (17-1) - (17-8) of Example 17 Classification was performed in the same manner.

The results are shown in Table 19 below. As shown in Table 19, the correct rate of relapse low risk group classification was very high in any combination pattern of a plurality of parameters.

Example 20: Increase rate of the number of eTreg cells, number of eTreg cells / CD8 ⁺ T cells, number of Tim-3 ⁺ eTreg cells / CD8 ⁺ T cells and Tim-3 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T Combination analysis of cell number Using the parameters obtained in Examples 1, 2, 10 and 11 (number of target patients: 21; of which 8 were relapsed and 13 were non-relapsed), a predetermined threshold corresponding to each parameter Based on the comparison, risk group classification was performed. More specifically, except that replaced the "PD-1 ⁺ eTreg cell number" and "Tim-3 ⁺ eTreg cell number" in Example 17 (17-1) - (17-8) of Example 17 Classification was performed in the same manner.

The results are shown in Table 20 below. As shown in Table 20, the correct rate of relapse low risk group classification was very high in any combination pattern of a plurality of parameters.

Example 21: Combination of the increase rate of the number of eTreg cells, the number of eTreg cells / CD8 ⁺ T cells, the number of TIGIT ⁺ eTreg cells / CD8 ⁺ T cells and the number of TIGIT ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells Analysis Using parameters obtained in Examples 1, 2, 12 and 13 (number of target patients: 21; of which 8 were relapsed and 13 were non-relapsed), for comparison with a predetermined threshold corresponding to each parameter Based on the risk group classification. More specifically, except that replaced the "PD-1 ^{+ eTreg} cell number" and "TIGIT ⁺ eTreg cell number" in Example 17 (17-1) - (17-8) in the same manner as in Example 17 Classification.

The results are shown in Table 21 below. As shown in Table 21, the correct rate of relapse low risk group classification was very high in any combination pattern of a plurality of parameters.

Example 22: Combination of the increase rate of the number of eTreg cells, the number of eTreg cells / CD8 ⁺ T cells, the number of ICOS ⁺ eTreg cells / CD8 ⁺ T cells and the number of ICOS ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells Analysis The parameters obtained in Examples 1, 2, 14, and 15 (number of target patients: 31; of which 11 were relapsed and 20 were non-relapsed) were compared with a predetermined threshold corresponding to each parameter. Based on the risk group classification. More specifically, except that replaced the "PD-1 ^{+ eTreg} cell number" and "ICOS ⁺ eTreg cell number" in Example 17 (17-1) - (17-8) in the same manner as in Example 17 Classification.

The results are shown in Table 22 below. As shown in Table 22, the correct rate of relapse low risk group classification was very high in any combination pattern of a plurality of parameters.

DESCRIPTION OF SYMBOLS 11 Prediction auxiliary | assistance apparatus 22 Measuring apparatus 33 Judgment apparatus 33a Computer main body 33b Input part 33c Display part 301 Receiving part 302 Memory | storage part 303 Calculation part 304 Judgment part 305 Output part 330 CPU
331 ROM
332 RAM
333 Hard disk 334 Input / output interface 335 Reading device 336 Communication interface 337 Image output interface 338 Bus 340 Recording medium 510, 520 Reagent kit 511, 521 First container 512, 522 Second container 513 Third container 514 Fourth container 515, 523 Document 516, 524 Packing box

Claims (23)

Measuring the number of eTreg cells in blood collected from a cancer patient;
Obtaining a value related to the number of eTreg cells based on the measurement results;
Classifying the patient into a low-risk relapse group based on the obtained value for the number of eTreg cells,
The cancer patient has a history of administration of tyrosine kinase inhibitors,
The method of assisting prediction of cancer recurrence, wherein the blood is blood collected 20 to 50 days after the administration of a tyrosine kinase inhibitor is stopped. Further comprising measuring the number of eTreg cells in blood collected 10 to 50 days prior to cessation of administration of the tyrosine kinase inhibitor,
The value for the number of eTreg cells is the rate of increase in the number of eTreg cells or the reciprocal of the rate of increase,
The rate of increase in the number of eTreg cells is expressed by the following formula:
(Number of eTreg cells in blood collected 20 to 50 days after cessation of administration of tyrosine kinase inhibitor) / (Number of eTreg cells in blood collected 10 to 50 days before cessation of administration of tyrosine kinase inhibitor)
Represented by
The reciprocal of the increase rate of the eTreg cell number is expressed by the following formula:
(Number of eTreg cells in blood collected 10 to 50 days before cessation of administration of tyrosine kinase inhibitor) / (Number of eTreg cells in blood collected 20 to 50 days after cessation of administration of tyrosine kinase inhibitor)
The method of claim 1, represented by: In the classification step, when the rate of increase in the number of eTreg cells is equal to or greater than a predetermined threshold, the patient is classified into a low risk group for recurrence, or in the classification step, the reciprocal of the rate of increase in the number of eTreg cells is a predetermined value. 3. The method of claim 2, wherein if the patient is below a threshold, the patient is classified into a low-risk group. CD8 ⁺ T cell count, PD-1 ⁺ CD8 ⁺ T cell count, PD-1 ⁺ eTreg cell count, CTLA-4 ⁺ eTreg cell count, LAG-3 ⁺ eTreg cell count in blood collected from the cancer patient Measuring at least one selected from the group consisting of Tim-3 ⁺ eTreg cell count, TIGIT ⁺ eTreg cell count and ICOS ⁺ eTreg cell count,
In the acquisition step, as the value related to the number of eTreg cells, the number of eTreg cells / CD8 ⁺ T cells, the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells, the number of PD-1 ⁺ eTreg cells / CD8 ⁺ T cells PD-1 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, CTLA-4 ⁺ eTreg cell count / CD8 ⁺ T cell count, CTLA-4 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count , LAG-3 ⁺ eTreg cells / CD8 ⁺ T cells, LAG-3 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells, Tim-3 ⁺ eTreg cells / CD8 ⁺ T cells, Tim-3 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells, TIGIT ⁺ eTreg cells / CD8 ⁺ T cells, TIGIT ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells, ICOS ⁺ eTreg cells / CD8 Any one parameter selected from the group consisting of ⁺ T cell count, ICOS ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, or
CD8 ⁺ T cell count / eTreg cell count, PD-1 ⁺ CD8 ⁺ T cell count / eTreg cell count, CD8 ⁺ T cell count / PD-1 ⁺ eTreg cell count, PD-1 ⁺ CD8 ⁺ T cell count / PD- 1 ⁺ eTreg cells, CD8 ⁺ T cells / CTLA-4 ⁺ eTreg cells, PD-1 ⁺ CD8 ⁺ T cells / CTLA-4 ⁺ eTreg cells, CD8 ⁺ T cells / LAG-3 ⁺ eTreg cells Count, PD-1 ⁺ CD8 ⁺ T cell count / LAG-3 ⁺ eTreg cell count, CD8 ⁺ T cell count / Tim-3 ⁺ eTreg cell count, PD-1 ⁺ CD8 ⁺ T cell count / Tim-3 ⁺ eTreg cell count Number, CD8 ⁺ T cell count / TIGIT ⁺ eTreg cell count, PD-1 ⁺ CD8 ⁺ T cell count / TIGIT ⁺ eTreg cell count, CD8 ⁺ T cell count / ICOS ⁺ eTreg cell count and PD-1 ⁺ CD8 ⁺ T cell count 2. The method of claim 1, wherein any one reciprocal parameter selected from the group consisting of number / ICOS ⁺ eTreg cell number is obtained.   The method according to claim 4, wherein, in the classification step, the patient is classified into a low recurrence risk group when the parameter is equal to or greater than a predetermined threshold value or when the reciprocal parameter is less than a predetermined threshold value. CD8 ⁺ T cell count, PD-1 ⁺ CD8 ⁺ T cell count, PD-1 ⁺ eTreg cell count, CTLA-4 ⁺ eTreg cell count in blood collected 20-50 days after cessation of administration of tyrosine kinase inhibitor Further comprising the step of measuring at least one selected from the group consisting of LAG-3 ⁺ eTreg cell count, Tim-3 ⁺ eTreg cell count, TIGIT ⁺ eTreg cell count and ICOS ⁺ eTreg cell count,
In the acquisition step, the following first parameter and second parameter are acquired as values relating to the number of eTreg cells:
First parameter: increase rate of the eTreg cell number; and second parameter: eTreg cell number / CD8 ⁺ T cell number, eTreg cell number / PD-1 ⁺ CD8 ⁺ T cell number, PD-1 ⁺ eTreg cell number / CD8 ⁺ T cell count, PD-1 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, CTLA-4 ⁺ eTreg cell count / CD8 ⁺ T cell count, CTLA-4 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, LAG-3 ⁺ eTreg cell count / CD8 ⁺ T cell count, LAG-3 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, Tim-3 ⁺ eTreg cell count / CD8 ⁺ T cell count , Tim-3 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, TIGIT ⁺ eTreg cell count / CD8 ⁺ T cell count, TIGIT ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, ICOS ⁺ eTreg 1 value selected from the group consisting of the number of cells / CD8 ⁺ T cells, the number of ICOS ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells,
The method according to claim 2, wherein, in the classification step, the cancer patient is classified into a low recurrence risk group when the first parameter is equal to or greater than a predetermined threshold value or the second parameter is equal to or greater than a predetermined threshold value. CD8 ⁺ T cell count, PD-1 ⁺ CD8 ⁺ T cell count, PD-1 ⁺ eTreg cell count, CTLA-4 ⁺ eTreg cell count in blood collected 20-50 days after cessation of administration of tyrosine kinase inhibitor Further comprising the step of measuring at least one selected from the group consisting of LAG-3 ⁺ eTreg cell count, Tim-3 ⁺ eTreg cell count, TIGIT ⁺ eTreg cell count and ICOS ⁺ eTreg cell count,
In the acquisition step, the following first reciprocal parameter and second reciprocal parameter are acquired as values relating to the number of eTreg cells:
First reciprocal parameter: reciprocal of the increase rate of the eTreg cell number; and second reciprocal parameter: CD8 ⁺ T cell number / eTreg cell number, PD-1 ⁺ CD8 ⁺ T cell number / eTreg cell number, CD8 ⁺ T cell number / PD-1 ⁺ eTreg cell count, PD-1 ⁺ CD8 ⁺ T cell count / PD-1 ⁺ eTreg cell count, CD8 ⁺ T cell count / CTLA-4 ⁺ eTreg cell count, PD-1 ⁺ CD8 ⁺ T cell count / CTLA-4 ⁺ eTreg cell count, CD8 ⁺ T cell count / LAG-3 ⁺ eTreg cell count, PD-1 ⁺ CD8 ⁺ T cell count / LAG-3 ⁺ eTreg cell count, CD8 ⁺ T cell count / Tim-3 ⁺ eTreg cell count, PD-1 ⁺ CD8 ⁺ T cell count / Tim-3 ⁺ eTreg cell count, CD8 ⁺ T cell count / TIGIT ⁺ eTreg cell count, PD-1 ⁺ CD8 ⁺ T cell count / TIGIT ⁺ eTreg cell count A value of 1 selected from the group consisting of CD8 ⁺ T cell count / ICOS ⁺ eTreg cell count and PD-1 ⁺ CD8 ⁺ T cell count / ICOS ⁺ eTreg cell count,
The method according to claim 2, wherein, in the classification step, the cancer patient is classified into a low-risk group when the first reciprocal parameter is less than a predetermined threshold value or the second reciprocal parameter is less than a predetermined threshold value. CD8 ⁺ T cell count, PD-1 ⁺ CD8 ⁺ T cell count, PD-1 ⁺ eTreg cell count, CTLA-4 ⁺ eTreg cell count in blood collected 20-50 days after cessation of administration of tyrosine kinase inhibitor Further comprising the step of measuring at least one selected from the group consisting of LAG-3 ⁺ eTreg cell count, Tim-3 ⁺ eTreg cell count, TIGIT ⁺ eTreg cell count and ICOS ⁺ eTreg cell count,
In the acquisition step, the following first parameter, second parameter, and third parameter are acquired as values relating to the number of eTreg cells:
First parameter: increase rate of the eTreg cell number; and second and third parameters: eTreg cell number / CD8 ⁺ T cell number, eTreg cell number / PD-1 ⁺ CD8 ⁺ T cell number, PD-1 ⁺ eTreg Number of cells / CD8 ⁺ T cells, PD-1 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells, CTLA-4 ⁺ eTreg cells / CD8 ⁺ T cells, CTLA-4 ⁺ eTreg cells / PD -1 ⁺ CD8 ⁺ T cells, LAG-3 ⁺ eTreg cells / CD8 ⁺ T cells, LAG-3 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells, Tim-3 ⁺ eTreg cells / CD8 ⁺ T cell count, Tim-3 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, TIGIT ⁺ eTreg cell count / CD8 ⁺ T cell count, TIGIT ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count ICOS ⁺ eTreg cell count / CD8 ⁺ T cell count, ICOS ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, any two values selected from the group consisting of:
In the classification step, when the first parameter is equal to or greater than a predetermined threshold, the second parameter is equal to or greater than a predetermined threshold, or the third parameter is equal to or greater than a predetermined threshold, the cancer patient is classified into a low recurrence risk group. The method according to claim 2. CD8 ⁺ T cell count, PD-1 ⁺ CD8 ⁺ T cell count, PD-1 ⁺ eTreg cell count, CTLA-4 ⁺ eTreg cell count in blood collected 20-50 days after cessation of administration of tyrosine kinase inhibitor Further comprising the step of measuring at least one selected from the group consisting of LAG-3 ⁺ eTreg cell count, Tim-3 ⁺ eTreg cell count, TIGIT ⁺ eTreg cell count and ICOS ⁺ eTreg cell count,
In the acquisition step, the following first reciprocal parameter, second reciprocal parameter, and third reciprocal parameter are acquired as values relating to the number of eTreg cells:
First reciprocal parameter: reciprocal of the increase rate of the eTreg cell number; and second reciprocal parameter and third reciprocal parameter: eTreg cell number / CD8 ⁺ T cell number, eTreg cell number / PD-1 ⁺ CD8 ⁺ T cell number, PD-1 ⁺ eTreg cells / CD8 ⁺ T cells, PD-1 ⁺ eTreg cells / PD-1 ⁺ CD8 ⁺ T cells, CTLA-4 ⁺ eTreg cells / CD8 ⁺ T cells, CTLA-4 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, LAG-3 ⁺ eTreg cell count / CD8 ⁺ T cell count, LAG-3 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, Tim-3 ⁺ eTreg cell count / CD8 ⁺ T cell count, Tim-3 ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count, TIGIT ⁺ eTreg cell count / CD8 ⁺ T cell count, TIGIT ⁺ eTreg cell count / PD-1 ⁺ The value of any two selected from the group consisting of CD8 ⁺ T cell count, ICOS ⁺ eTreg cell count / CD8 ⁺ T cell count, ICOS ⁺ eTreg cell count / PD-1 ⁺ CD8 ⁺ T cell count,
In the classification step, when the first reciprocal parameter is less than a predetermined threshold, the second reciprocal parameter is less than a predetermined threshold, or the third reciprocal parameter is less than a predetermined threshold, the cancer patient is in a low recurrence risk group. The method of claim 2, which is classified. Measuring the number of CD8 ⁺ T cells in the blood collected from the cancer patient;
Measuring the number of PD-1 ⁺ CD8 ⁺ T cells in blood collected from the cancer patient;
Measuring the number of eTreg cells in blood collected 10 to 50 days prior to cessation of administration of the tyrosine kinase inhibitor,
In the obtaining step, the increase rate of the number of eTreg cells, the number of eTreg cells / CD8 ⁺ T cells and the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells are obtained as values relating to the number of eTreg cells,
The rate of increase in the number of eTreg cells is expressed by the following formula:
(Number of eTreg cells in blood collected 20 to 50 days after cessation of administration of tyrosine kinase inhibitor) / (Number of eTreg cells in blood collected 10 to 50 days before cessation of administration of tyrosine kinase inhibitor)
Represented by
In the classification step, the rate of increase in the number of eTreg cells is greater than or equal to a predetermined threshold, the number of eTreg cells / CD8 ⁺ T cells is greater than or equal to a predetermined threshold, or the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells is greater than or equal to a predetermined threshold The method of claim 1, wherein the patient is classified into a low risk group for recurrence. Measuring the number of CD8 ⁺ T cells in the blood collected from the cancer patient;
Measuring the number of eTreg cells in blood collected 10 to 50 days prior to cessation of administration of the tyrosine kinase inhibitor,
In the acquisition step, as a value relating to the number of eTreg cells, an increase rate of the number of eTreg cells and the number of eTreg cells / CD8 ⁺ T cells are obtained,
The rate of increase in the number of eTreg cells is expressed by the following formula:
(Number of eTreg cells in blood collected 20 to 50 days after cessation of administration of tyrosine kinase inhibitor) / (Number of eTreg cells in blood collected 10 to 50 days before cessation of administration of tyrosine kinase inhibitor)
Represented by
The said classification | category process WHEREIN: When the increase rate of the number of eTreg cells is more than a predetermined threshold value, or when the number of eTreg cells / CD8 ⁺ T cell is more than a predetermined threshold value, the said patient is classified into the low recurrence risk group. The method described. Measuring the number of PD-1 ⁺ CD8 ⁺ T cells in blood collected from the cancer patient;
Measuring the number of eTreg cells in blood collected 10 to 50 days prior to cessation of administration of the tyrosine kinase inhibitor,
In the acquisition step, as a value related to the number of eTreg cells, an increase rate of the number of eTreg cells and the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells are obtained,
The rate of increase in the number of eTreg cells is expressed by the following formula:
(Number of eTreg cells in blood collected 20 to 50 days after cessation of administration of tyrosine kinase inhibitor) / (Number of eTreg cells in blood collected 10 to 50 days before cessation of administration of tyrosine kinase inhibitor)
Represented by
In the classification step, when the rate of increase in the number of eTreg cells is equal to or higher than a predetermined threshold or the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells is equal to or higher than a predetermined threshold, the patient is classified into a low-risk relapse group. The method of claim 1. Measuring the number of CD8 ⁺ T cells in the blood collected from the cancer patient;
Measuring the number of PD-1 ⁺ CD8 ⁺ T cells in blood collected from the cancer patient,
In the obtaining step, the number of eTreg cells / CD8 ⁺ T cells and the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells are obtained as values relating to the number of eTreg cells,
In the classification step, if the number of eTreg cells / CD8 ⁺ T cells is greater than or equal to a predetermined threshold or the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells is greater than or equal to a predetermined threshold, the patient is classified as a low risk group for recurrence The method of claim 1, wherein: Measuring the number of CD8 ⁺ T cells in the blood collected from the cancer patient;
Measuring the number of PD-1 ⁺ CD8 ⁺ T cells in blood collected from the cancer patient;
Measuring the number of eTreg cells in blood collected 10 to 50 days prior to cessation of administration of the tyrosine kinase inhibitor,
In the obtaining step, the increase rate of the number of eTreg cells, the number of eTreg cells / CD8 ⁺ T cells and the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells are obtained as values relating to the number of eTreg cells,
The rate of increase in the number of eTreg cells is expressed by the following formula:
(Number of eTreg cells in blood collected 20 to 50 days after cessation of administration of tyrosine kinase inhibitor) / (Number of eTreg cells in blood collected 10 to 50 days before cessation of administration of tyrosine kinase inhibitor)
Represented by
In the classification step, the rate of increase in the number of eTreg cells is equal to or greater than a predetermined threshold, the number of eTreg cells / CD8 ⁺ T cells is equal to or greater than a predetermined threshold, the rate of increase in the number of eTreg cells is equal to or greater than a predetermined threshold, and the number of eTreg cells / PD− If the number of 1 ⁺ CD8 ⁺ T cells is greater than or equal to a predetermined threshold, or the number of eTreg cells / CD8 ⁺ T cells is greater than or equal to a predetermined threshold and the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells is greater than or equal to a predetermined threshold, The method of claim 1, wherein the patient is classified into a low-risk group. Measuring the number of CD8 ⁺ T cells in the blood collected from the cancer patient;
Measuring the number of eTreg cells in blood collected 10 to 50 days prior to cessation of administration of the tyrosine kinase inhibitor,
In the acquisition step, as a value relating to the number of eTreg cells, an increase rate of the number of eTreg cells and the number of eTreg cells / CD8 ⁺ T cells are obtained,
The rate of increase in the number of eTreg cells is expressed by the following formula:
(Number of eTreg cells in blood collected 20 to 50 days after cessation of administration of tyrosine kinase inhibitor) / (Number of eTreg cells in blood collected 10 to 50 days before cessation of administration of tyrosine kinase inhibitor)
Represented by
The said classification | category process WHEREIN: When the increase rate of the number of eTreg cells is more than a predetermined threshold value, and the number of eTreg cells / CD8 ⁺ T cell is more than a predetermined threshold value, the said patient is classified into the low recurrence risk group. The method described. Measuring the number of PD-1 ⁺ CD8 ⁺ T cells in blood collected from the cancer patient;
Measuring the number of eTreg cells in blood collected 10 to 50 days prior to cessation of administration of the tyrosine kinase inhibitor,
In the acquisition step, as a value related to the number of eTreg cells, an increase rate of the number of eTreg cells and the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells are obtained,
The rate of increase in the number of eTreg cells is expressed by the following formula:
(Number of eTreg cells in blood collected 20 to 50 days after cessation of administration of tyrosine kinase inhibitor) / (Number of eTreg cells in blood collected 10 to 50 days before cessation of administration of tyrosine kinase inhibitor)
Represented by
In the classification step, when the rate of increase in the number of eTreg cells is a predetermined threshold or more and the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells is a predetermined threshold or more, the patient is classified into a low-risk relapse group. The method of claim 1. Measuring the number of CD8 ⁺ T cells in the blood collected from the cancer patient;
Measuring the number of PD-1 ⁺ CD8 ⁺ T cells in blood collected from the cancer patient,
In the obtaining step, the number of eTreg cells / CD8 ⁺ T cells and the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells are obtained as values relating to the number of eTreg cells,
If the number of eTreg cells / CD8 ⁺ T cells is greater than or equal to a predetermined threshold and the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells is greater than or equal to a predetermined threshold in the classification step, the patient is classified as a low risk group for recurrence The method of claim 1, wherein: Measuring the number of CD8 ⁺ T cells in the blood collected from the cancer patient;
Measuring the number of PD-1 ⁺ CD8 ⁺ T cells in blood collected from the cancer patient;
Measuring the number of eTreg cells in blood collected 10 to 50 days prior to cessation of administration of the tyrosine kinase inhibitor,
In the obtaining step, the increase rate of the number of eTreg cells, the number of eTreg cells / CD8 ⁺ T cells and the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells are obtained as values relating to the number of eTreg cells,
The rate of increase in the number of eTreg cells is expressed by the following formula:
(Number of eTreg cells in blood collected 20 to 50 days after cessation of administration of tyrosine kinase inhibitor) / (Number of eTreg cells in blood collected 10 to 50 days before cessation of administration of tyrosine kinase inhibitor)
Represented by
In the classification step, the rate of increase in the number of eTreg cells is a predetermined threshold or more, the number of eTreg cells / CD8 ⁺ T cells is a predetermined threshold or more, and the number of eTreg cells / PD-1 ⁺ CD8 ⁺ T cells is a predetermined number. The method of claim 1, wherein if the threshold is greater than or equal to the threshold, the patient is classified into a low-risk group.   The blood collected 20 to 50 days after the administration stop of the tyrosine kinase inhibitor is blood collected 25 to 42 days after the administration stop of the tyrosine kinase inhibitor. The method described in 1.   The blood collected 10 to 50 days before cessation of administration of the tyrosine kinase inhibitor is blood collected 15 to 44 days before cessation of administration of the tyrosine kinase inhibitor, The method according to any one of 14 to 16, 18 and 19.   21. The method of any one of claims 1-20, wherein the tyrosine kinase inhibitor is imatinib.   The method according to any one of claims 1 to 21, wherein the cancer is chronic myelogenous leukemia.   The said cancer patient is a patient who has been administered a tyrosine kinase inhibitor continuously for 3 years or more, and has shown complete molecular genetic complete remission for 2 years or more. The method according to claim 1.