CN111676290B

CN111676290B - Drug resistance molecular marker for acute myelogenous leukemia induced differentiation treatment and application thereof

Info

Publication number: CN111676290B
Application number: CN202010624681.4A
Authority: CN
Inventors: 王伟佳; 韩慧
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-07-02
Filing date: 2020-07-02
Publication date: 2021-03-02
Anticipated expiration: 2040-07-02
Also published as: CN111676290A

Abstract

The invention discloses a biomarker for assisting in judging the prognosis of induced differentiation therapy of an AML patient, wherein the biomarker is ICAT. Through the development and application of the ICAT diagnostic reagent and the diagnostic kit, the invention can lead the prognosis diagnosis of AML patients to be more convenient and feasible, and lays a foundation for clinicians to quickly and accurately master the illness state of the patients and the clinical treatment effect.

Description

Drug resistance molecular marker for acute myelogenous leukemia induced differentiation treatment and application thereof

Technical Field

The invention relates to a marker, in particular to a molecular marker for inducing differentiation and treating drug resistance of acute myelogenous leukemia and application thereof.

Background

Leukemia is a hematological disease due to dysfunction in differentiation and proliferation of blood precursor cells of myeloid and lymphoid origin. Research has shown that the incidence of leukemia in pediatric malignancies is the highest, reaching around 35%, and is considered as the "number one killer" in pediatric cancers under 14 years old. The leukemia incidence rate of children under 15 years old is 3-4/10 ten thousands of people all over the world, and about 5000 children in China develop leukemia every year. It not only brings great pain and economic burden to the family of the patient, but also brings heavy burden to the society. The most common types of leukemia are Acute Lymphocytic Leukemia (ALL) and Acute Myeloid Leukemia (AML). AML is a malignant disease of hematopoietic stem/progenitor cells of myeloid lineage, and the incidence rate of the malignant disease accounts for 30% of pediatric leukemia. The main characteristics are abnormal hyperplasia of primary and juvenile cells in bone marrow and peripheral blood, and the clinical manifestations are anemia, infection, fever, organ infiltration and metabolic abnormality. According to FAB typing criteria, AML is co-classified into 7 types, M1-M7, etc., namely M1: undifferentiated myelocytic leukemia; m2: partially differentiated myeloblastic leukemia; m3: acute promyelocytic leukemia; m4: acute myeloid, monocytic leukemia; m5: acute monocytic leukemia; m6: acute erythroleukemia or erythroleukemia; m7: acute megakaryocytic leukemia. The pathogenesis of AML is mostly related to the fusion protein of oncogenes, resulting from chromosomal shifts or inversions. With the use of combined chemotherapy and hematopoietic stem cell transplantation techniques, the survival rate of leukemias is currently greatly improved, but treatment-related mortality remains as high as 20%. At present, most of AML patients can achieve the purpose of controlling the disease deterioration or completely relieving by clinical chemotherapy means, but the result of realizing long-term relieving is not ideal, and the reasons that the initial treatment is ineffective or the effective later-stage relapse of the initial treatment is mostly chemotherapy resistance; and AML has a very strong clinical heterogeneity, resulting in a 5-year survival rate of only 26.6% for this class of patients. Many researches stay at the molecular mechanism level of cell drug resistance, and no practical and effective means for monitoring the drug resistance of AML chemotherapy in real time is found, so that the search for a new molecular marker is particularly important.

In the earlier research, the subject group adopts comparative proteomics technology, and uses all-trans-retinoic acid (ATRA) and sterol methanesulfonate (NSC 67657) to respectively induce HL60 cells to differentiate into a granular system and a mononuclear system, and finds that the differentiation of the mononuclear system specifically expresses a differential protein: catenin-associated protein 1(β -catenin-interacting protein-1, ICAT). ICAT protein is a key protein molecule in a Wnt signal path depended by beta-catenin protein (beta-catenin), and can bind to the beta-catenin protein to play a role in competitive inhibition of the beta-catenin protein, so that the activation of the whole Wnt/beta-catenin/Tcf signal path is influenced. ICAT has increased expression in the process of inducing HL60 cell monocyte lineage differentiation by NSC67657, and has no significant difference in expression in the granulosa lineage differentiation and non-treated cells. Earlier researches find that the Wnt/beta-catenin signal path regulated by ICAT significantly interferes with the chemotherapy expression of sterol drugs NSC67657 on HL60 cells, if exogenous ICAT protein is highly expressed, the sensitivity of HL60 cells on NSC67657 can be significantly improved, and if intracellular ICAT gene is silenced, the high-efficiency effect of the drugs can be significantly inhibited. Indicating that intracellular ICAT protein expression levels can interfere with chemotherapeutic effects. Interestingly, NSC67657 induced differentiation of THP-1 cells into the monocytic lineage and activated expression of the ICAT gene, but high expression of this gene alone was not sufficient to induce THP-I cell differentiation and did not increase the sensitivity of THP-1 cells to the pharmacological effects of NSC 67657. This suggests that ICAT protein may not be a target for NSC67657 to induce cell differentiation, or that ICAT promotes the monocytic differentiation of myeloid leukemia cells only at the granulocyte-monocyte progenitor stage without significant effect on tumor cells that entered the monocyte threshold.

Currently, prognosis of AML is mainly determined according to the age, karyotype, and other indicators of patients. However, even patients with the same age, karyotype, and other factors have different prognosis. Therefore, in order to obtain better therapeutic effect and improve the survival rate of patients, early judgment of prognosis index of AML is required to guide the formulation of an appropriate treatment regimen. Therefore, the method has important practical significance for searching effective drug resistance indexes of AML chemotherapy in real time. However, the currently available judgment indexes are seriously insufficient, and research on the level of the gene protein to find the indexes relevant to prognosis has become a research hotspot. There is currently a lack of effective biomarkers that can be used to predict the prognostic efficacy of AML patients.

Disclosure of Invention

It is an object of the present invention to provide a molecular marker for detecting AML cell or tissue samples to help determine the chemotherapy effect or clinical prognosis of AML patients.

The inventor finds that the differential protein ICAT specifically expressed in the mononuclear differentiation induced by NSC67657 can have good prognosis judgment effect on AML patients under the condition of not adopting NSC67657 for treatment, and even has good prognosis judgment effect on AML patients when adopting the grain differentiation induced by all-trans retinoic acid for treatment. This was unexpected from prior studies.

Specifically, the invention provides application of a biomarker, wherein the biomarker is ICAT gene or protein, in preparing a reagent for judging the prognosis of Acute Myelogenous Leukemia (AML) patients receiving induced differentiation therapy, and the reagent for inducing differentiation therapy is not NSC 67657.

Preferably, when ICAT is significantly highly expressed, it means that the patient has a good prognosis for receiving differentiation-inducing therapy, with a long survival time.

Preferably, said differentiation-inducing therapy is in particular: treating non-M3 patients with a standard dose DA (daunorubicin + cytarabine), DAE (daunorubicin + cytarabine + etoposide), CAG (pirarubicin + cytarabine) or IA (daunorubicin + cytarabine) regimen; m3 patients were treated with all-trans retinoic acid-induced differentiation.

Also provided is the use of a biomarker which is an ICAT gene or protein for the manufacture of a reagent for determining the prognosis of an induced differentiation therapy for a patient with Acute Myeloid Leukemia (AML) type M3, wherein the reagent for the induced differentiation therapy is not NSC 67657.

Preferably, wherein the expression level of the ICAT gene is detected by hybridization or PCR, the expression level of the ICAT protein is detected by ELISA method.

Also provided is a kit for determining the prognosis of Acute Myeloid Leukemia (AML) patients receiving differentiation-inducing therapy, which comprises a reagent for detecting the expression level of ICAT gene or protein, the differentiation-inducing therapy reagent being other than NSC 67657.

Also provided is a kit for determining the prognosis of a patient with Acute Myeloid Leukemia (AML) type M3 receiving differentiation-inducing therapy, the kit comprising an agent for detecting the expression level of an ICAT gene or protein, wherein the agent for inducing differentiation therapy is not NSC 67657.

Providing a method of use of the kit, characterized in that:

(1) collecting a blood sample of an individual to be detected;

(2) detecting the expression level of ICAT gene or protein in the blood sample.

The invention has the following beneficial effects:

(1) the invention develops a novel prognostic biomarker for induced differentiation therapy of AML patients, and no report on the use of the biomarker for induced differentiation therapy of AML patients, particularly differentiation therapy except NSC67657, has been provided before. The invention provides a new way for the prognosis of the induced differentiation treatment of AML patients.

(2) The invention takes blood as a detection object and has the advantages of convenience and sensitivity. By adopting the detection method of the invention, 3-5mL of peripheral blood is extracted, and the AML prognosis analysis can be rapidly completed by analyzing ICAT expression, and the whole detection process is non-invasive and has no negative effect on patients.

Drawings

FIG. 1 is the values of ICAT expression levels of each group in example 1.

FIG. 2 is the Log values of the expression amounts of ICAT in each group in example 1.

FIG. 3 is a graph showing the effect of the expression level of ICAT protein on the survival time of AML patients in example 2.

FIG. 4 is a graph showing the effect of ICAT protein expression level on survival time of AMLM3 patients in example 2.

Detailed Description

Example 1 comparison of expression of ICAT in AML patient Induction differentiation-treated groups

1. Selection and grouping of subjects: in the experiment, 37 confirmed AML patients collected from the hematology department in 2018 to 2019 in 12 months are selected, wherein 22 men are aged 9-86 years, the mean age is 44.95 years, 15 women are aged 15-67 years, and the mean age is 43.5 years. Diagnosis was performed by bone marrow cell morphology, cytochemical staining, cytogenetics, and detection of fusion genes of leukemia, according to diagnosis and treatment criteria established according to FAB typing. Taking the patient as an initial group before treatment, carrying out standard dose chemotherapy on the patient, and treating non-M3 patients by adopting standard dose DA (daunorubicin + cytarabine), DAE (daunorubicin + cytarabine + etoposide), CAG (pirarubicin + cytarabine) and IA (demethoxydaunorubicin + cytarabine) schemes; after 2 courses of treatment, the prognosis of all patients in the study is evaluated according to 'diagnosis and curative effect standard of hematopathy', and the patients are divided into Complete Remission (CR), Partial Remission (PR) and treatment failure (NR), wherein the CR patients are completely remission groups, the patients with prognosis not reaching CR are not completely remission groups, 25 non-malignant patients with hematological disorders (including 10 cases of hyperplastic anemia, 8 cases of ITP and 7 cases of multiple myeloma) with the damage of important organs such as heart, liver, lung and kidney in the same period of hospitalization are selected as control groups, meanwhile, other malignant patients (including breast cancer, colorectal cancer and the like) are selected as other tumor groups, and 13 patients with renal insufficiency are selected as renal insufficiency groups.

2. The experimental steps are as follows: before treatment, an ELISA method is used for detecting the expression condition of ICAT in serum/plasma of each group of patients, serum samples are collected after two treatment courses, and the proportion of primary cells in bone marrow of the patients, WBC, PLT and primary cell proportion in peripheral blood are counted.

The procedure for detecting ICAT expression in serum/plasma of each group of patients using ELISA method was as follows:

1) blood collection and treatment:

taking 3ml of venous blood clinically, and placing the venous blood in a test tube for standing for 30 min; centrifuging at 3000r/min for 5min after coagulation; taking the supernatant as the obtained serum; all sera were stored rapidly in-80 ℃ refrigerator within 30 minutes after acquisition and stored until use.

2) Detection of ICAT expression

A. Diluting the CIRP monoclonal antibody with 0.1M carbonate coating buffer (pH 9.6) to a protein concentration of 1-10 ug/ml;

B. 100ul of diluent is added into a reaction hole of a polystyrene plate;

standing overnight at C.4 deg.C;

D. removing liquid in the holes, and slightly spin-drying;

E. washing 3 times with PBS wash buffer containing 0.1% Tween-20 for 5 minutes each time (the same applies below for the wash solution);

F. adding 100ul of diluted serum into the reaction hole, and simultaneously arranging a standard hole, a blank hole and a sample hole to be detected;

G. gently shaking, covering a film, and incubating in a 37 ℃ incubator in a dark place for 1 hour;

H. pouring out liquid in the holes, and cleaning for 3 times by using a cleaning solution;

I. adding 100ul of freshly prepared enzyme-labeled antibody into each reaction hole;

J. gently shaking, covering a film, and incubating for 1 hour at 37 ℃ in a dark place;

K. pouring out liquid in the holes, and cleaning for 3 times;

l, adding a freshly prepared TMB substrate color developing solution into each reaction hole;

developing at M.37 ℃ in dark for 20 minutes;

n, adding 50ul of 2M sulfuric acid solution into each reaction hole to terminate the reaction; and reading the detection result (O.D. value) by a microplate reader under the dominant wavelength of 450 nm.

3. As a result:

1) ICAT expression in relation to clinical characteristics of AML patients (bone marrow primary cell proportion at first diagnosis, WBC number, PLT, LDH)

2) Comparison of expression levels of ICAT in AML groups

The expression levels of ICAT in 37 cases, 23 cases in CR group, 10 cases in PR group and 25 cases in Control group in each group are shown in FIGS. 1-2. It can be seen that the expression level of ICAT in the Complete Remission (CR) group was significantly higher than that in the Partial Remission (PR), control, and initial diagnosis groups (p < 0.01).

Example 2 evaluation of clinical value of ICAT in prognosis of therapy for inducing differentiation in AML patients

To verify the clinical utility value of ICAT in the prognosis of treatment of induced differentiation in AML patients, the following experiments were performed:

1. 47 AML patients were selected, treated in the manner of example 1 and tested for ICAT expression in serum/plasma. Follow-up was performed and patient treatment was recorded. The treatment mode is as above: standard dose chemotherapy is administered to patients, non-M3 patients are treated with standard dose DA (daunorubicin + cytarabine), DAE (daunorubicin + cytarabine + etoposide), CAG (pirarubicin + cytarabine), IA (idarubicin + cytarabine); the M3 patients are treated by all-trans retinoic acid induced differentiation, after 2 treatment courses, the prognosis of all patients in the study is evaluated according to the 'blood disease diagnosis and curative effect standard', and the prognosis is divided into Complete Remission (CR), Partial Remission (PR) and treatment failure (NR), wherein the CR patients are completely remission groups, and the patients with prognosis not reaching CR are not completely remission groups.

2. Survival analysis

Analyzing the result by using SPSS software, drawing an ROC curve and calculating and analyzing the area under the curve, comparing AUC of each index by adopting Z test of a single variable, and considering that the difference is statistically significant when P is less than 0.05; the Cut-off value of each index is determined by adopting a functional method, the diagnosis threshold value represented when the maximum value of 'sensitivity + specificity' is taken is the Cut-off value, and the clinical diagnosis performance evaluation indexes (sensitivity and specificity) are calculated according to the value and the gold standard (pathological diagnosis result). And (3) carrying out COX regression multi-factor model analysis on the person with P <0.05 in the one-factor variance analysis by adopting a Kalpan-Meier survival analysis method.

47 patients were followed for at least 2 years and died 6 in 25 cases in the low expression group within 2 years (24.0%, cause of death was 5 cases with drug resistance to the white blood pathogen, 1 case with infection after chemotherapy); however, only 2 deaths were observed within 2 years in the high expression group 22 cases (9.1%, the cause of death was 1 case of drug resistance to the white blood pathogen, and 1 case of infection after chemotherapy). The mortality rate in the high expression group was significantly lower than that in the low expression group (χ 2 ═ 5.015, P ═ 0.015), indicating that AML patients with high ICAT protein expression were more responsive to differentiation-inducing treatment. The effect of ICAT protein expression levels on survival time of AML patients is shown in figure 3.

In addition, 20M 3 patients included in 47 patients were also analyzed individually. 3 deaths within 2 years in 12 of 20M 3 patients in the low-expression group (25.0%, 2 cases with drug resistance to white blood pathogen, 1 case with infection after chemotherapy); however, only 1 of the 8 cases in the high expression group died within 2 years (12.5%, cause of death was infection after chemotherapy). The mortality rate in the high expression group was significantly lower than that in the low expression group (χ 2 ═ 5.089, P ═ 0.025), indicating that AML M3 patients with high ICAT protein expression were more responsive to trans-retinoic acid-induced differentiation therapy. The effect of ICAT protein expression levels on survival of AMLM3 type patients receiving trans-retinoic acid-induced differentiation therapy is shown in fig. 4.

By detecting the expression level of ICAT protein, the disease development or prognosis of AML patients can be judged, and a treatment scheme can be designed in a targeted manner, so that the blindness in treatment is reduced, and the cure rate of the patients is improved.

Claims

1. Use of an agent for detecting an ICAT protein for the preparation of an agent for judging the prognosis of an AML M3-type patient with acute myeloid leukemia receiving an induced differentiation therapy, which is not NSC67657 and which is specifically an all-trans retinoic acid induced differentiation therapy.

2. The use according to claim 1, wherein the expression level of ICAT protein is detected by ELISA method.