WO2020143500A1 - Leukemia marker and use thereof in assessment of leukemia prognosis - Google Patents

Abstract

Disclosed is a leukemia marker. The leukemia marker is a gene set forth in SEQ ID No. 1 or a gene having 85% to 99% homology thereto. The marker is applicable to the preparation of a product for predicting or diagnosing whether a patient is one with refractory myeloid leukemia, or sorting a patient with refractory myeloid leukemia, and for predicting or diagnosing whether a patient is one with myeloid leukemia who has been treated to the degree of complete bone marrow remission but shows the risk of recurrence, or sorting a patient with myeloid leukemia who has been treated to the degree of complete bone marrow remission but shows the risk of recurrence, and for predicting or assessing the time remaining for a patient with myeloid leukemia. Detection of the marker prior to chemotherapy can help determine responsiveness to chemotherapy, recurrence, and the prognostic survival of a patient, and nicely distinguish chemotherapy-sensitive, refractory, and recurrent patients, so as to overcome the shortcomings of existing risk stratification and achieve the purpose of further fine stratification of patients, effectively providing guidance for subsequent chemotherapy and transplantation treatment programs.

Description

Leukemia marker and its use in prognosis assessment of leukemia

This application requires the priority of the Chinese patent application submitted to the China Patent Office on January 10, 2018, with the application number 201910024579.8 and the invention titled "a leukemia marker and its use in the prognosis evaluation of leukemia". Incorporated by reference in this application.

Technical field

The invention relates to the field of in vitro diagnosis, in particular to a leukemia marker and its use in prognosis assessment of leukemia.

Background technique

Acute myeloid leukemia (AML) is a highly heterogeneous disease that is the most common type in adults, mostly elderly patients. The prognosis of AML is extremely poor, and the five-year survival rate is less than 27%. Even after complete remission of chemotherapy, 70-80% of patients relapse and become refractory. The clinical treatment of AML is based on the patient's risk stratification to determine the patient's induction treatment plan, consolidation chemotherapy plan and length of treatment, and whether hematopoietic stem cell transplantation should be actively performed after achieving complete remission. At present, the only clinically effective treatment methods for myeloid leukemia are chemotherapy and transplantation. The precise and accurate stratification of patients in diagnosis and prognosis evaluation is of great significance. It is related to the adjustment of chemotherapy regimen, determining the timing of transplantation for patients of appropriate age, and whether elderly patients can Supportive treatment and demethylation therapy are adopted; these decisions are closely related to the patient's survival time and quality of life.

At present, NCCN (National Comprehensive Cancer Network) risk stratification is mainly used globally, and some European countries adopt ELN (European Leukmia Net) risk stratification. These two stratifications are similar and are based on abnormal chromosomes in nucleated cells in bone marrow of various types of patients and several fusion genes and mutant genes with very clear clinical significance. The patients are classified into low, medium-risk (medium-low-risk, medium-high-risk) ), high-risk groups.

However, clinically, 40-50% of patients have normal chromosomes and negative fusion genes; some patients are unable to obtain information on mutated genes because they have not performed sequencing. These patients cannot be accurately stratified according to the current scheme, and can only be classified fuzzyly. Into the middle-risk group. In addition, although the current stratification has the function of indicating the patient's natural history and prognosis in the group, it indicates that the individual often has deviations, especially indicating the patient's refractory relapse and clinical outcomes. It is one of the most valuable guidance for treatment and prognosis to clearly indicate the refractory and recurrent outcome of patients before chemotherapy.

The limitations of current stratified diagnosis and treatment decisions are as follows:

Global current NCCN and ELN risk stratification. These two layers are basically the same, and are composed of abnormal chromosomes of various types of patients, several fusion genes with very clear clinical significance, and mutant genes. Mutant genes to assess chemotherapy sensitivity are obtained from scattered patient sequencing. The frequency of each mutation is only seen in about 10% of patients, while the fusion gene and chromosome assessment patients have a longer survival time. This leads to the following problems in the existing stratification: 1) About 40-50% of patients in the clinical are chromosomal normal, the fusion gene is negative, and the mutant gene has not been detected. . 2) The ability to predict the patient's chemotherapy responsiveness in advance is insufficient, and the indications for the patient's primary drug resistance, refractory, and recurring clinical outcomes are not clear, and often differ from the clinical outcomes; because the elderly often have poor tolerance to chemotherapy, this is The effect of elderly patients is particularly obvious. 3) It has the function of indicating the severity and prognosis of the disease in the population, but indicating that the individual often has deviations. 4) Not quantitative.

Therefore, there is an urgent need to find a marker that can accurately predict whether patients with myeloid leukemia have refractory and relapse risks before chemotherapy to solve the above-mentioned deficiencies in the prior art.

Summary of the invention

One aspect of the present invention is to provide a leukemia marker for the defect that the risk stratification of myeloid leukemia patients in the prior art is unclear and cannot clearly indicate whether the patient has a risk of refractory or relapse.

The technical solutions provided by the present invention are:

A leukemia marker, the leukemia marker is the gene shown in SEQ ID No. 1 or the gene having 85%-99% homology with the gene shown in SEQ ID No. 1, the marker is used for preparing prediction 2. Diagnose whether the patient is a patient with refractory myeloid leukemia, or sort products of patients with refractory myeloid leukemia.

The gene shown in SEQ ID No. 1 in the present invention is an essential gene for cell survival, and it plays an important role in the most basic biological processes of cells such as pre-mRNA shearing, RNA nuclear retention, cell transcription, and DNA repair. In the present invention, the gene shown in SEQ ID No. 1 is expressed in every human bone marrow, and is a gene expressed continuously in the cell. At present, there are very few reports about this gene in hematological diseases, and there is no related report about its refractory and relapse of myeloid leukemia, nor its use as a new prognostic evaluation and refractory relapse marker, which can be included in the risk of myeloid leukemia Layered reporting.

In the present invention, the gene shown in SEQ ID No. 1 includes the mRNA single-stranded sequence and its complementary sequence shown. At the same time, the marker of the present invention also includes the cDNA single-strand sequence obtained by reverse transcription of the gene shown in SEQ ID No. 1 and the sequence obtained by other transcription forms.

In the present invention, the gene shown in SEQ ID No. 1 may be derived from nucleated cells in peripheral blood and/or bone marrow of the patient.

Another aspect of the present invention is to provide a leukemia marker that is the gene shown in SEQ ID No. 1 or has 85%-99% homology with the gene shown in SEQ ID No. 1. Gene, this marker is used to prepare and predict whether the patient is a myeloid leukemia patient who has a risk of relapse after treatment to achieve complete remission of the bone marrow, or to sort a myeloid leukemia patient who has a risk of recurrence after treatment to achieve complete remission of the bone marrow. product.

The above-mentioned complete remission (CR) refers to the complete remission of bone marrow morphology according to the standards of the International Collaborative Group, specifically: the patient meets the morphological leukemia-free status, detaches from blood transfusion, and the original cells in the bone marrow aspiration smear <5% (at least count 200 nucleated cells), blasts without Auer bodies or extramedullary leukemia persist. In the peripheral blood neutrophil count> 1.0x10 ⁹ / L, platelet> 100x10 ⁹ / L.

In the present invention, the gene shown in SEQ ID No. 1 may be derived from nucleated cells in the peripheral blood and/or bone marrow of the patient.

The above "treated" refers to patients with myeloid leukemia undergoing risk stratification (NCCN-2016 and ELN-2016 version of risk stratification), and then undergoing routine clinical treatment, such as chemotherapy (including induction remission therapy, consolidation therapy, Maintenance treatment) or supportive treatment.

Another aspect of the present invention is to provide a leukemia marker that is the gene shown in SEQ ID No. 1 or has 85%-99% homology with the gene shown in SEQ ID No. 1. Gene, the marker is used to prepare products that predict or evaluate the prognosis of patients with myeloid leukemia.

The gene with 85%-99% homology with the gene shown in SEQ ID No. 1 may be 85%, 86%, 87%, 88%, 89%, 90% with the gene shown in SEQ ID No. 1 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homologous genes.

Another aspect of the present invention is to provide a marker composition, the marker composition comprising the above markers, the marker composition is used to prepare for predicting, diagnosing whether the patient is refractory and/or treated Patients with myeloid leukemia who have a risk of relapse after achieving complete bone marrow remission, or sorting patients with refractory and/or relapse risk after treatment to achieve complete remission of bone marrow, or predicting and evaluating the overall survival of patients with myeloid leukemia The product.

The above marker composition may be derived from peripheral blood and/or bone marrow cell samples. However, preferably, in one embodiment of the present invention, the above-mentioned genes are derived from nucleated cells in the bone marrow of the patient.

Preferably, the above marker composition further includes one or more selected from mutant genes, fusion genes or abnormal chromosomes involved in the onset and/or prognosis assessment of leukemia. More preferably, it is one or more selected from the group consisting of fusion genes, mutant genes, or abnormal chromosomes involved in the onset and/or prognosis of leukemia involved in the risk stratification of WHO and ELN. More preferably, it is one or more selected from PML-RARα or AML1-ETO fusion gene, RUNX1 or ASXL1 mutant gene, -5 or -7 abnormal chromosome.

In one embodiment of the invention, the level of any of the above biomarkers is determined by mass spectrometry. Preferably, it is determined by mass spectrometry chromatography (such as gas chromatography mass spectrometry (GC-MS) or liquid chromatography mass spectrometry (LC-MS)).

At the same time, the present invention can also use any suitable method to determine the level of any of the above biomarkers. Preferably, in another embodiment of the present invention, the level of the biomarker is measured by real-time fluorescence quantitative PCR.

In the present invention, the gene marker shown in SEQ ID No. 1 and the marker composition can be used to predict the transformation of patients with myeloid leukemia, for example, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome Acute myeloid leukemia. Preferably, in one embodiment of the present invention, the gene marker shown in SEQ ID No. 1 and the marker composition are used to predict patients with acute myeloid leukemia.

Another aspect of the present invention is to provide a kit including the product for detecting the gene marker shown in the above SEQ ID No. 1, and the above marker composition.

In the present invention, any suitable products and methods for detecting the above-mentioned genes may be used, which are considered to be included in the protection scope of the present invention. For example, polymerase chain reaction (PCR), such as qPCR; reverse transcription polymerase chain reaction (RT-PCR); in situ hybridization (FISH); transcription-mediated amplification (TMA); ligase chain reaction ( LCR); strand displacement amplification (SDA) and nucleic acid sequence-based amplification (NASBA); microarrays; Southern or Northern blotting; high-throughput sequencing methods.

Preferably, in one embodiment of the present invention, the product is a primer or a probe for detecting the above-mentioned leukemia marker or the above-mentioned marker composition.

More preferably, the product is a primer for detecting the above-mentioned leukemia marker, or the above-mentioned marker composition. In one embodiment of the present invention, the qPCR method is used to detect the target gene.

Preferably, the primers in the kit include an amplification primer pair of the internal reference GAPDH and an amplification primer pair of the gene marker shown in SEQ ID No. 1. More preferably, the amplification primer pair of the internal reference GAPDH is forward: 5'-CAACTACATGGTTTACATGTTC-3'; reverse: 5'-GCCAGTGGACTCCACGAC-3'; the gene marker shown in SEQ ID No. 1 The amplification primer pair is forward: 5'-GGATCCACGGGAAAGAGACA-3'; reverse: 5'-CACTCATGGTTGCTGGTGG-3'. Preferably, the kit further includes PCR reaction solution and kit instructions. The PCR reaction solution includes dNTP, Mg ²⁺ , Taq enzyme, fluorescent dye and buffer buffer, the fluorescent dye is SYBR Green II, and Taq enzyme is a hot-start enzyme.

In one embodiment of the present invention, the predicting the risk of myeloid leukemia patients includes the following steps:

Step 1) Determine the level Ct of the above-mentioned leukemia markers and each of the biomarkers in the above-mentioned marker composition in the sample from the patient;

Step 2) Detect the GAPDH level of the sample, and calculate the △Ct value according to △Ct=Ct-Ct _GAPDH . △Ct≥4.913 is a sensitive patient, and △Ct<4.913 is a refractory relapsed patient.

Preferably, the sample of the patient is a bone marrow nucleated cell of the patient.

The beneficial effects of the present invention are:

1) Because the markers of the present invention are expressed in nucleated cells of bone marrow of everyone, it is applicable to all AML patients of all types and ages;

2) The marker of the present invention can determine the responsiveness of patients to chemotherapy before chemotherapy, and can distinguish chemotherapy sensitive and refractory patients well; therefore, it can achieve further fine stratification of patients under the current risk stratification The purpose is to refer to the reactivity of chemotherapy.

3) Adding the marker stratification scheme of the present invention under the current risk stratification can prompt the hidden refractory and relapsed patients in the low-risk group to perform transplantation as soon as possible; high-risk, especially high-risk elderly patients are informed that they are refractory before chemotherapy Or, supportive therapy or demethylation therapy can be directly taken to avoid the side effects of ineffective chemotherapy and reduce the infection rate; these are necessary for patients to survive, prolong survival, and improve quality of life.

4) The detection of the markers of the present invention can be completed only by using the intermediate product cDNA of the fusion gene detection process of the patient's required test items, without the need for additional processing and preparation of specimens, and will not increase the patient's invasive specimen collection and specimen consumption. Patients save on medical costs.

5) This work is not only limited to the application significance in the field of diagnosis, but also has an important guiding role in the rational clinical use of drugs in the later period, the soothing treatment of malignant diseases in elderly patients, and the suggestion that the transplantation plan is more objective (quantified by test indicators).

BRIEF DESCRIPTION

FIG. 1 is a flowchart of a clinical research design scheme in an embodiment of the present invention;

2 is a graph showing the difference analysis results of the markers of the present invention among patients with different AML types;

Figure 3 is a graph showing the difference analysis results of the markers of the present invention under ELN2018 risk stratification;

4 is a graph showing the difference analysis results of the markers of the present invention among sensitive, refractory and relapsed patients;

FIG. 5 is a graph showing the difference analysis results of the patients of the present invention further subdivided under the current ELN2018 risk stratification;

6 is a graph of the receiver operating characteristic curve of the marker of the present invention;

7 is a graph showing the relationship between the marker levels of the present invention and the overall survival time of patients.

Sequence description

SEQ ID No. 1 is the nucleotide sequence of the leukemia marker of the present invention.

detailed description

The invention discloses a leukemia marker and its use in the prognosis evaluation of leukemia. Those skilled in the art can learn from this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, they are all considered to be included in the present invention, and it is obvious that the relevant personnel can deviate from the content, spirit and scope of the present invention Modify or appropriately modify and combine the contents described herein to implement and apply the technology of the present invention.

In the present invention, unless otherwise stated, the scientific and technical terms used herein have the meaning commonly understood by those skilled in the art. The following explains some related terms appearing in the present invention.

The term "acute myelogenous leukemia", or acute myelogenous leukemia, AML, is a malignant disease of myeloid hematopoietic stem/progenitor cells. Abnormal hyperplasia of primitive and naive myeloid cells in bone marrow and peripheral blood is the main feature. The clinical manifestations are anemia, bleeding, infection and fever, organ infiltration, metabolic abnormalities, etc. In most cases, the condition is critical and the prognosis is dangerous. If not treated in time Often can be life-threatening. Acute myeloid leukemia is divided into FA, AML morphological classification, and it is divided into M0, M1, M2, M3, M4, M5, M6 and M7 types.

The term "marker", also known as "biological marker", refers to a measurable indicator of a subject's biological state or condition. Such biomarkers can be any substance in the subject, such as nucleic acid markers (eg DNA), protein markers, cytokine markers, chemokine markers, carbohydrate markers, antigen markers, antibody markers Biomarkers, species markers (species/genus markers) and functional markers (KO/OG markers), etc., as long as they are related to the subject's specific biological state or condition (such as disease). The detection and evaluation of biomarkers are often used to examine normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions, and are useful in many scientific fields.

The terms "a", "an" and "the" are not intended to refer to a single entity only, but include general categories that can be illustrated using specific examples thereof.

The full English name of the term "QPCR" is Real-time Quantitative PCR Detection System. Real-time fluorescence quantitative nucleic acid amplification detection system, also called real-time quantitative gene amplification fluorescence detection system, referred to as QPCR.

The term "polymerase chain reaction", or PCR, uses DNA to become single-stranded when denatured at 95°C in vitro. At low temperatures (often around 60°C), primers and single-strands are combined according to the principle of base pairing , And then adjust the temperature to the optimal reaction temperature of DNA polymerase (about 72℃). DNA polymerase synthesizes the complementary chain along the direction of phosphate to five-carbon sugar (5'-3'), which is a specific Molecular biology technology of DNA fragments.

The term "sensitive" refers to clinical complete remission (CR) achieved within two standard treatment regimens, no recurrence within 18 months after CR, and no two or more relapses after CR.

The term "refractory" refers to the initial treatment of two cases that are ineffective after standard treatment; those who relapse 2 or more times; extramedullary leukemia persists.

The term "relapse" refers to those who underwent consolidation and intensive treatment after CR and relapsed within 6 months, 12 months, and 18 months. The recurrence is based on the recurrence of leukemia cells or blast cells in the bone marrow >5% after CR (except for other reasons such as bone marrow regeneration after consolidation chemotherapy) or extramedullary leukemia cell infiltration.

Those skilled in the art will understand that, except as outlined in the claims, the terminology herein is provided for a better understanding of the invention and is not intended to limit the invention.

In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with specific embodiments.

The clinical plan design of the present invention: routine clinical examination and genetic testing of the markers of the present invention are carried out after the patient has retained the specimen, the diagnosis is typed, the risk stratification is carried out on the patient, and clinical treatment is performed according to the guidelines. After the treatment, patients were grouped according to the clinical exposure outcomes (sensitivity, refractory, relapse). The relationship between the expression level of the marker of the present invention and the treatment outcome of the patient—sensitive, refractory, and relapsed was analyzed. The flow of clinical research design plan is shown in Figure 1. The risk stratification of patients was divided into NCCN-2016 and ELN-2016 versions of risk stratification; sensitive, refractory, and relapsed patients were grouped according to the Chinese diagnosis and treatment guidelines for relapsed/refractory AML (2017 version).

Example 1: Patient acceptance criteria and sample retention

Newly diagnosed patients with various types and ages of acute myeloid leukemia without chemotherapy. Exclude 1) patients with various infections; 2) patients with bleeding tendency, failure of anti-infection, and chemotherapy for various reasons; 3) patients with previous history of solid tumors and blood tumors, aplastic anemia. The patients underwent bone marrow aspiration, and while performing routine clinical examinations, they retained bone marrow specimens for each patient, extracted total RNA from bone marrow nucleated cells, reversed to cDNA, and stored frozen at -20°C.

Example 2: Isolation of bone marrow nucleated cells, total RNA extraction and reverse transcription

1. Isolate nucleated cells:

Place the bone-penetrating specimen of the patient at rest, layer the bone marrow plasma and cell components gently and then wash out the bone marrow plasma. Add the cell components and erythrocyte lysate to the lysate at a volume ratio of 1:4, invert and mix, and let stand at room temperature for 8-10 Minutes, inverting and mixing again several times during this period, and centrifuging to collect the cells after the solution is clear and transparent. 400 transparent TRIzol proportion lysed cells 5x10 ⁶ cells was added TRIzol lysate (ThermoFisher), RNA is extracted immediately or stored frozen at -80 ℃ subsequent to extracting.

2. Total RNA extraction:

TRIzol containing nucleated cells was mixed by shaking upside down, adding 1/5 volume of chloroform, mixing upside down for 1 minute, standing at room temperature for 5 minutes, centrifugation at 4°C, 13000 rpm for 15 minutes. Transfer the centrifuged supernatant carefully into a new centrifuge tube, add an equal volume of isopropanol, mix gently, and let stand at room temperature for 10 minutes. Centrifuge at 13000 rpm for 15 minutes at 4°C, discard the supernatant, and keep the pellet. Add 1 mL of 70% absolute ethanol to the precipitate, gently blow to mix, centrifuge at 4°C, 13000 rpm for 15 minutes, and gently aspirate the supernatant. After drying at room temperature, add appropriate amount of DEPC water to fully dissolve the precipitate, adjust the RNA to 200-500ng/μg, and immediately perform reverse transcription or freeze storage at -80℃ until use.

3. Reverse transcription:

After RNA was subjected to denaturation treatment at 70°C for 5 minutes and ice bath for 5 minutes, reverse enzyme was used with Promega Company. The reverse transcription system was configured on the ice bath, and reverse transcription was performed according to the system shown in Table 1. The configured reaction system is reverse transcribed on a PCR instrument. The reaction conditions are: 25°C for 5 minutes, 42°C for 1 hour, and 70°C for 15 minutes. After the reaction is completed, the cDNA is frozen at -20°C or immediately identified by real-time fluorescence quantitative PCR.

Table 1 Reverse transcription system

Example 3: Real-time quantitative PCR detection of target genes

1. Primer sequence

GAPDH (product length: 185bp)

Forward: 5’-CAACTACATGGTTTACATGTTC-3’ (22nt)

Backward: 5’-GCCAGTGGACTCCACGAC-3’ (18nt)

The marker of the present invention (product length: 148bp)

Forward: 5’-GGATCCACGGGAAAGAGACA-3’ (20nt)

Backward: 5’-CACTCATGGTTGCTGGTGG-3’ (19nt)

2. Real-time fluorescence quantitative PCR to amplify the target gene:

The reaction adopts Bio-rad real-time quantitative PCR instrument, and the reagents are used

qPCR Master Mix (Promega), according to the system shown in Table 2 to detect the expression levels of the markers and GAPDH gene in bone marrow specimens of each patient, and set a negative control without template. Reaction conditions: pre-denaturation at 95°C for two minutes; denaturation at 95°C for 15 seconds, annealing at 60°C for one minute for 40 cycles; finally 95°C for 15 seconds, 60°C for one minute, and 95°C for 15 seconds to end the reaction. The total reaction system is 25μL.

Table 2 PCR system

3. Results processing and statistical analysis:

According to the qPCR results, the GAPDH gene Ct value is used as an internal reference to correct the Ct value of the target gene (marker of the present invention), and the expression level of the comparable marker for each patient is indicated by △Dashui, △show, =Ct-Ct _GAPDH . According to WHO-2008 classification of acute myeloid leukemia, patients were divided into 8 groups, gr1: AML with t(15; 17)/PML RARα; gr2: AML with t(8; 21)/AML1-ETO; gr3: AML with inv(16)/CBFβ-MYH11; gr4:AML with minimal differentiaton and without maturation(FAB AML-M0,M1); gr5:AML with maturation(FAB AML-M2); gr6:AML with acute myelomonocytic leukemia(FAB AML -M4); gr7: AML with acute monoblastic/monocytic leukemia (FAB AML-M5); gr8: other AMLs (AML with myelodysplastic syndrome). One way ANOVA was used to analyze the difference in the target gene (marker of the present invention) ΔCt between the groups. The results are shown in Figure 2. The results indicate that the transcription level of the marker of the present invention is not related to AML typing. According to NCCN-2016 risk stratification and ELN stratification, they were divided into three groups: low-risk, medium-low-risk, medium-high-risk, and high-risk. One way ANOVA was used to analyze the differences in the target genes (markers of the present invention) between the groups. image 3. The results indicate that the transcription level of the marker of the present invention is not related to the current risk stratification.

According to the 2017 version of the Chinese diagnosis and treatment guidelines for relapsed/refractory AML, it is divided into three groups: sensitive, refractory and relapsed. The grouping criteria are shown in Table 3.

Table 3 Criteria for grouping sensitive, refractory and relapsed patients

The t-test was used to analyze the differences in the target genes (markers of the invention) between the two groups. The results are shown in Figure 4. The transcription level of the marker of the present invention is extremely significant between sensitive vs refractory and sensitive vs relapsed groups, P<0.0001; it can distinguish sensitive and refractory relapse patients well. Further, under the current stratification system, patients are divided into low-risk (low-risk and medium-low-risk) and high-risk (high-risk and medium-high-risk) layers. The transcription level of the markers of the present invention can still well distinguish the sensitivity and sensitivity of more patients. For the refractory relapse, there is a very significant difference between the groups, P <0.0001, the results are shown in Figure 5. The transcription level of the marker of the present invention can further refine the stratification of patients under the current stratification, and identify the reactivity of patients to chemotherapy before chemotherapy, which is an important supplement to the current stratification.

Example 4: Diagnosis of diagnostic efficacy

The value of the markers of the present invention detected by the patients with complete follow-up data (n=166) was used as the receiver operating characteristic curve (ROC curve), and the curve was calculated using the Youden method using R software and the cutoff value was calculated. According to the coordinate value, the specificity and sensitivity corresponding to the critical point are obtained. The result is shown in Fig. 6. The results show that the sensitivity is 83.3%, the specificity is 82.9%, the cutoff value of the marker ΔCt of the present invention is 4.913, and the area under the curve AUC is 0.88.

Example 5: Target gene transcription level and patient's overall survival (OS)

For patients with complete follow-up data (n=166), make a survival curve graph, make a tertile survival curve according to the detected marker value of the present invention, use GraphPad Prism software to draw a survival curve, and use Log-rank (Mantel-Cox) method to do Statistical analysis, the results are shown in Figure 7. The results show that the total survival time of patients has a statistically significant difference between different levels of the marker values detected by the present invention, P<0.0001.

The above is only a preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principles of the present invention, several improvements and retouches can be made. These improvements and retouches also It should be regarded as the protection scope of the present invention.

Claims (10)

1. A leukemia marker, characterized in that the leukemia marker is the gene shown in SEQ ID No. 1 or the gene having 85%-99% homology with the gene shown in SEQ ID No. 1, the marker It is used to prepare products for predicting and diagnosing whether patients are refractory myeloid leukemia patients, or to sort refractory myeloid leukemia patients.
2. A leukemia marker, characterized in that the leukemia marker is the gene shown in SEQ ID No. 1 or the gene having 85%-99% homology with the gene shown in SEQ ID No. 1, the marker It is used to prepare and predict whether the patient is a myeloid leukemia patient who has a risk of relapse after treatment to achieve complete remission of the bone marrow, or to sort a product of a myeloid leukemia patient who has a risk of recurrence after treatment to achieve complete remission of the bone marrow.
3. A leukemia marker, characterized in that the leukemia marker is the gene shown in SEQ ID No. 1 or the gene having 85%-99% homology with the gene shown in SEQ ID No. 1, the marker It is used to prepare products for predicting or evaluating the overall survival of patients with myeloid leukemia.
4. A marker composition, characterized in that the marker composition comprises the marker according to claim 1 or 2, the marker composition is used to prepare for predicting, diagnosing whether a patient is refractory and/or Patients with myeloid leukemia who have a risk of relapse after treatment to achieve complete remission of the bone marrow, or sort patients who are refractory and/or who have a risk of relapse after treatment to achieve complete remission of the bone marrow, or to predict and evaluate the total Lifetime products.
5. The marker composition according to claim 3, characterized in that the marker composition further comprises one or more selected from mutant genes, fusion genes or abnormal chromosomes involved in the onset and/or prognosis assessment of leukemia , Preferably one or more selected from fusion genes, mutant genes or abnormal chromosomes involved in the onset and/or prognosis of leukemia involved in WHO and ELN risk stratification, more preferably selected from PML-RARα or AML1 -One or more of ETO fusion gene, RUNX1 or ASXL1 mutant gene, -5 or -7 abnormal chromosome.
6. The leukemia marker according to any one of claims 1 to 3, or the marker composition according to claim 4 or 5, wherein the myeloid leukemia is acute myeloid leukemia.
7. A kit, characterized in that the kit includes a product for detecting the leukemia marker according to any one of claims 1-3, or the marker composition according to claim 4 or 5.
8. The kit according to claim 7, wherein the product is for detecting the leukemia marker according to any one of claims 1-3, or the marker composition according to claim 4 or 5. Primers or probes.
9. The leukemia marker according to any one of claims 1 to 3, the marker composition according to claim 4 or 5, or the kit according to claim 7, wherein the predicted risk of myeloid leukemia patients , Including the following steps:

   Step 1) Determine the level Ct of each biomarker in the leukemia marker according to any one of claims 1-3 and the marker composition according to claim 4 or 5 in a sample from the patient;

   Step 2) Detect the GAPDH level of the sample, and calculate the △Ct value according to △Ct=Ct _marker- Ct _GAPDH . △Ct≥4.913 is a sensitive patient, and △Ct<4.913 is a refractory relapsed patient.
10. The leukemia marker, marker composition or kit according to claim 9, characterized in that the sample is nucleated cells in the bone marrow of the patient.

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