Marker for prognosis evaluation of acute myelogenous leukemia and application of marker

Abstract

The invention belongs to the technical field of biology, and particularly relates to a marker for prognosis evaluation of acute myelogenous leukemia and application of the marker, the marker is HCK-positive mono-like cells, and the HCK-positive mono-like cells are determined when an antigen CD163, an antigen CD68, an antigen FCN1 and an antigen HCK are co-positive. The kit is used for detecting bone marrow or peripheral blood samples of adult acute myelogenous leukemia so as to assist in judging clinical prognosis conditions of patients with acute myelogenous leukemia.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a marker for prognosis assessment of acute myeloid leukemia and an application thereof. Background Art

[0002] Acute myeloid leukemia (AML) is a malignant clonal disease originating from hematopoietic stem cells, and its biological characteristics are highly heterogeneous. At present, the most important prognostic factor for AML is the cytogenetics-related results detected by conventional chromosome banding technology. However, due to the detection rate and the existence of some AML patients with normal karyotype, conventional cytogenetics technology has limitations as a prognostic assessment of leukemia residual disease. It is urgent to find other simple and universally applicable assessment methods in clinical practice, which is of great significance for the prognostic stratification of patients.

[0003] There are many factors that affect the prognosis of AML. The single morphological complete remission (CR) during or after the treatment can no longer be used as a single indicator to evaluate the treatment response. At present, the detection methods of minimal residual disease (MRD) mainly include polymerase chain reaction (PCR) and flow cytometry (FCM), as well as the developing next-generation sequencing (NGS) and digital PCR. Due to the differences in technical levels, differences in quality evaluation between laboratories, and the instability of standardized amplification systems, the detection of PCR fusion genes is only applicable to some AML patients. In addition, the qualitative / quantitative results of PCR cannot achieve a balanced match with the number of leukemia cells, so it is difficult to make an accurate assessment of minimal residual leukemia cells and patient prognosis. Compared with PCR technology, FCM has the characteristics of simple operation, fast detection speed, high specificity, and repeatable analysis. It can not only cover more than 90% of AML patients, but also can accurately quantify and quantify the number of leukemia cells on the basis of distinguishing between dead cells and live cells. Therefore, FCM is an important means of AML prognosis assessment, especially for AML patients without specific molecular markers. NGS and digital PCR are the most accurate MRD detection methods, which can clearly evaluate the residual leukemia cells of patients. However, these two technologies have high requirements for the professional skills and interpretation experience of the inspectors, and the detection cost is high, the standard operating system has not yet been widely used, and the results of different laboratories cannot be well matched and compared. Therefore, FCM detection of AML immunophenotype is not only important for the diagnosis and typing of AML patients, but also has important significance for AML MRD detection and patient treatment prognosis evaluation. At present, the CD antigens commonly used in clinical AML immunophenotypes are CD7, CD56, CD19, CD10, CD4, CD11b, CD11C, CD13, CD14, CD15, CD16, CD33, CD34, CD38, CD64, CD117, CD123, CD300e, MPO and HLA-DR. The main disadvantage is that the above antigens have certain requirements for the skills and experience of the inspectors, and the interpretation of the results contains some subjective factors.

[0004] When patients are admitted to the hospital, they need to undergo multiple parameters such as cell morphology, immunology, cytogenetics and MICM testing and analysis. According to the prognostic-related molecular genetic abnormalities in the results of real-time quantitative PCR, fluorescent in situ hybridization and chromosome karyotype analysis, the initial diagnosis and prognosis assessment of the patients are obtained. Patients in the three categories of "high risk", "intermediate risk" and "low risk" will receive targeted standardized early treatment plans. One course of treatment is 4 weeks. After two courses of treatment, the patients will show two states of "remission" and "incomplete remission". According to the remission status of the patient's disease, chemotherapy combined with allogeneic hematopoietic stem cell transplantation (Allo-HSCT) will be performed. After chemotherapy and transplantation, 35% to 40% of young patients (<60 years old) and 5% to 15% of elderly patients may be cured. However, more than half of young patients will still face relapse or refractory treatment, and most elderly and frail patients cannot tolerate conventional chemotherapy.

[0005] HCK (proto-oncogene) is a member of the SRC family of kinases. Reports suggest that HCK can be used for AML prognosis assessment, but different literatures show that HCK has different expression levels in AML patient samples and cannot be directly used for clinical AML patient prognosis assessment. Therefore, it is urgent to find other simple and universally applicable evaluation methods, which is of great significance for patient prognosis stratification. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a marker for prognosis assessment of acute myeloid leukemia and its application.

[0007] Application of a reagent for detecting the percentage of HCK-positive mono-like cells in the prognosis assessment of acute myeloid leukemia, wherein the HCK-positive mono-like cells are determined by co-positivity of antigen CD163, antigen CD68, antigen FCN1 and antigen HCK.

[0008] Preferably, the percentage of HCK-positive mono-like cells in the sample to mono-like cells is used to evaluate the prognosis of acute myeloid leukemia; the mono-like cells are determined when the antigen CD163, antigen CD68, and antigen FCN1 are co-positive.

[0009] Preferably, the sample is a bone marrow sample or a peripheral blood sample.

[0010] A kit for evaluating the prognosis of acute myeloid leukemia comprises a reagent for detecting the percentage of HCK-positive mono-like cells.

[0011] Preferably, the reagent includes a reagent for detecting the percentage of HCK-positive mono-like cells in a sample by flow cytometry.

[0012] Preferably, the reagents include anti-human CD163, anti-human CD68, anti-human FCN1, and anti-human HCK.

[0013] Preferably, the sample is a bone marrow sample or a peripheral blood sample.

[0014] Preferably, the acute myeloid leukemia includes newly diagnosed, relapsed or remitted acute myeloid leukemia.

[0015] Use of a reagent for detecting the percentage of HCK-positive mono-like cells in the preparation of the kit.

[0016] Based on the commonly used FCM detection and treatment in clinical practice, this study added HCK-related mono-like cell detection indicators: CD163, CD68 and FCN1. Through the detection of mono-like malignant cells, the patient's current and post-treatment prognosis was accurately evaluated based on the previous cut-off value. The sensitivity and specificity of AML prognosis evaluation in this study were 89.2% and 80.6%, respectively, the area under the ROC curve was 0.855, the range was 0.764-0.946, and P=0.046. Therefore, for patients with AML who are first diagnosed or re-examined, a more accurate prognosis assessment can be made for the patients based on the cut-off of flow cytometry HCK1=1.2. For patients with poor prognosis, the treatment plan can be improved or updated in time to enable patients to obtain a more rapid chance of remission.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The HCK-positive mono-like FCM test, with its advantages of simple operation and wide and reusable use, effectively avoids the instability of PCR technology, the high cost of NGS and digital PCR sequencing, and the experience requirements of testing personnel, so as to achieve stratified treatment and emergency intervention for AML. It can also provide precise targets for MRD-positive AML patients, thereby developing new targeted therapeutic drugs, reducing ineffective treatment of AML patients, and improving the treatment effect of relapsed and refractory AML patients.

[0019] The purpose of the present invention is to provide a marker with high sensitivity, strong specificity and good versatility for detecting bone marrow or peripheral blood samples of adult acute myeloid leukemia, so as to assist in judging the clinical prognosis of patients with acute myeloid leukemia. This is mainly based on the following problems: (1) Existing cytogenetic tests cannot effectively evaluate the prognosis of patients; (2) Gene sequencing and digital PCR technologies are expensive and have not yet been widely popularized; (3) SRC family kinase HCK can evaluate the prognosis of acute myeloid leukemia, but the direct evaluation value is very low. Therefore, new clinical prognostic markers are helpful for the clinical prognosis evaluation of patients with acute myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results are positive for flow cytometry. A is the cell population P1 after cell debris is removed in the first tube; B is the cell population P2 after adhesion cells are removed in the first tube; C is the cell population P3 after adhesion cells are further removed in the first tube; D is the P3 cell population marked with CD45; E is the cell population P4 that is co-positive for CD163 and FCN1 based on P3; F is the cell population that is co-positive for CD68, CD163, and FCN1, i.e., mono-like cells P5; G is the cell population P1 after cell debris is removed in the second tube; H is the cell population P2 after adhesion cells are removed in the second tube; I is the cell population P3 after adhesion cells are further removed in the second tube; J is all P3 cell populations marked with CD45; K is the replicated mono-like cell population P5; L is the mono-like cells P6 that are HCK-positive based on the P5 cell population;

[0021] Figure 2 ROC curves for mono-like, HCK1, and HCK2 indicators;

[0022] Figure 3 This is a stratification diagram of diluted bone marrow blood before and after centrifugation. DETAILED DESCRIPTION

[0023] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0024] Mono-like cells: All cells on the FCM machine are labeled with three antigens (FCN1, CD68, CD163) at the same time, that is, mono-like cells. This group of cells belongs to the mononuclear macrophage cluster, is in a relatively immature stage, and can differentiate into tumor-associated macrophages (TAM) and tissue-resident macrophages (TRM). Single-cell bioinformatics analysis found that mononuclear macrophages mainly include three types of cells: ① mono-like, with the main markers FCN1, CD68, and CD163; ② TAM, with the main markers ISG15, C1QC, and SPP1; ③ TRM, with the main markers LYVE and NLRP3. Data source: Chu X, Zhang Y, Cheng S. Heterogeneity of tumor-infiltrating myeloid cells in era of single-cell genomics. Chin J Cancer Res. 2022; 34(6): 543-553. doi: 10.21147 / j.issn.1000-9604.2022.06.01.

[0025] Example 1

[0026] 1. Collect clinical samples for flow cytometry testing

[0027] To analyze the differential expression of HCK in immune cells, bone marrow samples were collected from patients with newly diagnosed, relapsed, or remitted AML. Informed consent was obtained for sample collection, and all procedures were approved by the Institutional Research Ethics Committee of Guizhou Medical University.

[0028] Mononuclear cell isolation from clinical bone marrow samples

[0029] (1) Take fresh anticoagulated whole blood, add 1× PBS dilution buffer at a ratio of 1:1 to dilute the blood to reduce blood viscosity, mix gently, and set aside.

[0030] (2) Add an appropriate amount of Ficoll-Paque reagent, a mononuclear cell separation solution, into a 15 mL sterile centrifuge tube and slowly drip the diluted blood sample onto the surface of the separation solution (separation solution: diluted whole blood = 1:2), taking care to keep the interface between the two liquid surfaces clear.

[0031] (3) Centrifuge at 2500 r / min at room temperature for 20-30 min. Be careful to set slower acceleration and deceleration to avoid damaging the liquid surface stratification due to excessive acceleration or deceleration.

[0032] (4) After centrifugation, discard the plasma layer, carefully aspirate the PBMC buffy coat layer with a 1 mL pipette, and transfer the buffy coat layer to a 15 mL centrifuge tube. After centrifugation, the liquid levels in the tube are, from top to bottom, the diluted plasma layer, the PBMC layer, the separation liquid layer, and the red blood cell layer (see Figure 3 ).

[0033] (5) Add 10 mL of 1× PBS buffer to the centrifuge tube to resuspend the cells, centrifuge at 1500 rpm for 10 min at room temperature, and discard the supernatant.

[0034] (6) Add 1:500 red blood cell lysis buffer to the cell pellet, shake and mix, let stand for 5 min, centrifuge at 1500 rpm for 5 min at room temperature, discard the supernatant and repeat this step 1 to 2 times. The washed cells can be used for subsequent experiments.

[0035] 2. Establishment of streaming solution

[0036] The collected cells were incubated with the primary antibody on ice for 20 minutes in the dark, washed twice, and resuspended in PBS. The cells in the sample were then permeabilized with Cytofix / CytopermTM fixation / permeabilization solution. The intracellular proteins were labeled with unconjugated primary antibodies and corresponding secondary antibodies, and then labeled with conjugated primary antibodies, and finally detected on a BD flow cytometer.

[0037] For the first experiment on the machine, in addition to the antibody-stained tubes of the samples to be tested, a set of control tubes for adjusting the machine should be prepared:

[0038] (1) Blank control: no antibody, only cells

[0039] (2) Cell single staining control: A control in which only one antibody is added. If there are several fluorescent channels corresponding to the indicators to be measured, several tubes are made. The function of this control tube is to adjust the instrument compensation. In the current color scheme, there is compensation only between FITC and PE channels, so only these two tubes can be made. Because FITC is an intermediate label and a secondary antibody is added, it is recommended to use a highly expressed antibody labeled with FITC from other departments for single staining. PE is a marker of CD163 M2, and its proportion is relatively small. It can also be used for single staining with a highly expressed antibody labeled with PE from other departments. Bone marrow samples are relatively precious, and single staining can be replaced by blood samples.

[0040] The fluorescent conjugated primary antibodies are: anti-human CD163, anti-human CD68, and anti-human CD45. In addition, the unconjugated primary antibodies are: Phospho-LYN (Tyr397) / LCK (Tyr394) / HCK (Tyr411) / BLK (Tyr389) (E5L3D) rabbit monoclonal antibody, anti-human Ficolin-1 (FCN1), and the corresponding secondary antibodies are goat anti-rabbit IgG-APC antibody and goat anti-mouse IgG-AF488 antibody.

[0041] Flow cytometry analysis was performed on a BD FACSCanto: According to the fluorescence combination, the corresponding fluorescence channel on the instrument was selected; more samples could be collected on the machine, because the proportion of positive cells in the target cell population is relatively small, and the results will be better if more cells are collected, so it is best to collect as many cells as possible.

[0042] The details are as follows:

[0043] a. Program settings:

[0044] Due to the problem of setting the fluorescence channel of the laboratory flow cytometer, a 4-color scheme was used for detection in this study. If the flow cytometer uses a 10-color scheme or a 12-color scheme, FCN1, CD68, CD163, and HCK can be placed in the same tube for operation. CD45 is a good indicator for cell population division and is best added to the entire detection system.

[0045] (1) Other controls (cannot be replaced by other types of samples)

[0046] (2) Tube ①: Extracellular antibodies—CD45, CD163, intracellular antibodies—CD68, FCN1, and secondary antibody labeled with FCN1.

[0047] (3) Tube ②: Extracellular antibody - CD45, intracellular antibodies CD68, FCN1, HCK, FCN1 intermediate labelled secondary antibody, HCK intermediate labelled secondary antibody.

[0048] (4) Antibody dosage

[0049] Tube ①: CD45 (PC1): 5uL; CD163 (PE): 10uL; CD68 (APC): 4uL; FCN1: 1:22 dilution, 12.5uL; FCN1 intermediate label secondary antibody (FITC): 1:500 dilution, 100uL.

[0050] Tube ②: CD45 (PC1): 5uL; CD68 (APC): 4uL; FCN1: 1:22 dilution, 12.5uL; HCK: 1:400 dilution, 20uL; FCN1 intermediate label secondary antibody (FITC): 1:500 dilution, 100uL; HCK intermediate label secondary antibody: 1:500 dilution, 100uL.

[0051] First tube: P1 is the cell population after cell debris is removed; P2 is the cell population after adhesion cells are removed; C: P3 is the cell population after adhesion cells are further removed; P4 is the cell population with CD163 and FCN1 co-positive on the basis of P3; P5 is the cell population with CD68, CD163, and FCN1 co-positive on the basis of P4, i.e., mono-like cells;

[0052] The second tube: P1-P3, the meaning is the same as the first tube; P5 is the position of the mono-like cells in the flow cytometry result graph that completely matches the first tube, that is, the mono-like cell population is replicated; P6 is the HCK-positive mono-like cells based on the P5 cell population. Record the following target data:

[0053] mono: the percentage of P5 cell population to P3 cell population;

[0054] HCK1: HCK-positive mono-like cells, the percentage of P6 cell population to P5 cell population, recorded as HCK+mono-like cells;

[0055] HCK2: HCK-positive cell population, the percentage of all HCK-positive cells in the P1 cell population.

[0056] The results are shown in Table 1.

[0057] Table 1 Clinical data of patients

[0058]

[0059] Note: CR: complete remission; NO-CR: incomplete remission; FCM: flow cytometry; BM: bone marrow morphology; WBC: white blood cell; mono-like cells: CD68, CD163, FCN1 co-positive cells (P5); HCK-positive mono-like cells: CD68, CD163, FCN1, HCK co-positive cells (P7).

[0060] b. Staining steps (the entire staining process can be performed in a 5 mL flow cytometry tube)

[0061] (1) Take 100uL of cell suspension, the recommended cell number is 1-5*10^6, and the buffer used for the cell suspension is 1× PBS dilution buffer containing 1-5% serum.

[0062] (2) Add flow cytometry surface antibodies directly to tube ① for surface staining of CD45, CD14, CD16, and CD163, and add flow cytometry surface antibodies directly to tube ② for surface staining of CD45. Vortex or pipette to mix well and incubate at 4°C for 30 min or at room temperature (about 20°C) for 15 min in the dark.

[0063] (3) Add 2 mL of 1× PBS dilution buffer to each tube, vortex to mix, centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0064] (4) Resuspend the final cell pellet in 1× PBS dilution buffer to 100uL (generally, when discarding the supernatant, the residual liquid at the bottom of the reflux tube can be directly resuspended, which is about 100uL. If it is less, there is no need to make up).

[0065] (5) Fixation / Permeabilization solution: add 250uL Fixation / Permeabilization solution to the suspension, incubate at 4°C in dark for 20min; 1mL 1×Perm / Wash TM Buffer, vortex mix, centrifuge at 1500r / min for 5min, discard the supernatant; use 1×Perm / Wash TM Wash the cells once more with Buffer.

[0066] (6) The final cell pellet was washed with 1×Perm / Wash TM Resuspend in Buffer to 100uL (you can directly resuspend with the residual liquid, the volume is about 100uL), add intracellular antibody CD68 to tube ①, add intracellular staining antibodies FCN1 and HCK to tube ②, vortex or pipette to mix, and incubate at 4℃ for 40-60min or room temperature (about 20℃) for 20-30min in the dark.

[0067] (7) Add 2 mL of 1× PBS dilution buffer to each tube, vortex to mix, centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0068] (8) Resuspend the final cell pellet in 1×PBS dilution buffer to 100uL (you can directly resuspend it with the remaining liquid, the volume is about 100uL). Add FCN1 and HCK intermediate staining antibodies to tube ② and incubate at 4℃ for 30min.

[0069] (9) Add intracellular antibody CD68 to tube ②, vortex or pipette to mix, and incubate at 4°C for 40-60 min or at room temperature (about 20°C) for 20-30 min in the dark.

[0070] (10) Add 2 mL of 1× PBS dilution buffer to tube ①, vortex to mix, centrifuge at 1500 r / min for 5 min, and discard the supernatant. Resuspend the final cell pellet in 1× PBS dilution buffer to 300-500 uL, place at 4°C away from light, and wait for loading.

[0071] (11) Add 2 mL of 1× PBS dilution buffer to tube ②, vortex to mix, centrifuge at 400 g for 5 min, and discard the supernatant.

[0072] (12) Resuspend the final cell pellet in 1× PBS dilution buffer to 300-500uL and wait for loading.

[0073] It should be noted that for samples that cannot be put on the machine in time: there is no need to perform this operation on the next morning; if they are to be placed or left for a long time, this step is required; it is recommended that the preserved samples be put on the machine as soon as possible the next day, and theoretically they can be kept for 1 week, but the sooner the better; if they can be put on the machine on the same day, it is not recommended to store them additionally.

[0074] (1) After the entire staining process is completed, add 2 mL of 4% paraformaldehyde to the cell pellet.

[0075] (2) Mix by gently blowing and fix at 4℃ in the dark for 1 hour

[0076] (3) Add 2 mL of Stain Buffer, vortex to mix, centrifuge at 400 g for 5 min, and discard the supernatant.

[0077] (4) Add 300-500uL Stain Buffer to resuspend the cells and store at 4°C in the dark until the cells are detected by flow cytometry and the data are analyzed.

[0078] 3. Collection of flow cytometry result data and organization of patient clinical information.

[0079] According to the clinical data collected in the above table, the test data of "mono, HCK1, HCK2" and "treatment status" were respectively fitted with ROC curves, and the area under the curve (AUC) and the optimal cut-off value (cut-off value) were recorded.

[0080] 4. Draw the ROC curve.

[0081] According to the 2022 ELNAML guidelines and the "Guidelines for the Diagnosis and Treatment of Adult Acute Myeloid Leukemia (2018 Edition)", the prognosis of patients during follow-up was graded, with no remission, refractory, death, relapse, and complex abnormal karyotype involving ≥3 chromosomes as poor prognosis, and the rest as good prognosis. AML with poor prognosis was agreed to be identified as a "positive" sample, and "other AML" was defined as a "negative" sample.

[0082] A total of 73 patients were included in this study, 37 of whom had a poor prognosis (no-CR) and 36 had a good prognosis (CR). The mono, HCK1, and HCK2 test results of the positive and negative groups of patients were imported into SPSS (IBM SPSS Statistics 29.0.1.0) analysis software. After the "analysis-analysis-ROC analysis" step, the "non-parametric" distribution assumption was set with a confidence interval of 95% to draw the ROC curve. The "ROC curve", "area under the ROC curve", and "coordinates of the ROC curve" were obtained, and in the "coordinates of the ROC curve" table, the same row with the largest "Youden index = sensitivity + specificity - 1" was selected as the optimal critical value (cut-off) for this operation, the "sensitivity" in the same row was selected as the true positive rate (sensitivity) of this study, and the "specificity" in the same row was selected as the true negative rate (specificity) of this study. The "coordinates of the ROC curve" table is shown in Table 3.

[0083] 5. Results

[0084] (1) Figure 2 As shown in Table 2, under the ROC curves of mono, HCK1, and HCK2, the area under the curve of HCK+mono-like cells was the largest (AUC=0.855, P=0.046), with a confidence interval of 0.764-0.946. Compared with mono-like cells and HCK2 (HCK-positive cells), HCK1 has the best effect on the prognosis of AML patients.

[0085] (2) Select the row with the largest Youden index as the cut-off value, sensitivity, and specificity of the study. In this study, the cut-off value = 1.2, the Youden index max =0.697, sensitivity =0.892, specificity =0.806. Therefore, when HCK1 is selected with a cut-off value of 1.2, the sensitivity and specificity for AML "positive" samples are 89.2% and 80.6% respectively, and the area under the ROC curve is 0.855

[0086] (0.764-0.946, P=0.046). Therefore, for patients with AML who are newly diagnosed or re-diagnosed, a more accurate prognosis assessment can be performed based on the flow cytometric HCK1=1.2 cut-off. For patients with a poor prognosis, the treatment plan can be improved or updated in a timely manner to enable the patient to obtain a faster chance of remission.

[0087] Table 2 Area under the ROC curve of mono, HCK1, and HCK2 indicators

[0088]

[0089] Table 3 Coordinates of ROC curve

[0090]

[0091]

[0092]

[0093]

[0094]

[0095] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0096] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0097] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of a reagent for detecting the percentage of HCK-positive mono-like cells in the prognosis assessment of acute myeloid leukemia, characterized in that: The HCK-positive mono-like cells are determined when the antigen CD163, the antigen CD68, the antigen FCN1 and the antigen HCK are all positive.

2. The use according to claim 1, characterized in that: The prognosis of acute myeloid leukemia is evaluated by using the percentage of HCK-positive mono-like cells in the sample; the mono-like cells are determined when the antigen CD163, antigen CD68, and antigen FCN1 are co-positive.

3. The use according to claim 2, characterized in that: The sample is a bone marrow sample or a peripheral blood sample.

4. A kit for evaluating the prognosis of acute myeloid leukemia, characterized in that: The kit comprises a reagent for detecting the percentage of HCK-positive mono-like cells described in claim 1.

5. The kit according to claim 4, characterized in that The reagents include reagents for detecting the percentage of HCK-positive mono-like cells in a sample by flow cytometry.

6. The kit according to claim 5, characterized in that The reagents include anti-human CD163, anti-human CD68, anti-human FCN1, and anti-human HCK.

7. The kit according to claim 4, characterized in that The sample is a bone marrow sample or a peripheral blood sample.

8. The kit according to claim 4, characterized in that The acute myeloid leukemia includes newly diagnosed, relapsed or remitted acute myeloid leukemia.

9. Use of a reagent for detecting the percentage of HCK-positive mono-like cells in the preparation of a kit according to any one of claims 4 to 8.

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