A marker for prognosis evaluation of acute myeloid leukemia and application thereof

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a marker for prognosis evaluation of acute myeloid leukemia and application thereof, wherein the marker is HCK-positive mono-like cells, the HCK-positive mono-like cells are determined by co-positivity of antigens CD163, antigen CD68, antigen FCN1 and antigen HCK, and the application provides a marker with high sensitivity, strong specificity and good universality, which is used for detecting adult acute myeloid leukemia bone marrow or peripheral blood samples, so as to assist in judging the clinical prognosis of patients with acute myeloid leukemia.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a marker for prognosis evaluation of acute myeloid leukemia and application thereof. BACKGROUND

[0002] Acute myeloid leukemia (AML) is a kind of malignant clonal disease originated from hematopoietic stem / progenitor cells, and its biological characteristics have high heterogeneity. At present, the most important prognostic factor of AML is the cytogenetic related results detected by conventional chromosome banding technology. However, due to the detection rate and the existence of part of normal karyotype AML patients, the conventional cytogenetic technology has limitations as a prognostic evaluation of leukemia residual disease, and it is clinically urgent to find other simple and easy methods which can be widely applied to evaluate the prognosis of patients, and it is of great significance to stratify the prognosis of patients.

[0003] There are many factors affecting the prognosis of AML. Complete remission (CR) alone can not be used as an indicator to evaluate the therapeutic response during the treatment or at the end of the treatment. At present, the main methods for detecting minimal residual disease (MRD) are polymerase chain reaction (PCR) and flow cytometry (FCM), and next generation sequencing (NGS) and digital PCR are in development. Due to the differences in technical level, the differences in quality evaluation between laboratories, and the instability of the standard amplification system, the detection of PCR fusion genes is only suitable for some AML patients. Moreover, the qualitative / quantitative results of PCR cannot match the number of leukemia cells, so it is difficult to accurately evaluate the residual leukemia cells and the prognosis of patients. Compared with PCR technology, FCM has the characteristics of simple operation, fast detection speed, high specificity, and reproducible analysis. It can not only cover more than 90% of AML patients, but also can accurately distinguish between dead cells and live cells and quantify the number of leukemia cells. Therefore, FCM is an important means of AML prognosis evaluation, especially for AML patients without specific molecular markers. NGS and digital PCR, as the most accurate methods for MRD detection, can clearly evaluate the residual leukemia cells of patients. However, these two techniques require high professional skills and interpretation experience of the testers, and the detection cost is high. The standard operation system has not yet reached widespread application, and the results of different laboratories cannot be well matched and compared. Therefore, FCM detection of AML immunophenotype not only plays an important role in the diagnosis and typing of AML patients, but also has important significance for MRD detection and prognosis evaluation of AML patients. At present, the commonly used CD antigens related to AML immunophenotype in clinical practice include CD7, CD56, CD19, CD10, CD4, CD11b, CD11C, CD13, CD14, CD15, CD16, CD33, CD34, CD38, CD64, CD117, CD123, CD300e, MPO, and HLA-DR. The main shortcomings are that the above antigens require certain technical and experience of the testers, and the results interpretation contains some subjective factors.

[0004] At the time of admission, the patient needs to be detected and analyzed by multiple parameters such as cell morphology, immunology, cytogenetics and molecular biology (MICM), and the initial diagnosis and prognosis evaluation of the patient are obtained according to the prognosis-related molecular genetic abnormalities in the results of real-time quantitative PCR, fluorescence in situ hybridization and chromosome karyotype analysis. The patients of 'high risk','medium risk' and 'low risk' will obtain the targeted standardized early treatment plan. Four weeks is a course of treatment, and after two courses of treatment, the patient will present'remission' and 'incomplete remission'. According to the remission state of the patient, the patient will be treated by chemotherapy combined with allogeneic hematopoietic stem cell transplantation (Allo-HSCT). After chemotherapy and transplantation treatment, 35%-40% of young patients (<60 years old) and 5%-15% of old patients can be cured. However, more than half of the young patients will face recurrence or refractory, and the old and weak patients cannot tolerate conventional chemotherapy.

[0005] HCK (proto-oncogene) is a member of the SRC family kinase, and it has been reported that HCK can be used for AML prognosis evaluation, but HCK has different expression levels in AML patient samples in different literatures, and cannot be directly used for clinical AML patient prognosis evaluation. Therefore, it is of great significance to find other simple and easy methods for prognosis evaluation and prognosis stratification of patients. SUMMARY

[0006] To solve the above technical problems, the present application provides a marker for acute myeloid leukemia prognosis evaluation and application thereof.

[0007] The application of the reagent for detecting the percentage of HCK-positive mono-like cells in the prognosis evaluation of acute myeloid leukemia, wherein the HCK-positive mono-like cells are determined by co-positive of antigens CD163, CD68, FCN1 and HCK.

[0008] Preferably, the percentage of HCK-positive mono-like cells in the sample is used for prognosis evaluation of acute myeloid leukemia; the mono-like cells are determined by co-positive of antigens CD163, CD68 and FCN1.

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

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

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

[0012] Preferably, the reagent comprises 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 comprises newly diagnosed, relapsed, or remission acute myeloid leukemia.

[0015] The reagent for detecting the percentage of HCK-positive mono-like cells is used for preparing the kit.

[0016] In the present study, the detection of HCK-positive mono-like cells was added to the FCM detection of the treatment commonly used in clinic: CD163, CD68, and FCN1. The detection of the malignant cells of mono-like and the cut-off value of the early stage were used to accurately evaluate the prognosis of the patients before and after the treatment. The sensitivity and specificity of the prognosis evaluation of AML in the present 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 the patients with newly diagnosed or recurrent AML, the cut-off of HCK1=1.2 of flow cytometry can be used to more accurately evaluate the prognosis of the patients. For the patients with poor prognosis, the treatment scheme can be improved or updated in time, so that the patients can obtain a more rapid remission probability.

[0017] Compared with the prior art, the present application has the beneficial effects that:

[0018] The FCM detection of HCK-positive mono-like cells has the advantages of simple operation, wide and repeated use, and good avoidance of the problems of unstable PCR technology, high cost of NGS and digital PCR sequencing, and experience requirements of the detection personnel, so that the AML hierarchical treatment and rescue intervention can be achieved, the precise target for the AML patients with MRD positivity can be provided, new targeted therapeutic drugs can be developed, the ineffective treatment of AML patients can be reduced, and the treatment effect of the patients with relapsed and refractory AML can be improved.

[0019] The purpose of the present application is to provide a marker with high sensitivity, strong specificity and good universality, which is used for detecting adult acute myeloid leukemia bone marrow or peripheral blood samples, so as to assist in judging the clinical prognosis of patients with acute myeloid leukemia. The main problems are as follows: (1) the existing cytogenetic detection cannot effectively evaluate the treatment prognosis of patients; (2) gene sequencing and digital PCR technology are high in cost and have not been widely popularized; (3) SRC family kinase HCK can evaluate the prognosis of acute myeloid leukemia, but the direct evaluation value is low. Therefore, a new clinical prognosis marker is helpful for the clinical prognosis evaluation of patients with acute myeloid leukemia. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 For flow positive results, A is the cell population P1 after removing cell debris in the first tube; B is the cell population P2 after removing adherent cells in the first tube; C is the cell population P3 after further removing adherent cells in the first tube; D is the P3 cell population labeled by CD45; E is the co-positive cell population P4 of CD163 and FCN1 based on P3; F is the cell population P5 co-positive of CD68, CD163 and FCN1, that is, mono-like cells; G is the cell population P1 after removing cell debris in the second tube; H is the cell population P2 after removing adherent cells in the second tube; I is the cell population P3 after further removing adherent cells in the second tube; J is the P3 cell population labeled by CD45; K is the replicated mono-like cell population P5; L is the mono-like cell P6 positive for HCK based on the P5 cell population;

[0021] Figure 2 ROC curve of mono-like, HCK1 and HCK2 indicators;

[0022] Figure 3 Layering diagram before and after centrifugation of diluted bone marrow blood. DETAILED DESCRIPTION

[0023] The specific embodiments of the present application are described in detail below, but it should be understood that the protection scope of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative labor are within the protection scope of the present application. The experimental methods described in the embodiments of the present application are all conventional methods unless otherwise specified.

[0024] Mono-like cells: All cells on the FCM machine were labeled with three antigens (FCN1, CD68, CD163) at the same time, namely mono-like cells. This group of cells belongs to the monocyte macrophage cluster and is in a relatively immature stage, which can differentiate into tumor-associated macrophages (TAM) and tissue-resident macrophages (TRM). Single-cell bioinformatics analysis found that monocyte macrophages mainly contain three cells, ① mono-like, the main markers are FCN1, CD68, and CD163; ② TAM, the main markers are ISG15, C1QC, and SPP1; ③ TRM, the main markers are 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 detection

[0027] To analyze the differential expression of HCK in immune cells, clinical AML bone marrow samples from initial diagnosis, recurrence or remission were collected. The collection of samples was obtained with informed consent, and all procedures were approved by the Institutional Research Ethics Committee of Guizhou Medical University.

[0028] Isolation of mononuclear cells from clinical bone marrow samples

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

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

[0031] (3) Centrifuge at 2500 r / min for 20-30 min at room temperature, and pay attention to set a slower acceleration and deceleration to avoid breaking the liquid layer due to excessive speed.

[0032] (4) After centrifugation, the plasma layer is discarded, and the PBMC white membrane layer is carefully taken with a 1 mL pipette and transferred to a 15 mL centrifuge tube. After centrifugation, the liquid level in the tube from top to bottom is dilution plasma layer, PBMC layer, separation liquid layer, and red blood cell layer (see Figure 3 ).

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

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

[0035] 2. Flow scheme establishment

[0036] Incubate the collected cells with the primary antibody on ice in the dark for 20 minutes, wash twice, and resuspend in PBS. Then break the membrane of the cells in the sample with Cytofix / CytopermTM fixation / permeation solution. Label the intracellular proteins with uncoupled primary antibodies and corresponding secondary antibodies, and then label the coupled primary antibodies. Finally, detect on a BD flow cytometer.

[0037] For the first time, in addition to the antibody full staining tube of the sample to be tested, a set of control tubes for instrument adjustment is needed:

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

[0039] (2) Cell single staining control: add only one antibody, corresponding to the index to be tested in several fluorescence channels, do several tubes. This control tube functions to adjust the instrument compensation. In the current color matching scheme, only between FITC and PE channels there is compensation, so only these two tubes can be done. Since FITC is a method of adding secondary antibodies to intercalating agents, it is recommended to use high expression antibodies labeled with FITC from other departments for single staining. PE is the marker of CD163 M2, which itself accounts for a small proportion, and high expression antibodies labeled with PE from other departments can also be used for single staining. Bone marrow samples are relatively valuable, and single staining can be replaced by blood samples.

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

[0041] Flow cytometry analysis on BD FACSCanto: according to the fluorescence combination, select the corresponding fluorescence channel on the instrument; collect more samples on the machine, because the positive cell population in the target cell population accounts for a small proportion, more cell collection results will be better, and the amount of collection should be appropriate.

[0042] Specifically as follows:

[0043] a, scheme setting:

[0044] Because of the problem of fluorescence channel setting of laboratory flow cytometry, 4-color scheme is used for detection in this study. If the flow cytometry uses 10-color scheme or 12 scheme, FCN1, CD68, CD163 and HCK can be operated in the same tube. CD45 is a good indicator for cell population division, and it is best to add to the whole detection system.

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

[0046] (2) Tube ①: extracellular antibody-CD45, CD163, intracellular antibody-CD68, FCN1, FCN1 inter-marker secondary antibody.

[0047] (3) Tube ②: extracellular antibody-CD45, intracellular antibody CD68, FCN1, HCK, FCN1 inter-marker secondary antibody, HCK inter-marker 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 inter-marker 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 inter-marker secondary antibody (FITC): 1:500 dilution, 100uL; HCK inter-marker secondary antibody: 1:500 dilution, 100uL.

[0051] The first tube: P1 is the cell population after removing cell debris; P2 is the cell population after removing adherent cells; C: P3 is the cell population after further removing adherent cells; P4 is the CD163 and FCN1 co-positive cell population based on P3; P5 is the CD68, CD163 and FCN1 co-positive cell population based on P4, that is, mono-like cells;

[0052] Second tube: P1-P3, same as the first tube; P5 is the mono-like cell population in the flow cytometry result map that is completely matched to the first tube, i.e. replicates the mono-like cell population; P6 is the mono-like cell that is HCK positive based on the P5 cell population. The following target data is recorded:

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

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

[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 Patient clinical data

[0058]

[0059] Note: CR: complete remission; NO-CR: not complete remission; FCM: flow cytometry; BM: bone marrow morphology; WBC: white blood cell; mono-like cell: CD68, CD163, FCN1 co-positive cell (P5); HCK positive mono-like cell: CD68, CD163, FCN1, HCK co-positive cell (P7).

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

[0061] (1) Take 100 uL of cell suspension, which is recommended to contain 1-5*10^6 cells. The buffer used for the cell suspension is 1-5% serum-containing 1xPBS dilution buffer.

[0062] (2) Directly add flow cytometry surface antibodies for surface staining CD45, CD14, CD16, CD163 to tube ①, and directly add flow cytometry surface antibodies for surface staining CD45 to tube ②, vortex or blow to mix, and then 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 1xPBS dilution buffer to each tube, vortex to mix, centrifuge at 1500 r / min 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 refluxed tube can be directly resuspended, which is about 100uL. No need to add more if it is insufficient).

[0065] (5) Fixation / Permeabiization solution: Add 250 μL of Fixation / Permeabiization solution to the suspension, incubate at 4°C in the dark for 20 min; 1 mL of 1×Perm / Wash TM Buffer, vortex to mix, centrifuge at 1500 rpm for 5 min, discard supernatant; use 1×Perm / Wash TM The cells were washed once more with the buffer.

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

[0067] (7) Add 2 mL of 1×PBS dilution buffer to each tube, vortex to mix, centrifuge at 1500 r / min 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 in the residual liquid, the volume of which is approximately 100uL). Add FCN1 and HCK interstitial staining antibodies to tube ② and 4℃ for 30min.

[0069] (9) Add intracellular antibody CD68 to tube ②, vortex or blow to mix well, and incubate at 4℃ for 40-60 min or at room temperature (around 20℃) 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℃ in the dark, and wait for it to be used.

[0071] (11) Add 2 mL of 1×PBS dilution buffer to tube ②, vortex to mix, centrifuge at 400g 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 it to be loaded onto the machine.

[0073] Note: Sample preservation when unable to go on machine in time: No need to do this operation for samples going on machine early next morning; do this step if samples need to be placed or stored for a long time; recommended to put samples on machine as soon as possible the next day, theoretically 1 week, but the sooner the better; if samples can be put on machine the same day, it is not recommended to store them additionally.

[0074] (1) Add 2 mL of 4% paraformaldehyde to the cell precipitate after the completion of the entire staining process

[0075] (2) Gently blow and mix, fix at 4°C in the dark for 1 h

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

[0077] (4) Add 300-500 uL of Stain Buffer to resuspend the cells, store at 4°C in the dark, wait for flow cytometry detection and data analysis.

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

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

[0080] 4. ROC curve drawing.

[0081] According to the 2022 ELNAML guidelines and "Adult Acute Myeloid Leukemia Diagnosis and Treatment Specifications (2018 Edition)", the prognosis of patients during follow-up was classified. Unrelieved, refractory, death, recurrence, and complex abnormal karyotype involving ≥3 chromosomes were determined as poor prognosis, and the rest were good prognosis. AML with poor prognosis was agreed to be "positive" sample, and "the rest of AML" was defined as "negative" sample.

[0082] This study included 73 patients, with 37 having poor prognosis (no-CR) and 36 having good prognosis (CR). The mono, HCK1, and HCK2 test results of both positive and negative groups were imported into SPSS (IBM SPSS Statistics 29.0.1.0) analysis software. After the "Analyze-Analyze-ROC Analysis" steps, with a non-parametric distribution assumption and a 95% confidence interval, ROC curves were plotted. The "ROC curve," "region under the ROC curve," and "ROC curve coordinates" were obtained. In the "ROC curve coordinates" table, the row with the largest "Youden index = sensitivity + specificity - 1" value, marked "greater than or equal to this value is positive," was selected as the optimal cut-off value for this calculation. The "sensitivity" value in the same row was selected as the true positive rate, and the "specificity" value in the same row was selected as the true negative rate. The "ROC curve coordinates" table is shown in Table 3.

[0083] 5. Results

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

[0085] (2) The row with the highest Youden index was selected as the cut-off value, sensitivity, and specificity values ​​for the study. In this study, the cut-off value = 1.2, and the Youden index... max =0.697, sensitivity =0.892, specificity =0.806. Therefore, when HCK1 is selected at this cut-off value of 1.2, the sensitivity and specificity for diagnosing 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 newly diagnosed or re-diagnosed with AML, a more accurate prognostic assessment can be made based on a flow cytometry cutoff of HCK1=1.2. For patients with a poor prognosis, treatment plans can be improved or updated in a timely manner, increasing the chances of faster remission.

[0087] Table 2 shows the regions under the ROC curves of mono, HCK1, and HCK2 indicators.

[0088]

[0089] Table 3 Coordinates of ROC curves

[0090]

[0091]

[0092]

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[0094]

[0095] It is to be understood that the numerical ranges recited in the claims are intended to include every integer value within the range and any fraction of the values within the range. In other words, whenever a numerical range is recited, it is intended to include every possible combination of the numbers within the range. It is to be understood that the present application is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present application will be limited only by the appended claims.

[0096] Although the preferred embodiments of the application have been described, those skilled in the art will recognize that many modifications and variations of the preferred embodiments could be made without departing from the spirit and scope of the application. It is therefore intended that the appended claims encompass all such modifications and variations as falling within the scope of the application. Accordingly, many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the application. Therefore, the scope of the application is not to be limited to the specific embodiments disclosed herein, but is to be accorded the full scope consistent with the language of the claims, wherein reference to an element in the singular is not intended to mean "one and only one" unless specifically so stated, but rather "one or more." Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an overly literal sense unless expressly so defined herein.

[0097] Obviously, many modifications and variations of the present application are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims and their equivalents, the application can be practiced otherwise than as specifically described.

Claims

1. The use of a reagent for detecting HCK-positive mono-like cells in the preparation of a kit for prognostic assessment of acute myeloid leukemia using the percentage of HCK-positive mono-like cells in a sample, characterized in that... The HCK-positive mono-like cells are identified when they are co-positive for antigens CD163, CD68, FCN1, and HCK. The mono-like cells were identified when antigens CD163, CD68, and FCN1 were co-positive. The reagents include anti-human CD163, anti-human CD68, anti-human FCN1, and anti-human HCK.

2. The application according to claim 1, characterized in that, The sample is a bone marrow sample or a peripheral blood sample.

3. The application according to claim 1, characterized in that, The reagents include those for detecting HCK-positive mono-like cells in a sample using flow cytometry.

4. The application according to claim 1, characterized in that, The acute myeloid leukemia mentioned includes newly diagnosed, relapsed, or remission acute myeloid leukemia.

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