Biomarkers for predicting disease progression and prognosis in patients with myeloproliferative tumors

Abstract

The invention provides a biological marker for predicting disease progress and prognosis of patients with myeloproliferative tumors, namely myeloid-derived suppressor cells (MDSC). The kit is used for predicting disease progression and prognosis analysis of patients with myeloproliferative tumors, and has high prediction accuracy, high consistency and high clinical net benefit. The invention also provides a method for detecting the biological marker for predicting the disease progress and prognosis of the myeloproliferative tumor patient. The content of the biological marker is detected by using a mouse anti-human CD33 monoclonal antibody, a mouse anti-human Lin monoclonal antibody and a mouse anti-human HLA-DR monoclonal antibody through a flow cytometry. The kit is suitable for prognosis evaluation and early risk factor intervention of diagnosed patients with myeloproliferative tumors.

Description

Technical Field

[0001] The present invention relates to the field of hematological tumor disease progression and prognosis, and in particular to a biological marker for predicting disease progression and prognosis of myeloproliferative tumor patients. Background Art

[0002] Myeloproliferative neoplasms (MPNs) are a group of diseases that originate from abnormal clonal proliferation of hematopoietic stem cells, are characterized by excessive proliferation of one or more myeloid lineages, and are accompanied by unique neoplastic constitutional symptoms. Classic myeloproliferative neoplasms include polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF). The pathogenesis of MPNs is still unclear, and there is no effective treatment. At the same time, the unique neoplastic constitutional symptoms of MPNs also suggest that there are more important genes and immune regulatory mechanisms that jointly cause the onset of MPNs. Tumor immunosuppression plays a very important role in the onset of myeloproliferative neoplasms. Myeloid-derived suppressor cells (MDSCs) are a newly discovered type of major suppressor cells involved in the formation of the tumor immune microenvironment. More and more evidence shows that myeloid-derived suppressor cells play a negative immunoregulatory role in myeloproliferative neoplasms.

[0003] The interaction between tumor genes and the immunosuppressive microenvironment plays a very important role in the pathogenesis of hematological tumors. In the occurrence of MPNs, the activation of the JAK / STATs signaling pathway is not the only initiating factor causing the disease, and the immunosuppressive tumor microenvironment plays a very important role (Z. Lu et al. Nature, 2020). Myeloid-derived suppressor cells (MDSCs) are the main suppressive cells involved in the formation of the tumor immune microenvironment. It is a group of phenotypically immature myeloid cells at different stages of differentiation. During the differentiation of hematopoietic stem cells into mature granulocytes, macrophages or dendritic cells, they are stimulated by a variety of inflammatory mediators, cytokines and chemokines, resulting in the blockage of the differentiation of immature myeloid cells, forming a cell population with significant immunosuppressive and tumorigenic activity (F. Veglia et al. Nature immunology, 2018). MDSCs need to be activated before they can exert their immunosuppressive effects. Cytokines including IL-6, CCL2, IL-1β, SCF, TNF-α, etc. are involved in the proliferation, recruitment and activation of MDSCs, leading to the proliferation and metastasis of tumor cells (K. Gonda et al. Annals of Oncology, 2012), and are negatively correlated with the overall survival of cancer patients (K. Nakamura et al. Cancer cell, 2018). Summary of the invention

[0004] The purpose of the present invention is to provide a biological marker for predicting disease progression and prognosis of patients with myeloproliferative neoplasms in response to the deficiencies of the prior art, and to provide a simple and intuitive detection method based on clinical characteristics for the biological marker, which can predict the poor prognosis and disease progression of MPN patients and achieve early evaluation and intervention.

[0005] The technical solution adopted by the present invention is as follows: The present invention provides a biological marker for predicting disease progression and prognosis of patients with myeloproliferative neoplasms, wherein the biological marker is myeloid-derived suppressor cells.

[0006] As a second aspect, the present invention also provides an application of the biological marker in predicting disease progression and prognosis of patients with myeloproliferative neoplasms.

[0007] As a third aspect, the present invention also provides a method for detecting biological markers for predicting disease progression and prognosis of patients with myeloproliferative neoplasms, using specific antibodies to detect the content by flow cytometry. The specific antibodies used are as follows:

[0008] (1) Mouse anti-human CD33 monoclonal antibody (APC fluorescent label), used to label CD33+ cells;

[0009] (2) Mouse anti-human Lin monoclonal antibody (FITC fluorescent label), used to label Linage- cells;

[0010] (3) Mouse anti-human HLA-DR monoclonal antibody (PerCP fluorescent labelled), used to label HLA-DR- cells.

[0011] As a fourth aspect, the present invention also provides a reagent for detecting the biological marker in the preparation of a product for predicting disease progression and prognosis of patients with myeloproliferative neoplasms, wherein the reagent is a flow cytometry detection reagent, and the flow cytometry marker of the biological marker is Lin - HLA-DR - CD33 + .

[0012] The beneficial effects of the present invention are as follows:

[0013] (1) The present invention uses myeloid-derived suppressor cells (MDSC) as a biological marker for predicting disease progression and prognosis analysis in patients with myeloproliferative neoplasms, with high predictive accuracy, high consistency, and high clinical net benefit.

[0014] (2) The present invention provides a method for detecting and predicting the biological markers, using mouse anti-human CD33 monoclonal antibody, mouse anti-human Lin monoclonal antibody, mouse anti-human HLA-DR monoclonal antibody to detect their content through flow cytometry, which is simple, intuitive, and convenient, and has good application prospects.

[0015] (3) The present invention is suitable for prognosis assessment and early risk factor intervention for patients diagnosed with myeloproliferative neoplasms. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the flow cytometry detection diagram of MDSC in primary myelofibrosis patients and ordinary people;

[0017] Figure 2 This is the flow cytometry detection of MDSC in patients with essential thrombocythemia and polycythemia vera;

[0018] Figure 3 This is the flow cytometry detection of MDSCs in patients with myelofibrosis after polycythemia vera and patients with myelofibrosis after essential thrombocythemia;

[0019] Figure 4Comparison of the number of MDSCs in patients with different types of myeloproliferative neoplasms, including: (a) is a comparison of the number of MDSCs in the bone marrow of patients with primary myelofibrosis and ordinary people, (b) is a comparison of the number of MDSCs in the bone marrow of patients with polycythemia vera and ordinary people, (c) is a comparison of the number of MDSCs in the bone marrow of patients with essential thrombocythemia and ordinary people, (d) is a comparison of the number of MDSCs in the bone marrow of patients with post-polycythemia vera myelofibrosis and ordinary people, and (e) is a comparison of the number of MDSCs in the bone marrow of patients with post-essential thrombocythemia myelofibrosis and ordinary people;

[0020] Figure 5 The incidence of splenomegaly and systemic symptoms in patients with myelofibrosis with different MDSC numbers, among which (a) is the incidence of systemic symptoms in patients with different MDSC numbers. Patients with MDSC numbers greater than 20% are more likely to develop systemic symptoms than those with MDSC numbers between 5% and 20% (p<0.001). (b) is the incidence of splenomegaly in patients with different MDSC numbers. Patients with MDSC numbers greater than 20% are more likely to develop splenomegaly than those with MDSC numbers less than 20% (p<0.001).

[0021] Figure 6 Figure 2 shows the leukemia conversion, thrombosis and mortality of myelofibrosis patients with different MDSC numbers. (a) shows the leukemia conversion rate of patients with different MDSC numbers. Patients with MDSC numbers > 20% are more likely to have leukemia conversion than those with MDSC numbers < 20% (p < 0.001). (b) shows the thrombosis incidence rate of patients with different MDSC numbers. Patients with MDSC numbers > 20% are more likely to have thrombosis than those with MDSC numbers < 5% (p < 0.05). (c) shows the overall survival rate of patients with different MDSC numbers. Patients with MDSC numbers > 5% have a lower overall survival rate than those with MDSC numbers < 5%. DETAILED DESCRIPTION

[0022] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0023] The present invention analyzes the clinical and laboratory data of patients with myeloproliferative neoplasms and finds that Figure 4 As shown in (ae), compared with normal people, the number of MDSCs in the bone marrow of patients with myeloproliferative neoplasms is significantly increased, and no difference in MDSC levels between different subtypes has been found so far.

[0024] The present invention studies the incidence of splenomegaly and systemic symptoms in patients with myelofibrosis with different MDSC numbers, such as Figure 5As shown in (a), patients with MDSC counts greater than 20% were more likely to develop systemic symptoms than those with MDSC counts between 5% and 20% (p < 0.001). Figure 5 As shown in (b), patients with MDSC counts > 20% were more likely to develop splenomegaly than those with MDSC counts < 20% (p < 0.001).

[0025] In addition, the present invention studied the leukemia, thrombosis and mortality of myelofibrosis patients with different MDSC numbers and found that Figure 6 As shown in (a), patients with MDSC counts > 20% were more likely to undergo leukemic transformation than those with MDSC counts < 20% (p < 0.001). Figure 6 (b) showed that patients with MDSC counts > 20% were more likely to develop thrombosis than those with MDSC counts < 5% (p < 0.05). Figure 6 (c) shows that the overall survival rate of patients with MDSC count > 5% is lower than that of patients with MDSC count < 5%.

[0026] It can be concluded that the leukemia transformation rate, thrombotic event incidence rate and probability of combination with other tumors in patients with myeloproliferative neoplasms with high MDSC levels are significantly higher than those in patients with low MDSC levels, indicating that patients with high MDSC levels have a poor prognosis.

[0027] In summary, the present invention confirms that myeloid-derived suppressor cells (MDSC) can be used as a biological marker to predict disease progression and prognosis in patients with myeloproliferative neoplasms.

[0028] Accordingly, the present invention also provides a method for detecting biological markers for predicting disease progression and prognosis of patients with myeloproliferative neoplasms, which is convenient for sample collection, simple and rapid in detection method, and the operator only needs to master basic cell analysis techniques, and has the advantages of easy promotion and strong practicality. The details are as follows:

[0029] (1) Draw 3 mL of fresh bone marrow fluid into a sodium heparin anticoagulant tube and filter the bone marrow fluid with a 200-mesh filter;

[0030] (2) Add 5 μL of each of the antibodies Lin-FITC, HLA-DR PerCP, and CD33 APC to the bone marrow fluid and gently shake to mix so that the antibodies and bone marrow can fully combine;

[0031] (3) Incubate the test tube with antibody and bone marrow at 4°C in the dark for 30 min.

[0032] (4) After incubation, centrifuge at 1500 rpm for 5 min and discard the supernatant; add 1 mL of phosphate buffered saline (PBS) after centrifugation, gently shake to mix, and centrifuge at 1500 rpm for 5 min, discard the supernatant, and repeat this step once;

[0033] (5) Add 200-300 μL PBS to resuspend into a cell suspension, and filter the cell suspension through a 400-mesh filter;

[0034] (6) On-line analysis: Flow cytometry (FACS-Calibur) was used to detect at least 50,000 cells per tube. Lin - HLA-DR - CD33 + MDSC. Using the flow cytometry software Cell Quest, we first set gates to circle out single-nuclear and multinuclear cells R1 based on the distribution of the side and forward scatter plots, and then circle out Lin in R1. - HLA-DR - Cell population R2, and then calculate CD33 in R2 + The cell ratio is Lin - HLA-DR - CD33 + The proportion of MDSCs.

[0035] It should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Without making creative work, any modification, equivalent replacement, improvement, etc., shall be included in the protection scope of the present invention.

Claims

1. A biological marker for predicting disease progression and prognosis in patients with myeloproliferative neoplasms, characterized in that: The biological marker is myeloid-derived suppressor cells.

2. A use of the biological marker as claimed in claim 1 in predicting disease progression and prognosis of patients with myeloproliferative neoplasms.

3. A method for detecting and predicting the biological marker according to claim 1, characterized in that: Its content is detected by flow cytometry using specific antibodies, including: Mouse anti-human CD33 monoclonal antibody, fluorescently labeled with APC, used to label CD33 + cell; Mouse anti-human Lin monoclonal antibody, labeled with FITC fluorescence, used to label Linage - cell; Mouse anti-human HLA-DR monoclonal antibody, fluorescently labeled with PerCP, for labeling HLA-DR - cell.

4. Use of a reagent for detecting the biological marker according to claim 1 in the preparation of a product for predicting disease progression and prognosis of patients with myeloproliferative neoplasms.

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