Biomarker for diagnosing transformation from myeloproliferative tumor to acute leukemia and application of biomarker

Abstract

The invention relates to the field of biological medicine, and discloses a biomarker for diagnosing transformation of myeloproliferative tumors to acute leukemia and application of the biomarker. The biomarker is HMGA1, and the application refers to the application of the HMGA1 in preparation of a kit for diagnosing transformation from myeloproliferative tumors to acute leukemia. The HMGA1 is found to serve as a single marker, the performance of the HMGA1 in the aspect of predicting conversion from MPN to sAML is superior to that of an existing clinical scoring system and traditional markers such as CD34 / CD117, a longer early warning window is provided for clinic, and timely intervention treatment is facilitated.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and more specifically, to a biomarker for diagnosing the transformation of myeloproliferative neoplasms to acute leukemia and its application. Background Art

[0002] Myeloproliferative neoplasms (MPNs) are a group of clonal disorders originating from hematopoietic stem cells, primarily including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The natural history of MPNs carries the risk of transformation to secondary acute myeloid leukemia (sAML), a leading cause of mortality in MPN patients. Once MPNs transform to sAML, the prognosis is extremely poor, with a median survival of less than six months and poor response to traditional treatment options.

[0003] Currently, clinical risk assessment for MPN to sAML transformation mainly relies on a series of clinical and hematological parameters, such as the International Prognostic Scoring System (IPSS) for myelofibrosis, the Dynamic International Prognostic Scoring System (DIPSS), and its subsequent versions that integrate molecular markers (such as MIPSS70+). Although these scoring systems can stratify patients for risk to a certain extent, their accuracy and timeliness in predicting sAML transformation are still limited. They mainly reflect the current state of the disease and it is difficult to fully capture the key molecular events and dynamic clonal evolution processes that drive disease progression, which may lead to delayed clinical intervention (such as allogeneic hematopoietic stem cell transplantation) and miss the optimal treatment window.

[0004] At the molecular level, the occurrence of MPN is closely related to mutations in driver genes such as JAK2, CALR, and MPL. Transformation to sAML is a more complex clonal evolution process, often accompanied by more complex genomic abnormalities, such as the acquisition of high-risk mutations such as TP53, ASXL1, SRSF2, EZH2, and IDH1 / 2. However, the presence or absence of these mutations alone is not reliable for predicting leukemic transformation. For example, although TP53 mutations are recognized as a strong signal for transformation and poor prognosis, they may also exist at a low allele frequency in patients in the chronic phase before transformation, and the precise association between their dynamic changes and the risk of transformation is not yet fully understood. Therefore, genetic testing at a single time point is not sufficient to accurately predict the risk of transformation in all patients.

[0005] In addition, immunophenotypic markers commonly used in clinical practice, such as CD34 and CD117, are mainly used to identify and quantify primitive cells in the bone marrow. According to the diagnostic criteria of the World Health Organization (WHO), sAML can be diagnosed when the proportion of primitive cells reaches 20%. However, in the early stages of MPN transformation to sAML, the proportion of primitive cells may not have increased significantly, and these markers are often lost in differentiated malignant clones, so their sensitivity as early warning markers is insufficient.

[0006] Therefore, there is an urgent need in this field for a new biomarker that can reflect clonal evolution and transformation potential earlier and more accurately to help clinicians identify high-risk patients, thereby conducting more active monitoring and preemptive intervention to improve patient prognosis. Summary of the Invention

[0007] This application aims to address the inadequate accuracy, timeliness, and sensitivity of existing methods for predicting MPN-to-sAML transformation. Specifically, the technical problem addressed by this application is to provide a new method and biomarker that can early and reliably identify MPN patients at high risk of transformation, thereby providing a longer early warning window for timely intervention and treatment.

[0008] In order to achieve the above-mentioned invention objectives, this application adopts the following scheme:

[0009] High Mobility Group A1 (HMGA1) is a non-histone chromatin structural protein that regulates gene expression by altering chromatin conformation. It plays a crucial role during embryonic development, but is typically silent in normal adult terminally differentiated tissues. Numerous studies have demonstrated that HMGA1 re-expression is a common feature of tumors. HMGA1 is abnormally overexpressed in a variety of solid tumors and high-grade hematologic malignancies, and its elevated expression is closely associated with tumor aggressiveness, stem cell characteristics, drug resistance, and poor patient prognosis. Recent studies have further revealed that HMGA1, as a chromatin regulator, induces transcriptional networks associated with GATA2 and cell proliferation during the progression of MPNs. However, its clinical value as an early diagnostic marker for MPN-to-sAML transformation has yet to be systematically elucidated and validated.

[0010] Based on the above, in a first aspect of the present application, a biomarker for diagnosing the transformation of myeloproliferative neoplasms to acute leukemia is provided, wherein the biomarker is HMGA1.

[0011] In a second aspect of the present application, there is provided a use of HMGA1 in preparing a kit for diagnosing transformation of myeloproliferative neoplasms to acute leukemia.

[0012] Furthermore, the test sample is a bone marrow sample, a peripheral blood sample or a derivative thereof; the derivative includes but is not limited to total nucleated cells, mononuclear cells, CD34+ cells, plasma, serum isolated from the sample, or proteins and nucleic acids extracted therefrom.

[0013] Furthermore, the application refers to determining the transformation of myeloproliferative neoplasms into acute leukemia by detecting the expression level of HMGA1.

[0014] Furthermore, the expression level of HMGA1 is determined by detecting the level of HMGA1 protein.

[0015] Furthermore, the level of the HMGA1 protein is determined by an immunological detection method, which is immunohistochemistry or immunocytochemistry.

[0016] Furthermore, detecting the level of HMGA1 protein includes determining the percentage of HMGA1-positive cells in the total number of selected cells; comparing the detected percentage of HMGA1-positive cells with a preset threshold value, and when the percentage is higher than the threshold value, diagnosing the transformation of myeloproliferative neoplasm to acute leukemia.

[0017] Furthermore, the HMGA1 positive cell percentage threshold is above 40%.

[0018] Furthermore, the reference value of the HMGA1 positive cell percentage is 40.78%.

[0019] In a third aspect of the present application, a kit for diagnosing the transformation of myeloproliferative neoplasms to acute leukemia is provided, comprising a reagent for detecting the expression level of HMGA1 protein in an individual biological sample.

[0020] Furthermore, the reagent is an antibody that specifically binds to the HMGA1 protein.

[0021] Furthermore, the antibody is a rabbit monoclonal antibody.

[0022] In a fourth aspect of the present application, a method for in vitro diagnosis or prediction of transformation of a myeloproliferative neoplasm (MPN) to secondary acute myeloid leukemia (sAML) is provided, the method comprising the steps of:

[0023] a) obtaining biological samples from individuals with or suspected of having MPN;

[0024] b) detecting the expression level of high mobility group protein A1 (HMGA1) in the biological sample;

[0025] c) comparing the detected HMGA1 expression level with a preset reference value;

[0026] Wherein, if the detected HMGA1 expression level is higher than the reference value, it indicates that the individual has a higher risk of MPN transforming to sAML or has already undergone transformation.

[0027] In summary, this application has the following beneficial effects:

[0028] 1. High Predictive Accuracy: This study found that HMGA1, as a single marker, outperformed existing clinical scoring systems and traditional markers such as CD34 / CD117 in predicting MPN to sAML transformation. Receiver operating characteristic (ROC) curve analysis based on a large cohort study demonstrated that the area under the curve (AUC) for HMGA1 IHC testing reached 0.96, demonstrating extremely high diagnostic efficacy.

[0029] 2. Early Warning: Upregulation of HMGA1 is an early event throughout the malignant clonal evolution of MPNs, and its expression level increases before a significant increase in the proportion of clinically identifiable blasts. This enables the methods of the present invention to provide a longer warning window, buying valuable time for clinical intervention. For example, studies have found that in some patients whose blast proportion has not yet reached the diagnostic criteria for sAML (<20%) but whose HMGA1 expression has increased significantly, the majority progress to sAML within a short period of time (e.g., within a year).

[0030] 3. Wide applicability: Unlike CD34 / CD117, which are limited to primitive cells, HMGA1 is expressed in all stages of malignant clones, from progenitor cells to relatively mature ones. It can more comprehensively cover the entire tumor cell population, thereby more reliably reflecting tumor burden and disease aggressiveness.

[0031] 4. The method is simple and easy to promote: The present invention mainly relies on the mature IHC technology, which is a routine test item in the pathology department of most hospitals. It does not require complicated equipment or operations and is easy to promote and standardize in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The transcript levels of HMGA1 in peripheral blood mononuclear cells (PBMCs) and CD34+ stem cells of healthy controls and patients were gradually upregulated during the progression of MPN to sAML, suggesting that it may serve as a potential diagnostic marker for sAML.

[0033] Figure 2HMGA1 nuclear protein expression levels can serve as a robust diagnostic biomarker for leukemic transformation in MPNs. Representative images show that HMGA1 protein is primarily expressed in megakaryocytes during the chronic phase (ET, PV) and gradually increases during the fibrotic phase (MF). It is also highly expressed in sAML samples.

[0034] Figure 3 :The expression level of HMGA1 nuclear protein gradually increases in samples from patients with secondary fibrosis or acute leukemia transformation. Figure 3 (a) shows that during the 11-year follow-up, the megakaryocytes of this PV patient had abnormal morphology, reduced lobulation, and increased HMGA1 protein levels. Figure 3 (b) shows that during the 3-year follow-up of this patient, squeezed distribution of leukemia cells with abnormally high expression of HMGA1 appeared.

[0035] Figure 4 :HMGA1 positive rate>40.78% can be used as a high-risk indicator for sAML transformation. Figure 4 a(i) shows the ROC curve of the HMGA1 IHC score for distinguishing sAML from non-sAML MPNs, demonstrating its high diagnostic efficacy. Figure 4 a(ii) shows that among 15 patients with high HMGA1 expression and blast rate < 20% who were followed up for one year, 7 patients transformed into secondary leukemia during the follow-up period (median time 6 months, interquartile range 4-9 months).

[0036] Figure 5 :HMGA1 is better than traditional standards in predicting the transformation of sAML. Figure 5 a(i-ii) shows an example where HMGA1 upregulation precedes morphological changes in the original cells. In both 5a(i) and (ii), HMGA1 immunohistochemical staining showed abnormal nuclear expression before leukemic transformation. Degenerative granulocyte morphology (neutrophil loss, granular thickening, vacuolization, and other pathological features associated with early leukemic transformation) was present in the cell smear before leukemic transformation, which is one of the characteristics before leukemic transformation.

[0037] Figure 6 :HMGA1 is specifically overexpressed in bone marrow biopsy samples of sAML. Figure 6 a shows the multiple immunofluorescence results of HMGA1, CD34 and CD117 in chronic phase patients (ET, PV and PMF) and blast crisis patients (sAML), showing the high expression of CD34, CD117 and HMGA1 in sAML patients. Figure 6be demonstrated that HMGA1 is superior to CD34 and CD117 for the diagnosis of sAML, and the results were due to the disease heterogeneity and different disease processes in sAML patients.

[0038] Figure 7 :This figure shows a case of ET developing into secondary fibrosis and then finally transforming into secondary leukemia, which illustrates the practicality of HMGA1 for sAML risk assessment. As shown in the figure, HMGA1 is highly expressed in early leukemia transformation, but the expression of two leukemia stem cell indicators CD34 is mainly due to angiogenesis, while the positive expression of CD117 appears in the late stage of leukemia transformation. This figure explains Figure 6 The staining results vary among patients due to different disease heterogeneity and progression status. DETAILED DESCRIPTION

[0039] The technical solutions and effects of the present application are further described in detail below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.

[0040] Example 1: Correlation analysis between HMGA1 expression level and MPN disease progression in clinical samples

[0041] 1.1 Patients and samples

[0042] This example enrolled 240 individuals, including 162 confirmed MPN patients (57 with essential thrombocythemia vera, 33 with polycythemia vera, and 51 with myelofibrosis) and 21 patients with sAML transformed from MPN. A control group included 78 patients with suspected MPN but ultimately diagnosed with a non-MPN hematologic disorder. All diagnoses met the WHO fifth edition criteria and were centrally reviewed by pathologists. The study received institutional approval and patient informed consent.

[0043] 1.2 Immunohistochemistry (IHC) testing of bone marrow biopsy samples

[0044] Formalin-fixed, paraffin-embedded (FFPE) bone marrow biopsy tissue blocks were obtained from the above patients, and 4 μm thick tissue sections were prepared.

[0045] The IHC staining steps are as follows:

[0046] 1) Dewaxing and hydration: The sections were baked in a 60°C oven for 2 hours, then placed in xylene and graded alcohol (absolute ethanol, 95% ethanol, 75% ethanol) in sequence, and finally rinsed with water.

[0047] 2) Antigen retrieval: Place the sections in EDTA buffer (pH 9.0) and heat-repair at 95-100°C for 20-30 minutes.

[0048] 3) Block endogenous peroxidase: Incubate with 3% hydrogen peroxide solution at room temperature for 15 minutes.

[0049] 4) Blocking: Block with 5% normal goat serum at room temperature for 30 minutes.

[0050] 5) Primary antibody incubation: Add HMGA1 rabbit monoclonal antibody (Abcam, Cat#ab129153, dilution ratio 1:5000) and incubate at 4°C overnight.

[0051] 6) Secondary antibody incubation: Add biotinylated goat anti-rabbit secondary antibody and incubate at room temperature for 30 minutes.

[0052] 7) HRP complex incubation: add streptavidin-horseradish peroxidase (HRP) complex dropwise and incubate at room temperature for 30 minutes.

[0053] 8) Color development: Use a DAB color development kit for color development, and control the color development time under a microscope.

[0054] 9) Counterstaining, dehydration, transparency, and mounting: Counterstain the cell nuclei with hematoxylin, dehydrate with graded alcohols, transparentize with xylene, and finally mount the slides with neutral gum.

[0055] 1.3 Interpretation of results

[0056] Staining results were observed under a microscope at 400x magnification. HMGA1 protein was primarily localized in the cell nucleus, and the presence of brownish-yellow granules in the nucleus was considered a positive signal. Two independent pathologists performed double-blind review of the slides. At least 500 nucleated cells were counted in at least three representative high-power fields, and the percentage of HMGA1-positive cells was calculated.

[0057] 1.4 Statistical analysis and results

[0058] IHC staining results showed that the percentage of HMGA1-positive cells increased significantly with MPN disease progression. In non-MPN controls, HMGA1 expression was extremely low. In chronic-phase MPN patients, the average HMGA1-positive rate was 16.03% ± 8.43% in the ET / PV group and 32.58% ± 12.88% in the MF group. In the sAML group, the average positive rate increased significantly to 63.21% ± 18.04% (P < 0.0001).

[0059] Fifteen patients with chronic MPN who had high HMGA1 expression (based on the cutoff value determined by ROC analysis described below) but a blast percentage of <20% were followed up and found that 7 (46.7%) of them progressed to sAML within 12 months, with a median warning time of 6 months. This suggests that high HMGA1 expression is a strong signal for impending leukemic transformation.

[0060] Example 2: Performance evaluation of HMGA1 as a diagnostic marker for sAML

[0061] 2.1 ROC Curve Analysis

[0062] To evaluate the efficacy of HMGA1 IHC testing in diagnosing MPN to sAML transformation, the HMGA1 IHC percentage data of 162 MPN patients (including 21 sAML patients) obtained in Example 1 were subjected to receiver operating characteristic (ROC) curve analysis, taking clinically confirmed sAML (primitive cells ≥ 20%) as the gold standard.

[0063] 2.2 Results

[0064] The results of ROC curve analysis showed that the area under the curve (AUC) of the HMGA1 IHC score for distinguishing sAML from non-sAML was 0.96 (95% CI: 0.94-0.99), demonstrating extremely high diagnostic accuracy.

[0065] The Youden's index determined the optimal diagnostic cut-off value, which was 40.78% of HMGA1-positive cells. At this cut-off point, the sensitivity for diagnosing sAML was 90.48% and the specificity was 89.36%.

[0066] This result suggests that whether the percentage of HMGA1-positive cells is higher than 40.78%, for example, can be used as a clear cutoff to efficiently identify high-risk patients.

[0067] Example 3: Comparison of HMGA1 with other markers

[0068] The study also compared HMGA1 with traditional markers CD34 and CD117. Multiple immunofluorescence (mIF) results showed that in sAML samples, HMGA1 was widely expressed in malignant clones, while the expression of CD34 and CD117 was more limited to primitive cell subsets. In a longitudinal follow-up case from ET to sAML, it was observed that the expression of HMGA1 increased earlier than the significant upregulation of CD117, and its coverage of cells was wider than that of CD34. This explains why HMGA1 showed superior diagnostic performance than traditional markers in ROC analysis.

[0069] Example 4: Construction of diagnostic kit

[0070] Based on the findings of the present invention, a kit for diagnosing or predicting the transformation of MPN to sAML can be constructed.

[0071] The kit may contain the following components:

[0072] 1. HMGA1-specific antibody: Preferably, a rabbit monoclonal antibody, such as ab129153 from Abcam, or other antibodies with similar binding properties and specificity. This antibody has been optimized for IHC staining of FFPE tissue sections.

[0073] 2. Detection reagents: including HRP-labeled secondary antibody, DAB color development solution, etc.

[0074] 3. Buffer: such as antigen retrieval buffer, washing buffer, etc.

[0075] 4. Positive and negative controls: For example, sections of cell lines with known high expression of HMGA1 (such as HEL cells) can be used as positive controls, and sections of tissues with known negative expression can be used as negative controls.

[0076] 5. Instructions for use: Detailed description of the IHC procedure, interpretation criteria (including positive cell morphology, counting methods, etc.), and clear reference values ​​for risk assessment, for example: "If the percentage of HMGA1-positive cells in the sample is >40.78%, it indicates a high risk of MPN transforming to sAML."

[0077] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A biomarker for diagnosing the transformation of myeloproliferative neoplasms to acute leukemia, characterized in that: The biomarker is HMGA1.

2. Application of HMGA1 in the preparation of a kit for diagnosing the transformation of myeloproliferative neoplasms to acute leukemia.

3. The use according to claim 2, characterized in that The test sample is a bone marrow sample, a peripheral blood sample or a derivative thereof; the derivative includes but is not limited to total nucleated cells, mononuclear cells, CD34+ cells, plasma, serum separated from the sample, or proteins and nucleic acids extracted therefrom.

4. The use according to claim 2, characterized in that The application refers to determining the transformation of myeloproliferative neoplasms into acute leukemia by detecting the expression level of HMGA1.

5. The use according to claim 4, characterized in that The expression level of HMGA1 is determined by detecting the level of HMGA1 protein.

6. The use according to claim 5, characterized in that The level of the HMGA1 protein is determined by an immunological detection method, such as immunohistochemistry or immunocytochemistry.

7. The use according to claim 6, characterized in that Detecting the level of HMGA1 protein includes determining the percentage of HMGA1-positive cells in the total number of selected cells; comparing the detected percentage of HMGA1-positive cells with a preset threshold value; when the percentage is higher than the threshold value, diagnosing the transformation of myeloproliferative neoplasm to acute leukemia.

8. The use according to claim 7, characterized in that The HMGA1 positive cell percentage threshold is above 40%.

9. A kit for diagnosing transformation of myeloproliferative neoplasms into acute leukemia, characterized in that: Included are reagents for detecting the expression level of HMGA1 protein in a biological sample of an individual.

10. The kit according to claim 9, characterized in that The reagent is an antibody that specifically binds to the HMGA1 protein.

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