A sugar chain marker combination for diagnosing waldenstrom macroglobulinemia and application thereof

Abstract

The application discloses a sugar chain marker combination for diagnosing Waldenstrom's macroglobulinemia and application thereof, and the marker combination comprises the following N-sugar chains: cFA2_2, cFA2B, cFA2(6)G1, cFA2(3)G1, A2G2, cFA2B(3)G1, cFA2G2, cFA2BG2, A3G3_2 and aFA3G3_2. By analyzing the change of N-sugar chains on glycoprotein in blood, the application finds that specific N-sugar chain marker combination has significant correlation with WM. The marker combination has high sensitivity and high specificity in WM diagnosis, can be rapidly detected through fluorescence labeling technology, has a non-invasive advantage, can be used for early discovery and accurate diagnosis, and is helpful to improve the cure rate and survival rate of patients. In addition, the discovery of the bisecting biantennary N-sugar chain combination provides a new potential treatment target for WM, and has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a combination of glycan biomarkers for diagnosing Waldenström macroglobulinemia and their applications. Background Technology

[0002] Waldenström macroglobulinemia (WDM) Macroglobulinemia (WM) is a rare, indolent B-cell lymphoma, accounting for 90%–95% of lymphoplasmacytic lymphomas (LPL). It is characterized by lymphoplasmacytic cell infiltration in the bone marrow and abnormally elevated levels of monoclonal immunoglobulin M (IgM) in the serum. Although targeted therapies (such as BTK inhibitors ibrutinib and zanubrutinib) have significantly improved patient prognosis in recent years, the 5-year overall survival rate for high-risk patients remains below 40%, and traditional prognostic models rely on clinical parameters (such as age and β2-microglobulin) and lack in-depth integration of molecular biomarkers. Furthermore, traditional bone marrow biopsy is invasive, and subsequent sample pretreatment for flow cytometry and gene mutation screening is complex and time-consuming. Therefore, exploring disease mechanisms and developing novel, non-invasive biomarkers and therapeutic targets is urgently needed.

[0003] Glycosylation, as a key post-translational modification of proteins, plays a crucial role in hematologic malignancies by regulating immunoglobulin function, cell signal transduction, and the tumor microenvironment. For example, in patients with multiple myeloma, the galactose content on serum immunoglobulin G (IgG) is significantly lower than in patients without bone disease, while the sialic acid content in IgG is significantly lower than in healthy individuals. During the onset of bone disease, serum sialic acid on IgG is significantly reduced. In mouse models, increasing sialic acid on IgG through sialic acid precursors can improve bone loss; therefore, regulation based on IgG glycosylation is a potential therapeutic strategy for multiple myeloma. Similarly, the abnormal accumulation of monoclonal IgM in the serum of WM patients may be accompanied by altered glycosylation patterns, but related research has not yet been conducted.

[0004] Existing research indicates that targeting glycosylation-related pathways holds therapeutic potential in hematologic malignancies. However, studies on the correlation between N-glycans and globulin-induced muscular dystrophy (WM) are currently lacking. Nevertheless, given the importance of glycosylation in protein function and disease progression, investigating changes in glycosylation patterns in the serum of WM patients may reveal disease mechanisms or provide new therapeutic targets. Furthermore, it holds promise for identifying disease-specific glycosylation biomarkers, offering new strategies for early diagnosis, prognostic stratification, and targeted therapy. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present application provides a sugar chain marker combination for diagnosing Waldenstrom's macroglobulinemia and application thereof, by detecting the change of N-glycan on glycoprotein in blood, establishing N-glycan marker combination and analysis screening system to diagnose WM patients, the method has the advantages of non-invasiveness, high sensitivity and high specificity, and is beneficial to early diagnosis of WM patients.

[0006] The present application is realized by the following technical solutions:

[0007] A sugar chain marker combination for diagnosing Waldenstrom's macroglobulinemia, comprising a combination of one or more of the following N-glycan markers: cFA2_2, cFA2B, cFA2(6)G1, cFA2(3)G1, A2G2, cFA2B(3)G1, cFA2G2, cFA2BG2, A3G3_2, aFA3G3_2; wherein the cFA2(6)G1 and the cFA2(3)G1 are isomers.

[0008] The application of the above-mentioned sugar chain marker combination in the preparation of products for diagnosing Waldenstrom's macroglobulinemia.

[0009] Preferably, the sugar chain marker combination comprises a combination of G0, G1, G2, G3, B, cF and aF;

[0010] The G0 combination comprises cFA2_2 and cFA2B.

[0011] The G1 combination comprises cFA2(6)G1, cFA2(3)G1 and cFA2B(3)G1.

[0012] The G2 combination comprises A2G2, cFA2G2 and cFA2BG2.

[0013] The G3 combination comprises A3G3_2 and aFA3G3_2.

[0014] The B combination comprises cFA2B, cFA2B(3)G1 and cFA2BG2.

[0015] The cF combination comprises cFA2_2, cFA2B, cFA2(6)G1, cFA2(3)G1, cFA2B(3)G1, cFA2G2 and cFA2BG2.

[0016] The aF combination comprises aFA3G3_2.

[0017] Preferably, the product comprises a reagent or kit for detecting the content of the sugar chain marker combination in a sample.

[0018] Preferably, the sample is whole blood, serum or plasma of a subject.

[0019] A kit for diagnosing Waldenstrom's macroglobulinemia, comprising reagents for detecting the content of the above-mentioned sugar chain marker combination in a sample.

[0020] Preferably, the reagents comprise:

[0021] Reagent A: NH4HCO3 at a concentration of 2-15 mM is added to a SDS solution with a mass concentration of 3-5%;

[0022] Reagent B1: glycoside exoglycosidase solution at a concentration of 2-6 U / μL;

[0023] Reagent B2: glycoside endoglycosidase at a concentration of 2-6 U / μL;

[0024] Reagent C: ddH2O;

[0025] Reagent D: a mixture of a fluorescent labeling solution at 1-20 mM mixed with a DMSO solution at 1-1.5 M.

[0026] Preferably, the volume ratio of reagent A, reagent B1, reagent B2, reagent C, and reagent D is (1-10):(1-10):(1-10):(20-120):(1-10).

[0027] The above-mentioned sugar chain marker combination as a therapeutic target for screening or preparing a drug for preventing and / or treating Waldenstrom's macroglobulinemia.

[0028] Preferably, the sugar chain marker combination comprises a B combination comprising cFA2B, cFA2B(3)G1, and cFA2BG2.

[0029] The beneficial effects of the present application are as follows:

[0030] (1) The present application first discovers and verifies N-glycan marker combinations specifically related to Waldenstrom's macroglobulinemia (WM) in blood, breaking through the limitations of traditional reliance on bone marrow biopsy and IgM quantitative detection, and providing a new strategy for non-invasive diagnosis of WM. By detecting the abnormal expression of specific N-glycans in samples, the early diagnosis efficiency can be significantly improved, especially for patients with hidden symptoms or who cannot tolerate invasive examinations.

[0031] (2) The detection method developed based on the N-glycan marker combination provided by the present application exhibits excellent sensitivity and specificity in clinical verification, and can accurately distinguish WM from healthy people, effectively reducing the risk of misdiagnosis. This technical breakthrough provides a reliable tool for early detection and early intervention of WM, and is expected to improve the survival prognosis of high-risk patients.

[0032] (3) The biantennary N-glycan combination (B combination) discovered in the present application is not only a diagnostic marker, but its abnormal expression also suggests that it may be involved in the disease progression of WM. This finding provides a theoretical basis for the development of new therapies targeting glycosylation modification. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 N-glycan profile of blood samples of healthy subjects (A) and WM patients (B) in Example 1;

[0034] Figure 2 ROC curve of N-glycan marker combination G0, G1, G2, G3, B, cF and aF for the diagnosis of WM patients in the training set of Example 1;

[0035] Figure 3 ROC curve of N-glycan marker combination G0, G1, G2, G3, B, cF and aF for the diagnosis of WM patients in the validation set of Example 2

[0036] Figure 4 N-glycan profile of blood samples of healthy subjects (A), paired WM baseline patients (B) and WM post-treatment patients (C) in Example 3. DETAILED DESCRIPTION

[0037] The present application will be further described in detail below with reference to the accompanying drawings and specific examples.

[0038] Unless otherwise specified, the technical means used in the following examples are conventional means known to those skilled in the art. The experimental methods not specified in the specific conditions are conventional methods in the art.

[0039] The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0040] Example 1

[0041] In the early stage of the present application, blood samples of healthy subjects and WM patients were collected, and the N-glycan markers obtained were subjected to data processing by using the scheme of the present example for glycan extraction, separation and detection. Through t-test between groups, 7 significantly different N-glycan marker combinations (each consisting of 10 N-glycans) were found, and through ROC curve analysis, N-glycan marker combinations for the diagnosis of WM were further screened.

[0042] Based on the technical platform of capillary electrophoresis for detecting N-glycans, N-glycan detection was performed on blood samples of subjects to obtain N-glycan profile and collect N-glycan data. The specific steps are as follows:

[0043] 1. Sample detection

[0044] Blood samples of healthy subjects and WM patients, including whole blood, serum or plasma, were collected and randomly divided into two groups, training set and validation set, before detection. The experimental detection and data processing analysis in this embodiment are based on the samples randomly divided into the training set before experimental detection.

[0045] In this embodiment, 79 blood samples of healthy subjects and 55 blood samples of WM subjects were collected from Nanjing Drum Tower Hospital, a total of 134 samples (the time period involved was from January 2018 to June 2021), and the above WM patient samples all underwent the recommended examination in the clinical diagnosis and treatment guidelines, and the detection results met the WM clinical diagnosis standard. The following experiments have been reported to the ethics committee and approved.

[0046] 2. Instruments and equipment

[0047] Capillary electrophoresis analyzer, automatic biochemical analyzer, PCR and centrifuge.

[0048] 3. Detection reagents

[0049] Reagent A: 2-15 mM NH4HCO3 added to a 3-5% SDS solution by mass concentration;

[0050] Reagent B1: 2-6 U / μL glycoside exoglycosidase solution;

[0051] Reagent B2: 2-6 U / μL glycoside endoglycosidase solution;

[0052] Reagent C: ddH2O;

[0053] Reagent D: 1-20 mM fluorescent labeling solution (8-aminopyrene-1, 3, 6-trisulfonic acid trisodium salt) mixed with 1-1.5 M DMSO solution.

[0054] 4. N-glycan profile detection and collection

[0055] (1) Release of N-glycan

[0056] Add 4-10 μL of reagent A to 2-10 μL of sample, denature at 95-100°C for 10-25 min, cool to 4-6°C, add 4-10 μL of 1:1 volume premixed reagent B1 and reagent B2, react at 30-40°C for 1-3 h, and add 50-70 μL of reagent C.

[0057] (2) Labeling of N-glycan

[0058] Take 4-10 μL of the sample in step (1) and dry at 60-80 °C for 30-40 min, then add 2-5 μL of reagent D and react at 75-95 °C for 1-2 h, and finally add 50-70 μL of reagent C.

[0059] (3) Detection of N-glycan chain and collection of spectrum

[0060] Take 4-10 μL of the N-glycan chain sample prepared in step (2) and place it in an ABI special 96-well plate, then detect it by ABI 3500 sequencer to obtain the N-glycan spectrum.

[0061] As shown in Figure 1 , the N-glycan marker combination consists of 10 N-glycan chains, namely P1 (cFA2_2, non-galactosylated α-1, 6 core fucosylated biantennary N-glycan), P2 (cFA2B, non-galactosylated α-1, 6 core fucosylated bisecting biantennary N-glycan), P3 (cFA2(6)G1, single branched galactosylated α-1, 6 core fucosylated biantennary N-glycan), P4 (cFA2(3)G1, single branched galactosylated α-1, 6 core fucosylated biantennary N-glycan), P5 (A2G2, galactosylated biantennary N-glycan), P5a (cFA2B(3)G1, single branched galactosylated α-1, 6 core fucosylated bisecting biantennary N-glycan), P6 (cFA2G2, galactosylated α-1, 6 core fucosylated biantennary N-glycan), P7 (cFA2BG2, galactosylated α-1, 6 core fucosylated bisecting biantennary N-glycan), P8 (A3G3_2, galactosylated triantennary N-glycan), and P9 (aFA3G3_2, galactosylated α-1, 3 branched fucosylated triantennary N-glycan); wherein P3 (cFA2(6)G1) and P4 (cFA2(3)G1) are isomers.

[0062] 5. Data processing

[0063] The detection results of the above-obtained 10 N-glycan chains are quantitatively processed. Specifically, the relative content of each peak in the N-glycan spectrum is calculated by dividing the height of each peak by the sum of the heights of all peaks to obtain the peak quantification data of the N-glycan chain.

[0064] 6. Data analysis

[0065] The 10 N-glycan chains obtained above are divided into the following 7 combinations: G0 (non-galactosylated biantennary N-glycan combination, including P1 and P2), G1 (single branched galactosylated biantennary N-glycan combination, including P3, P4 and P5a), G2 (galactosylated biantennary N-glycan combination, including P5, P6 and P7), G3 (galactosylated triantennary N-glycan combination, including P8 and P9), B (bisecting biantennary N-glycan combination, including P2, P5a and P7), cF (α-1, 6 core fucosylated biantennary N-glycan combination, including P1, P2, P3, P4, P5a, P6 and P7) and aF (α-1, 3 branched fucosylated triantennary N-glycan combination, including P9).

[0066] The peak quantification results of the N-glycan chains obtained above are analyzed for N-glycan marker combinations. Specifically, inter-group t-test significance difference analysis of N-glycan marker combinations in healthy human subjects and WM patients, and ROC curve analysis.

[0067] (1) Significance difference analysis

[0068] The inter-group t-test significance difference analysis of the above 7 N-glycan marker combinations in healthy human subjects and WM patients is shown in Table 1 below.

[0069] Table 1 Significance difference analysis

[0070] Sugar chain marker combinations Healthy controls (N=79) WM (N=55) P value G0 5.12±1.48 6.08±2.55 0.0073 G1 8.33±1.41 9.46±3.46 0.0102 G2 76.24±2.81 79.56±5.34 ＜0.0001 G3 10.30±1.80 4.91±2.33 ＜0.0001 B 5.52±0.86 30.51±13.99 ＜0.0001 cF 32.88±3.74 63.01±14.35 ＜0.0001 aF 0.36±0.08 0.14±0.07 ＜0.0001

[0071] Table 1 shows that the above 7 N-glycan marker combinations have inter-group significance differences (P < 0.05) in healthy human subjects and WM patients.

[0072] (2) ROC curve analysis

[0073] The combined diagnostic value of the above glycan marker combinations is detected by receiver operating characteristic curve (ROC) analysis and calculation of the area under the ROC curve (AUC), and the AUC is the area under the receiver operating curve, which is an indicator of diagnostic performance or accuracy, and the closer the AUC is to 1, the better the diagnostic performance.

[0074] As Figure 2The image shows the ROC curve analysis of the training set for the N-glycan biomarker combinations used to diagnose WM. The results show that the AUC values ​​of the seven N-glycan biomarker combinations for diagnosing WM patients are as follows: G0 (0.586), G1 (0.576), G2 (0.767), G3 (0.960), B (1.000), cF (0.991), and aF (0.979). Among them, the AUC values ​​of N-glycan biomarker combinations G3, B, cF, and aF all exceed 0.950, indicating that they have good diagnostic performance in the diagnosis of WM.

[0075] In a preferred embodiment, based on the data analysis of the training set mentioned above, the bisecting two-antenna N-glycan combination (B combination) has the best performance in diagnosing clinical WM patients, with an AUC value of 1.000 and both sensitivity and specificity reaching 100%.

[0076] Example 2

[0077] 1. Test Sample

[0078] Blood samples, including whole blood, serum, or plasma, were collected from healthy controls and WM patients. Before testing, the samples were randomly divided into two groups: a training set and a validation set. The experimental testing and data processing analysis in this embodiment are based on the samples randomly assigned to the validation set before the experimental testing.

[0079] This embodiment collected blood samples from 34 healthy subjects and 24 WM subjects from Nanjing Drum Tower Hospital, totaling 58 samples (covering the period from July 2021 to December 2022). All WM patient samples underwent the examinations recommended in the clinical practice guidelines, and the test results met the clinical diagnostic criteria for WM. The following experiments have been filed and approved by the ethics committee.

[0080] 2. Instruments and equipment

[0081] Same as Example 1.

[0082] 3. Test reagents

[0083] Same as Example 1.

[0084] 4. N-glycan map detection and acquisition

[0085] Same as Example 1.

[0086] 5. Data Processing

[0087] Same as Example 1.

[0088] 6. Data Analysis

[0089] Will as Figure 1The 10 N-glycan chains (P1, P2, P3, P4, P5, P5a, P6, P7, P8, P9) shown are divided into 7 combinations (G0, G1, G2, G3, B, cF, aF) as shown in Example 1. The peak quantification results of the N-glycan chains obtained above are analyzed for N-glycan marker combinations.

[0090] The diagnostic value of the above N-glycan marker combinations is detected by receiver operating characteristic (ROC) analysis and calculation of the area under the ROC curve (AUC), which is the area under the receiver operating curve and is an indicator of diagnostic performance or accuracy. The closer the AUC is to 1, the better the diagnostic performance.

[0091] As shown in Figure 3 The ROC curve analysis of the N-glycan marker combinations for diagnosing WM is shown in the validation set, and the results show that the AUC values of the above 7 N-glycan marker combinations for diagnosing WM patients are: G0 (0.665), G1 (0.559), G2 (0.775), G3 (0.971), B (1.000), cF (0.994) and aF (0.518). Among them, the AUC values of N-glycan marker combinations G3, B and cF all exceed 0.950, indicating that they have good diagnostic performance in diagnosing WM.

[0092] A preferred scheme is based on the data analysis of the above validation set, and the performance of the bisected biantennary N-glycan combination (B combination) for diagnosing clinical WM patients is the best, with an AUC value of 1.000, and the sensitivity and specificity are both 100%.

[0093] Example 3

[0094] Based on the experimental results of Examples 1 and 2, the discrimination effect of the bisected biantennary N-glycan combination (B combination) in paired WM baseline patients and WM post-treatment patients is analyzed, as follows:

[0095] 1. Test samples

[0096] Blood samples of healthy subjects, WM baseline and WM post-treatment patients are collected, including whole blood, serum or plasma. The paired WM baseline and WM post-treatment patients involved in this embodiment are the same patient. In the design of the research scheme, the patient is divided into the WM baseline (initial diagnosis) and the WM post-treatment stage at the time of blood sample collection, i.e., the same patient has undergone blood sample collection at these two stages.

[0097] This example collects 32 blood samples from healthy subjects in Nanjing Gulou Hospital, 58 blood samples from WM subjects (divided into 29 WM baseline patient blood samples and 29 paired WM post-treatment patient blood samples), a total of 90 (involving the period from January 2023 to January 2025), the above WM patient samples all received the recommended examination in the clinical diagnosis and treatment guidelines, the test results meet the WM clinical diagnosis standard, and they have been treated. The following experiments have been reported and approved by the ethics committee.

[0098] 2. Instruments and equipment

[0099] The same as Example 1.

[0100] 3. Detection reagent

[0101] The same as Example 1.

[0102] 4. N-glycan profile detection and collection

[0103] The same as Example 1.

[0104] 5. Data processing

[0105] The same as Example 1.

[0106] 6. Data analysis

[0107] The P2, P5a and P7 peak quantification results in the bisecting biantennary N-glycan combination (B combination) were analyzed.

[0108] As shown in Figure 4 , the N-glycan profiles and data of healthy subjects (A in Figure 4 ), paired WM baseline patients (B in Figure 4 ) and WM post-treatment patients (C in Figure 4 ) were compared and analyzed. The results showed that in the bisecting biantennary N-glycan combination (B combination), the relative contents of the involved N-glycan markers P2 (cFA2B, bisecting biantennary N-glycan without galactosyl α-1, 6 core fucosylation), P5a (cFA2B(3)G1, bisecting biantennary N-glycan with single branched chain galactosyl α-1, 6 core fucosylation) and P7 (cFA2BG2, bisecting biantennary N-glycan with galactosyl α-1, 6 core fucosylation) were all significantly reduced in WM post-treatment patients.

[0109] The experimental results of this example show that the bisecting biantennary N-glycan combination (B combination) is significantly reduced in WM post-treatment patients, suggesting that it may serve as a new potential therapeutic target for WM patients.

[0110] The application screens a new N-glycan marker combination for the clinical auxiliary diagnosis of WM patients by detecting the correlation between the changes of N-glycan on glycoprotein in blood samples and diseases, and can be used for monitoring WM baseline patients and WM patients after treatment. The results of the above examples show that the detection method of the application has high sensitivity and high specificity for WM patients, which can help WM patients to be detected and treated early, and improve the overall cure rate and survival rate of patients.

[0111] The above-described embodiments are only a part of the embodiments of the application, rather than all the embodiments. The detailed description of the embodiments of the application is not intended to limit the scope of the claimed application, but only represents selected embodiments of the application. The protection scope of the application is the scope claimed in the claims, and all other embodiments obtained by those of ordinary skill in the art based on the embodiments in the application without creative labor fall within the protection scope of the application.

Claims

1. The application of a combination of glycan biomarkers in the preparation of products for diagnosing Waldenström macroglobulinemia, characterized in that, The glycan marker combination is a combination of G3, B, or cF; The G3 combination is composed of N-glycan chains A3G3_2 and aFA3G3_2; The B group consists of N-glycan chains cFA2B, cFA2B(3)G1, and cFA2BG2; The cF combination consists of N-glycans cFA2_2, cFA2B, cFA2(6)G1, cFA2(3)G1, cFA2B(3)G1, cFA2G2, and cFA2BG2.

2. The application according to claim 1, characterized in that, The glycan marker combination is combination B.

3. The application according to claim 1, characterized in that, The product includes reagents or kits for detecting the content of the combination of glycan markers in a sample.

4. The application according to claim 3, characterized in that, The sample is the subject's whole blood, serum, or plasma.

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