Application of Plasma Exosomal Protein LAMP2 as a Biomarker for the Clinical Diagnosis of Neuroblastoma

Abstract

This invention belongs to the field of biomedicine, specifically relating to biomarkers and their applications for the clinical diagnosis of high-risk neuroblastoma. A product for the clinical diagnosis of high-risk neuroblastoma includes a reagent for detecting the expression level of the biomarker, which is a plasma exosomal protein called LAMP2. By detecting LAMP2 expression levels, this method can complement existing clinical indicators, enabling precise stratification and treatment of neuroblastoma, and also provides important guidance for the prognosis of children with tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of plasma exosome protein LAMP2 as a biomarker for the clinical diagnosis of neuroblastoma. Background Art

[0002] Neuroblastoma (NB) is the most common extracranial solid tumor in infants and young children, with an incidence of 10.2 cases per million children under 15 years of age. Characterized by insidious onset, high malignancy, and a high incidence of metastasis to the bone marrow, bones, and distant organs, it accounts for 15% of all childhood cancer deaths and is considered the "king of childhood tumors." In my country, over 50% of children with NB have already progressed to high-risk disease at the time of initial diagnosis, and the five-year survival rate is less than 50%.

[0003] The survival rate of children with NB is significantly positively correlated with the rate of early screening and diagnosis. The delayed age of first diagnosis for NB in children is a significant clinical issue, primarily due to the relatively slow early screening of suspected cases. Therefore, the discovery of more sensitive and effective molecular markers for NB early screening will further enhance the diagnosis and treatment of children with NB.

[0004] Exosomes are a type of extracellular matrix microcapsule ranging in size from 30 to 150 nm. Tumor cells, as non-blood-derived cells, can secrete exosomes. The secreted exosomes enter body fluids such as blood, saliva, urine, and breast milk, reaching other cells and tissues through the circulatory system, exerting remote regulatory effects. As a vesicular structure, exosomes carry biological substances (such as DNA, RNA, and proteins) with important biological significance. Many molecules have been shown to be involved in the development of important diseases such as tumorigenesis and can serve as early diagnostic markers for tumors. Summary of the Invention

[0005] The present invention provides the use of plasma exosome protein LAMP2 as a biomarker for high-risk neuroblastoma.

[0006] This invention uses proteomics technology to explore the differential proteins in the plasma and plasma exosomes of children with NB, explore the key regulatory molecules for the occurrence and development of NB tumors and new molecular markers that can be used as clinical diagnosis, which has important theoretical significance and potential application value.

[0007] The present invention provides the use of plasma exosome protein LAMP2 as a biomarker in the preparation of a diagnostic preparation for high-risk neuroblastoma. The expression level of plasma exosome protein LAMP2 in high-risk neuroblastoma is significantly reduced.

[0008] The diagnostic preparation is used to detect LAMP2 expression in plasma exosomes of a subject; comparing the detected value with a reference value; if LAMP2 is significantly lower than the reference value, it indicates that the subject has high-risk neuroblastoma. The reference value is the expression level of LAMP2 in plasma exosomes of a normal child or a child with low-risk neuroblastoma. The subject is known or suspected to harbor tumor cells.

[0009] In the present invention, the term LAMP2 refers to lysosomal-associated membrane protein 2, including the LAMP2 gene and the protein it encodes, as well as its homologs, mutations, and isoforms. The term encompasses full-length, unprocessed LAMP2 as well as any form of LAMP2 derived from cellular processing. Preferably, the LAMP2 is a human protein with a UniProt ID of P13473.

[0010] In the present invention, the term "exosomes" refers to a class of extracellular matrix microcapsules with a size between 30-150nm. Tumor cells, as a non-hematogenous cell, can secrete exosomes. The secreted exosomes will enter body fluids such as blood, saliva, urine, and milk, and reach other cells and tissues through the circulatory system, producing a remote regulatory effect. As a vesicle structure, exosomes carry biological substances (such as DNA, RNA, and proteins, etc.) with important biological significance. Many molecules have been shown to be involved in the occurrence of important diseases such as tumorigenesis, and can be used as early diagnostic markers for tumors.

[0011] In the present invention, the term "biomarker" refers to an indicator that can be detected in a sample, such as a predictive, diagnostic and / or prognostic indicator. A biomarker can serve as an indicator of a specific disease or disorder (e.g., cancer) subtype characterized by specific molecular, pathological, histological and / or clinical characteristics. In some embodiments, a biomarker is a gene. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and / or RNA), changes in polynucleotide copy number (e.g., DNA copy number), polypeptides, polypeptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrates and / or glycolipid-based molecular markers.

[0012] In the present invention, the term "expression level" generally refers to the amount of a biomarker in a biological sample. "Expression" generally refers to the process by which information (e.g., genetically encoded and / or epigenetic) is converted into a structure that exists and operates in a cell. Therefore, "expression" as used in the present invention can refer to transcription into polynucleotides, translation into polypeptides, polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of polypeptides). Fragments of transcribed polynucleotides, translated polypeptides, or polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of polypeptides) should also be considered expressed, whether they are derived from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of polypeptides (e.g., by proteolysis).

[0013] According to a specific embodiment of the present invention, a product can be used for the clinical diagnosis of high-risk neuroblastoma, and the product includes a reagent for detecting the expression level of a biomarker, and the biomarker is LAMP2 in plasma exosome protein.

[0014] The present invention provides use of a reagent for detecting the expression level of plasma exosome protein LAMP2 in the preparation of a diagnostic product for diagnosing high-risk neuroblastoma.

[0015] Preferably, the diagnostic product is a chip or a kit.

[0016] In the present invention, the term "chip" refers to a gene chip, also known as a DNA chip or a biochip, which fixes probe molecules on a support and hybridizes with labeled sample molecules, and obtains the number and sequence information of the sample molecules by detecting the hybridization signal intensity of each probe molecule.

[0017] Wherein, the diagnostic product also includes a negative control, a positive control, primers, probes or antibodies.

[0018] As used herein, the term "primer" refers to a single-stranded polynucleotide capable of hybridizing to a nucleic acid and allowing polymerization of a complementary nucleic acid (generally by providing a free 3'-OH group).

[0019] In the present invention, the term "antibody" is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies) and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0020] At the same time, the present invention provides a kit for diagnosing high-risk neuroblastoma, which includes a detection reagent for the expression level of the plasma exosome protein LAMP2.

[0021] Preferably, the detection reagent further includes a negative control, a positive control, a primer, a probe or an antibody.

[0022] Beneficial effects of the present invention:

[0023] The present invention uses proteomics technology to mine differential proteins in the plasma and plasma exosomes of children with NB, and uses LAMP2 protein expression levels to identify high-risk NB. It has good external validity for identifying high-risk NB, is universal and applicable, and has good predictive performance.

[0024] The present invention found that LAMP2 is lowly expressed in the exosomes of high-risk NB and is negatively correlated with clinical progression; in the plasma exosomes of children with newly diagnosed NB, low expression of LAMP2 is positively correlated with M-stage NB, high-risk NB, large primary tumor lesions, and bone marrow metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 Shows the screening process for the differentially expressed protein LAMP2 in plasma exosomes of children with neuroblastoma.

[0027] Figure 2 The relationship between LAMP2 expression in the public database GSE62564 and high-risk group, event-free survival, and overall survival is shown;

[0028] A. LAMP2 expression was low in tumor tissues of children with high-risk NB (P < 0.001);

[0029] B. Children with low LAMP2 expression in tumor tissue had a lower event-free survival rate (P < 0.05);

[0030] C. Children with low expression of LAMP2 in tumor tissue had poor overall survival rate (P<0.0001).

[0031] Figure 3 The expression level of LAMP2 in the validation set samples was verified by ELISA experiment.

[0032] Figure 4 The ROC curve of the validation set showing the classification of high-risk or low-risk NB based on LAMP2 protein expression level. DETAILED DESCRIPTION

[0033] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0034] In the present invention, high-risk NB children are grouped according to the International NB Risk Group (INRG), and the patients' pre-treatment risk is stratified based on factors such as diagnosis age, INRGSS stage, histological type, MYCN gene, etc. High-risk children have a poor prognosis, and the 5-year event-free survival rate is less than 50%.

[0035] Children with low-risk NB: According to the International NB Risk Group (INRG) pre-treatment risk stratification of NB, low-risk children have a better prognosis, with a 5-year event-free survival rate of more than 75%.

[0036] Event-free survival: Event-free survival (EFS) refers to the time from the start of randomization (or the start of treatment in a single-arm trial) to the first occurrence of any of the following events: disease progression that is incapable of surgical treatment, local or distant recurrence, death from any cause, etc.

[0037] Overall survival: OS refers to the time from randomization (or the start of treatment in a single-arm trial) to death from any cause.

[0038] M stage: According to the International NB Risk Group (INRG) staging system, any primary tumor is accompanied by distant lymph node, bone marrow, liver, skin and / or other organ metastasis (except Ms stage).

[0039] Non-M stage: According to the International NB Risk Group (INRG) staging, excluding the M stage NB staging, including L1 stage, L2 stage and Ms stage.

[0040] Example 1 Screening for differentially expressed LAMP2 protein in plasma exosomes of children with neuroblastoma

[0041] 1.1 Extraction of exosomes from plasma

[0042] In the initial phase of this study, plasma samples were collected from 30 high-risk and 30 low-risk children with NB who had not undergone surgical resection or chemotherapy. Plasma samples were also collected from 30 children with non-tumor or non-infectious diseases that had no potential impact on plasma components (e.g., tongue tie, strabismus, phimosis, scoliosis, etc.) as a healthy control group. All samples were used to extract plasma exosomes using the Invitrogen Total Exosome Isolation (from plasma) kit (Cat. No. 4484450). The extraction method was as follows:

[0043] (1) Take 1 ml of fresh whole blood, centrifuge at 2,000 g for 10 min at room temperature, aspirate the plasma, and store it in a -80°C refrigerator;

[0044] (2) Take the sample at -80℃, melt it in a water bath at room temperature, and place it on ice;

[0045] (3) Centrifugation at 2,000 g for 20 min at room temperature to remove cells and debris, and transfer the supernatant to a new EP tube;

[0046] (4) Centrifuge at 10,000 g for 20 min at room temperature, transfer the supernatant to a new EP tube, and place on ice (record the plasma volume V1);

[0047] (5) Add PBS (V2 = V1 * 0.5) and vortex mix for at least 30 seconds;

[0048] (6) Add Exosome Precipitation Reagent: V3 = 0.2*(V1 + V2), vortex mix until the liquid becomes cloudy;

[0049] (7) Let stand at room temperature for 10 minutes;

[0050] (8) Centrifuge at 10,000 g for 5 min at room temperature, carefully discard the supernatant, and retain the pellet at the bottom of the tube;

[0051] (9) Centrifuge at 10,000 g for 1 min at room temperature, carefully discard the supernatant, and retain the pellet at the bottom of the tube;

[0052] (10) Resuspend the pellet in PBS and mix well to avoid air bubbles. V4 = 0.3 * V1;

[0053] (11) One-third of the exosome sample was directly frozen for electron microscopy and particle number analysis;

[0054] (12) Add 2× Zwitter lysis buffer (cocktail added in advance) according to the remaining exosome volume in a 1:1 ratio and sonicate on ice for 1 h;

[0055] (13) 12,000 g, 20 min, 4°C, take 40 μl, add 5*loading 10 μl, 98°C, 5 min.

[0056] (14) Protein quantification was performed using the BCA method, and absorbance was measured at 562 nm. 100 μg of protein was collected for subsequent mass spectrometry.

[0057] 1.2 Pre-processing of plasma exosome protein proteomics

[0058] The filtration-assisted sample preparation (FASP) method was used for plasma exosome protein proteomics pre-processing. The steps are as follows:

[0059] (1) Reduction: Add DDT at a final concentration of 20 mM to 100 μl of plasma exosome protein lysate (containing 100 μg of exosome protein), vortex, centrifuge briefly, and heat at 95°C for 5 minutes. Equilibrate to room temperature.

[0060] (2) Alkylation: Add IAM to a final concentration of 50 mM, vortex, spin briefly, and then react in the dark for 45 minutes.

[0061] (3) Equilibration of membrane: 30K membrane was pre-washed twice with 200 μl Tris (4°C × 14,000 g × 5 min), centrifuged until there was no liquid in the tube, and the liquid was discarded.

[0062] (4) Sample loading: Transfer the protein sample to a 30K membrane and centrifuge at 14,000 g for 30 min at 4°C until there is no liquid in the tube and a small crescent shape appears on the membrane.

[0063] (5) Washing: Add 200 μl of 20 mM Tris, shake and mix, incubate at 4°C × 14,000 g × 30 min until there is no liquid in the cannula, repeat 3 times.

[0064] (6) Cleaning the cannula: Wash the lower tube twice with 20 mM Tris.

[0065] (7) Enzymatic hydrolysis: Add enzyme at a mass ratio of sample: enzyme = 50:1, add Tris to 30 μl, shake to mix, and throw the liquid onto the membrane. Place the test tube on a float in a beaker filled with tap water, microwave on high for 1 min, twice, changing the tap water in between, and incubate in a 37°C water bath overnight.

[0066] (8) Collect the enzymatic hydrolysate: Centrifuge at 14,000 g until the membrane is clean and free of liquid. The liquid below is the enzymatic hydrolysate.

[0067] (9) The BCA method was used to determine the peptide concentration.

[0068] 1.3 Independent Data Acquisition (DIA) Mass Spectrometry Analysis

[0069] 2 μg of peptide was taken from each sample and spiked with iRT standard peptide at a volume ratio of 1:20. Each sample was tested by DIA mass spectrometry once.

[0070] Chromatographic separation was performed using an UltiMate 3000 UPLC system at a flow rate of 1.5 μl / min. Buffer A consisted of 0.1% formic acid in water and buffer B consisted of 0.1% formic acid in acetonitrile (80% acetonitrile). Peptide samples were directly applied to a chromatographic column (length: 50 cm, inner diameter: 75 μm) for mass spectrometry detection.

[0071] The liquid phase separation gradient was as follows: 0 min-50 min, linear gradient of solution B from 1% to 20%; 50 min–55 min, linear gradient of solution B from 20% to 30%; 55 min-56 min, linear gradient of solution B from 30% to 50%; 55 min-56 min, linear gradient of solution B from 30% to 50%; 56 min-60 min, linear gradient of solution B from 50% to 90%.

[0072] The samples after liquid chromatography separation were detected by mass spectrometry using an Orbitrap Exploris 480 high-resolution mass spectrometer (ThermoScientific).

[0073] Detection mode: Positive ionization, primary mass spectrometry scan range: 350-1200 m / z, mass spectrometry resolution: 120,000, AGC target: Custom, Maximum IT: 50 ms, Data type: Profile. MS2 acquisition settings: 60 acquisition windows, secondary resolution: 30,000, AGC target: Custom, Maximum IT: 50 ms, Data type: Centroid. HCD collision energy: 30%.

[0074] 1.4 Mass spectrometry data processing

[0075] Mass spectrometry data were processed using Spectronaut Pulsar (18.0) software, and the Swiss Prothuman database (20,386 sequences, released June 2022) was used. A direct search was used for retrieval. Qualitative and quantitative parameters were: Precursor Q value: 0.01, Protein Q value: 0.01, peak area-based MS1-level quantification, Cross-Run Normalization (Cross-Run Normalization), MaxLFQ (Max LFQ), and IDPicker (Inference Algorithm).

[0076] Table 1 Screening results of proteins with reduced expression between the two groups

[0077]

[0078] As shown in the table above, among the numerous proteins, LAMP2 was a protein with reduced expression in the three groups, thus LAMP2 was identified as a candidate down-regulated differential protein for investigation.

[0079] Example 2

[0080] The public database GSE62564 was used to investigate the relationship between LAMP2 expression and the grouping and survival of children with NB.

[0081] The public database GSE62564 uses RNA-seq (RNA sequencing, transcriptome sequencing technology) technology to sequence and analyze NB samples to form a gene expression matrix, whose values represent the expression abundance of the corresponding genes in the corresponding samples, reflecting the expression level of the genes.

[0082] In the public database GSE62564, there were 176 high-risk children with a median LAMP expression of 4.160, and 322 non-high-risk children with a median expression of 4.493. The t-test showed a P value < 0.001, indicating that LAMP2 was lowly expressed in tumor tissues of high-risk NB children. Figure 2 -A;

[0083] The LAMP2 gene expression was analyzed by receiver operating characteristic (ROC) analysis based on the dichotomous outcome of death. The point with the maximum Youden index (sensitivity + specificity - 1) was selected as the cutoff value, and the data of the children were divided into high expression (368 patients) and low expression (130 patients).

[0084] like Figure 2-B, The 5-year event-free survival rate of NB children with high LAMP2 expression was 66.47%, while that of NB children with low LAMP2 expression was 51.15%. The event-free survival rate was even lower in children with low LAMP2 expression in tumor tissue (P < 0.05).

[0085] like Figure 2 -C, The 5-year overall survival rate of NB children with high LAMP2 expression was 88.17%, while the 5-year overall survival rate of NB children with low LAMP2 expression was 70.99%. The overall survival rate of NB children with low LAMP2 expression was worse (P < 0.0001).

[0086] Example 3: ELISA test to verify the expression level of LAMP2 in the validation set samples

[0087] The present invention also collected plasma samples as a validation set for the proteomics results, including 15 healthy controls, 9 low-risk NB children, and 15 high-risk NB children. After the plasma exosome proteins were extracted, ELISA experiments were performed for verification.

[0088] The ELISA experimental process is as follows:

[0089] (1) Prepare reagents: dilute the concentrated washing solution with distilled water at a ratio of 1:20; thoroughly mix substrate solutions A and B at a ratio of 1:1 by volume and use within 15 minutes; remove the pre-coated enzyme labeling strips and set aside.

[0090] (2) Set up standard wells, 0 value wells, blank wells and sample wells. Add 50 μL of standard of different concentrations to each standard well, add 50 μL of sample diluent to the 0 value wells, add nothing to the blank wells, and add 50 μL of the sample to be tested to the sample wells.

[0091] (3) Add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to the standard wells, 0 value wells, and sample wells, except for the blank wells.

[0092] (4) Cover the reaction plate with a sealing film and incubate in a 37°C water bath or incubator in the dark for 60 min.

[0093] (5) Remove the sealing film, discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let it stand for 20 seconds, shake off the washing solution, pat dry on absorbent paper, and repeat this process 5 times.

[0094] (6) Mix substrates A and B thoroughly at a 1:1 volume ratio and add 100 μL of the substrate mixture to all wells. Cover the reaction plate with a sealing film and incubate in a 37°C water bath or incubator in the dark for 15 min.

[0095] (7) Add 50 μL of stop solution to all wells and read the absorbance (OD value) of each well on a microplate reader at a wavelength of 450 nm.

[0096] (8) Calculation of results: With the concentration of the standard as the horizontal axis and the corresponding absorbance (OD value) as the vertical axis, computer software was used to fit the four-parameter logistic curve (4-pl) to create a standard curve equation. The concentration of the sample was calculated using the equation based on the absorbance (OD value) of the sample.

[0097] like Figure 3 As shown in the data, the median LAMP2 expression levels in the control group, low-risk group, and high-risk group were 0.139, 0.133, and 0.086, respectively. Compared with the control group and low-risk group, the t-test result P value was <0.05, indicating that the LAMP2 expression in the high-risk group was significantly decreased.

[0098] Thus, it was demonstrated that LAMP2 expression levels were significantly reduced in high-risk NB children.

[0099] Example 4: Drawing an ROC curve for distinguishing high-risk or low-risk NB based on LAMP2 protein expression level in the validation set

[0100] ROC curves were drawn based on LAMP2 protein expression levels to identify high-risk NB or low-risk NB. Figure 4 As shown in the figure, the AUC value of the ROC curve was 0.8519, P = 0.0046.

[0101] It can be seen that through external validation, LAMP2 protein expression level has good external validity for identifying high-risk NB, is universal and applicable, and has good predictive performance.

[0102] Example 5 Investigating the Correlation between LAMP2 Expression and Clinical Progression

[0103] The patients were divided into a high LAMP2 expression group and a low LAMP2 expression group using the median LAMP2 expression level of 0.107 in Example 4 as the cutoff value. The chi-square test was used for analysis.

[0104] Table 2 Analysis of LAMP2 expression and clinical characteristics in children with NB

[0105]

[0106]

[0107] LDH: Lactate dehydrogenase; NSE: Neuron-specific enolase; * P<0.05, chi-square test.

[0108] As shown in Table 2, among children with low plasma exosomal LAMP2 expression, the proportion of children in M stage was significantly higher than that in non-M stage children (91.7% vs. 33.3%, P = 0.011), and the proportion of children in the high-risk group was significantly higher than that in the non-high-risk group (83.3% vs. 33.3%, P = 0.013).

[0109] Among children with low plasma exosomal LAMP2 expression, the proportion of children with large primary tumors was significantly higher than that of children with small primary tumors (0.0% vs. 45.5% vs. 54.5%, P < 0.001), the proportion of children with elevated NSE was significantly higher than that of children with normal values (8.3% vs. 8.3% vs. 83.4%, P = 0.037), and the proportion of children with bone marrow metastasis was significantly higher than that without bone marrow metastasis (75.0% vs. 25.0%, P = 0.014). These results demonstrate that low LAMP2 expression in plasma exosomes of NB is positively correlated with the high malignancy and clinical progression of NB.

[0110] In the plasma exosomes of children with newly diagnosed NB, low LAMP2 expression was positively correlated with M-stage NB, high-risk NB, large primary tumor lesions, and bone marrow metastasis.

[0111] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. Application of a reagent for detecting the expression level of plasma exosomal protein LAMP2 in the preparation of a diagnostic product for diagnosing high-risk neuroblastoma.

2. The use according to claim 1, characterized in that The diagnostic product is a chip or a kit.

3. The use according to claim 2, characterized in that The diagnostic product may also include a negative control, a positive control, primers, probes or antibodies.

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