Application of plasma exosome protein LAMP2 as clinical diagnosis biomarker of neuroblastoma

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a biomarker for clinical diagnosis of high-risk neuroblastoma and application of the biomarker. The invention relates to a product for clinical diagnosis of high-risk neuroblastoma, the product comprises a reagent for detecting the expression level of a biomarker, and the biomarker is a plasma exosome protein LAMP2. By detecting the LAMP2 expression level, existing clinical indexes can be supplemented, accurate layering and accurate treatment of neuroblastoma are achieved, and meanwhile the kit has important guiding significance for prognosis judgment of tumor children.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an application of plasma exosome protein LAMP2 as a biomarker for 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 per child under 15 years old. The disease is characterized by insidious onset, high malignancy, and high risk of metastasis to the bone marrow, bones, and distant organs. It accounts for 15% of all childhood cancer deaths and is the "king of childhood tumors." In my country, more than 50% of children with NB have progressed to high-risk types at the time of their first visit, and the 5-year survival rate is less than 50%.

[0003] The survival rate of children with NB is significantly positively correlated with the early screening and diagnosis rate of the disease. The delayed age of first diagnosis of NB in ​​children is an important problem currently faced by the clinic, mainly due to the relatively delayed early screening of suspected cases. Therefore, exploring and discovering more sensitive and effective molecular markers for early screening of NB will further improve the diagnosis and treatment of children with NB.

[0004] Exosomes are a type of extracellular matrix microcapsules with a size ranging from 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, reach other cells and tissues through the circulatory system, and produce remote regulatory effects. As a vesicle structure, the biological substances carried by exosomes (such as DNA, RNA and proteins, etc.) have important biological significance. Many molecules have been proven to be involved in the occurrence of important diseases such as tumorigenesis, and can be used 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] The present invention uses proteomics technology to mine differentially expressed proteins in the plasma and plasma exosomes of children with NB, explores key regulatory molecules for the occurrence and development of NB tumors, and can be used as new molecular markers for 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 use of the diagnostic preparation includes: detecting the expression of LAMP2 in the plasma exosomes of a subject; comparing the detected value with the reference value, LAMP2 is significantly lower than the reference value, indicating that the subject suffers from high-risk neuroblastoma. The reference value level is the expression level of LAMP2 in the plasma exosomes of a normal child or a child with low-risk neuroblastoma. The subject is known or suspected to contain tumor cells.

[0009] In the present invention, the term LAMP2 is lysosomal associated membrane protein 2, including LAMP2 genes and proteins encoded therein and homologs, mutations and isoforms thereof. The term encompasses full-length, unprocessed LAMP2 and any form of LAMP2 derived from processing in cells. Preferably, the LAMP2 is a human protein with a UniProt ID of P13473.

[0010] In the present invention, the term "exosome" 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, reach other cells and tissues through the circulatory system, and produce remote regulatory effects. As a vesicle structure, the biological substances carried by exosomes (such as DNA, RNA, and proteins, etc.) have important biological significance. Many molecules have been proven 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 features. 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., genetic coding and / or epigenetic) is converted into structures that exist and operate in cells. Therefore, "expression" as used in the present invention may 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 that can be used for the clinical diagnosis of high-risk neuroblastoma 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 preparing 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 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, a primer, a probe or an antibody.

[0018] As used herein, the term "primer" refers to a single-stranded polynucleotide that is 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 (eg, 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, wherein the kit comprises 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 utilizes proteomics technology to mine differentially expressed proteins in the plasma and plasma exosomes of children with NB, and utilizes LAMP2 protein expression levels to identify high-risk NB. This method 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 was lowly expressed in the exosomes of high-risk NB and negatively correlated with clinical progression; in the plasma exosomes of children with newly diagnosed NB, the low expression of LAMP2 was 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative work.

[0026] Figure 1 Shown is the screening process for LAMP2, a differentially expressed protein in plasma exosomes of childhood neuroblastoma.

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

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

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

[0030] C. The overall survival rate of children with low expression of LAMP2 in tumor tissue was poor (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 LAMP2 protein expression level in the validation set was used to divide NB into high-risk and low-risk groups. DETAILED DESCRIPTION

[0033] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to 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 pre-treatment risk of patients 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 pre-treatment risk stratification of NB by the International NB Risk Group (INRG), 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, any primary tumor is accompanied by distant lymph nodes, bone marrow, liver, skin and / or other organ dissemination (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 protein LAMP2 in plasma exosomes of children with neuroblastoma

[0041] 1.1 Extraction of exosomes from plasma

[0042] In the early stage of this invention, 30 plasma samples were collected from high-risk NB children and low-risk NB children who did not undergo surgical resection of the tumor and chemotherapy. At the same time, 30 plasma samples from children with non-tumor and non-infectious diseases that have no potential impact on plasma components (such as tongue tie, strabismus, phimosis, scoliosis, etc.) were collected as a healthy control group. All samples were extracted from plasma exosomes using Invitrogen's Total Exosome Isolation (from plasma) kit (Cat. No.: 4484450), and 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 plasma, and store in a -80°C refrigerator;

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

[0045] (3) Centrifuge 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 to mix for at least 30 seconds;

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

[0049] (7) Leave at room temperature for 10 min;

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

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

[0053] (11) Take 1 / 3 of the exosome sample and freeze it directly 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 taken 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, 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, and react in the dark for 45 min.

[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 20 mM Tris, vortex 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, flick the liquid onto the membrane, place the test tube on a float in a beaker of tap water, microwave on high for 1 min, twice, changing to tap water in between, and incubate in a 37°C water bath overnight.

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

[0067] (9) The peptide concentration was determined by the BCA method.

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

[0069] 2 μg of peptide was taken from each sample and mixed 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 a 1.5 μl / min flow rate UPLC system UltiMate 3000. Buffer: Solution A was 0.1% formic acid in water, and solution B was 0.1% formic acid in acetonitrile (80% acetonitrile). The peptide sample directly entered the chromatographic analysis column (length: 50 cm, inner diameter: 75 μm) for mass spectrometry detection.

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

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

[0073] Detection mode: positive ion, primary mass spectrometry scanning 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] Spectronaut Pulsar (18.0) software was used for mass spectrometry data processing, and the database used was Swiss Prothuman database (20,386 sequences, 2022.6 released). The search method was direct search. Qualitative and quantitative parameter settings: Precursor Qvalue: 0.01, Protein Qvalue: 0.01, quantification based on peak area MS1 level, correction method: Cross-Run Normalization, quantification method: MaxLFQ, Inference Algorithm: IDPicker.

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

[0077]

[0078] As shown in the table above, among many proteins, LAMP2 was a protein with reduced expression in the three groups, so 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 that the P value was <0.001, indicating that LAMP2 was lowly expressed in tumor tissues of high-risk NB children. Figure 2 -A;

[0083] The ROC analysis of LAMP2 gene expression was performed based on the binary classification of the patient's survival outcome (death or not). The point with the largest Youden index (sensitivity + specificity - 1) was selected as the cutoff value, and the children's data were divided into two groups: 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 of children with low LAMP2 expression in tumor tissue was even lower (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 that 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: Using ELISA to verify the expression of LAMP2 in the validation set samples

[0087] The present invention also collected plasma samples as a validation set for 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 validation.

[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 solution A and substrate solution B at a ratio of 1:1 by volume and use within 15 minutes of mixing; remove the pre-coated ELISA 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 the standard wells, 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 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 substrate A and substrate B in a 1:1 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 450 nm microplate reader.

[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 create a standard curve equation using four-parameter logistic curve fitting (4-pl). 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 figure, the median expression of LAMP2 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 the low-risk group, the t-test result P value was <0.05, indicating that the expression of LAMP2 in the high-risk group was significantly reduced.

[0098] Thus, it was demonstrated that the expression level of LAMP2 was significantly reduced in children with high-risk NB.

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

[0100] ROC curve was drawn based on LAMP2 protein expression level to identify high-risk NB or low-risk NB. The results are as follows Figure 4 As shown, the AUC value of the obtained 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 median expression level of LAMP2 in Example 4, 0.107, was used as the cutoff value to divide the patients into a LAMP2 high expression group and a low expression group. 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 expression of plasma exosomal LAMP2, the proportion of children in M ​​stage was significantly higher than that in non-M stage (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 expression of plasma exosome LAMP2, the proportion of children with large primary tumor volume was significantly higher than that of children with small primary tumor volume (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). All of the above prove that low expression of LAMP2 in NB plasma exosomes is positively correlated with the high malignancy and clinical progression of NB.

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

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

Claims

1. Use of plasma exosome protein LAMP2 as a biomarker in the preparation of a diagnostic preparation for high-risk neuroblastoma, characterized in that: The expression level of plasma exosomal protein LAMP2 was significantly reduced in high-risk neuroblastoma.

2. Application of reagents for detecting the expression level of plasma exosomal protein LAMP2 in the preparation of diagnostic products for diagnosing high-risk neuroblastoma.

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

4. The use according to claim 3, characterized in that: The diagnostic product also includes a negative control, a positive control, a primer, a probe or an antibody.

5. A kit for diagnosing high-risk neuroblastoma, characterized in that: The kit comprises a detection reagent for detecting the expression level of the plasma exosome protein LAMP2 according to claim 2.

6. The kit according to claim 5, characterized in that The detection reagents also include negative controls, positive controls, primers, probes or antibodies.

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