Serum biomarker for monitoring glioma prognosis by mass spectrometry and application thereof

Abstract

The invention discloses a serum biomarker for monitoring glioma prognosis through a mass spectrometry technology and application of the serum biomarker. The marker comprises at least one of CORO1A, H2AC20, H2BC21, HPX, IGHA1, IGHA2, JCHAIN and SNCA. The invention also discloses an application of the biomarker in preparation of a product for prognosis monitoring of tumors, and a detection method of the serum biomarker. According to the invention, a plurality of potential biomarkers are screened out, and important guiding significance can be provided for diagnosis and prognosis monitoring of high-grade glioblastoma. Molecular-level real-time dynamic information can be provided, and inflammation and tumor activity can be directly distinguished through specific molecules; analyzing an immune-tumor interaction mechanism; high-frequency non-invasive monitoring is realized, and an accurate intervention window is guided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a serum biomarker for monitoring prognosis of glioma by mass spectrometry and application thereof. BACKGROUND

[0002] Glioblastoma (GBM) is a common malignant tumor in the central nervous system, which has the characteristics of high invasiveness, wide infiltration and poor prognosis. The conventional surgical resection and postoperative radiotherapy and chemotherapy still cannot improve the poor prognosis, and it has the characteristics of high recurrence rate and mortality. CAR-T (Chimeric Antigen Receptor-T) cell immunotherapy has achieved good results in hematological tumors, providing a new direction for the treatment of solid tumors. Glioblastoma (GBM) chimeric antigen receptor T-cell (CAR-T) cell therapy provides new hope for improving the treatment effect of glioblastoma, but there is still a lack of biomarkers to monitor treatment response and evaluate efficacy.

[0003] At present, the clinical evaluation of CAR-T treatment for tumors mainly relies on magnetic resonance imaging (Magnetic resonance imaging, MRI) and CT imaging to evaluate disease progression and treatment effect. Although imaging (especially MRI) is still the gold standard for GBM prognosis evaluation, its sensitivity is insufficient, its specificity is limited, and its functional resolution is low when evaluating dynamic immunotherapy such as CAR-T. The main manifestations are: it is easy to misjudge the cytokine release syndrome (Cytoline release syndrome, CRS) or the extensive edema phenomenon caused by CAR-T induced immune cell infiltration as tumor progression; it cannot capture microscopic changes in the early stage (<1 week) of treatment; single imaging parameters are difficult to comprehensively evaluate tumor heterogeneity (such as coexistence of proliferation / immunity / metabolism) and the like.

[0004] Therefore, for CAR-T treatment of tumors, especially glioblastoma, there is still a lack of effective means for prognosis monitoring. SUMMARY

[0005] Non-targeted mass spectrometry is a comprehensive and high-throughput protein analysis method, which can simultaneously detect and quantify a large number of protein samples by mass spectrometry.

[0006] To solve the above technical problems, the purpose of the present application is to provide a serum biomarker for monitoring the prognosis of glioma by mass spectrometry technology and application, which is the first time to dynamically monitor the serum samples of patients with CAR-T treatment of glioblastoma, aiming to find serum biomarkers that can effectively judge the curative effect of CAR-T treatment.

[0007] The purpose of the present application is achieved by the following technical solutions: In a first aspect, the present application provides a biomarker for prognosis monitoring of tumors, comprising at least one of: CORO1A (crown protein 1A), H2AC20 (H2A group protein C20), H2BC21 (H2B group protein C21), HPX (hemoglobin binding protein), IGH A1 (immunoglobulin heavy constant alpha 1), IGH A2 (immunoglobulin heavy constant alpha 2), JCHAIN (immunoglobulin J chain), SNCA (alpha-synuclein).

[0008] As some specific embodiments of the present application, the biomarker comprises at least two of: CORO1A, H2AC20, H2BC21, HPX, IGH A1, IGH A2, JCHAIN, SNCA.

[0009] As some specific embodiments of the present application, the biomarker comprises: CORO1A, H2AC20, H2BC21, HPX, IGH A1, IGH A2, JCHAIN, SNCA.

[0010] As some specific embodiments of the present application, the biomarker is derived from a serum biological sample. Serum, as a sample easily obtained in clinical detection, can avoid trauma while monitoring treatment.

[0011] As some specific embodiments of the present application, the tumor comprises glioma.

[0012] As some specific embodiments of the present application, the glioma comprises glioblastoma.

[0013] As some specific embodiments of the present application, the prognosis monitoring comprises prognosis monitoring after immunotherapy of tumors by CAR-T therapy.

[0014] In a second aspect, the present application provides use of the biomarker as described in any one of the above in the preparation of a product for prognosis monitoring of tumors.

[0015] As some specific embodiments of the present application, the product comprises at least one of a kit, a detection card, a test strip, and a detection chip.

[0016] In a third aspect, the present application provides a method for detecting the biomarker as described in any of the above aspects, wherein the serum biomarker is detected by high performance liquid chromatography tandem mass spectrometry.

[0017] As some embodiments of the present application, the method for detecting comprises the following steps: S1, pretreating the serum sample; S2, desalting, washing, eluting and concentrating using a solid phase extraction column; S3, performing liquid chromatography tandem mass spectrometry analysis.

[0018] As some embodiments of the present application, in step S1, the pretreatment comprises: S11, mixing and incubating the serum sample with a surfactant solution; S12, sequentially adding dithiothreitol (DTT) and iodoacetamide (IAA) and incubating; S13, adding trypsin and incubating.

[0019] As some embodiments of the present application, in step S11, the surfactant is selected from any one of RapiGest, SDS or ProteaseMAX, preferably RapiGest; and / or, the temperature of the incubation is 30-70°C and the time is 30-60 min, and the protein is denatured by co-incubating the serum sample with the surfactant.

[0020] As some embodiments of the present application, in step S12, the temperature of the incubation is 37°C and the time is 30 min; dithiothreitol and iodoacetamide are added to perform reduction and alkylation reactions.

[0021] As some embodiments of the present application, in step S13, the temperature of the incubation is 35-40°C and the time is 1-18 h, and the proteolysis is completed.

[0022] As some embodiments of the present application, in step S2, the desalting step is performed using a solid phase extraction column to remove salt impurities; and / or, the washing liquid used for washing comprises 1-10% (v / v) methanol aqueous solution containing 0.1-5% (v / v) formic acid or 1-10% (v / v) acetonitrile aqueous solution containing 0.1-5% (v / v) formic acid, and weakly bound impurities are washed away by washing; and / or, the eluent used for elution is 60-100% (v / v) acetonitrile aqueous solution containing 0.1-5% (v / v) formic acid; and / or, the concentration comprises blow-drying under nitrogen or concentrating by vacuum centrifugation at room temperature.

[0023] As some specific embodiments of the present application, in step S3, a nanoliter liquid chromatography system Nano-LC system combined with a mass spectrometer is used to perform liquid chromatography tandem mass spectrometry analysis, and the sample is analyzed by liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) in a positive ion mode through an EASY nLC system and an Orbitrap fusion fluorescence mass spectrometer.

[0024] Compared with the prior art, the present application has the following beneficial effects: (1) The present application first detects the serum sample of a high-grade glioblastoma patient after CAR-T treatment by non-targeted mass spectrometry technology, analyzes the dynamic change pattern of proteins in the serum, and screens out a plurality of potential biomarkers, which can play an important guiding significance for the diagnosis and prognosis monitoring of high-grade glioblastoma.

[0025] (2) It can provide real-time dynamic information at the molecular level, and can directly distinguish inflammation and tumor activity through specific molecules; analyze the immune-tumor interaction mechanism (such as complement activation and inflammation pathway); realize high-frequency non-invasive monitoring, and guide precise intervention window. BRIEF DESCRIPTION OF DRAWINGS

[0026] Other features, objects and advantages of the present application will become more apparent through reading the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 It is a Wayne diagram of the number of proteins in serum at different time points after treatment; Figure 2 It is a GSEA enrichment analysis result graph of KEGG gene set; Figure 3 It is a GSEA enrichment analysis result graph of GO gene set; Figure 4 It is the number of differential proteins (log2 |FC|>1) at different time points in serum; Figure 5 It is a STEM analysis result graph of differential proteins in serum; Figure 6 It is the time pattern classification and functional annotation of differential expression proteins. DETAILED DESCRIPTION

[0027] The application will be described in detail below with specific embodiments. The following examples will help those skilled in the art to further understand the application, but do not limit the application in any form. It should be noted that for those skilled in the art, without departing from the concept of the application, a number of modifications and improvements can be made. These are within the scope of the present application.

[0028] Example 1 Patients with recurrent glioblastoma who underwent CAR-T treatment in the Fourth Affiliated Hospital of Suzhou University from November 1, 2023 to April 1, 2024 were selected as research subjects. The inclusion criteria included: (1) age > 18 years old; (2) KPS > 60; (3) previously biopsy or histopathologically confirmed high-grade glioma (WHO grade 4), after comprehensive treatment, imaging examination showed continued progression or recurrence; (4) expected survival time greater than 3 months; (5) no other central nervous system diseases and no other types of tumors, no coagulation dysfunction diseases, normal bone marrow reserve function and normal liver and kidney function. The qualified subjects were intracerebroventricularly or Ommaya sac CAR-T infusion, every 4 weeks as a treatment cycle, once on the first day of each cycle. CRS was graded according to the American Society of Transplantation and Cellular Therapy (ASTCT) consensus grading system, and all subjects developed grade 1-2 CRS on days 3 and 4 after treatment, with symptoms such as fever, headache, and vomiting. According to the selection criteria, 3 of the patients enrolled in this study showed persistent clinical benefit after CAR-T infusion, with a survival duration of more than 12 months. All patients were diagnosed with glioblastoma (WHO, grade 4), with an average age of 57.3 years (range 50-62 years).

[0029] Example 2 The day of CAR-T cell infusion was defined as day 0 (D0), and peripheral blood samples were collected 2 hours before the patient received CAR-T treatment, then every 24 hours, a total of 18 samples were collected. Peripheral blood samples were collected every 24 hours, i.e. on days 0, 1, 2, 3, 4, and 5 (D1-D5), for a total of 18 samples. 3 ml of peripheral blood was collected in a sterile collection tube, centrifuged at 3000 g for 15 minutes at 4°C, and the serum was collected and stored in a new sterile centrifuge tube for LC-MS / MS identification by Yuqing Technology (Shanghai) Co., Ltd.

[0030] Serum samples (10 μL) were treated with RapiGest surfactant solution to remove high abundant proteins, followed by incubation with DTT and IAA reduction alkylation. Trypsin digestion was performed at 37 °C overnight. The resulting peptides were desalted, washed, eluted, and concentrated using SPEC18 columns. The samples were analyzed using an EASY nLC system and an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) in positive ion mode.

[0031] Peptide fragments and proteins were identified from raw MS / MS data by QuestHT search engine using UniProt human protein database in Proteome Discoverer (Thermo Scientific, 2.4) software. The precursor m / z and fragment m / z tolerances were set to 10 ppm and 0.02 Da, respectively. Static modification was set to carbamidomethylation of cysteine, while dynamic modifications included oxidation of methionine, phosphorylation of serine, threonine, and tyrosine, and methylation of lysine, arginine, and glutamine.

[0032] The number of protein species detected at different time points was shown by a Venn diagram. Gene Set Enrichment Analysis (GSEA) was performed for all proteins, and |NSE| > 1, p < 0.05, FDR < 0.25 were considered to be significantly related.

[0033] Differentially expressed proteins at different time points were determined, and proteins detected only in a single patient were excluded. Time change pattern analysis was performed using Short Time-series Expression Miner (STEM) software, with log2 |Fold Change| > 1 as the change indicator and p < 0.05 as statistically significant. The differences in protein abundance in serum at different time points were compared. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) were used to annotate genes showing similar trends. The above statistical analysis was performed using the OmicShare platform (https: / / www.omicsmart.com).

[0034] Example 3 1. The Venn diagram showed that a total of 444 proteins were detected, of which 310 proteins were detected at all time points after treatment Figure 1 Among them, the most proteins were detected at D2 after treatment (n = 409), and the least proteins were detected at D5 (n = 356), which was relatively close to that before treatment (D0) (n = 415).

[0035] 2、GSEA results show that complement and coagulation cascade pathways are significantly enriched in serum ( Figure 2 ), including inflammatory factors such as C1QA, C2, SERPIND1, etc. In addition, the highest enrichment in the GO gene set is the extracellular matrix region ( Figure 3 ). Taking log2 |FC|>1 as the initial screening indicator of differential proteins, we found that the proteomic changes in serum were mainly concentrated in the 2nd, 4th and 5th days after treatment, with 209, 237 and 268 proteins down-regulated, respectively, and fewer up-regulated proteins ( Figure 4 ).

[0036] 3、STEM trend analysis results show that 3 of the 20 modules are significantly enriched, namely modules 0, 8 and 14. Among them, module 0 shows a linear decreasing trend, and the number of enriched proteins is the most ( Figure 5 ). Functional annotation and classification of proteins in different modules show that proteins in the three modules are significantly enriched in complement and coagulation cascade pathways, Staphylococcus aureus infection, cholesterol metabolism, systemic lupus erythematosus and other pathways closely related to immune inflammation. GO annotation results show that these proteins are mainly located in the extracellular region, with molecular functions including adhesion and binding, and are involved in biological processes such as immune defense ( Figure 6 ). It is worth noting that module 14 rapidly rises on the first day after treatment, and then there is a continuous fluctuation and decline, which is consistent with the clinical response of patients (all patients have varying degrees of fever on the first day, and the treatment response is concentrated on the 3rd and 4th days). These results further indicate that the differential proteins may show a non-linear change trend in the CAR-T treatment response process, and continuous dynamic monitoring is of great significance for immunotherapy.

[0037] 4、Further statistical analysis of the differential proteins in module 14 (p<0.05), proteins that show a "first rise and then fall" in all patients are selected as candidate markers, a total of 8 proteins, namely CORO1A (coronin 1A), H2AC20 (H2A group protein C20), H2BC21 (H2B group protein C21), HPX (hemoglobin binding protein), IGH A1 (immunoglobulin heavy constant alpha 1), IGH A2 (immunoglobulin heavy constant alpha 2), JCHAIN (immunoglobulin J chain), SNCA (alpha-synuclein). These proteins show similar time change patterns in all patients with persistent clinical benefit, and can provide some guidance for prognosis monitoring of CAR-T treatment of glioblastoma.

[0038] Example 4 - ROC analysis The results of ROC analysis are shown in Table 1 below, and the results in Table 1 show that the serum biomarkers of the application have good overall discrimination before and after treatment (AUC = 0.77 ± 0.10). These proteins are mainly involved in the process of inflammation / immune defense, showing good prognosis stratification ability.

[0039] Table 1 ROC analysis of serum candidate biomarkers

[0040] The application first detects the serum samples of patients with high-grade glioblastoma after CAR-T treatment through non-targeted mass spectrometry technology, analyzes the dynamic change pattern of proteins in the serum, and screens out a variety of potential biomarkers, which can play an important guiding significance in the diagnosis and prognosis monitoring of high-grade glioblastoma.

[0041] The specific embodiments of the application are described above. It should be understood that the application is not limited to the specific embodiments described above, and various modifications or changes can be made by those skilled in the art within the scope of the claims, which does not affect the essential content of the application.

Claims

1. A biomarker for prognosis monitoring of a tumor, characterized in that, Including at least one of the following: CORO1A, H2AC20, H2BC21, HPX, IGHA1, IGHA2, JCHAIN, SNCA.

2. The biomarker according to claim 1, characterized in that, The biomarkers are derived from serum biological samples.

3. The biomarker according to claim 1, characterized in that, The tumors include gliomas.

4. The biomarker according to claim 3, characterized in that, The gliomas include glioblastomas.

5. The biomarker according to claim 1, characterized in that, The prognostic monitoring includes prognostic monitoring after tumor immunotherapy via CAR-T therapy.

6. The use of a biomarker as described in any one of claims 1-5 in the preparation of a product for prognostic monitoring of tumors.

7. The application according to claim 6, characterized in that, The product includes at least one of the following: reagent kit, test card, test strip, and test chip.

8. A method for detecting a biomarker as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Pre-processing of serum samples; S2. Desalting, washing, elution and concentration are performed using a solid-phase extraction column; S3. Perform liquid chromatography-tandem mass spectrometry analysis.

9. The detection method according to claim 8, characterized in that, In step S1, the preprocessing includes: S11. Mix the serum sample with the surfactant solution and incubate. S12, add dithiothreitol and iodoacetamide sequentially and incubate; S13. Add trypsin and incubate.

10. The detection method according to claim 8, characterized in that, In step S2, the washing solution used for washing includes a 1-10% methanol aqueous solution containing 0.1-5% formic acid, or a 1-10% acetonitrile aqueous solution containing 0.1-5% formic acid. And / or, the eluent used for elution is a 60-100% aqueous solution of acetonitrile containing 0.1-5% formic acid; And / or, the concentration includes drying under nitrogen or concentration by vacuum centrifugation.

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