Group of urine metabolites for diagnosing brain stem glioma H3K27M gene mutation state

Abstract

The invention discloses a group of urine metabolites for diagnosing BSG (brain stem glioma) H3K27M gene mutation states, urine of BSG patients is detected through non-targeted LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry), metabolites related to the H3K27M gene mutation states are screened, and content differences of Nomilin, Lys-Leu (lysine-leucine) and Hawkinsin in the BSG patients with different H3K27M mutation states are found to be obvious; further verifying the content change of the H3K27M gene mutation related metabolite in the urine of the BSG patient by using targeted LC-MS / MS; the research confirms that the combination of Nomilin, lysine-leucine (Lys-Leu) and Hawkinsin in the urine can be used for predicting the gene mutation state of the brain stem glioma H3K27M, and the gene mutation state of the brain stem glioma H3K27M can be used for predicting the gene mutation state of the brain stem glioma H3K27M.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a group of urinary metabolites for diagnosing the H3K27M gene mutation status in brainstem gliomas. Background Technology

[0002] Brainstem gliomas (BSGs) are gliomas located in the midbrain, pons, and medulla oblongata. Surgical resection is quite challenging due to the vital functions of the brainstem. Furthermore, BSGs often carry a somatic gain-of-function mutation called the H3K27M mutation, which results in the substitution of methionine for lysine at position 27 of histone H3, leading to a highly malignant phenotype. [1] Clinically, BSGs with the H3K27M mutation are among the most deadly malignant brain tumors, with a median overall survival of 10-12 months and a two-year survival rate of less than 10%, due to the limited effectiveness of current treatments, including surgery, radiotherapy, and chemotherapy. In contrast, BSG patients without this gene mutation generally have a much better prognosis, with a median overall survival of approximately five years after surgical resection plus radiotherapy and / or chemotherapy. Therefore, early identification of mutation status in H3 is crucial for clinical decision-making, such as treatment selection and prognostic prediction.

[0003] Previous reports have indicated that H3K27M mutations are associated with tumor location and patient age, but these clinical information alone cannot accurately predict H3K27M mutation status. [2][3] Currently, the conventional method for determining mutation status still relies on invasive brain biopsies or surgical resection.

[0004] With advancements in next-generation sequencing technology, liquid biopsy strategies, specifically detecting circulating tumor DNA (ctDNA) in cerebrospinal fluid (CSF), have proven effective in predicting H3K27M mutation status. [4][5] However, lumbar puncture for CSF collection is not always feasible due to the risk of high intracranial pressure. Radiomics-based methods using magnetic resonance imaging (MRI) have shown potential for predicting H3K27M mutation status. [6] However, radiomics models based on conventional MRI sequences are not always reliable, and models with high diagnostic performance require specialized MRI sequences. [7][8] This limits its clinical application. We also investigated the relationship between 11C-methionine positron emission tomography (PET) imaging features and the H3K27M mutation, but were unable to establish a connection. [9] However, the vital functions of the brainstem significantly limit the application of these methods and hinder repeated testing for purposes such as therapeutic monitoring. Therefore, the development of minimally invasive or non-invasive methods is urgently needed.

[0005] Metabolic dysregulation is a hallmark of cancer, playing a crucial role in the development and progression of most malignant tumors, including gliomas and brainstem gliomas (BSGs). H3K27M mutations are also deeply involved in the metabolic reprogramming of BSG cells, maintaining a low H3K27me3 state, leading to tumor initiation and progression. Furthermore, mounting evidence suggests that tumors not only induce local metabolic changes within tumor tissue but also trigger systemic metabolic alterations. These systemic changes can be detected in plasma / serum or urine using mass spectrometry-based metabolomics analysis, holding great potential for identifying non-invasive biomarkers for molecular diagnostics and treatment monitoring. Therefore, the identification of metabolite biomarkers in blood and urine has received considerable attention in cancer research. In gliomas, many metabolites distinct from those in patients and healthy controls have been identified in blood or urine samples. Some metabolites have demonstrated high diagnostic accuracy in differentiating glioma samples from healthy control samples, showing the feasibility of using metabolomics analysis in blood and urine for glioma diagnosis. However, these studies primarily involve gliomas located in the supratentorial brain region and are not specific to BSGs.

[0006] However, there are currently no studies on the impact of H3K27M mutations on blood or urine metabolomics analysis or on identifying non-invasive metabolite biomarkers for predicting H3K27M mutations.

[0007] References:

[0008] [1] Wu G, Broniscer A, McEachron TA et al. Somatic histone H3 alterations in pediatric intrinsic pontine gliomas and non-brainstemglioblastomas. Nat Genet. 2012; 44(3): 251-253.

[0009] [2]Wang Y, Pan C, Xie M et al. Adult diffuse intrinsic pontine glioma: clinical, radiological, pathological, molecular features, and treatments of96patients. J Neurosurg. 2022; 137(6):1628-1638.

[0010] [3]Chen LH,Pan C,Diplas BH et al.The integrated genomic andepigenomic landscape of brainstem glioma.Nat Commun.2020;11(1):3077

[0011] [4]Pan C,Diplas BH,Chen X et al.Molecular profiling of tumors of thebrainstem by sequencing of CSF-derived circulating tumor DNA.ActaNeuropathol.2019;137(2):297-306.

[0012] [5]Pentsova EI,Shah RH,Tang J et al.Evaluating Cancer of the CentralNervous System Through Next-Generation Sequencing of Cerebrospinal Fluid.JClin Oncol.2016;34(20):2404-15.

[0013] [6]Pan C,Liu J,Tang J et al.A machine learning-based prediction modelof H3K27M mutations in brainstem gliomas using conventional MRI and clinicalfeatures.Radiother Oncol.2019;130:172-179.

[0014] [7]Zhuo Z,Qu L,Zhang P et al.Prediction of H3K27M-mutant brainstemglioma by amide proton transfer-weighted imaging and its derivedradiomics.Eur J Nucl Med Mol Imaging.2021;48(13):4426-4436.

[0015] [8]Yang N, Xiao

[0016] [9]Zhao Summary of the Invention

[0017] To address the challenges in diagnosing H3K27M gene mutation status in brainstem gliomas, this application utilizes non-targeted LC-MS / MS to detect H3K27M gene mutation-related metabolites in the urine of BSG patients. The study revealed significant differences in the levels of nomilin, lysine-leucine (Ly-Leu), and Hawkinsin among BSG patients with different H3K27M mutation statuses. Targeted LC-MS / MS mass spectrometry was used to further validate the changes in the levels of H3K27M gene mutation-related metabolites in the urine of BSG patients. The study confirmed that the combination of nomilin, lysine-leucine (Ly-Leu), and Hawkinsin in urine can be used to predict the H3K27M gene mutation status in brainstem gliomas. To achieve the above objectives, the specific technical solution of this invention is as follows:

[0018] Firstly, urinary metabolites are detected and metabolites related to the H3K27M gene mutation status in BSG patients are screened.

[0019] Urine samples from 112 untreated, newly diagnosed brainstem glioma (BSG) patients (72 with H3K27M mutation and 40 with H3K27M wild-type) were analyzed using non-targeted liquid chromatography-tandem mass spectrometry (LC-MS / MS). Data-dependent acquisition (DDA), a non-targeted mass spectrometry acquisition method, was used to detect and screen for metabolites related to the H3K27M gene mutation status in BSG patients.

[0020] Results: A total of 166 differentially expressed metabolites were screened from urine. Further screening revealed that the levels of nomilin, lysine-leucine, and Hawkinsin differed significantly and statistically in BSG patients with different H3K27M mutation states.

[0021] Secondly, targeted LC-MS / MS mass spectrometry analysis was used to further verify the changes in the content of H3K27M gene mutation-related metabolites in the urine of BSG patients.

[0022] The urine of 94 untreated newly diagnosed brainstem glioma patients (55 H3K27M mutant and 39 H3K27M wild-type) was further analyzed using targeted LC-MS / MS mass spectrometry to verify the changes in the content of H3K27M gene mutation-related metabolites in the urine of BSG patients.

[0023] Results: Compared with patients with wild-type brainstem glioma H3K27M, patients with H3K27M gene mutations had upregulated levels of nomilin, lysine-leucine, and Hawkinsin in their urine.

[0024] Thirdly, the diagnostic efficacy of individual combinations of nomiline, lysine-leucine, and hopkinsin in predicting H3K27M gene mutation status in patients with brainstem gliomas (BSGs) was evaluated.

[0025] Urine samples were collected from 94 newly diagnosed, untreated patients with brainstem gliomas (55 H3K27M mutants and 39 H3K27M wild-types) at Beijing Tiantan Hospital. The samples were randomly assigned to the training and testing sets at a ratio of 2:1.

[0026] The levels of nomilin, lysine-leucine, and Hawkinsin in urine were determined by targeted LC-MS / MS mass spectrometry analysis and corrected for creatinine.

[0027] The diagnostic efficacy of nomiline, lysine-leucine, and hopkinsin alone in predicting H3K27M gene mutation status in patients with brainstem gliomas (BSGs) was evaluated, with ROC-AUC values ​​of 0.7119, 0.6275, and 0.6345, respectively.

[0028] Establish a combined model of nomiline, lysine-leucine and hopkinsin, and evaluate the diagnostic efficacy of the combined model of nomiline, lysine-leucine and hopkinsin;

[0029] Results: The ROC-AUC values ​​for the training and test sets were 0.7457 and 0.7449, respectively.

[0030] Conclusion: The combined model of nomiline, lysine-leucine and hopkinsin can be used to predict the H3K27M gene mutation status in patients with brainstem gliomas (BSGs).

[0031] Compared with the prior art, the beneficial effects of this application are as follows:

[0032] 1. This application is the first to discover that, compared with patients with wild-type H3K27M brainstem gliomas, patients with H3K27M gene mutations have upregulated levels of nomilin, lysine-leucine, and Hawkinsin in their urine;

[0033] 2. For the first time, a combination of biomarkers in urine samples was used to predict the H3K27M gene mutation status in brainstem gliomas. Attached Figure Description

[0034] Figure 1 PCA classification diagram of urine metabolomics in patients with brainstem gliomas;

[0035] Figure 2 Non-targeted mass spectrometry acquisition method, differential expression map of nomilin, lysine-leucine and Hawkinsin in urine samples between H3K27M mutant (MUT) patients and H3K27M wild-type (WT) patients;

[0036] Figure 3 Targeted mass spectrometry analysis: differential expression of nomilin, lysine-leucine, and Hawkinsin in urine samples between H3K27M mutant (MUT) and wild-type (WT) patients;

[0037] Figure 4 , three ROC curves of individual metabolites predicting H3K27M mutation status in brainstem gliomas;

[0038] Figure 5 , three ROC curves for predicting the H3K27M mutation status of brainstem gliomas using combinations of metabolites are shown, where Trainingset is the training set and Testset is the test set. Detailed Implementation

[0039] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0041] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0042] Reagents and Materials

[0043]

[0044] instrument

[0045]

[0046] Example 1: Detection of urinary metabolites and screening for metabolites related to H3K27M gene mutation status in BSG patients

[0047] Using non-targeted liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis and data-dependent acquisition (DDA) as a non-targeted mass spectrometry acquisition method, urinary metabolites were detected and metabolites related to the H3K27M gene mutation status in BSG patients were screened.

[0048] I. Methods

[0049] (I) Sample

[0050] Urine samples were collected from 112 untreated, newly diagnosed brainstem glioma patients (72 with H3K27M mutation and 40 with H3K27M wild type), and plasma samples were also collected from the patients. The patients were divided into two groups according to mutation type for the experiment. The samples were obtained from Beijing Tiantan Hospital.

[0051] All enrolled patients subsequently underwent surgery or puncture biopsy to obtain tumor tissue. The tumor tissue was then used for IHC staining to determine the H3K27M mutation status, which is a common clinical diagnostic method. Patients who are H3K27M positive and H3K27me3 negative are considered mutant, while those who are not are considered wild-type. For those who do not match, gene sequencing was performed to determine the H3K27M mutation type.

[0052] (II) Collection of human urine samples and extraction of metabolites

[0053] Urine samples were collected using sterile centrifuge tubes and centrifuged at 4000×g for 15 minutes at 4°C to remove precipitates. The supernatant was collected, and 200 μL of the sample was added to 200 μL of acetonitrile, vortexed for 30 seconds, and then frozen at -40°C for 30 minutes. The mixture was centrifuged at 14,000×g for 10 minutes. The resulting supernatant was collected, vacuum dried, and stored at -80°C. Before analysis, the sample was reconstituted with 2% acetonitrile and filtered through a 10 kDa molecular weight cutoff ultracentrifuge filter before being transferred to an autosampler as the test sample.

[0054] (III) LC-MS / MS analysis of the sample to be tested

[0055] 1. Chromatographic conditions:

[0056] Column: Waters HSS C18, 1.8 μm, 3.0 mm × 100 mm;

[0057] mobile phase

[0058] Mobile phase A: composed of 0.1% formic acid and 99.9% water;

[0059] Mobile phase B: Acetonitrile;

[0060] Flow rate: 0.5 mL / min;

[0061] Injection volume: 10 μL;

[0062] Column temperature: 45℃;

[0063] Gradient elution, elution procedure:

[0064]

[0065] ★The percentages of mobile phase A and mobile phase B are volume percentages.

[0066] 2. Mass spectrometry conditions

[0067] The scan range is 100-1000 m / z, with a resolution of 60 K. The automatic gain control (AGC) target is set to 1×10⁻⁶. 6 The maximum injection time is 100 ms. The high-energy collisional dissociation (HCD) fragmentation settings are 20, 35, and 60.

[0068] 3. Mass spectrometry data analysis:

[0069] For label-free metabolomics analysis, raw data files were processed using Progenesis QI software (Waters, Milford, MA, USA). Data were matched against the HMDB and Metlin databases. Subsequently, data were preprocessed using the MetaboAnalyst (6.0) metabolomics analysis platform (www.metaboanalyst.ca), including missing value estimation, median normalization, and logarithmic transformation to enhance the comparability of features. Variables with missing values ​​in 50% of the samples were removed and not subjected to further statistical analysis.

[0070] II. Results

[0071] like Figure 1 As shown, patients with the H3K27M mutant (MUT) and those with the H3K27M wild-type (WT) exhibited a high degree of differentiation. Differential metabolites were defined as those with p < 0.05 and a fold change (FC) > 1.5; a total of 166 differential metabolites were identified in the urine.

[0072] like Figure 2 As shown, further screening revealed that the levels of nomilin, lysine-leucine (Lys-Leu), and Hawkinsin differed significantly among BSG patients with different H3K27M mutation states. Compared to H3K27M wild-type patients, the expression of nomilin, Lys-Leu, and Hawkinsin in the urine of H3K27M mutant patients was elevated and statistically significant.

[0073] Example 2: Further verification of the changes in the content of H3K27M gene mutation-related metabolites in the urine of BSG patients using targeted LC-MS / MS mass spectrometry analysis.

[0074] I. Sample

[0075] Urine samples were collected from 94 newly diagnosed, untreated patients with brainstem gliomas (55 H3K27M mutants and 39 H3K27M wild-type), along with their urinary creatinine levels. The samples were obtained from Beijing Tiantan Hospital.

[0076] All enrolled patients subsequently underwent surgery or puncture biopsy to obtain tumor tissue. The tumor tissue was then used for IHC staining to determine the H3K27M mutation status, which is a common clinical diagnostic method. Patients who are H3K27M positive and H3K27me3 negative are considered mutant, while those who are not are considered wild-type. For those who do not match, gene sequencing was performed to determine the H3K27M mutation type.

[0077] II. Methods

[0078] (I) Preparation of standard curve samples

[0079] 1. Prepare the standard curve working solution

[0080] Take Nomilin reference standard and dissolve it in double-distilled water to prepare a 250 μmol / L Nomilin standard curve working solution;

[0081] Take Lys-Leu reference standard and dissolve it in double-distilled water to prepare a 250 μmol / L Lys-Leu standard curve working solution;

[0082] Prepare a 250 μmol / L Hawkinsin standard curve working solution by dissolving Hawkinsin reference standard in double-distilled water;

[0083] 2. Prepare standard curve samples

[0084] Double-distilled water and the working solution of the Nomilin standard curve were mixed in an appropriate ratio to prepare Nomilin standard curve samples. The concentrations of Nomil in the Nomilin standard curve samples were 0.061, 0.244, 0.977, 3.906, 15.625, 62.5, and 250 μmol / L, respectively.

[0085] Double-distilled water and Lys-Leu standard curve working solution were mixed in an appropriate ratio to prepare Lys-Leu standard curve samples. The concentrations of Lys-Leu in the Lys-Leu standard curve samples were 0.061, 0.244, 0.977, 3.906, 15.625, 62.5, and 250 μmol / L, respectively.

[0086] Double-distilled water and Hawkinsin standard curve working solution were mixed in an appropriate ratio to prepare Hawkinsin standard curve samples. The concentrations of Hawkinsin in the Hawkinsin standard curve samples were 0.061, 0.244, 0.977, 3.906, 15.625, 62.5, and 250 μmol / L, respectively.

[0087] (II) Collection of urine samples and extraction of metabolites from patients with brainstem gliomas

[0088] Urine samples from patients with brainstem gliomas were collected using sterile centrifuge tubes. The samples were centrifuged at 4000×g for 15 minutes at 4°C to remove precipitates. The supernatant was collected, and 200 μL of the sample was added to 200 μL of acetonitrile, vortexed for 30 seconds, and then frozen at -40°C for 30 minutes. The mixture was centrifuged at 14,000×g for 10 minutes. The resulting supernatant was collected, vacuum dried, and stored at -80°C.

[0089] Before analysis, the vacuum-dried and stored sample was reconstituted with 2% acetonitrile, filtered through a 10kDa molecular weight cutoff ultracentrifugal filter, and then transferred to an autosampler to obtain the sample to be tested.

[0090] (III) Targeted LC-MS / MS analysis of the test samples and standard curve samples.

[0091] 1. Chromatographic conditions

[0092] Column: Waters HSS C18, 1.8 μm, 3.0 mm × 100 mm;

[0093] mobile phase

[0094] Mobile phase A: composed of 0.1% formic acid and 99.9% water;

[0095] Mobile phase B: composed of 0.1% formic acid and 99.9% acetonitrile;

[0096] Flow rate: 0.4 mL / min;

[0097] The contents of Nomilin, Lys-Leu and Hawkinsin in the test sample were measured. The test sample and the standard curve sample were injected separately, and the injection volume of each sample was 10 μL.

[0098] Column temperature: 45℃;

[0099] Gradient elution, elution procedure:

[0100]

[0101] ★The percentages of mobile phase A and mobile phase B are volume percentages.

[0102] 2. Mass spectrometry conditions

[0103]

[0104]

[0105] 3. Detection parameters

[0106]

[0107] (iv) Quantitative analysis of the concentrations of Nomilin, Lys-Leu, and Hawkinsin in the test sample was performed using the external standard-standard curve method.

[0108] A linear regression was performed with the mass spectrometric abundance of Nomil in the Nomil standard curve samples as the ordinate and the concentration of Nomil in the Nomil standard curve samples as the abscissa to obtain the Nomil standard curve. The concentration of Nomil in the sample to be tested was then calculated by substituting the mass spectrometric abundance of Nomil into the Nomil standard curve.

[0109] A linear regression was performed with the mass spectrometric abundance of Lys-Leu in the Lys-Leu standard curve sample as the ordinate and the concentration of Lys-Leu in the Lys-Leu standard curve sample as the abscissa to obtain the Lys-Leu standard curve. The concentration of Nomilin in the sample was then calculated by substituting the mass spectrometric abundance of Lys-Leu in the test sample into the Lys-Leu standard curve.

[0110] A linear regression was performed with the mass spectrometric abundance of Hawkinsin in the Hawkinsin standard curve sample as the ordinate and the concentration of Hawkinsin in the Hawkinsin standard curve sample as the abscissa to obtain the Hawkinsin standard curve. The mass spectrometric abundance of Hawkinsin in the sample to be tested was then substituted into the Hawkinsin standard curve to calculate the concentration of Hawkinsin in the sample to be tested.

[0111] III. Results

[0112] The raw data files were processed using Analyst 1.6.2 to obtain the mass spectrometry abundance values ​​of Nomilin, Lys-Leu, and Hawkinsin;

[0113] The mass spectrometry abundance values ​​of Nomilin, Lys-Leu, and Hawkinsin in the test sample were converted into concentration values ​​using a standard curve, corrected using the corresponding creatinine concentration values ​​in the test sample, and then logarithmically transformed for further statistical analysis.

[0114] The creatinine content in urine was tested by our hospital's laboratory using a creatinine assay kit (sarcosine oxidase method) and an instrument: Beckman Coulter AU5800 fully automated biochemical analyzer.

[0115] Statistical results are as follows Figure 3 As shown, compared with patients with wild-type brainstem glioma H3K27M, patients with H3K27M gene mutations had upregulated levels of nomilin, lysine-leucine, and Hawkinsin in their urine.

[0116] Example 3: Diagnostic efficacy of the combination of nomiline, lysine-leucine, and hopkinsin in predicting H3K27M gene mutation status in patients with brainstem gliomas (BSGs).

[0117] I. Sample

[0118] Urine samples from 94 newly diagnosed, untreated patients with brainstem gliomas (55 H3K27M mutants and 39 H3K27M wild-type) were collected from Beijing Tiantan Hospital and randomly assigned to the training and test sets at a ratio of 2:1.

[0119] II. Quantitative determination of the contents of nomiline, lysine-leucine and hopkinsin

[0120] The levels of nomilin, lysine-leucine, and Hawkinsin in urine were analyzed by targeted LC-MS / MS mass spectrometry (corrected for creatinine), as described in Example 2.

[0121] The creatinine content in urine was tested by our hospital's laboratory using a creatinine assay kit (sarcosine oxidase method) and an instrument: Beckman Coulter AU5800 fully automated biochemical analyzer.

[0122] III. Diagnostic efficacy of nomiline, lysine-leucine, and hopkinsin alone in predicting H3K27M gene mutation status in patients with brainstem gliomas (BSGs)

[0123] The sensitivity and specificity of three urinary metabolites—nomilin, lysine-leucine, and hormonin—in predicting H3K27M gene mutation status in patients with brainstem gliomas (BSGs) are shown in Table 1. Figure 4 .

[0124] Table 1. Nomiline, lysine-leucine, and hopkinin as biomarkers of H3K27M gene mutation status

[0125] Sensitivity Specificity AUC Nomilin 78.18％ 53.85％ 0.7119 Lysine-Leucine 52.73％ 74.36％ 0.6275 Hawkinsin 60.00％ 69.23％ 0.6345

[0126] From Table 1 and Figure 4 The results showed that the three urinary metabolites, nomiline, lysine-leucine, and hopkinsin, had slightly poor sensitivity and specificity in predicting the H3K27M gene mutation status in patients with brainstem gliomas (BSGs) when used alone.

[0127] IV. Diagnostic efficacy of the combination of nomiline, lysine-leucine, and hopkinsin in predicting H3K27M gene mutation status in patients with brainstem gliomas (BSGs)

[0128] (I) Combination model of nomiline, lysine-leucine and hormonin

[0129] A combined model of nomiline, lysine-leucine, and hopkinsonine was established using binary logistic regression, as follows:

[0130] Model score = -2.720 × Log10 (Nomilin / Creatine)-0.120×Log 10 (Lys-Leu / Creatine)-0.440×Log 10 (Hawkinsin / Creatine) - 18.07

[0131] in the formula

[0132] Nomilin: refers to the content of nomilin, unit: μmol / L;

[0133] Creatine: refers to the content of creatinine, unit: μmol / L;

[0134] Lys-Leu: refers to the content of lysine-leucine (Lys-Leu), unit: μmol / L;

[0135] Hawkinsin: refers to Hawkinsin, unit: μmol / L.

[0136] Result determination

[0137] A model score ≤0 indicates an H3K27M gene mutation status.

[0138] A model score > 0 indicates the wild state of the H3K27M gene.

[0139] (II) Diagnostic efficacy of the combined model of nomiline, lysine-leucine and hookinin

[0140] The combined model of nomiline, lysine-leucine, and hopkinsin was analyzed using the R package "pROC" (version 1.15.0) to perform receiver operating characteristic (ROC) analysis. The area under the curve (AUC) was calculated to evaluate the accuracy, sensitivity, and specificity of the combined model in predicting the H3K27M gene mutation status in patients with brainstem gliomas (BSGs).

[0141] See results Figure 5 ,

[0142] The sensitivity, specificity, and ROC-AUC of the training set were 75.00%, 53.85%, and 0.7457, respectively.

[0143] The sensitivity, specificity, and ROC-AUC of the test set were 52.63%, 76.92%, and 0.7449, respectively.

[0144] Conclusion: The combined model of nomiline, lysine-leucine and hopkinsin can be used to predict the H3K27M gene mutation status in patients with brainstem gliomas (BSGs).

[0145] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The application of a group of metabolite compositions from the urine of brainstem glioma patients in the preparation of reagents and chips for predicting H3K27M mutation status, characterized in that, The metabolite composition is Nomilin, Lys-Leu, and Hawkinsin.

2. The application according to claim 1, characterized in that, The combinatorial model of the metabolite composition is as follows: Model score = -2.720 × Log 10 (Nomilin / Creatine)-0.120×Log 10 (Lys-Leu / Creatine)-0.440×Log 10 (Hawkinsin / Creatine) - 18.07 in the formula Nomilin: refers to the content of nomilin, unit: μmol / L; Creatine: refers to the content of creatinine, unit: μmol / L; Lys-Leu: refers to the content of lysine-leucine (Lys-Leu), unit: μmol / L; Hawkinsin: refers to Hawkinsin, unit: μmol / L; Result determination A model score ≤0 indicates an H3K27M gene mutation status. A model score > 0 indicates the wild state of the H3K27M gene.

3. The application according to claim 1, characterized in that, Quantitative detection of Nomilin, Lys-Leu, and Hawkinsin was performed using targeted LC-MS / MS mass spectrometry.

4. The application according to claim 3, characterized in that, The method for targeted LC-MS / MS mass spectrometry analysis is as follows: (1) Preparation of standard curve samples A. Preparation of standard curve working solution Take Nomil reference standard and dissolve it in double-distilled water to prepare a 250 μmol / L Nomil standard curve working solution; Take Lys-Leu reference standard and dissolve it in double-distilled water to prepare a 250 μmol / L Lys-Leu standard curve working solution; Prepare a 250 μmol / L Hawkinsin standard curve working solution by dissolving Hawkinsin reference standard in double-distilled water; B. Prepare standard curve samples Double-distilled water and the working solution of the Nomilin standard curve were mixed in an appropriate ratio to prepare Nomilin standard curve samples. The concentrations of Nomil in the Nomilin standard curve samples were 0.061, 0.244, 0.977, 3.906, 15.625, 62.5, and 250 μmol / L, respectively. Double-distilled water and Lys-Leu standard curve working solution were mixed in an appropriate ratio to prepare Lys-Leu standard curve samples. The concentrations of Nomilin in the Lys-Leu standard curve samples were 0.061, 0.244, 0.977, 3.906, 15.625, 62.5, and 250 μmol / L, respectively. Double-distilled water and Hawkinsin standard curve working solution were mixed in an appropriate ratio to prepare Hawkinsin standard curve samples. The concentrations of Hawkinsin in the Hawkinsin standard curve samples were 0.061, 0.244, 0.977, 3.906, 15.625, 62.5, and 250 μmol / L, respectively. (2) Collection of urine samples and extraction of metabolites from patients with brainstem gliomas Urine samples from patients with brainstem gliomas were collected using sterile centrifuge tubes and centrifuged at 4000×g for 15 minutes at 4°C to remove urine sediment. Collect the supernatant, add the sample (200 μL) to acetonitrile (200 μL), vortex for 30 seconds, and then freeze at -40°C for 30 minutes; centrifuge the mixture at 14,000 × g for 10 minutes; collect the obtained supernatant, vacuum dry it and store it at -80°C; Before analysis, the vacuum-dried and stored sample was reconstituted with 2% acetonitrile, filtered through a 10kDa molecular weight cutoff ultracentrifugal filter, and then transferred to an autosampler to obtain the sample to be tested. (3) Targeted LC-MS / MS analysis of the test sample and the standard curve sample. Chromatographic conditions Column: Waters HSS C18, 1.8 μm, 3.0 mm × 100 mm; mobile phase Mobile phase A: composed of 0.1% formic acid and 99.9% water; Mobile phase B: composed of 0.1% formic acid and 99.9% acetonitrile; Flow rate: 0.4 mL / min; The contents of Nomilin, Lys-Leu and Hawkinsin in the test sample were measured. The test sample and the standard curve sample were injected separately, and the injection volume of each sample was 10 μL. Column temperature: 45℃; Gradient elution, the elution procedure is as follows: From 0 to 1 minute, phase A was 98% and phase B was 2%. Within 1–3 minutes, phase A decreased from 98% to 85%, while phase B increased from 2% to 15%. Within 3–5 minutes, phase A decreased from 85% to 50%, while phase B increased from 15% to 50%. In 5–10 minutes, phase A decreased from 50% to 5%, while phase B increased from 50% to 95%. 10–11 min, maintain phase A at 5% and phase B at 95%; Within 11–15 minutes, phase A increased from 5% to 98%, while phase B decreased from 95% to 2%. The mass spectrometry conditions for the LC-MS / MS analysis are as follows: High-purity nitrogen was used as the curtain gas, collision gas, and ESI ion source to perform selective reaction monitoring (SRM) in positive ion mode. Mass spectrometry parameters: Spray voltage 4500V, temperature 450℃, collision energy 18V, ion source gas 1: 50psi; ion source gas 2: 50psi; curtain gas: 55psi; Collision gas: High; Mass spectrometry acquisition time: 15.00 min; Q1 / Q3 resolution: Unit / Unit; Interval time: 5.007 ms; (4) The concentrations of Nomilin, Lys-Leu, and Hawkinsin in the test sample were quantitatively analyzed using the external standard-standard curve method. A linear regression was performed with the mass spectrometric abundance of Nomil in the Nomil standard curve samples as the ordinate and the concentration of Nomil in the Nomil standard curve samples as the abscissa to obtain the Nomil standard curve. The concentration of Nomil in the sample to be tested was then calculated by substituting the mass spectrometric abundance of Nomil into the Nomil standard curve. A linear regression was performed with the mass spectrometric abundance of Lys-Leu in the Lys-Leu standard curve sample as the ordinate and the concentration of Lys-Leu in the Lys-Leu standard curve sample as the abscissa to obtain the Lys-Leu standard curve. The concentration of Nomilin in the sample was then calculated by substituting the mass spectrometric abundance of Lys-Leu in the test sample into the Lys-Leu standard curve. The Hawkinsin standard curve was obtained by performing linear regression with the mass spectrometric abundance of Hawkinsin in the Hawkinsin standard curve sample as the ordinate and the concentration of Hawkinsin in the Hawkinsin standard curve sample as the abscissa. The concentration of Hawkinsin in the sample is obtained by substituting the mass spectrometry abundance of Hawkinsin in the sample into the Hawkinsin standard curve.

5. The application according to claim 4, characterized in that, During LC-MS / MS analysis, the m / z of the monitoring ions Q1 for nomilin was 515.5, and the m / z of Q3 was 411.2 / 393.2; the m / z of the monitoring ions Q1 for lysine-leucine was 260.3, and the m / z of Q3 was 242.9; and the m / z of the monitoring ions Q1 for Hawkinsin was 292.3, and the m / z of Q3 was 238 / 209.8 / 167.

1.

6. The application according to claim 1, characterized in that, Compared to H3K27M wild-type patients, the expression of nomilin, lysine-leucine, and Hawkinsin was increased in the urine of H3K27M mutant patients.

7. The application according to claim 6, characterized in that, The nomilin, lysine-leucine, and Hawkinsin were identified by non-targeted LC-MS / MS analysis and multiple reaction monitoring (MRM).

Images