A panel of urinary metabolites for diagnosing the h3k27m mutation status of brainstem glioma

Abstract

The application discloses a group of urine metabolites for diagnosing H3K27M gene mutation state of brain stem glioma, and relates to non-target LC-MS / MS detection of urine of brain stem glioma (BSG) patients and screening of H3K27M gene mutation state related metabolites, finding that nomilin, lysine-leucine (Lys-Leu) and Hawkinsin have obvious content differences in BSG patients with different H3K27M mutation states; the content changes of H3K27M gene mutation related metabolites in urine of BSG patients are further verified by using targeted LC-MS / MS; and research confirms that the combination of nomilin, lysine-leucine (Lys-Leu) and Hawkinsin in urine can be used for predicting the H3K27M gene mutation state of brain stem glioma.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a group of urine metabolites for diagnosing the H3K27M gene mutation state of brain stem glioma. BACKGROUND

[0002] Brain stem gliomas (BSGs) are gliomas located in the midbrain, pons and medulla. Due to the important functions of the brain stem, surgical resection is quite challenging. In addition, BSGs often carry a somatic gain-of-function mutation, known as the H3K27M mutation, which results in the substitution of lysine at position 27 of histone H3 with methionine, leading to a highly malignant phenotype [1] . Clinically, BSGs with the H3K27M mutation are one of the most lethal brain malignancies, with a median overall survival of 10-12 months and a two-year survival rate of less than 10%, because the current treatment modalities, including surgery, radiotherapy and chemotherapy, have limited effects. In contrast, BSG patients without this genetic mutation generally have much better prognosis, with a median overall survival of about five years after receiving surgical resection plus radiotherapy and / or chemotherapy. Therefore, early identification of the mutation status in H3 is crucial for clinical decision making, such as treatment selection and prognosis prediction.

[0003] Previous reports have shown that the H3K27M mutation is associated with tumor location and patient age, but these clinical information alone cannot accurately predict the H3K27M mutation status [2][3] . Currently, the conventional method to determine the mutation status still relies on invasive brain biopsy or surgical resection.

[0004] With the advancement of second-generation sequencing technology, liquid biopsy strategies, i.e. detecting circulating tumor DNA (ctDNA) in cerebrospinal fluid (CSF), have been shown to be effective in predicting the H3K27M mutation status [4][5] . However, lumbar puncture for CSF collection is not always feasible due to the risk of high intracranial pressure. Magnetic resonance imaging (MRI)-based radiomics methods show potential in predicting the H3K27M mutation status [6] . However, radiomics models based on conventional MRI sequences are not always reliable, and models with high diagnostic performance require special MRI sequences [7][8] , limiting their clinical application. We also investigated the relationship between 11C-methionine positron emission tomography (PET) imaging features and H3K27M mutation, but failed to establish a link [9] . However, the important functions of the brain stem greatly limit the application of these methods and hinder repeated testing for purposes such as treatment monitoring. Therefore, it is urgent to develop minimally invasive or non-invasive methods.

[0005] Metabolic dysregulation is a hallmark of cancer and plays a key role in the development and progression of most malignancies, including gliomas and BSGs. H3K27M mutation is also deeply involved in metabolic reprogramming of BSG cells, maintaining a low H3K27me3 state, leading to tumor initiation and progression. In addition, there is growing evidence that tumors not only cause local metabolic changes within tumor tissue, but also trigger systemic metabolic changes. These systemic changes can be detected in plasma / serum or urine by mass spectrometry-based metabolomics analysis, which has great potential in identifying non-invasive biomarkers for molecular diagnosis and treatment monitoring. Therefore, the identification of metabolite biomarkers in blood and urine has received extensive attention in cancer research. In gliomas, many metabolites have been identified in blood or urine samples that are different between patients and healthy controls. Some metabolites show high diagnostic accuracy in distinguishing glioma samples from healthy control samples, showing the feasibility of using metabolomics analysis in blood and urine for glioma diagnosis. However, these studies mainly involve gliomas located in the supratentorial brain region and are not specific to BSGs.

[0006] However, there is currently no research on the impact of H3K27M mutation on blood or urine metabolomics analysis or the identification of non-invasive metabolite biomarkers for predicting H3K27M mutation.

[0007] References:

[0008] [1] Wu G, Broniscer A, McEachron TA et al. Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas. 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 of 96 patients. J Neurosurg. 2022; 137(6): 1628-1638.

[0010] [3] Chen LH, Pan C, Diplas BH et al. The integrated genomic and epigenomic 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 the brainstem by sequencing of CSF-derived circulating tumor DNA. Acta Neuropathol. 2019; 137(2): 297-306.

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

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

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

[0015] [8] Yang N, Xiao X, Gu G et al. Diffusion MRI-based connectomics features improve the noninvasive prediction of H3K27M mutation in brainstem gliomas. Radiother Oncol. 2023; 186: 109789.

[0016] [9] Zhao X, Li D, Qiao Z et al. 11C-methionine PET imaging characteristics in children with diffuse intrinsic pontine gliomas and relationship to survival and H3 K27M mutation status. Eur J Nucl Med Mol Imaging. 2023; 50(6): 1709-1719. SUMMARY

[0017] In view of the problems in the diagnosis of H3K27M gene mutation status of brainstem glioma, the application detects the urine of brainstem glioma (BSG) patients by non-targeted LC-MS / MS and screens H3K27M gene mutation status related metabolites, finds that nomilin, lysine-leucine (Lys-Leu) and Hawkinsin have obvious content differences in BSG patients with different H3K27M mutation states, and further verifies the content changes of H3K27M gene mutation related metabolites in the urine of BSG patients by targeted LC-MS / MS mass spectrometry analysis technology. The study confirms that the combination of nomilin, lysine-leucine (Lys-Leu) and Hawkinsin in urine can be used to predict the H3K27M gene mutation status of brainstem glioma. In order to achieve the above purpose, the specific technical scheme of the application is as follows:

[0018] First aspect, detecting urine metabolites and screening H3K27M gene mutation status related metabolites of BSG patients

[0019] 112 urine samples from patients with brainstem glioma (BSG) (72 H3K27M mutant, 40 H3K27M wild type) were analyzed by non-targeted liquid chromatography-tandem mass spectrometry (LC-MS / MS) to detect metabolites related to H3K27M mutation status in BSG patients through data-dependent acquisition (DDA) method.

[0020] Results: 166 differential metabolites were screened out in urine, and after further screening, nomilin, lysine-leucine and Hawkinsin were found to have significant differences in content in BSG patients with different H3K27M mutation status, and the differences were statistically significant.

[0021] Second aspect, further verify the content changes of H3K27M gene mutation related metabolites in urine of BSG patients by targeted LC-MS / MS mass spectrometry analysis technology

[0022] 94 urine samples from patients with brainstem glioma (BSG) (55 H3K27M mutant, 39 H3K27M wild type) were further verified by targeted LC-MS / MS mass spectrometry analysis technology to verify the content changes of H3K27M gene mutation related metabolites in urine of BSG patients.

[0023] Results: Compared with H3K27M wild type patients with brainstem glioma, nomilin, lysine-leucine and Hawkinsin were up-regulated in H3K27M mutant patients.

[0024] Third aspect, evaluate the diagnostic efficiency of nomilin, lysine-leucine and Hawkinsin alone in predicting H3K27M gene mutation status in patients with brainstem glioma (BSGs)

[0025] 94 urine samples from patients with brainstem glioma (BSG) (55 H3K27M mutant, 39 H3K27M wild type) were randomly divided into training set and test set according to the ratio of 2:1, and the samples were from Beijing Tiantan Hospital.

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

[0027] To evaluate the diagnostic performance of nomilin, lys-leu and Hawkinsin alone in predicting the H3K27M mutation status of brain stem glioma (BSGs) patients, the ROC-AUC was 0.7119, 0.6275, and 0.6345, respectively;

[0028] A combination model of nomilin, lys-leu and Hawkinsin was established to evaluate the diagnostic performance of the combination model of nomilin, lys-leu and Hawkinsin;

[0029] Results: The ROC-AUC of the training set and the test set was 0.7457 and 0.7449, respectively;

[0030] Conclusion: The combination model of nomilin, lys-leu and Hawkinsin can be used to predict the H3K27M mutation status of brain stem glioma (BSGs) patients.

[0031] Compared with the prior art, the beneficial effects of the present application are:

[0032] 1. The present application first found that nomilin, lys-leu and Hawkinsin were up-regulated in the urine of H3K27M gene mutation patients compared with brain stem glioma H3K27M wild-type patients;

[0033] 2. The present application first predicts the H3K27M mutation status of brain stem glioma through a combination of biomarkers in urine samples. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 , PCA classification chart of brain stem glioma patient urine metabolomics;

[0035] Figure 2 , non-targeted mass spectrometry acquisition method, nomilin (Nomilin), lys-leu and Hawkinsin in urine samples in H3K27M mutant (MUT) patients and H3K27M wild-type (WT) patients Content difference expression chart;

[0036] Figure 3 , targeted mass spectrometry analysis, nomilin (Nomilin), lys-leu and Hawkinsin in urine samples in H3K27M mutant (MUT) patients and wild-type (WT) patients Content difference expression chart;

[0037] Figure 4 , Three ROC curve of 1 metabolite alone in predicting the H3K27M mutation status of brain stem glioma;

[0038] Figure 5 , Three ROC curve of the combination of metabolites for predicting the H3K27M mutation status of brainstem glioma, wherein the Training set is the training set, and the Test set is the test set. DETAILED DESCRIPTION

[0039] The following examples are intended to illustrate the present application but not to limit the scope of the present application. If not specifically mentioned, the technical means used in the examples are the conventional means well known to those skilled in the art.

[0040] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0041] All materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0042] Reagents and materials

[0043]

[0044] Instruments

[0045]

[0046] Example 1, detecting urine metabolites and screening BSG patient H3K27M gene mutation status related metabolites

[0047] Using non-targeted liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis, through the non-targeted mass spectrometry acquisition method of data-dependent acquisition (Data dependent acquisition, DDA), urine metabolites were detected and BSG patient H3K27M gene mutation status related metabolites were screened.

[0048] I. Method

[0049] (I) Sample

[0050] The urine of 112 cases of untreated newly diagnosed brainstem glioma patients (72 cases of H3K27M mutant type and 40 cases of H3K27M wild type) was collected, and the patient's plasma sample was also collected. The samples were divided into two groups according to the mutation type for experiment, and the samples were from Beijing Tiantan Hospital.

[0051] The enrolled patients were subsequently subjected to surgery or puncture biopsy to obtain tumor tissue. IHC staining was performed on the tumor tissue to determine the H3K27M mutation status. This is a general method for clinical diagnosis. H3K27M positive and H3K27me3 negative are mutant type, and vice versa are wild type. Those who are not consistent are subjected to gene sequencing 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 the urine samples were centrifuged at 4000 x g for 15 min at 4 °C to remove the urine sediment. The supernatant was collected, and the sample (200 μL) was added to acetonitrile (200 μL), vortexed for 30 s, and then frozen at -40 °C for 30 min. The mixture was centrifuged at 14,000 x g for 10 min. The resulting supernatant was collected, vacuum dried, and stored at -80 °C. Prior to analysis, the sample was reconstituted with 2% acetonitrile and filtered through an ultra-centrifugal filter with a 10 kDa molecular weight cutoff before being transferred to the autosampler as the sample to be analyzed.

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

[0055] 1. Chromatographic conditions:

[0056] Chromatographic column: Waters HSS C18, 1.8 μm, 3.0 mm x 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 °C;

[0063] Gradient elution, elution program:

[0064]

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

[0066] 2. Mass spectrometry conditions

[0067] The scan range was 100-1000 m / z, and the resolution was 60 K. The automatic gain control (AGC) target was set to 1 x 10 6 , and the maximum injection time was 100 ms. The high-energy collisional dissociation (HCD) fragmentation was set to 20, 35, and 60.

[0068] 3. Analysis of mass spectrometry data:

[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. Preparation of standard curve working solution

[0080] Take the Nomilin control, dissolve with double distilled water to prepare 250 μmol / L Nomilin standard curve working solution;

[0081] Take Lys-Leu control, dissolve with double distilled water to prepare 250 μmol / L Lys-Leu standard curve working solution;

[0082] Take Hawkinsin control, dissolve with double distilled water to prepare 250 μmol / L Hawkinsin standard curve working solution;

[0083] 2. Preparation of standard curve sample

[0084] Double distilled water and Nomilin standard curve working solution are mixed in appropriate proportions to prepare Nomilin standard curve sample. The concentration of Nomilin in the Nomilin standard curve sample is 0.061, 0.244, 0.977, 3.906, 15.625, 62.5, 250 μmol / L, respectively;

[0085] Double distilled water and Lys-Leu standard curve working solution are mixed in appropriate proportions to prepare Lys-Leu standard curve sample. The concentration of Lys-Leu in the Lys-Leu standard curve sample is 0.061, 0.244, 0.977, 3.906, 15.625, 62.5, 250 μmol / L, respectively;

[0086] Double distilled water and Hawkinsin standard curve working solution are mixed in appropriate proportions to prepare Hawkinsin standard curve sample. The concentration of Hawkinsin in the Hawkinsin standard curve sample is 0.061, 0.244, 0.977, 3.906, 15.625, 62.5, 250 μmol / L, respectively;

[0087] (II) Collection and metabolite extraction of brainstem glioma patient urine samples

[0088] Brainstem glioma patient urine samples were collected using sterile centrifuge tubes. The urine samples were centrifuged at 4000 x g for 15 minutes at 4°C to remove the urine precipitate. The supernatant was collected and the sample (200 μL) was added to acetonitrile (200 μL), vortexed for 30 seconds, and then frozen at -40°C for 30 minutes. The mixture was centrifuged at 14,000 x g for 10 minutes. The resulting supernatant was collected and stored at -80°C after vacuum drying.

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

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

[0091] 1. Chromatographic conditions

[0092] Chromatographic column: Waters HSS C18, 1.8 μm, 3.0 mm x 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 tested, and the test sample and the standard curve sample were injected, with an injection amount of 10 μL each;

[0098] Column temperature: 45°C;

[0099] Gradient elution, elution program:

[0100]

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

[0102] 2. Mass spectrometric conditions

[0103]

[0104]

[0105] 3. Detector parameters

[0106]

[0107] (IV) Quantitative analysis of the concentrations of nomilin, Lys-Leu and Hawkinsin in the test sample according to the external standard-standard curve method

[0108] The mass spectral abundance of Nomilin in the Nomilin standard curve sample is taken as the vertical coordinate, and the concentration of the Nomilin standard curve sample is taken as the horizontal coordinate, linear regression is performed to obtain the Nomilin standard curve; the mass spectral abundance of Nomilin in the sample to be tested is substituted into the Nomilin standard curve to calculate the concentration of Nomilin in the sample to be tested,

[0109] The mass spectral abundance of Lys-Leu in the Lys-Leu standard curve sample is taken as the vertical coordinate, and the concentration of the Lys-Leu standard curve sample is taken as the horizontal coordinate, linear regression is performed to obtain the Lys-Leu standard curve; the mass spectral abundance of Lys-Leu in the sample to be tested is substituted into the Lys-Leu standard curve to calculate the concentration of Nomilin in the sample to be tested,

[0110] The mass spectral abundance of Hawkinsin in the Hawkinsin standard curve sample is taken as the vertical coordinate, and the concentration of the Hawkinsin standard curve sample is taken as the horizontal coordinate, linear regression is performed to obtain the Hawkinsin standard curve; the mass spectral abundance of Hawkinsin in the sample to be tested is 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 file is processed using Analyst 1.6.2 to obtain the mass spectral abundance values of Nomilin, Lys-Leu, and Hawkinsin;

[0113] The mass spectral abundance values of Nomilin, Lys-Leu, and Hawkinsin in the sample to be tested are converted to concentration values using the standard curve, corrected using the creatinine concentration values in the corresponding sample to be tested, and then logarithmically transformed for further statistical analysis;

[0114] The content of creatinine in urine is detected by the laboratory of the hospital, using a creatinine assay kit (sarcosine oxidase method) for detection, and the instrument is Beckman Coulter AU5800 full-automatic biochemical analyzer;

[0115] The statistical results are shown in Table Figure 3 Compared with patients with wild-type H3K27M brainstem glioma, the levels of Nomilin, Lys-Leu, and Hawkinsin were up-regulated in patients with H3K27M gene mutation.

[0116] Example 3, Diagnostic efficiency of combination of Nomilin, Lys-Leu, and Hawkinsin in predicting H3K27M gene mutation status of patients with brainstem glioma (BSGs)

[0117] I. Samples

[0118] Urine samples from 94 patients with newly diagnosed brainstem glioma (55 H3K27M mutant, 39 H3K27M wild type) were randomly divided into training set and test set at a ratio of 2:1.

[0119] II. Quantitative detection of nomilin, lysine-leucine and Hawkinsin content

[0120] The content of nomilin, lysine-leucine and Hawkinsin in urine was detected by targeted LC-MS / MS mass spectrometry (creatinine correction), and the method is shown in Example 2.

[0121] The content of creatinine in urine was detected by the laboratory of our hospital using a creatinine detection kit (creatinine oxidase method), and the instrument was Beckman Coulter AU5800 automatic biochemical analyzer.

[0122] III. Diagnostic performance of nomilin, lysine-leucine and Hawkinsin alone in predicting the H3K27M gene mutation status of brainstem glioma (BSGs) patients

[0123] The sensitivity and specificity of nomilin, lysine-leucine and Hawkinsin in predicting the H3K27M gene mutation status of brainstem glioma (BSGs) patients alone are shown in Table 1 and Figure 4 .

[0124] Table 1. Nomilin, lysine-leucine and Hawkinsin as biomarkers for H3K27M gene mutation status

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

[0126] From the results in Table 1 and Figure 4 , it can be seen that the sensitivity and specificity of nomilin, lysine-leucine and Hawkinsin in predicting the H3K27M gene mutation status of brainstem glioma (BSGs) patients alone are slightly poor.

[0127] IV. Diagnostic performance of nomilin, lysine-leucine and Hawkinsin combination in predicting the H3K27M gene mutation status of brainstem glioma (BSGs) patients

[0128] (I) Nomilin, lysine-leucine and Hawkinsin combination model

[0129] A combination model of nomilin, lysine-leucine and Hawkinsin was established using binary logistic regression as follows:

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

[0131] In the formula

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

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

[0134] Lys-Leu: refers to the content of Lys-Leu, unit: pmol / L;

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

[0136] Result determination

[0137] Model score ≤ 0 is H3K27M gene mutation state;

[0138] Model score > 0 is H3K27M gene wild type.

[0139] (II) Diagnostic performance of the combination model of Nomilin, Lys-Leu and Hawkinsin

[0140] The combination model of Nomilin, Lys-Leu and Hawkinsin uses R package "pROC" (version 1.15.0) to perform receiver operating characteristic (ROC) analysis, calculate the area under the curve (AUC) to evaluate the accuracy of the combination model in predicting the H3K27M gene mutation state of brain stem glioma (BSGs) patients, and its sensitivity and specificity.

[0141] The results are shown in Figure 5 ,

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

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

[0144] Conclusion: The combination model of Nomilin, Lys-Leu and Hawkinsin can be used to predict the H3K27M gene mutation state of brain stem glioma (BSGs) patients.

[0145] While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. It is therefore intended that whatever lies within the scope of the application be covered by the appended claims.

Claims

1. Use of a combination of metabolites in urine of a group of patients with brainstem gliomas in the manufacture of a reagent for predicting the H3K27M mutation status, characterized in that, The metabolite composition is Nomilin, Lys-Leu and Hawkinsin.

2. Use according to claim 1, characterized in that, The combination model of the metabolite composition is: Model score = -2.720 x Log10(Nomilin / Creatine) - 0.120 x Log10(Lys-Leu / Creatine) - 0.440 x Log10(Hawkinsin / Creatine) - 18.07 In the formula Nomilin refers to the content of Nomilin, unit: μmol / L; Creatine refers to the content of Creatine, unit: μmol / L; Lys-Leu refers to the content of Lys-Leu, unit: μmol / L; Hawkinsin refers to Hawkinsin, unit: μmol / L; Result determination Model score ≤ 0 is H3K27M gene mutation state; Model score > 0 is H3K27M gene wild type.

3. Use according to claim 1, characterized in that, The quantitative detection of Nomilin, Lys-Leu and Hawkinsin is carried out by targeted LC-MS / MS mass spectrometry.

4. Use according to claim 3, characterized in that, The method of targeted LC-MS / MS mass spectrometry is: (1) Preparation of standard curve sample A, preparation of standard curve working solution Take Nomilin control, dissolve with double distilled water to prepare 250 μmol / L Nomilin standard curve working solution; Take Lys-Leu control, dissolve with double distilled water to prepare 250 μmol / L Lys-Leu standard curve working solution; Take Hawkinsin control, dissolve with double distilled water to prepare 250 μmol / L Hawkinsin standard curve working solution; B, preparation of standard curve sample Double distilled water and Nomilin standard curve working solution are mixed in appropriate proportion to prepare Nomilin standard curve sample, and the concentration of Nomilin in Nomilin standard curve sample is 0.061, 0.244, 0.977, 3.906, 15.625, 62.5, 250 μmol / L respectively; Double distilled water and Lys-Leu standard curve working solution are mixed in appropriate proportion to prepare Lys-Leu standard curve sample, and the concentration of Nomilin in Nomilin standard curve sample is 0.061, 0.244, 0.977, 3.906, 15.625, 62.5, 250 μmol / L respectively; Double distilled water and Hawkinsin standard curve working solution are mixed in appropriate proportion to prepare Hawkinsin standard curve sample, and the concentration of Hawkinsin in Hawkinsin standard curve sample is 0.061, 0.244, 0.977, 3.906, 15.625, 62.5, 250 μmol / L respectively; (2) Collection of brain stem glioma patient urine sample and extraction of metabolites The urine sample of the brain stem glioma patient was collected in a sterile centrifuge tube. The urine sample was centrifuged at 4000 x g for 15 minutes at 4°C to remove the urine precipitate; The supernatant was collected, 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 x g for 10 minutes; the resulting supernatant was collected, vacuum dried, and stored at -80°C; Before analysis, the vacuum-dried and stored sample was reconstituted with 2% acetonitrile and filtered through a 10 kDa molecular weight cutoff ultrafiltration filter before being transferred to an autosampler, thereby obtaining the sample to be tested; (3) Targeted LC-MS / MS analysis of the sample to be tested and the standard curve sample Chromatographic conditions Chromatographic column: Waters HSS C18, 1.8 µm, 3.0 mm x 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 sample to be tested were measured, and the sample to be tested and the standard curve sample were injected, with an injection volume of 10 µL each; Column temperature: 45°C Gradient elution with the following elution program: 0-1 min, A phase 98%, B phase 2%; 1-3 min, A phase from 98% to 85%, B phase from 2% to 15%; 3-5 min, A phase from 85% to 50%, B phase from 15% to 50%; 5-10 min, A phase from 50% to 5%, B phase from 50% to 95%; 10-11 min, A phase remains 5%, B phase remains 95%; 11-15 min, A phase from 5% to 98%, B phase from 95% to 2%; The mass spectrometry conditions for the LC-MS / MS analysis are as follows: High-purity nitrogen was used as the gas curtain gas and collision gas, and the ion source was ESI, which was operated in positive ion mode for selected reaction monitoring (SRM); Mass spectrometry parameters: Spray voltage 4500 V, temperature 450°C, collision energy 18 V, ion source gas 1: 50 psi; ion source gas 2: 50 psi; gas curtain gas: 55 psi; Collision gas: High; mass spectrometry collection time: 15.00 min; Q1 / Q3 resolution: Unit / Unit; intermittent time: 5.007 ms; (4) Quantitative analysis of the concentrations of Nomilin, Lys-Leu, and Hawkinsin in the sample to be tested according to the external standard-standard curve method The mass spectrometry abundance of Nomilin in the Nomilin standard curve sample was taken as the vertical coordinate, and the concentration of the Nomilin standard curve sample was taken as the horizontal coordinate, and linear regression was performed to obtain the Nomilin standard curve. The mass spectrometry abundance of Nomilin in the sample to be tested was substituted into the Nomilin standard curve to calculate the concentration of Nomilin in the sample to be tested, The mass spectrometry abundance of Lys-Leu in the Lys-Leu standard curve sample is taken as the ordinate, and the concentration of the Lys-Leu standard curve sample is taken as the abscissa, linear regression is performed to obtain the Lys-Leu standard curve; the mass spectrometry abundance of Lys-Leu in the sample to be tested is substituted into the Lys-Leu standard curve to calculate the concentration of Nomilin in the sample to be tested, The mass spectrometry abundance of Hawkinsin in the Hawkinsin standard curve sample is taken as the ordinate, and the concentration of the Hawkinsin standard curve sample is taken as the abscissa, linear regression is performed to obtain the Hawkinsin standard curve; The mass spectrometry abundance of Hawkinsin in the sample to be tested is substituted into the Hawkinsin standard curve to calculate the concentration of Hawkinsin in the sample to be tested.

5. Use according to claim 4, characterized in that, During the LC-MS / MS analysis process, the monitoring ion Q1 of the nomilin has an m / z of 515.5, and the Q3 has an m / z of 411.2 / 393.2; the monitoring ion Q1 of the lysine-leucine Lys-Leu has an m / z of 260.3, and the Q3 has an m / z of 242.9; the monitoring ion Q1 of the Hawkinsin has an m / z of 292.3, and the Q3 has an m / z of 238 / 209.8 / 167.

1.

6. Use according to claim 1, characterized in that, Compared with H3K27M wild-type patients, the expression of nomilin, lysine-leucine Lys-Leu and Hawkinsin in the urine of H3K27M mutant patients is increased.

7. Use according to claim 6, characterized in that, The nomilin, lysine-leucine Lys-Leu and Hawkinsin are identified by multiple reaction monitoring MRM through non-targeted LC-MS / MS analysis.

8. Use of the composition of metabolites in urine of a group of patients with brainstem gliomas in the preparation of a chip for predicting the H3K27M mutational status, characterized in that, The metabolite composition is nomilin, lysine-leucine Lys-Leu and Hawkinsin. The mass spectrometry abundance of Lys-Leu in the Lys-Leu standard curve sample is taken as the ordinate, and the concentration of the Lys-Leu standard curve sample is taken as the abscissa, linear regression is performed to obtain the Lys-Leu standard curve; the mass spectrometry abundance of Lys-Leu in the sample to be tested is substituted into the Lys-Leu standard curve to calculate the concentration of Nomilin in the sample to be tested, The mass spectrometry abundance of Hawkinsin in the Hawkinsin standard curve sample is taken as the ordinate, and the concentration of the Hawkinsin standard curve sample is taken as the abscissa, linear regression is performed to obtain the Hawkinsin standard curve; The mass spectrometry abundance of Hawkinsin in the sample to be tested is substituted into the Hawkinsin standard curve to calculate the concentration of Hawkinsin in the sample to be tested. During the LC-MS / MS analysis process, the monitoring ion Q1 of the nomilin has an m / z of 515.5, and the Q3 has an m / z of 411.2 / 393.2; the monitoring ion Q1 of the lysine-leucine Lys-Leu has an m / z of 260.3, and the Q3 has an m / z of 242.9; the monitoring ion Q1 of the Hawkinsin has an m / z of 292.3, and the Q3 has an m / z of 238 / 209.8 / 167.

1. Compared with H3K27M wild-type patients, the expression of nomilin, lysine-leucine Lys-Leu and Hawkinsin in the urine of H3K27M mutant patients is increased. The nomilin, lysine-leucine Lys-Leu and Hawkinsin are identified by multiple reaction monitoring MRM through non-targeted LC-MS / MS analysis. The metabolite composition is nomilin, lysine-leucine Lys-Leu and Hawkinsin.

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