Biomarker compositions and their use in detection of mgt promoter methylation

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a biomarker composition and application thereof in MGMT promoter methylation detection. The biomarker composition comprises: phospholipids (L5PL) in LDL-5 subcomponent, creatinine, and ornithine. The application uses multiple biomarkers to determine whether a glioblastoma patient carries MGMT promoter methylation, has good sensitivity and specificity, is less traumatic to the patient, can overcome individual differences, and provides a new solution for prognosis evaluation of glioblastoma.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to biomarker compositions and their application in the detection of MGMT promoter methylation. Background Technology

[0002] Glioblastoma multiforme (GBM) is a highly malignant and rapidly growing brain tumor, known for its aggressiveness and high mortality rate. With the increasing aging of the global population, the number of glioma patients is rising annually. According to the World Health Organization (WHO) classification system, these tumors are divided into grades I and II (low-grade gliomas), grade III (anaplastic gliomas), and grade IV (glioblastomas). Because gliomas tend to infiltrate normal brain tissue, complete surgical resection is extremely difficult; therefore, efforts to improve the extent of surgical resection are ongoing. Furthermore, significant progress has been made in chemotherapy, immunotherapy, molecularly targeted drugs, and radiotherapy, which have improved the prognosis of glioma patients.

[0003] The O-6-methylguanine-DNA methyltransferase (MGMT) gene, located on chromosome 10q26, encodes a protein responsible for repairing alkylation damage in DNA, particularly damage caused by chemotherapeutic agents such as temozolomide. Studies have shown that CpG island methylation in the MGMT promoter region can silence this gene, thereby enhancing the sensitivity of tumor cells to alkylating agents in chemotherapy. Research has found that glioma patients with MGMT promoter methylation often exhibit better treatment response and longer survival when treated with temozolomide. Specifically, the median overall survival in the unmethylated MGMT group was approximately 12 to 15 months, while the median overall survival in the methylated MGMT group was extended to approximately 21 to 27 months. This difference suggests that MGMT promoter methylation status can serve as an important biomarker for predicting chemotherapy efficacy and can help guide the selection of personalized treatment strategies.

[0004] Currently, the detection of MGMT promoter methylation status mainly relies on surgically obtained tissue samples using methods such as quantitative real-time PCR or bisulfite sequencing. However, these invasive procedures not only increase the surgical risk for patients but may also lead to complications such as brain swelling or neurological impairment. Therefore, developing non-invasive methods for detecting MGMT promoter methylation is of great significance for the prognostic assessment and optimization of treatment plans for glioblastoma. Although imaging techniques such as magnetic resonance imaging (MRI), diffusion-weighted imaging (DWI), perfusion imaging, spectroscopic analysis (MRS), and positron emission tomography (PET) combined with specific tracers have been used to assist in determining MGMT promoter methylation status, these methods have shown some potential in predicting MGMT methylation status, especially at the level of changes in macroscopic structure and metabolic activity. However, at the molecular level, their sensitivity and specificity remain limited, and they cannot replace direct molecular biological detection methods (such as methylation-specific PCR and bisulfite sequencing). Summary of the Invention

[0005] To overcome the above-mentioned shortcomings of the prior art, the present invention provides a biomarker composition and its application in the detection of MGMT promoter methylation.

[0006] The biomarker composition provided by this invention enables efficient and non-invasive detection of the methylation status of the MGMT promoter. The application of this biomarker composition can not only provide clinicians with more accurate diagnostic information but also support personalized treatment decisions, thereby improving patients' quality of life and life expectancy.

[0007] According to a first aspect of the present invention, a biomarker composition is provided comprising phospholipids (L5PL), creatinine, and ornithine from the LDL-5 subfraction. The biomarker composition provided by the present invention is a group of lipid metabolism biomarkers. The present invention can help to rapidly and accurately identify whether glioblastoma patients carry MGMT promoter methylation through a combination of multiple biomarkers.

[0008] According to a second aspect of the present invention, the present invention provides a detection reagent for a biomarker composition, which can be used to detect the metabolic level of the biomarker composition.

[0009] In some embodiments, when the above-described detection reagent is used to detect the metabolic level of the biomarker composition, the sample to be detected includes the serum or plasma of the subject.

[0010] According to a third aspect of the present invention, the present invention provides the application of the biomarker composition and the detection reagent of the biomarker composition in the preparation of a glioblastoma patient MGMT promoter methylation detection product.

[0011] According to a fourth aspect of the present invention, the present invention provides the use of the biomarker composition and the detection reagent of the biomarker composition in the preparation of a prognostic assessment product for glioblastoma patients.

[0012] According to a fifth aspect of the present invention, a kit is provided that includes a detection reagent for a biomarker composition.

[0013] According to a sixth aspect of the present invention, the present invention provides a detection model for MGMT promoter methylation in glioblastoma patients, wherein the following formula is used to determine whether glioblastoma patients carry MGMT promoter methylation;

[0014] mutation index=W0+W1X1+W2X2+...+W n X n Where n is the number of biomarkers, and n takes the value of 3; the mutation index is calculated using the Sigmoid function 1 / (1+e (-x) Normalization yields the risk coefficient RS, i.e., RS = 1 / (1+e) (-x) If RS ≥ threshold, then glioblastoma patients are considered to carry MGMT promoter methylation; if RS < threshold, then glioblastoma patients are considered to carry wild-type MGMT promoter; X1, X2, and X3 are the metabolic levels of phospholipids, creatinine, and ornithine in LDL-5 subfractions, respectively; W1, W2, and W3 are the weighting coefficients of phospholipids, creatinine, and ornithine in LDL-5 subfractions, respectively; W1 is 0.094771481, W2 is 6.644420484, W3 is 18.03435936; W0 is -2.711341715; the threshold is 0.50.

[0015] According to a seventh aspect of the present invention, the present invention provides a method for constructing a detection model for MGMT promoter methylation in glioblastoma patients. The method includes the following steps: collecting MGMT promoter methylation samples and MGMT promoter wild-type samples from glioblastoma patients, performing plasma metabolite detection, screening for significantly differentially expressed metabolites, modeling using a logistic regression machine learning algorithm, selecting an optimal model, and choosing the model with the largest AUC on the test set among the optimal models as the optimal screening model, which is the detection model; the metabolites include phospholipids, creatinine, and ornithine in the LDL-5 subfraction.

[0016] Compared with the prior art, the present invention has the following beneficial effects and advantages:

[0017] (1) The present invention provides a biomarker composition that can help diagnose whether the MGMT promoter of glioblastoma patients is methylated;

[0018] (2) The present invention provides a detection reagent and a diagnostic kit, which can complete the detection by obtaining only serum or plasma. It is convenient to sample, has low detection cost, and is suitable for the prognostic assessment of glioblastoma patients.

[0019] (3) The biomarker composition provided by the present invention can effectively overcome individual differences among patients and improve detection sensitivity and specificity by combining multiple biomarkers. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. It should be noted that any processes or parameters not specifically described in detail below are those that can be understood or implemented by those skilled in the art with reference to the prior art.

[0021] Example 1: Model Building

[0022] EDTA plasma samples were collected from glioblastoma patients. Methylation information of the MGMT promoter was obtained using methylation sequencing. Patients were divided into methylated and unmethylated groups based on the MGMT promoter methylation status. A total of 34 samples were collected from the unmethylated group and 23 samples from the methylated group.

[0023] Equipment, materials and reagents:

[0024] (1) Instrument: Nuclear magnetic resonance spectrometer (600MHz) (provided by Bruker Biospin AG);

[0025] (2) Main reagents: NMR lipid buffer (purchased from Bruker Biospin AG, model name Plasmabufer), NMR tubes (purchased from Bruker Biospin AG, model name NMR Tubes / cap3)

[0026] Sample testing:

[0027] 1. Remove each plasma sample from the freezer and wait for it to thaw completely. Then, take 340 μL of each plasma sample and mix it with 340 μL of NMR lipid buffer (Bruker Plasma buffer) at a 1:1 ratio. After mixing thoroughly, take 600 μL of the mixture and place it in a 5 mm NMR tube.

[0028] 2. Load the NMR tube from step 1 into the autosampler and use the plasma detection program of the nuclear magnetic resonance spectrometer (600MHz) to obtain the detection spectrum;

[0029] 3. The spectra detected in step 2 were normalized using the QuantRef management system built into the Topspin software of the 600MHz NMR spectrometer to normalize the intensity of the spectra to a proton concentration of mmol / L. Chemical shifts were corrected using the bimodal signals of TSP (sodium 3-trimethylsilylpropionate) and alanine at 1.48 ppm. Qualitative analysis of the detected spectra was performed based on the Bruker NMR library built into the 600MHz NMR spectrometer. Metabolites were quantified using the integral of the signal group at specific chemical shift positions to obtain metabolite content data in the sample.

[0030] 4. The obtained metabolite content data (114 blood lipid indicators and 39 small molecule metabolite indicators) were screened for differential indicators, including the following steps:

[0031] a) Fill in the minimum detection limit (LOD) for each indicator with empty values;

[0032] b) Take the median of each index in each group (unmethylated group, methylated group) and label it as Mn_pos and Mn_neg, respectively;

[0033] c) Take the ratio of the medians of the two groups for each indicator as the difference multiple Fn = Mn_pos / Mn_neg;

[0034] d) Perform a t.test on the values ​​of each indicator in the two groups to obtain pn;

[0035] e) Screen for indicators with Fn≥1.5 or Fn≤0.67 and pn<0.05;

[0036] 5. Three indicators met the differentiation criteria: phospholipids (L5PL), creatinine, and ornithine in the LDL-5 subfraction. The screening criteria are shown in Table 1.

[0037] Table 1. Values ​​of Difference Indicators

[0038] FC (fold change) p Creatinine 1.62 0.0467 Phospholipids (L5PL) 1.77 0.0286 Ornithine 3.70 0.0318

[0039] 6. Based on the sample detection data of the three indicators, a model was built using logistic regression. The optimal model was selected, and the model with the largest AUC on the test set (AUC = 0.96) was chosen as the optimal screening model, which is the detection model. The mutation index is defined as: mutation index = W0 + W1X1 + W2X2 + W3X3, where W1, W2, and W3 are the weight coefficients of phospholipids (L5PL), creatinine, and ornithine in the LDL-5 subfraction, respectively; W1, W2, and W3 are 0.094771481, 6.644420484, and 18.03435936, respectively, and their corresponding biomarkers are phospholipids (L5PL), creatinine, and ornithine in the LDL-5 subfraction, respectively; X1, X2, and X3 are the metabolic level detection values ​​of phospholipids (L5PL), creatinine, and ornithine in the LDL-5 subfraction, respectively; and the constant W0 is -2.711341715.

[0040] 7. Apply the function 1 / (1+e) to the mutation index Sigmoid of each sample. (-x) Normalization yields the risk coefficient RS = 1 / (1+e) (-x) The RS threshold was set at 0.5. When determining whether a glioblastoma patient carries MGMT promoter methylation or assessing the prognosis of glioblastoma patients, if the patient's RS ≥ the threshold, the patient is considered to carry MGMT promoter methylation and has a good prognosis; if the RS < the threshold, the patient is considered to carry the wild-type MGMT promoter and has a relatively poor prognosis.

[0041] Example 2: Sample Detection and Verification

[0042] 1. Methylation sequencing was used to detect the methylation status of the MGMT promoter in 15 patients with glioblastoma; EDTA plasma samples were also obtained from these 15 patients.

[0043] 2. The risk coefficient RS of 15 patients was obtained by following steps 1, 2, 3, 6, and 7 in Example 1;

[0044] 3. Determine if the patient's RS is greater than 0.5 to determine the methylation status of the MGMT promoter in the patient's sample. If RS ≥ 0.5, the MGMT promoter in the sample is methylated. If RS < threshold, the MGMT promoter in the sample is not methylated.

[0045] By collecting samples from 15 patients, the RS values ​​were obtained and compared with the methylation status of the MGMT promoter detected by methylation sequencing. The results are shown in Table 2 below.

[0046] Table 2. Patient testing results and MGMT promoter methylation detection results

[0047]

[0048]

[0049] Statistical results showed that among the 15 validated patient samples collected, 6 samples were positive for MGMT promoter methylation and 9 samples were negative when detected by methylation sequencing. When evaluated using this method, 14 samples were correct. One positive sample scored 0.47, close to the threshold of 0.5, and was judged as negative by this method. The overall accuracy rate of the small validation cohort was 93%.

[0050] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. The application of a detection reagent for a biomarker composition in the preparation of a product for detecting MGMT promoter methylation in glioblastoma patients, characterized in that, The biomarker composition comprises phospholipids, creatinine, and ornithine from the LDL-5 subfraction; the detection reagent is used to detect the metabolic level of each biomarker in the above biomarker composition in serum or plasma.

2. The application of a detection reagent for a biomarker composition in the preparation of a prognostic assessment product for glioblastoma patients, characterized in that, The biomarker composition comprises phospholipids, creatinine, and ornithine from the LDL-5 subfraction; the detection reagent is used to detect the metabolic level of each biomarker in the above biomarker composition in serum or plasma.

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