CfDNA methylation marker related to muscular tension after glioblastoma operation and radiotherapy and chemotherapy and prediction model of cfDNA methylation marker

Abstract

The invention relates to the field of biomedical application, in particular to a group of cfDNA methylation markers related to muscular tension after glioblastoma operation and chemoradiotherapy treatment as well as a prediction model and application of the cfDNA methylation markers. The invention provides a novel cfDNA methylation marker, and the novel cfDNA methylation marker has innovativeness and specificity. Noninvasive detection is realized, and clinical application and dynamic monitoring are facilitated. An accurate prediction model and a standardization tool are developed, and standardization and individualization of treatment effect monitoring are promoted. The method has wide application value and can be used for treatment effect evaluation, prognosis prediction and treatment strategy optimization. A novel molecular mechanism is disclosed, and scientific basis and reference are provided for tumor research. Due to the advantages, the invention has important application prospect and scientific value in the fields of clinical diagnosis and treatment and scientific research of glioblastoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedical applications, and in particular to a group of cfDNA methylation markers related to muscle tension after surgery and radiotherapy and chemotherapy for glioblastoma, as well as a prediction model and application thereof. Background Art

[0002] Muscle tension refers to the tension of muscles at rest and the resistance of muscles to passive stretching. It is the basis for maintaining body posture and completing movement and is regulated by the nervous system.

[0003] Glioma is divided into low-grade glioma (LGG) and high-grade glioma (HGG) according to pathological characteristics and malignancy. High-grade glioma (HGG) includes: Glioblastoma Multiforme (GBM): WHO grade IV, the highest malignancy; Anaplastic Astrocytoma: WHO grade III; Anaplastic Oligodendroglioma: WHO grade III.

[0004] Glioblastoma (GBM) is the most common and most aggressive primary malignant tumor of the central nervous system, with a low survival rate and a lack of effective clinical treatments. The current standard treatment is maximal surgical resection followed by chemoradiotherapy. However, even after active treatment, the prognosis of GBM patients remains poor, with a median survival time of less than 15 months, and a high risk of recurrence due to the infiltration of rapidly growing invasive GBM cells in the tumor into the normal brain parenchyma surrounding the lesion. In addition, many patients are diagnosed only when symptoms develop, often missing the best time for treatment. Therefore, it is crucial to develop effective biomarkers to monitor GBM.

[0005] Although biopsy is the standard method for diagnosis, it is difficult to repeatedly obtain tumor tissue due to the location of GBM in the central nervous system, and it cannot provide real-time disease monitoring data. In addition, it may not fully represent the heterogeneity of the tumor, affecting the accuracy of the prediction results. Therefore, a method for real-time diagnosis of tumors and monitoring their progression and recurrence is urgently needed in the clinic.

[0006] Liquid biopsy, especially the analysis of cfDNA (cell-free DNA), offers significant advantages and promising applications in the field of cancer detection and monitoring. ctDNA (circulating tumor DNA) in cfDNA contains tumor-specific genetic information and can therefore be used as a substitute for tumor DNA for diagnosis and prognosis monitoring. In GBM, cerebrospinal fluid is a rich source of ctDNA, but the continuous collection of cerebrospinal fluid is invasive, which hinders its clinical application. Blood collection is basically non-invasive, and the detection of plasma ctDNA is easier to operate clinically. However, due to the limitation of the blood-brain barrier, the content of plasma ctDNA in patients with CNS tumors is much lower than that in solid tumors, so the sensitivity and specificity of somatic mutation detection based on GBM plasma ctDNA are low. Although specific detection methods based on whole exome sequencing (WES) of matched tissues can effectively improve the sensitivity of mutations, this tumor-informed method is often expensive and has a long experimental cycle, which hinders its practical clinical application. Methylation detection is low-cost and can be detected by PCR-based experimental methods with a short reporting time, which is more suitable for hospital auxiliary diagnosis and clinical routine molecular projects. In addition, ctDNA methylation can trace the tissue origin of tumors, and methylation signals are much richer than gene mutation signals, which can improve the sensitivity of tumor detection. Therefore, in the scenario of recurrence monitoring of GBM patients after treatment, the ctDNA content is low, the methylation pattern has a greater advantage, and has a better application prospect.

[0007] Chinese patent document CN116790755A discloses a marker for glioma detection and its application. The marker is TEC with methylation at CpG site. The marker is specifically used for early screening and prognosis monitoring of glioma. It provides a variety of TEC with methylation at different CpG sites as markers for glioma, which has high diagnostic value and provides a new auxiliary diagnosis method for glioma, making the diagnosis of glioma more convenient. In addition, a methylation-based risk score model was constructed through LASSO Cox regression as the cause of poor outcomes in patients with glioma.

[0008] However, there are no reports on cfDNA methylation markers related to muscle tone after surgery, chemoradiotherapy and radiotherapy for glioblastoma, as well as their prediction models and applications. Summary of the invention

[0009] The purpose of the present invention is to provide a group of cfDNA methylation markers related to muscle tone after surgery and chemoradiotherapy for glioblastoma, its prediction model, and its application in the prediction of muscle tone status after surgery and chemoradiotherapy.

[0010] The first aspect of the present invention provides a set of cfDNA methylation markers related to muscle tone after glioblastoma surgery and radiotherapy and chemotherapy, which are composed of the following three methylation sites located on the target genes PCDHGA5, DNAJC5B, and PCDHGA5 respectively: chr5:141419792, chr8:66069461, chr5:141441923.

[0011] The second aspect of the present invention provides the use of a reagent for detecting the methylation level of the methylation marker as described above in the preparation of a kit for predicting the muscle tension status after surgery and radiotherapy and chemotherapy for glioblastoma.

[0012] In a third aspect of the present invention, a kit is provided for predicting the muscle tension status after surgery and radiotherapy and chemotherapy for glioblastoma, wherein the kit comprises a reagent for detecting the methylation level of the methylation marker as described above in a biological sample.

[0013] Furthermore, the reagents for detecting the methylation level of the methylation marker include any one or more reagents used in the following methylation detection methods: PCR based on bisulfite conversion (such as methylation-specific PCR), DNA sequencing (such as pyrophosphate sequencing, bisulfite sequencing, whole genome methylation sequencing, simplified bisulfite sequencing), DNA enrichment detection method, methylation-sensitive restriction endonuclease analysis, fluorescence quantification method, methylation-sensitive high-resolution melting curve method, methylation chip method, mass spectrometry (such as flight mass spectrometry), or a combination thereof.

[0014] Furthermore, the reagents also include: bisulfite and its derivatives, PCR buffer, polymerase, dNTP, primers, probes, methylation-sensitive or -insensitive restriction endonucleases, enzyme digestion buffer, fluorescent dyes, fluorescence quenchers, fluorescent reporters, exonucleases, alkaline phosphatase, internal standards, and controls.

[0015] Furthermore, the prediction of muscle tension after surgery and radiotherapy and chemotherapy for glioblastoma is based on the results of methylation level detection of biological samples from the subject.

[0016] Furthermore, the biological sample is blood.

[0017] The present invention also provides a method for predicting muscle tension after surgery and radiotherapy and chemotherapy for glioblastoma, which specifically comprises the following steps:

[0018] (a) Detecting the methylation levels of three methylation sites chr5:141419792, chr8:66069461, and chr5:141441923 in the sample to be tested;

[0019] (b) The predicted score is calculated according to the following formula:

[0020]

[0021] When Prob is greater than 0.29, muscle tone recovery is poor, and when Prob is less than or equal to 0.29, muscle tone recovery is unchanged or improved.

[0022] The fourth aspect of the present invention provides a set of cfDNA methylation markers related to muscle tone after glioblastoma surgery and chemoradiotherapy as described above, and reagents for detecting the methylation levels of the methylation markers as described above, for use in establishing a prediction system for muscle tone status after glioblastoma surgery and chemoradiotherapy.

[0023] In a fifth aspect of the present invention, a prediction system for muscle tension status after surgery and radiotherapy and chemotherapy for glioblastoma is provided, the prediction system comprising:

[0024] A processor and a memory, the memory being coupled to the processor, the memory storing instructions which, when executed by the processor, use the following steps:

[0025] The methylation levels of the three methylation sites chr5:141419792, chr8:66069461, and chr5:141441923 in the sample to be tested are entered into the following calculation formula:

[0026]

[0027] When Prob is greater than 0.29, muscle tone recovery is poor, and when Prob is less than or equal to 0.29, muscle tone recovery is unchanged or improved.

[0028] A sixth aspect of the present invention provides a use of the prediction system as described above in the preparation of a product for predicting the muscle tension status after surgery and radiotherapy and chemotherapy for glioblastoma.

[0029] According to a seventh aspect of the present invention, a computer-readable storage medium is provided, on which a computer program is stored, and when the computer program is executed by a processor, the following steps are performed:

[0030] The methylation levels of the three methylation sites chr5:141419792, chr8:66069461, and chr5:141441923 in the sample to be tested are entered into the following calculation formula:

[0031]

[0032] When Prob is greater than 0.29, muscle tone recovery is poor, and when Prob is less than or equal to 0.29, muscle tone recovery is unchanged or improved.

[0033] In an eighth aspect, the present invention provides a product for predicting the muscle tension status after surgery and radiotherapy and chemotherapy for glioblastoma, comprising:

[0034] Reagents for detecting the methylation levels of three methylation sites chr5:141419792, chr8:66069461, and chr5:141441923 in the sample to be tested;

[0035] and the prediction system for muscle tension status after glioblastoma surgery and radiotherapy and chemotherapy, or the computer-readable storage medium.

[0036] In the ninth aspect of the present invention, there is provided a methylation marker, a kit, a prediction system, a computer-readable storage medium, and a product for predicting the muscle tension status after surgery and chemoradiotherapy for glioblastoma as described above, for use in the preparation of a product for monitoring the tumor control effect or neurological function recovery after surgery and chemoradiotherapy for glioblastoma.

[0037] The recovery of muscle tone can be used as an indicator to evaluate the recovery of neurological function in patients with glioblastoma (GBM) after surgery and radiotherapy and chemotherapy. Poor muscle tone recovery indicates poor tumor control or severe neurological damage. No change or improvement in muscle tone recovery indicates good tumor control or mild neurological damage.

[0038] The advantages and beneficial effects of the present invention are:

[0039] 1. The present invention provides a set of new cfDNA methylation markers for evaluating GBM, which are innovative and specific for predicting muscle tension status after surgery and chemoradiotherapy.

[0040] 2. It realizes non-invasive detection, which is convenient for clinical application and dynamic monitoring.

[0041] 3. Developed accurate prediction models and standardized tools to promote the standardization and individualization of treatment effect monitoring.

[0042] 4. It has wide application value and can be used for treatment effect evaluation, prognosis prediction and treatment strategy optimization.

[0043] 5. It reveals a new molecular mechanism and provides a scientific basis and reference for tumor research. The present invention has important application prospects and scientific value in the clinical diagnosis and treatment and scientific research fields of glioblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1. Statistical box plot of the methylation rates of the three screened methylation sites in the training set between the groups with and without muscle tone changes (P value calculated using the Wilcox rank sum test).

[0045] Figure 2 . Statistical box plot of the methylation rates of the three screened methylation sites in the test set between the groups with and without muscle tone changes (P value calculated using the Wilcox rank sum test).

[0046] Figure 3 . ROC curve of the model’s predictive effectiveness in the training set.

[0047] Figure 4 . ROC curve of the model’s predictive effectiveness in the test set.

[0048] Figure 5 . Distribution of methylation rates of each methylation site in the blood of the good and poor muscle tone groups after surgery.

[0049] Figure 6 . ROC curve of the model's predictive power in postoperative blood samples. DETAILED DESCRIPTION

[0050] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0051] Example:

[0052] 1. Experimental Materials and Methods

[0053] 1. Research subjects

[0054] Samples from 34 glioblastoma patients from Baoshan District Renhe Hospital were collected, including 27 tissue samples, 34 preoperative blood samples, and 32 postoperative blood samples. The clinical and pathological data of all subjects were obtained from the medical records of each patient. All patients enrolled in this study have given informed consent, and Baoshan District Renhe Hospital has approved the relevant research.

[0055] 2. Treatment plan and sample collection

[0056] We used the clinical pathological diagnosis report as the standard to judge the patient's muscle tone. Before surgery, none of the patients in the study had received chemotherapy, immunotherapy drugs or radiotherapy. All patients underwent glioblastoma resection, and then started chemoradiotherapy 2-4 weeks after surgery according to their physical condition after surgery. The chemotherapy drug was temozolomide capsules (TMZ). During synchronous chemoradiotherapy, TMZ was 75 mg / m2 / d for 42 consecutive days; during adjuvant chemotherapy, TMZ was 150-200 mg / m2 / d, taken orally for 5 consecutive days, repeated every 28 days, for a total of 6 cycles. Radiotherapy uses three-dimensional conformal radiation therapy (3D-CRT) or intensity modulated radiation therapy (IMRT) technology for local brain irradiation. The gross tumor volume (GTV) is the visible lesion after surgery. The GTV is expanded outward by 1-2 cm to obtain the clinical target volume (CTV), and the CTV is expanded outward by 0.3-0.5 cm to obtain the plan target volume (PTV). The radiation dose is DT: 60 Gy, divided into 30 times, 2.0 Gy each time, 5 days a week, and the treatment is completed in 6 weeks.

[0057] Sample collection: Tumor tissue samples (wax block tissue or 15 unprocessed 3μm thick paraffin sections) were collected from GBM patients after surgery. Before surgery, 10 ml of peripheral blood was collected from GBM patients in a free DNA collection tube filled with protective solution. The plasma was stored in a -20°C cold storage or refrigerator after secondary centrifugation on the same day. Seven days after surgery, 10 mL of peripheral blood was collected again, and the plasma was stored after the same centrifugation. The third blood collection was performed 3-6 months after the patient's surgery and radiotherapy and chemotherapy treatment, and the process was the same as before.

[0058] 3. Methylation Sequencing

[0059] Tissue DNA and plasma cfDNA were extracted, and the DNA concentration was controlled by Qubit® 4.0 fluorescent dye method, which was required to be above 0.5 ng / μL. Agarose gel electrophoresis was used for quality control, and there was no obvious genomic DNA contamination (if there was genomic DNA contamination that could be identified by agarose gel electrophoresis, the cfDNA fragment region needed to be cut and recovered). After extraction, the qualified cfDNA was stored in a -20 ℃ cold storage or refrigerator for future use.

[0060] Then, the tumor tissue DNA was subjected to simplified methylation (RRBS) sequencing, and the extracted tissue DNA was digested with MspⅠ, and then the end repair was performed using a combination system of T4 DNA polymerase, polynucleotide kinase, Taq enzyme, etc., and then the template DNA was connected to the methylated complete adapter under the action of T4 ligase (high concentration PEG buffer), and then the library was determined to be within the range of 190-270 bp after double rounds of magnetic bead sorting, and then sodium sulfite conversion (Zymo Research EZ DNAMethylation-Gold series kit, product number D5005) was performed, and finally the conversion product was PCR amplified using short adapter primers. The final post-PCR purified product was quantified using the Qubit®4.0 fluorescent dye method, with a concentration of not less than 20 ng / μL, and the main band of the library was distributed between 190-270 bp by agarose gel electrophoresis quality control.

[0061] Whole genome methylation sequencing (WGBS) was performed on blood cfDNA. 25 ng of cfDNA samples were converted using a commercial methylation kit (Zymo Research EZ DNA Methylation-Gold series kit, catalog number D5005) according to the instructions, and then quantified using the Qubit®4.0 fluorescent dye method, with a concentration of not less than 1 ng / μl. 1% Lambda DNA (for subsequent conversion rate calculation) was added to the converted cfDNA, and the library was constructed using a micro DNA kit based on terminal transferase single-stranded DNA adapter ligation (Abclonal methylation library construction kit, catalog number RK20220). The final PCR-purified product was quantified using the Qubit®4.0 fluorescent dye method, with a concentration of not less than 30 ng / μL. The main band of the library was distributed around 310 bp by agarose gel electrophoresis quality control. The libraries were mixed in equal molar numbers and added to the Illumina NovaSeq 6000 platform S4 chip flow cell for PE150 sequencing. After sequencing, the data were split according to the double-end independent index and uploaded to the laboratory server.

[0062] The initial methylation data were subjected to analysis procedures such as removal of adapters, data quality control, genome alignment, CpG site identification, coverage depth analysis, methylation degree quantification, and site annotation. Medical record inquiries and telephone follow-up were used to understand the postoperative conditions of the patients.

[0063] 2. Experimental Results

[0064] 1. Clinical information

[0065] Muscle tension is closely related to the patient's quality of life, and the change of this physiological index can be used as a quantitative indicator to evaluate the effect of surgical treatment. We extracted the changes in muscle tension of each patient before and after surgery as the endpoint information, where 0 means that the muscle tension remains unchanged or improves after surgery, and 1 means that the muscle tension deteriorates after surgery.

[0066] Detailed clinical information is shown in Table 1.

[0067] Table 1. Information on tissue samples and muscle tension changes of each patient

[0068]

[0069] 2. Site selection

[0070] Table 2. Important HGG-related hypermethylation sites reported in the literature

[0071]

[0072] Table 3. HGG methylation sites associated with changes in muscle tone

[0073]

[0074] When constructing a prediction model for changes in muscle tone in patients, the present invention refers to the hypermethylated genes related to the prognosis and recurrence of HGG in existing literature (Table 2), ensuring that the sites selected by the model are related to the occurrence and development of HGG, making the characteristics of the model relevant to the disease and improving the interpretability of the model. In the process of constructing the model, the present invention extracts the methylation rate information of the sites in this area of ​​the preoperative blood, and compares the methylation rate information of each site in each sample of the training set, and compares the changes in methylation rate between the muscle tone change group and the unchanged group. After comparison and screening, 3 key sites were found (Table 3), and these three sites were used for logistic regression modeling.

[0075] 3. Model Results

[0076] Figure 1 Box plots showing the methylation rates of the three methylation sites selected in the training set between the groups with and without muscle tone changes (P values ​​were calculated using the Wilcox rank sum test). Figure 2 The box plots for the methylation rates of the three methylation sites selected in the test set between the groups with and without muscle tone changes (P value was calculated using the Wilcox rank sum test). The results showed that the methylation rates of the three methylation sites were significantly different between the pre-treatment samples of the two groups of patients.

[0077] The model for predicting muscle force changes was trained using a logistic regression model, and the methylation rate information of the three sites was used as the model input information. After constructing the logistic regression linear model (Formula I), the ROC curves of the model's predictive effectiveness in the training set and the test set were plotted. Preoperative blood samples (34) were used as the training set, and tissue samples (27) were used as the test set.

[0078] Formula I:

[0079]

[0080] The calculated Prob for the best prediction in the training set was 0.29, that is, a Prob greater than 0.29 indicates poor muscle tone recovery, and a Prob less than or equal to 0.29 indicates no change or improvement in muscle tone recovery.

[0081] In the training set, the sensitivity was 1, the specificity was 0.84, and the ROC curve area (AUC) was 0.956 (95% confidence interval was 0.894-1.000), which performed well ( Figure 3 ). In the test set, the sensitivity was 0.375, the specificity was 0.947, and the ROC curve area (AUC) was 0.921 (95% confidence interval was 0.813-1.000), which was not significantly lower than that of the training set, indicating that the model was not overfitting and was scalable ( Figure 4 ).

[0082] 4. Results of the 32-sample model after surgery + chemoradiotherapy

[0083] Figure 5 The methylation rate distribution diagram of each methylation site in the blood of the good and poor muscle tone groups after surgery. It can be seen that there is no significant difference in the methylation rate distribution of the three sites after surgery. Figure 6 The ROC curve of the model's predictive effectiveness in postoperative blood samples shows that the model's AUC is only 0.611. These results indicate that surgical treatment itself has affected the distribution of methylation ratios at the three sites of the patient, and also indirectly indicate that these sites are closely related to the patient's treatment.

[0084] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A set of cfDNA methylation markers related to muscle tone after surgery and chemoradiotherapy for glioblastoma, characterized by: It consists of the following three methylation sites located on the target genes PCDHGA5, DNAJC5B, and PCDHGA5 respectively: chr5:141419792, chr8:66069461, and chr5:141441923.

2. Use of a reagent for detecting the methylation level of the methylation marker as claimed in claim 1 in the preparation of a kit for predicting the muscle tension status after surgery and radiotherapy and chemotherapy for glioblastoma.

3. A kit for predicting muscle tension after surgery and radiotherapy and chemotherapy for glioblastoma, characterized in that: The kit comprises a reagent for detecting the methylation level of the methylation marker as claimed in claim 1 in a biological sample.

4. The kit according to claim 3, characterized in that The biological sample is blood.

5. Use of a group of cfDNA methylation markers related to muscle tone after surgery and chemoradiotherapy for glioblastoma as described in claim 1, and reagents for detecting the methylation level of the methylation markers as described in claim 1 in establishing a prediction system for muscle tone status after surgery and chemoradiotherapy for glioblastoma.

6. A prediction system for muscle tension after surgery and radiotherapy and chemotherapy for glioblastoma, characterized in that: The prediction system comprises: A processor and a memory, the memory being coupled to the processor, the memory storing instructions which, when executed by the processor, use the following steps: The methylation levels of the three methylation sites chr5:141419792, chr8:66069461, and chr5:141441923 in the sample to be tested are entered into the following calculation formula:

7. When Prob is greater than 0.29, muscle tone recovery is poor, and when Prob is less than or equal to 0.29, muscle tone recovery is unchanged or improved.

8. Use of the prediction system as claimed in claim 6 in the preparation of a product for predicting the muscle tension status after surgery and radiotherapy and chemotherapy for glioblastoma.

9. A computer-readable storage medium having a computer program stored thereon, characterized in that: The computer program, when executed by a processor, uses the following steps: The methylation levels of the three methylation sites chr5:141419792, chr8:66069461, and chr5:141441923 in the sample to be tested are entered into the following calculation formula:

10. When Prob is greater than 0.29, muscle tone recovery is poor, and when Prob is less than or equal to 0.29, muscle tone recovery is unchanged or improved.

11. A product for predicting muscle tension after surgery and radiotherapy and chemotherapy for glioblastoma, characterized in that: include: Reagents for detecting the methylation levels of three methylation sites chr5:141419792, chr8:66069461, and chr5:141441923 in the sample to be tested; and the prediction system of claim 6, or the computer-readable storage medium of claim 8.

12. An application of the methylation marker according to claim 1, the kit according to claim 3, the prediction system according to claim 6, the computer-readable storage medium according to claim 8, and the product according to claim 9 in the preparation of a monitoring product for predicting the tumor control effect or neurological function recovery after surgery and chemoradiotherapy for glioblastoma.

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