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CN-115701453-B - Molecular marker and kit for auxiliary diagnosis of cancer

CN115701453BCN 115701453 BCN115701453 BCN 115701453BCN-115701453-B

Abstract

The invention discloses a molecular marker and a kit for auxiliary diagnosis of cancer. The invention provides application of a substance for detecting the methylation level of a BRD4 gene in preparation of a product, wherein the product is used for at least one of assisting in diagnosing cancer or predicting cancer risk, assisting in distinguishing benign nodules from cancers, assisting in distinguishing different subtypes of cancer, assisting in distinguishing different stages of cancer, assisting in distinguishing different cancers, and determining whether an object to be detected has an inhibition or promotion effect on occurrence of the cancer, wherein the cancer can be lung cancer or breast cancer. The invention discovers the hypomethylation phenomenon of BRD4 gene in the blood of lung cancer and breast cancer patients, and has important scientific significance and clinical application value for improving the early diagnosis and treatment effects of lung cancer and breast cancer and reducing the death rate.

Inventors

  • ZHANG ZHENG
  • ZHANG JING
  • DI FEIFEI

Assignees

  • 南京腾辰生物科技有限公司

Dates

Publication Date
20260505
Application Date
20210802

Claims (8)

  1. 1. Use of a substance for detecting the methylation level of a BRD4 gene for the preparation of a product for aiding in the differentiation of lung cancer from a cancer-free control; The methylation level of the BRD4 gene is the methylation level of the CpG site shown in any one of the following (e 1) - (e 2) in the BRD4 gene: (e1) All CpG sites on the DNA fragment shown in SEQ ID No. 4; (e2) All CpG sites on both DNA fragments shown in SEQ ID No.2 and SEQ ID No. 4.
  2. 2. Use of a substance for detecting the methylation level of the BRD4 gene and a medium describing a mathematical model building method and/or a use method for the preparation of a product for the purpose of assisting in distinguishing lung cancer from a cancer-free control; the mathematical model is obtained according to a method comprising the following steps: (A1) Detecting BRD4 gene methylation levels of n1 type a samples and n2 type B samples, respectively; (A2) Taking BRD4 gene methylation level data of all samples obtained in the step (A1), establishing a mathematical model by a two-classification logistic regression method according to classification modes of A type and B type, and determining a threshold value of classification judgment; the using method of the mathematical model comprises the following steps: (B1) Detecting the methylation level of BRD4 genes of a sample to be detected; (B2) Substituting the BRD4 gene methylation level data of the sample to be detected obtained in the step (B1) into the mathematical model to obtain a detection index, comparing the detection index with the threshold value, and determining whether the type of the sample to be detected is A type or B type according to the comparison result; the type a sample and the type B sample are lung cancer samples and non-cancer controls; The methylation level of the BRD4 gene is the methylation level of the CpG site shown in any one of the following (e 1) - (e 2) in the BRD4 gene: (e1) All CpG sites on the DNA fragment shown in SEQ ID No. 4; (e2) All CpG sites on both DNA fragments shown in SEQ ID No.2 and SEQ ID No. 4.
  3. 3. The application of the medium recorded with the mathematical model establishing method and/or the using method in the preparation of products for assisting in distinguishing lung cancer from non-cancer control; the mathematical model is obtained according to a method comprising the following steps: (A1) Detecting BRD4 gene methylation levels of n1 type a samples and n2 type B samples, respectively; (A2) Taking BRD4 gene methylation level data of all samples obtained in the step (A1), establishing a mathematical model by a two-classification logistic regression method according to classification modes of A type and B type, and determining a threshold value of classification judgment; the using method of the mathematical model comprises the following steps: (B1) Detecting the methylation level of BRD4 genes of a sample to be detected; (B2) Substituting the BRD4 gene methylation level data of the sample to be detected obtained in the step (B1) into the mathematical model to obtain a detection index, comparing the detection index with a threshold value, and determining whether the type of the sample to be detected is A type or B type according to a comparison result; the type a sample and the type B sample are lung cancer samples and non-cancer controls; The methylation level of the BRD4 gene is the methylation level of the CpG site shown in any one of the following (e 1) - (e 2) in the BRD4 gene: (e1) All CpG sites on the DNA fragment shown in SEQ ID No. 4; (e2) All CpG sites on both DNA fragments shown in SEQ ID No.2 and SEQ ID No. 4.
  4. 4. The method according to claim 1 to 3, wherein the substance for detecting the methylation level of the BRD4 gene is a primer set.
  5. 5. The method of claim 4, wherein the primer set is any one of (1) primer set D, (2) primer set B and primer set D; The primer pair B is a primer pair consisting of a primer B1 and a primer B2, wherein the primer B1 is single-stranded DNA shown by SEQ ID No.7 or 11 th-35 th nucleotide of the SEQ ID No.7, and the primer B2 is single-stranded DNA shown by SEQ ID No.8 or 32 nd-56 th nucleotide of the SEQ ID No. 8; the primer pair D is a primer pair consisting of a primer D1 and a primer D2, the primer D1 is single-stranded DNA shown by SEQ ID No.11 or 11-35 nucleotides of the SEQ ID No.11, and the primer D2 is single-stranded DNA shown by SEQ ID No.12 or 32-56 nucleotides of the SEQ ID No. 12.
  6. 6.A system, comprising: (D1) Reagents and/or instrumentation for detecting BRD4 gene methylation levels; (D2) A device comprising a unit X and a unit Y; The unit X is used for establishing a mathematical model and comprises a data acquisition module, a data analysis processing module and a model output module; The data acquisition module is configured to acquire BRD4 gene methylation level data of n 1A type samples and n 2B type samples obtained by (D1) detection; The data analysis processing module is configured to receive BRD4 gene methylation level data of n 1A type samples and n 2B type samples sent by the data acquisition module, establish a mathematical model through a two-classification logistic regression method according to classification modes of the A type and the B type, and determine a threshold value of classification judgment; The model output module is configured to receive the mathematical model sent by the data analysis processing module and output the mathematical model; The unit Y is used for determining the type of the sample to be detected and comprises a data input module, a data operation module, a data comparison module and a conclusion output module; the data input module is configured to input (D1) the BRD4 gene methylation level data of the tested person obtained by detection; the data operation module is configured to receive BRD4 gene methylation level data of the testee sent by the data input module, and substitutes the BRD4 gene methylation level data of the testee into the mathematical model established by the data analysis processing module in the unit X, so as to calculate a detection index; The data comparison module is configured to receive the detection index sent by the data operation module and compare the detection index with the threshold value determined by the data analysis processing module in the unit X; The conclusion output module is configured to receive the comparison result sent by the data comparison module and output a conclusion of whether the type of the sample to be tested is A type or B type according to the comparison result; the type a sample and the type B sample are lung cancer samples and non-cancer controls; The methylation level of the BRD4 gene is the methylation level of the CpG site shown in any one of the following (e 1) - (e 2) in the BRD4 gene: (e1) All CpG sites on the DNA fragment shown in SEQ ID No. 4; (e2) All CpG sites on both DNA fragments shown in SEQ ID No.2 and SEQ ID No. 4.
  7. 7. The system of claim 6, wherein the reagent for detecting the methylation level of the BRD4 gene is a primer combination.
  8. 8. The system of claim 7, wherein the primer set is any one of (1) primer set D, (2) primer set B and primer set D; The primer pair B is a primer pair consisting of a primer B1 and a primer B2, wherein the primer B1 is single-stranded DNA shown by SEQ ID No.7 or 11 th-35 th nucleotide of the SEQ ID No.7, and the primer B2 is single-stranded DNA shown by SEQ ID No.8 or 32 nd-56 th nucleotide of the SEQ ID No. 8; the primer pair D is a primer pair consisting of a primer D1 and a primer D2, the primer D1 is single-stranded DNA shown by SEQ ID No.11 or 11-35 nucleotides of the SEQ ID No.11, and the primer D2 is single-stranded DNA shown by SEQ ID No.12 or 32-56 nucleotides of the SEQ ID No. 12.

Description

Molecular marker and kit for auxiliary diagnosis of cancer Technical Field The invention relates to the biomedical field, in particular to a molecular marker and a kit for auxiliary diagnosis of cancers. Background Lung cancer is a malignant tumor that occurs in the epithelium of bronchial mucosa, and is the cancer with highest morbidity and mortality worldwide. In recent years, despite new progress in diagnostic methods, surgical techniques, chemotherapeutic drugs, etc., the overall 5-year survival rate of lung cancer patients is only 16%, mainly because most lung cancer patients have been transferred at the time of diagnosis, and have lost the opportunity for radical surgery. The study shows that the prognosis of lung cancer is directly related to stage, the survival rate of lung cancer in stage I for 5 years is 83%, the survival rate in stage II is 53%, the survival rate in stage III is 26%, and the survival rate in stage IV is 6%. Therefore, the key to reducing mortality in lung cancer patients is early diagnosis and early treatment. The current methods for diagnosing lung cancer mainly include (1) imaging methods such as chest X-ray and low-dose spiral CT. However, early lung cancer is difficult to detect by chest X-rays. Although low-dose spiral CT can find nodules in the lung, the false positive rate is high and can reach 96.4%, and unnecessary psychological burden is brought to a checked person. At the same time, chest X-rays and low dose helical CT are not suitable for frequent use due to radiation. In addition, imaging methods are also often affected by equipment and physician experience, and effective film reading time. (2) Cytologic methods such as sputum cytology, bronchoscopy brushing or biopsy, bronchoalveolar lavage cytology, and the like. Sputum cytology examination bronchoscopy lower brush piece or bronchoscopy lower brush piece biopsy was less sensitive to peripheral lung cancer. Meanwhile, the operation of brushing a piece under a bronchoscope or taking a biopsy and performing cytological examination on bronchoalveolar lavage fluid is complicated, and the comfort level of a physical examination person is poor. (3) The currently used serum tumor markers are carcinoembryonic antigen (CEA), carbohydrate antigen (CA 125/153/199), cytokeratin 19 fragment antigen (CYFRA 21-1), neuron Specific Enolase (NSE) and the like. These serum tumor markers have limited sensitivity to lung cancer, typically only 30-40%, and even lower for stage I tumors. Furthermore, tumor specificity is also limited, and is affected by many benign lesions, such as benign tumors, inflammation, degenerative diseases, and the like. At present, the tumor markers are mainly used for screening malignant tumors and rechecking tumor treatment effects. Therefore, there is a need to further develop a highly efficient and specific early diagnosis technique for lung cancer. Currently, the most effective method of internationally accepted pulmonary nodule diagnosis is chest low dose helical CT screening. However, the low-dose helical CT has high sensitivity, and a large number of nodules can be found, but it is difficult to determine whether or not the subject is benign or malignant. Among the nodules found, the malignant proportion was still less than 4%. Currently, clinical identification of benign and malignant lung nodules requires long-term follow-up, repeated CT examination, or invasive examination methods relying on biopsy sampling of lung nodules (including chest wall fine needle biopsy, bronchoscopy biopsy, thoracoscopy or open chest lung biopsy), and the like. CT guided or ultrasound guided transthoracic biopsy has higher sensitivity, but has lower diagnosis rate for <2cm nodules, 30-70% missed diagnosis rate, and higher pneumothorax and hemorrhage incidence rate. The incidence rate of the aspiration biopsy complications of the bronchoscope needle is relatively low, but the diagnosis rate of the surrounding nodules is limited, the diagnosis rate of the nodules less than or equal to 2cm is only 34%, and the diagnosis rate of the nodules greater than 2cm is 63%. Surgical excision has a high diagnostic rate and can directly treat the node, but can cause a transient decline in patient lung function, and if the node is benign, the patient performs unnecessary surgery, resulting in excessive medical treatment. Therefore, there is a strong need for new in vitro diagnostic molecular markers to aid in the identification of pulmonary nodules, while reducing the rate of missed diagnosis and minimizing unnecessary punctures or surgeries. Breast cancer is a malignant tumor caused by uncontrolled proliferation of mammary epithelial cells. Breast cancer is one of the most common malignant tumors of women worldwide, and the incidence rate is the first place of female malignant tumors. The survival rate of breast cancer is related to the class and stage of the tumor. The 5-year survival prognosis for early stage breast cancer is generally h