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Methylated biomarker or combination thereof for detecting breast cancer and application

A biomarker and methylation technology, which can be used in biochemical equipment and methods, microbial determination/inspection, recombinant DNA technology, etc., and can solve problems such as increased breast cancer risk

Pending Publication Date: 2021-02-23
ANCHORDX MEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The latest research finds 72 new common genetic variants that increase the risk of breast cancer in women, bringing the number of genetic mutations currently known to be associated with breast cancer to 177

Method used

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  • Methylated biomarker or combination thereof for detecting breast cancer and application
  • Methylated biomarker or combination thereof for detecting breast cancer and application
  • Methylated biomarker or combination thereof for detecting breast cancer and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] 1. Plasma cfDNA or tissue extraction and methylation library construction

[0055] 1.1 Extraction of blood cfDNA or tissue DNA.

[0056] The specific operation steps of plasma DNA extraction were carried out according to the instructions of MagMAX™ Cell-Free DNA Isolation Kit of Life Company. The extraction steps of tissue DNA were carried out according to the DNeasy Blood&Tissue Kit operating instructions of QIAGEN;

[0057] 1.2 Conversion

[0058] The extracted cfDNA (10ng) or tissue DNA (50ng) was subjected to bisulfite conversion to deaminate the unmethylated cytosine in the DNA and convert it to uracil, while the methylated cytosine remained unchanged to obtain For the DNA after bisulfite conversion, the specific operation of the conversion was carried out according to the instructions of the EZ DNA Methylation-Lightning Kit of Zymo Research.

[0059] 1.3 End Repair

[0060] 1.3.1 Add the following reagents to the transformed 17ul sample for reaction:

[0061]...

Embodiment 2

[0125] This example discloses a combination of methylation-specific biomarkers for the diagnosis of breast cancer. The plasma samples of 74 cases of mammography and color Doppler ultrasound without abnormality and the plasma samples of 155 cases of breast cancer patients were used in the Different groups of methylation level differences screened out 66 methylation biomarkers related to breast cancer, and verified the differential methylation combination model in normal and breast cancer plasma samples with independent data sets, specifically Process: LASSO and Random Forest were used to perform marker screening and model building in the training set and test set, respectively, including 52 healthy individuals and 108 plasma cfDNA samples from breast cancer patients. 42 methylation markers were screened by the LASSO method, 50 methylation markers were screened by the random forest method, and finally a total of 26 methylation markers were further screened. Finally, in the indep...

Embodiment 3

[0131] Using the information of 26 methylation markers (SEQ ID NO.1-26) among them, 22 cases of mammography and color Doppler ultrasound without abnormal normal people and 47 cases of breast cancer patient plasma samples (among them, 0 phase 3 cases, I 12 cases in stage II, 22 cases in stage II, 8 cases in stage III, and 2 cases in stage IV) were subjected to random combination test, and logistic regression model was used for modeling and analysis, and four markers (cg23035715, cg26371731, cg04541368 and cg13973436) were further screened. The data analysis method is consistent with Example 2, Top1 (cg23035715) and Top1 / 2 / 3 / 4 (cg23035715, cg26371731, cg04541368 and cg13973436) at 98% specificity (22 / 22), the breast cancer of verification set 0 The detection sensitivities of stage I were 100% (3 / 3) and 100% (3 / 3), the detection sensitivities of breast cancer stage I were 91.67% (11 / 12) and 91.67% (11 / 12), and breast cancer II The detection sensitivities of stage III were 90.90% ...

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Abstract

The invention relates to a methylated biomarker and combination thereof for detecting breast cancer and application. The methylated biomarker for detecting the breast cancer is at least one selected from 26 kinds of genome fragments of blood plasma cfDNA methylation-modified modes. The invention further relates to a kit for detecting the methylated biomarker or combination thereof. According to the application, a co-methylation characteristic of a plurality of methylation cytosines on at least one genome fragment is subjected to conjoint analysis and serves as a biomarker for judging onset ofthe breast cancer, the accuracy is far higher than that of detection by other blood plasma biomarkers, and the occurrence of false positive and false negative in breast cancer detection can be lowered.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a methylation biomarker for detecting breast cancer, its combination and application. Background technique [0002] According to the China Cancer Registry Annual Report, the incidence of female breast cancer is at a low level in the 0-24 age group, gradually increases after the age of 25, reaches a peak in the 50-54 age group, and gradually decreases after the age of 55. A large number of studies have been done in Europe and the United States on the susceptibility genes of breast cancer. Currently, BRCA-1, BRCA-2, p53, PTEN, etc. are known. Breast cancers related to these gene mutations are called hereditary breast cancer. cancer, accounting for 5% to 10% of all breast cancers. "Breast cancer 21 gene detection" not only provides 1-5 years and 5-year recurrence risk prediction, but also can be used as the only multi-gene detection to predict the benefit of chemotherapy an...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/154
Inventor 赵德志杨婷王军张显玉庞达陈志伟范建兵
Owner ANCHORDX MEDICAL CO LTD