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WO2021244423A1 - Biomarqueur méthylé pour la détection du cancer du sein et son utilisation - Google Patents

Biomarqueur méthylé pour la détection du cancer du sein et son utilisation Download PDF

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WO2021244423A1
WO2021244423A1 PCT/CN2021/096741 CN2021096741W WO2021244423A1 WO 2021244423 A1 WO2021244423 A1 WO 2021244423A1 CN 2021096741 W CN2021096741 W CN 2021096741W WO 2021244423 A1 WO2021244423 A1 WO 2021244423A1
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chr1
methylation
breast cancer
sites
biomarker
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赵德志
杨婷
张显玉
庞达
陈志伟
范建兵
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AnchorDx Medical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a methylation biomarker for detecting breast cancer and its application.
  • cfDNA is a small fragment of free nucleic acid DNA in peripheral blood, derived from the metabolism and apoptosis of normal cells or tumor cells, and contains genetic information such as somatic mutations and DNA methylation.
  • Liquid Biopsy the technology for the occurrence and development of diseases can be mastered, which is called Liquid Biopsy. Compared with traditional tissue biopsy, liquid biopsy has many advantages such as speed, convenience, and less damage.
  • Methylation is an important modification of DNA, which means that under the catalysis of DNA methyltransferase, S-adenosylmethionine (SAM) is used as the methyl donor to transfer the methyl group.
  • SAM S-adenosylmethionine
  • DNA methylation in mammals mainly occurs on the C of the CpG dinucleotide, producing 5-methylcytosine (5mC).
  • 5mC 5-methylcytosine
  • 60% to 90% of the (CpG) in the human genome are methylated, and unmethylated CpG forms clusters of CpG islands, which are mainly located in the promoter and exon regions of genes, with a length of 300 to 3,000 bp.
  • Abnormal methylation near or in the promoter region generally inhibits transcription and silences the expression of related genes.
  • the abnormal hypermethylation of tumor suppressor gene and DNA repair gene promoter region makes tumor suppressor gene silencing and repair gene inactivation, thereby losing the inhibitory effect on tumorigenesis, which is also the main direction of current tumor methylation research.
  • DNA methylation is one of the earliest apparent modification pathways discovered, and its abnormal methylation patterns are highly specific in different tumor tissues.
  • the characteristic methylation "fingerprint" pattern can be used for early diagnosis and staging of cancer, curative effect evaluation, recurrence monitoring and prognosis judgment, etc.
  • breast cancer is the first among women, with an annual incidence of about 304,000.
  • the incidence of breast cancer in women is relatively low in the age range of 0-24 years, gradually rising after 25 years of age, reaching a peak in the age range of 50-54 years, and gradually decreasing after 55 years of age.
  • European and American countries have done a lot of research, now known as BRCA-1, BRCA-2, as well as p53, PTEN, etc.
  • Breast cancer related to these gene mutations is called hereditary breast Cancer, which accounts for 5% to 10% of all breast cancers.
  • the breast cancer 21 gene test not only provides prediction of the risk of recurrence after 1-5 years and 5 years, but also serves as the only multi-gene test to predict the benefit of chemotherapy and endocrine therapy for patients with estrogen receptor-positive invasive breast cancer.
  • Breast cancer 70 gene detection also plays an important role in early breast cancer diagnosis.
  • the current breast cancer genetic detection method is a kind of non-invasive detection, but it needs to be based on the detection of the tissue removed during the original operation (mastectomy, mastectomy, or core puncture biopsy).
  • Breast cancer patients undergo DNA methylation changes in the early stages of tumorigenesis. Some studies have shown that the frequency or density of methylation gradually increases from normal breast to benign lesions, and then to carcinoma in situ or invasive carcinoma.
  • One of the objectives of the present invention is to provide a methylation biomarker for breast cancer.
  • a methylation biomarker for breast cancer detection is a hypomethylated cg23035715 fragment, the sequence is shown in SEQ ID NO.1.
  • the cg23035715 fragment includes any at least 3 of the following 15 methylation sites and a combination of co-methylation patterns above the methylation modification CpG site, and the 15 methylation sites
  • the points are: chr1:223302630, chr1:223302635, chr1:223302651, chr1:223302662, chr1:223302700, chr1:223302710, chr1:223302753, chr1:223302762, chr1:223302764, chr1:223302793, chr1:223302797, chr1:223302793 , Chr1:223302807, chr1:223302809 and chr1:223302825.
  • the at least 3 methylation modification CpG site and above co-methylation pattern combinations include methylation from chr1:223302630, chr1:223302635, chr1:223302651 and chr1:223302662 positions Modify at least one of the CpG sites.
  • the at least 3 methylation modification CpG sites or more of the co-methylation pattern combination includes methylation from chr1:223302630, chr1:223302635, chr1:223302651 and chr1:223302662 positions At least two modified CpG sites.
  • the at least 3 methylation modification CpG sites or more of the co-methylation pattern combination includes methylation from chr1:223302630, chr1:223302635, chr1:223302651 and chr1:223302662 positions At least three modified CpG sites.
  • the at least 3 methylation modified CpG sites or more of the co-methylation pattern combination includes chr1: 223302630, chr1: 223302635, chr1: 223302651 and chr1: 223302662 methylation modified CpG At least one site; and at least two of the following methylation modified CpG sites: chr1:223302700, chr1:223302710, chr1:223302753, chr1:223302762 and chr1:223302764.
  • the at least 3 methylation modification CpG sites or more co-methylation pattern combinations include methylation modifications at chr1:223302630, chr1:223302635, chr1:223302651 and chr1:223302662 positions At least two of the CpG sites; and at least one of the CpG sites including methylation modification at the following positions: chr1:223302700, chr1:223302710, chr1:223302753, chr1:223302762 and chr1:223302764.
  • 4 of the 15 methylation sites are methylated to modify the combination of co-methylation patterns above the CpG site: chr1: 223302630, chr1: 223302635, chr1: 223302651 and 223302700.
  • 5 of the 15 methylation sites are methylated to modify the combination of co-methylation patterns above the CpG site: 223302630, chr1:223302635, chr1:223302651, chr1:223302662 and chr1:223302700.
  • the invention also provides the application of the methylation biomarker in the preparation of a breast cancer detection kit.
  • Another object of the present invention is to provide the application of the above-mentioned methylation biomarker in the preparation of a kit for early diagnosis of breast cancer.
  • Another object of the present invention is to provide a kit for early diagnosis of breast cancer.
  • a kit for the early diagnosis of breast cancer comprising a reagent for detecting that the co-methylation mode of the methylation biomarker in the sample is hypomethylation
  • the sample is peripheral blood, such as whole blood or plasma or serum.
  • the reagents are based on the use of pyrosequencing method, bisulfite conversion sequencing method, methylation chip method, qPCR method, digital PCR method, second-generation sequencing method, third-generation sequencing method, whole genome Reagents used for methylation sequencing method, DNA enrichment detection method, simplified bisulfite sequencing technology, HPLC method, MassArray, methylation-specific PCR, or a combination thereof.
  • the breast cancer is early breast cancer.
  • the inventors of the present invention found that the low co-methylation rate corresponding to the cg23035715 fragment is very related to breast cancer (especially early breast cancer), and can be used as a methylation biomarker for breast cancer diagnosis. Furthermore, 15 methylation sites of the cg23035715 fragment in blood cfDNA of breast cancer patients were also found, including chr1:223302630, chr1:223302635, chr1:223302651, chr1:223302662, chr1:223302700, chr1:223302710, chr1 : 223302753, chr1:223302762, chr1:223302764, chr1:223302793, chr1:223302797, chr1:223302802, chr1:223302807, chr1:223302809 and chr1:223302825 There are at least 3 co-methylation modifications above CpG sites
  • Fig. 1 is a schematic diagram of the comparison result of the co-methylation rate of the cg23035715 fragment in plasma cfDNA of 74 healthy people and 155 breast cancer patients in Example 2.
  • Figure 2 is a schematic diagram of the comparison results of the co-methylation rate of the plasma cfDNA of 74 healthy people and 155 breast cancer patients in Table 1 in the eight random patterns of Table 1.
  • Fig. 3 is the ROC curve (a) of the co-methylation pattern of top1 in the independent verification set in Example 3 and the sensitivity and specificity in different breast cancer stages (b).
  • Figure 4 is the ROC curve (a) of the co-methylation pattern of top3 in the independent verification set in Example 3 and the sensitivity and specificity in different breast cancer stages (b).
  • Figure 5 is the ROC curve (a) of the co-methylation mode combination of top1/2/3 in the independent verification set in Example 3 and the sensitivity and specificity in different breast cancer stages (b).
  • Figure 6 is the ROC curve (a) of the combination of the co-methylation mode of top1/2/3 and the four random co-methylation modes in the independent verification set in Example 3 (a) and the sensitivity and specificity in different breast cancer stages ( b).
  • Figure 7 is the ROC curve (a) of the 69 co-methylation mode combinations in the independent verification set in Example 3 and the sensitivity and specificity (b) in different breast cancer stages.
  • the "plurality” mentioned herein means two or more.
  • “And/or” describes the association relationship of the associated objects, indicating that there can be three types of relationships, for example, A and/or B, which can mean: A alone exists, A and B exist at the same time, and B exists alone.
  • the character “/” generally indicates that the associated objects before and after are in an "or” relationship.
  • This application relates to the discovery of a hypomethylated CpG island (CpG island) used for cell-free DNA (cfDNA) in the plasma cells of breast cancer (Breast Cancer, BC) patients.
  • CpG island used for cell-free DNA (cfDNA) in the plasma cells of breast cancer (Breast Cancer, BC) patients.
  • the forms of all combinations at the CpG site By studying the differences in methylation modification of plasma methylation-modified genome fragments in breast cancer patients and healthy people at various stages, it is found that the plasma genome fragment does have a combination pattern of low methylation rate.
  • the Cancer Genome Atlas (TCGA) database strictly compares and analyzes the information of methylation modified CpG sites in breast cancer and adjacent tissues, and screens specific methylation sites in breast cancer.
  • DML differential methylation locus
  • DMR differential methylation regions
  • Step 1 Breast cancer-specific methylation markers selected based on TGCA breast cancer and para-cancer methylation chip data;
  • Step 2 Plasma cfDNA extraction and methylation database construction of healthy people and breast cancer patients;
  • Step three breast cancer specific panel targeted hybridization to capture methylation pre-library
  • Step 4 Quantify the target captured library, and perform next-generation sequencing on the computer
  • Step 5 Data preprocessing to obtain the real reads data (bam file) captured by the probe;
  • Step 6 Analysis of methylation data, screening breast cancer and normal control plasma cfDNA DML and DMR.
  • This embodiment discloses the above-mentioned screening method for predicting differential methylation markers of ctDNA in breast cancer.
  • the detailed scheme is shown in Figure 1, which specifically includes the following steps:
  • the extracted cfDNA (10ng) is subjected to bisulfite conversion, and the unmethylated cytosine in the DNA is deaminated and converted into uracil, while the methylated cytosine remains unchanged to obtain the bisulfite conversion.
  • the specific operation of the transformation is carried out in accordance with the EZ DNA Methylation-Lightning Kit instruction of Zymo Research.
  • Component Volume (ul) The reaction volume of the previous step 20 H2O 4 MSB1 Buffer 8 MSR1 Reagent 2 MSR5 Reagent 2 MSE1 Enzyme 2 MSE5 Enzyme 2 total capacity 40
  • the hybrid capture kit is xGen Lockdown Reagents from IDT, and the specific operation is performed in accordance with the instructions.
  • co-methylation rate all co-methylated reads/(non-co-methylated reads + co-methylated reads), that is, the number of fragments of all co-methylated combinations in the fragment region is removed by the fragment All numbers measured by next-generation sequencing.
  • P-vlaue is the wilcoxn test, p ⁇ 0.001 is statistically significant; diff is defined as the average methylation rate (mea_Malignant) of plasma cfDNA of breast cancer patients in all samples minus the plasma cfDNA of healthy people The average methylation rate (mean-Normal) of this group in all healthy samples.
  • AUC area under the curve
  • ROC Receiveiver Operating Characteristic
  • the plasma cfDNA of breast cancer patients is based on the DNA fragment unit reference sequence cg23035715, as shown in SEQ ID NO. 1, which includes 15 methylation modified CpG sites, including chr1:223302630, chr1:223302635, chr1:223302651, chr1:223302662, chr1:223302700, chr1:223302710, chr1:223302753, chr1:223302762, chr1:223302764, chr1:223302793, chr1:223302:797, chr1:223302802, chr1:223302807, chr1:223302802 223302809 and chr1:223302825, as shown in the sequence of SEQ ID NO.1. Further, the co-methylation combination of at least three methylation modification sites described above forms a hypomethylation state.
  • One of the detection methods includes the following steps:
  • the sequencing data is compared with a reference sequence, and the methylation result of the target site in the sequencing data is determined based on the comparison result;
  • the reference sequence is the reference sequence cg23035715 of the cfDNA fragment unit in human plasma as a benchmark;
  • the sequencing is performed by a second-generation sequencing method or a third-generation sequencing method.
  • SEQ ID NO.1 ATA CCATGTGGTCAGCA ACTTCTCCA TCTCCTCTTTCTCTTCTTCCTGCAGAGTGAGCCCAC GTTCACCA TTGCTCTTGCCCCTAGCCCACCAGGCCCTGGCCCTCAGCAG GACCT GC GTTTCTGGACTAGGAAGGCCCTGCTCC AC ACC GAG CTGCTGCCCTCTAC
  • the fragment of cg23035715 is 195bp long, Represents 15 CpG sites.
  • the co-hypomethylation sites include chr1:223302630, chr1:223302635, chr1:223302651, chr1:223302662, chr1:223302700, chr1:223302710, chr1:223302753 , Chr1: 223302762, chr1: 223302764, chr1: 223302793, chr1: 223302797, chr1: 223302802, chr1: 223302807, chr1: 223302809 and chr1: 223302825 combinations of at least 3 CpG sites, a total of 69 co-
  • Figure 2 is a selection of 8 combinations in Table 1. Each co-methylated fragment compares the plasma cfDNA co-methylation rate of 74 healthy people and 155 breast cancer patients. From the figure It can be seen from 2 that there is a hypomethylation modification pattern in the plasma cfDNA of each co-methylated fragment breast cancer patient population.
  • the 15 sites of a reference sequence of cg23035715 gene intron DNA fragment unit in plasma cfDNA with a total of 197 bp are described as a simplified form, chr1:223302630, chr1:223302635, chr1:223302651, chr1 15 Chrs of: 223302662, chr1:223302700, chr1:223302710, chr1:223302753, chr1:223302762, chr1:223302764, chr1:223302793, chr1:223302797, chr1:223302802, chr1:223302807, chr1:223302809 and chr1:223G825
  • C and T to represent the above 15 sites
  • C represents the C of the methylated CpG site in the above 15 sites
  • T represents the C of the unmethylated CpG site
  • 15 sites are CCCTCTTTTTTTTTT, CCCCCTTTTTTTTTT, CCTCCTTTTTTTTTT, CCCCTTCTTTTTTTT, CCTTTTCTTTTTTTT, CTCCTTTTTTTTTTT, CCTTCTTTTTTTTTT, CCTCTTCTTTTTTTT and CCTTTTTCTTTTTTT and other 69 combinations of methylated C and unmethylated C and unmethylated.
  • the methylation rates of 69 combinations of co-methylation patterns are significantly lower than those of normal healthy human plasma cfDNA, as shown in Figure 1, and AUC> Above 0.8, the 3 co-methylation pattern combinations of 1-3 in Table 1 all contain the combination of the first 4 CpG sites among the 15 CpG sites, including chr1:223302630, chr1:223302635, chr1: 223302651 and chr1:223302662, participated in the formation of the combination: CCTCTTTTTTTTTTT, CCCCTTTTTTTTTTTTTTT and CCCTTTTTTTTTTTTTT.
  • This form is expressed as a simplified form of the reference sequence of the plasma cfDNA fragment unit with a total of 197bp bases, where C represents the C of the methylated CpG site, T represents the C of the unmethylated CpG site, and the middle base
  • C represents the C of the methylated CpG site
  • T represents the C of the unmethylated CpG site
  • the middle base The base sequence is omitted and not shown, and the sequence is shown in Sequence Table 1 in Example 2.
  • An independent verification set consisting of tissue samples from 22 normal subjects with no abnormalities in mammography and color Doppler ultrasound, and plasma samples from 47 breast cancer patients, to further verify the single combination of CCTCTTTTTTTTTTT co-methylation pattern fragments formed by the first 4 CpG sites
  • the detection sensitivity of phase 0 of the verification set is 55% (2/3)
  • the detection sensitivity of phase I is 36.67% (4/12)
  • the detection sensitivity of phase II is 47.5%.
  • the detection sensitivity of phase III is 22.91% (2/8)
  • the detection sensitivity of phase IV is 27.5% (1/2)
  • the overall sensitivity of the overall detection is 40.18% (19/47)
  • the AUC is 0.8680 respectively, see Figure 3a-3b.
  • the detection sensitivity of phase 0 of the verification set is 100% (3/3), and the detection sensitivity of phase I is 100% (3/3).
  • the detection sensitivity is 100% (12/12)
  • the detection sensitivity of phase II is 68.18% (15/22)
  • the detection sensitivity of phase III is 50% (4/8)
  • the detection sensitivity of phase IV is 100%.
  • the overall sensitivity of the overall detection is 76.59% (36/47)
  • the AUC is 0.9163 respectively, as shown in Figure 4a-4b. From the results, it can be seen that the CCCTTTTTTTTTTTT mode combination has high sensitivity for early breast cancer diagnosis.
  • the detection sensitivity of phase 0 of the verification set was all Is 100% (3/3), the detection sensitivity of phase I is 91.67% (11/12), the detection sensitivity of phase II is both 81.82% (18/22), and the detection sensitivity of phase III is 75% (6 /8), the detection sensitivity of stage IV is 100% (2/2), the overall sensitivity of the overall detection is 85.11% (36/47), and the AUC is 0.9623 respectively. See Figure 5a-5b. From the results, there are three types The overall sensitivity of the model combination for breast cancer diagnosis has been improved, and it is still better for early diagnosis, and the ROC curve stability is better.
  • the detection sensitivity of phase 0 of the verification set is 100% (3/3)
  • the detection sensitivity of phase I is 91.67% (11/12)
  • the detection sensitivity of phase II is both
  • the sensitivity is 95.45% (21/22)
  • the detection sensitivity of stage III is 87.5% (7/8)
  • the sensitivity of stage IV detection is 100% (2/2)
  • the overall sensitivity of the overall detection is 93.62% (44/ 47)
  • AUC is 0.9671, respectively, see Figure 6a-6b.
  • the detection sensitivity of phase 0 of the verification set is 100% (3/3), and the detection sensitivity of phase I is 100% (12 /12), the detection sensitivity of phase II is 95.45% (21/22), the detection sensitivity of phase III is 87.5% (7/8), the detection sensitivity of phase IV is 100% (2/2), the overall detection The overall sensitivity is 95.74% (41/47), and the AUC is 0.9691, respectively, as shown in Figure 7a-7b. From the results, the 69 combinations have high sensitivity for early breast cancer diagnosis, the overall sensitivity is the highest, and the ROC curve stability is the best .
  • the 15 methylation sites include the following combinations of at least 3 or more methylation modified CpG sites: including chr1: 223302630, chr1:223302635, chr1:223302651, chr1:223302662, chr1:223302700, chr1:223302710, chr1:223302753, chr1:223302762, chr1:223302764, chr1:223302793, chr1:223302797, chr1:223302802, chr1:223302302, 807
  • the reference sequence cg23035715 of the plasma cfDNA fragment unit is the benchmark, including 15 CpG sites.
  • the first 4 CpG sites chr1: At least one of 223302630, chr1: 223302635, chr1: 223302651, chr1: 223302662 is more likely to be involved in the co-methylation pattern combination of the plasma cfDNA generation methylation rate of breast cancer patients.
  • the more common or more relevant are 2 of them CpG sites or 3 methylation modified CpG sites participate in the co-methylation mode combination.
  • the 4 methylation sites are combined with the following 5-99 methylation sites, and there are at least 3 methyl groups The probability of chemically modified CpG sites participating in the co-methylation mode combination is also greater.
  • the first column is that the cg23035715 fragment is 197bp long and has 15 CpG sites.
  • the co-methylation mode of at least 3 methylation modification sites is defined as a co-methylated read, including 69 kinds of total Combination of methylation patterns.
  • the second and third columns represent the average co-methylation rate of a single co-methylation pattern fragment in plasma cfDNA of breast cancer patients and healthy people, respectively.
  • the negative number of Diff means that the co-methylation rate of the combination in breast cancer patients is lower than that of normal healthy people.
  • P-value is wilcoxn test (signed rank test).
  • AUC measures the ability of a single co-methylation pattern to distinguish breast cancer patients from healthy people.
  • CCCTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT Expressed in a combined form, not the actual base.
  • the reference sequence of the plasma cfDNA fragment unit has a total of 197 bp bases, C represents the C of the methylated CpG site, and T represents the unmethylated pG site Point C, the base sequence in the middle is omitted.

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Abstract

L'invention concerne un fragment cg23035715 d'un biomarqueur méthylé pour la détection du cancer du sein. L'invention concerne également le fait que l'expression de méthylation faible d'une combinaison de 3 sites CpG ou plus est au moins présente dans 15 sites de méthylation du fragment cg23035715 dans l'ADN acellulaire sanguin d'un patient atteint d'un cancer du sein. Le biomarqueur peut être utilisé pour détecter le cancer du sein. L'invention concerne en outre un kit de détection du biomarqueur méthylé qui est utilisé pour détecter le cancer du sein.
PCT/CN2021/096741 2020-06-01 2021-05-28 Biomarqueur méthylé pour la détection du cancer du sein et son utilisation Ceased WO2021244423A1 (fr)

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CN113930516B (zh) * 2021-12-17 2022-04-19 北京迈基诺基因科技股份有限公司 宫颈癌相关基因甲基化的引物、试剂盒、模型及构建方法
CN113943817B (zh) * 2021-12-20 2022-04-19 北京迈基诺基因科技股份有限公司 宫颈癌癌化水平评估模型及构建方法
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