WO2021233329A1 - 用于检测乳腺癌的甲基化生物标记物或其组合和应用 - Google Patents

用于检测乳腺癌的甲基化生物标记物或其组合和应用 Download PDF

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WO2021233329A1
WO2021233329A1 PCT/CN2021/094588 CN2021094588W WO2021233329A1 WO 2021233329 A1 WO2021233329 A1 WO 2021233329A1 CN 2021094588 W CN2021094588 W CN 2021094588W WO 2021233329 A1 WO2021233329 A1 WO 2021233329A1
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breast cancer
methylation
combination
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赵德志
杨婷
王军
张显玉
庞达
陈志伟
范建兵
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广州市基准医疗有限责任公司
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    • 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
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    • 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

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  • the invention belongs to the field of biotechnology, and specifically relates to a methylation biomarker for detecting breast cancer and a combination and application thereof.
  • breast cancer susceptibility genes European and American countries have done a lot of research, now known as BRCA-1, BRCA-2, as well as p53, PTEN, etc.
  • 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.
  • 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 played an important role in the early diagnosis of breast cancer. The latest research findings have found that 72 new common gene mutations will increase the risk of breast cancer in women, which has increased the number of gene mutations currently known to be associated with breast cancer to 177. Many people may carry common variant genes. A single gene mutation has a relatively small risk of causing cancer. However, the more relevant variant genes in a woman's body, the greater the risk of breast cancer.
  • 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 (lumpectomy, mastectomy, or core puncture biopsy).
  • cfDNA cell-free DNA
  • cfDNA cell-free DNA
  • Liquid Biopsy By detecting disease-specific cfDNA fragments, the technology to master the occurrence and development of diseases is called Liquid Biopsy. Compared with traditional tissue biopsy, it has many advantages such as speed, convenience, and less damage.
  • a study of 640 cases of various tumors from the team of Bert Vogelstein and Kenneth Kinzler found that more than 75% of advanced pancreatic cancer, ovarian cancer, colorectal cancer, bladder cancer, gastroesophageal cancer, melanoma cancer, and hepatocellular carcinoma As well as patients with head and neck cancer, the presence of ctDNA can be detected.
  • ctDNA is a kind of biomarker with wide applicability, sensitivity and specificity, which can be used in the clinical and research of various and many different types of cancer.
  • Professor Lu Yuming proved the technical and theoretical feasibility of liquid biopsy instead of tissue biopsy through cfDNA whole-genome methylation sequencing; in 2017, Professor Zhang Kun’s team used ctDNA methylation to quantitatively describe tumor burden and tumor origin.
  • ctDNA map The Turner team used high-throughput sequencing to find breast cancer-specific somatic mutation sites and monitored their dynamic changes, proving that ctDNA detection can detect tumor recurrence and metastasis earlier than CT.
  • One of the objectives of the present invention is to provide a methylation biomarker or a combination thereof that can be used for breast cancer detection.
  • a methylation biomarker for breast cancer detection or a combination thereof selected from at least one of the following methylation biomarkers:
  • At least one of the following methylation biomarkers is selected:
  • a combination of methylation biomarkers for breast cancer detection including marker cg23035715, and at least one selected from the following markers cg26371731, cg04541368 and cg13973436.
  • the methylation biomarker combination for breast cancer detection includes the marker cg23035715, and the following three markers: cg26371731, cg04541368 and cg13973436.
  • the methylation biomarker combination for breast cancer detection further includes at least one selected from the following biomarkers: cg16304215, cg20072171, cg08402365, cg21501525, cg22778178, cg08599259, cg25566568 , cg15634980, cg07458308, cg01348584, cg14140881, cg25756435, cg00594560, cg08279008, cg09760908, cg18087672, cg14868703, cg17632299, cg18786873, cg20631750, cg25924096, cg24615528, cg22889755, cg23524195, cg20861607, cg22435300, cg25504443, cg14351528, cg25824543,
  • the methylation biomarker combination for breast cancer detection includes markers cg23035715, cg26371731, cg04541368, cg13973436, cg16304215, cg20072171, cg08402365, cg21501525, cg22778178, cg08599259, cg25566568, cg15634980, cg07458308, cg01348584, cg14140881, cg25756435, cg00594560, cg08279008, cg09760908, cg18087672, cg14868703, cg17632299, cg18786873, cg20631750, cg25924096, cg24615528, cg22889755, cg23524195, cg20861607, cg22435300, cg25504443
  • the sequence of the marker cg23035715 is shown in SEQ ID NO. 1
  • the sequence of cg26371731 is shown in SEQ ID NO. 2
  • the sequence of cg04541368 is shown in SEQ ID NO. 3
  • the sequence of cg13973436 As shown in SEQ ID NO.4, the sequence of cg16304215 is shown in SEQ ID NO.5, the sequence of cg20072171 is shown in SEQ ID NO.6, the sequence of cg08402365 is shown in SEQ ID NO.7, and the sequence of cg21501525 is shown in SEQ.
  • sequence of cg22778178 is shown in ID NO.8, the sequence of cg22778178 is shown in SEQ ID NO.9, the sequence of cg08599259 is shown in SEQ ID NO.10, the sequence of cg25566568 is shown in SEQ ID NO.11, and the sequence of cg15634980 is shown in SEQ ID NO. 12, the sequence of cg07458308 is shown in SEQ ID NO. 13, the sequence of cg01348584 is shown in SEQ ID NO. 14, the sequence of cg14140881 is shown in SEQ ID NO. 15, and the sequence of cg25756435 is shown in SEQ ID NO. 16.
  • sequence of cg00594560 is shown in SEQ ID NO.17
  • sequence of cg08279008 is shown in SEQ ID NO.18
  • sequence of cg09760908 is shown in SEQ ID NO.19
  • sequence of cg18087672 is shown in SEQ ID NO.20.
  • sequence of cg14868703 is shown in SEQ ID NO.21
  • sequence of cg17632299 is shown in SEQ ID NO.22
  • sequence of cg18786873 is shown in SEQ ID NO.23
  • sequence of cg20631750 is shown in SEQ ID NO.24
  • sequence of cg25924096 is shown in The sequence is shown in SEQ ID NO.25
  • sequence of cg15321298 is shown in SEQ ID NO.26
  • sequence of cg22889755 is shown in SEQ ID NO.27
  • sequence of cg23524195 is shown in SEQ ID NO.28
  • sequence of cg20861607 is shown in 29,
  • sequence of cg22435300 is shown in SEQ ID NO.30
  • sequence of cg25504443 is shown in SEQ ID NO.31
  • sequence of cg14351528 is shown in SEQ ID NO.32
  • sequence of cg25824543 is shown in SEQ ID 33
  • sequence of cg14825633 is shown in SEQ ID NO. 45
  • sequence of cg11901043 is shown in SEQ ID NO. 46
  • sequence of cg24504927 is shown in SEQ ID 47
  • sequence of cg21962423 is shown in SEQ ID NO.48
  • sequence of cg14135814 is shown in SEQ ID NO.49
  • sequence of cg01070209 is shown in SEQ ID NO.50
  • sequence of cg14841828 is shown in SEQ ID NO.
  • sequence of cg04947764 is shown in SEQ ID NO.52
  • sequence of cg01397141 is shown in SEQ ID NO.53
  • sequence of cg00436496 is shown in SEQ ID NO.54
  • sequence of cg01832036 is shown in SEQ ID NO.55.
  • sequence of cg27125093 is shown in SEQ ID NO.56
  • sequence of cg21319323 is shown in SEQ ID NO.57
  • sequence of cg06916239 is shown in SEQ ID NO.58
  • sequence of cg11667451 is shown in SEQ ID NO.59.
  • sequence of cg03355998 is shown in SEQ ID NO.60
  • sequence of cg19019849 is shown in SEQ ID NO.61
  • sequence of cg22009488 is shown in SEQ ID NO.62
  • sequence of cg15012484 is shown in SEQ ID NO.63
  • sequence of cg20817483 The sequence is shown in SEQ ID NO.64
  • sequence of cg21254450 is shown in SEQ ID NO.65
  • sequence of cg23134869 is shown in SEQ ID NO.66.
  • Another object of the present invention relates to the application of the above-mentioned methylation biomarker combination in the preparation of a kit for detecting breast cancer.
  • Another object of the present invention is to provide a kit for detecting breast cancer.
  • a kit for detecting breast cancer includes a reagent for detecting the methylation status of the methylation biomarker or a combination thereof in a sample to be tested.
  • the breast cancer is breast cancer of different stages, including stages 0, I, II, III, and IV according to different TNMs.
  • the detection method adopted by the kit is the pyrosequencing method, the bisulfite conversion sequencing method, the methylation chip method, the qPCR method, the digital PCR method, the second-generation sequencing method, and the third-generation sequencing method.
  • Method whole genome methylation sequencing method, DNA enrichment detection method, simplified bisulfite sequencing technology, HPLC method, MassArray, methylation-specific PCR, or a combination of reagents used.
  • the sample to be tested is plasma, serum or blood.
  • the present invention discloses plasma gene methylation markers or a combination thereof for early diagnosis of breast cancer. Specifically, the study obtained genomic fragments with obvious abnormal methylation modification patterns in breast cancer plasma cfDNA, namely cg23035715, cg26371731, cg04541368, cg13973436, cg16304215, cg20072171, cg08402365, cg21501525, cg22778178, cg08599259, cg25566568, cg15634980, cg07458308, cg01348584, cg14140881, cg25756435, cg00594560, cg08279008, cg09760908, cg18087672, cg14868703, cg17632299, cg18786873, cg20631750, cg25924096, cg24615528, cg22
  • the present invention jointly analyzes the co-methylation characteristics of multiple methylated cytosines on at least one genome segment as a biomarker for judging the onset of breast cancer. The accuracy is far greater than the detection of other plasma biomarkers, reducing false positives and The occurrence of false negatives.
  • Figure 1 is the ROC curve of the 26 methylation marker combinations in the training set in Example 2.
  • Figure 2 shows the ROC curve of 26 methylation marker combinations and the sensitivity and specificity of different stages of breast cancer in the independent verification set in Example 2.
  • Figure 3 is the ROC curve of the combination of 42 methylation markers screened by LASSO in the independent verification set and 50 methylation markers screened by Random Forest in Example 2.
  • Figure 4 shows the sensitivity and specificity of the combination of 42 (a) and 50 (b) methylation markers screened by Random Forest in the independent verification of centralized LASSO screening in Example 2 in different breast cancer stages.
  • Figure 5 is the ROC curve (a) of the combination of SEQ ID NO.1-26 markers in the independent verification set of Example 2 and random selection of other SEQ ID NO.27-66 markers (a) and the sensitivity of different breast cancer stages , Specificity (b).
  • Fig. 6 is the ROC curve of the combination of methylation markers of SEQ ID NO. 1 or SEQ ID NO. 1-4 in the independent verification set in Example 3.
  • Fig. 7 shows the sensitivity and specificity of different breast cancer stages in a random combination of SEQ ID NO.1-4 methylation markers in the independent verification set in Example 3.
  • Figure 8 is the ROC curve (a) of the independent verification set of SEQ ID NO.1 markers and randomly selected SEQ ID NO.5-26 markers in Example 3 (a) and the sensitivity and specificity of different breast cancer stages ( b).
  • Figure 9 shows the ROC curve (a) of the SEQ ID NO.1 marker in the independent verification set and randomly selected SEQ ID NO.27-66 markers in Example 3 (a) and the sensitivity and specificity (b) of different breast cancer stages. ).
  • Figure 10 is the ROC curve (a) of the independent verification set of SEQ ID NO.1-4 markers and random selection of SEQ ID NO.5-26 markers in Example 3 (a) and the sensitivity and specificity of different breast cancer stages Sex (b).
  • Figure 11 is the ROC curve (a) of the independent verification set of SEQ ID NO.1-4 markers and randomly selected SEQ ID NO.27-66 markers in Example 3 and the sensitivity and specificity of different breast cancer stages (b).
  • the "plurality” mentioned in the present invention 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.
  • the present invention relates to the 66 selected breast cancer-related methylation biomarkers cg23035715, cg26371731, cg04541368, cg13973436, cg16304215, cg20072171, cg08402365, cg21501525, cg22778178, cg08599259, cg25566568, cg15634980, cg07458308, cg01348584, cg14140881, cg25756435, cg00594560, cg08279008, cg09760908, cg18087672, cg14868703, cg17632299, cg18786873, cg20631750, cg25924096, cg24615528, cg22889755, cg23524195, cg20861607, cg22435300
  • the present invention specifically includes the following steps for the screening method for predicting ctDNA differential methylation markers of breast cancer.
  • the specific operation steps of plasma DNA extraction are carried out in accordance with Life's MagMAXTM Cell-Free DNA Isolation Kit operating instructions.
  • the extraction steps of tissue DNA are carried out in accordance with the DNeasy Blood&Tissue Kit operating instructions of QIAGEN;
  • the extracted cfDNA (10ng) or tissue DNA (50ng) is subjected to bisulfite conversion to deaminate the unmethylated cytosine in the DNA into uracil, while the methylated cytosine remains unchanged.
  • the specific operation of the conversion 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.
  • the co-methylation rate of a certain methylation marker referred to herein is actually the sum of the respective methylation rates of all methylation combinations in a specific region of the marker.
  • P-vlaue is the wilcoxn test, and p ⁇ 0.001 is statistically significant; we also calculated the individual AUC values of all 26 markers, and AUC (area under the curve) is under the ROC (Receiver Operating Characteristic) curve The area is used to judge the ability of the combination to diagnose breast cancer. The closer the AUC is to 1, the better.
  • This embodiment discloses a methylation-specific biomarker combination for diagnosing breast cancer, using plasma samples from 74 normal people with no abnormalities in mammography and color Doppler ultrasound and plasma samples from 155 breast cancer patients.
  • the differences in methylation levels of different groups screened out 66 methylation biomarkers related to breast cancer, and the differential methylation combined model was validated in an independent data set in normal and breast cancer plasma samples.
  • Process: LASSO and Random Forest were used for marker screening and model establishment in the training set and test set, respectively, including plasma cfDNA samples of 52 healthy people and 108 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.
  • the 66 methylation biomarkers are: cg23035715, cg26371731, cg04541368, cg13973436, cg16304215, cg20072171, cg08402365, cg21501525, cg22778178, cg08599259, cg25566568, cg15634980, cg14435458, cg14435, and 458308 , cg00594560, cg08279008, cg09760908, cg18087672, cg14868703, cg17632299, cg18786873, cg20631750, cg25924096, cg24615528, cg22889755, cg23524195, cg20861607, cg22435300, cg25504443, cg14351528,
  • the 26 methylation biomarkers (markers) of the intersection of the above two algorithms were detected.
  • the detection sensitivity of phase I is 60% (3/5)
  • the detection sensitivity of phase I is 80% (32/40)
  • the detection sensitivity of phase II is 95.45% (42/44)
  • the detection sensitivity of phase III is 94.74% (18 /19)
  • the overall detection sensitivity is 87.9% (95/108)
  • the AUC is 0.9839.
  • stage IV The detection sensitivity of stage IV is 100%(2/2) and 0%(0/2), respectively.
  • the overall sensitivity of detection is respectively They are 87.23% (43/47) and 59.57% (28/47), and the overall AUC are 0.9709 and 0.9497, respectively. See Figure 3 and Figure 4 for details.
  • cg22889755, cg23524195 and cg20861607 are random Three selected from the other 40 methylation markers ((SEQ ID NO.27-66)), with a specificity of 98% (22/22), the detection sensitivity of phase 0 is 100% (3/ 3), the detection sensitivity of phase I is 83.33% (10/12), the detection sensitivity of phase II is 86.36% (19/22), the detection sensitivity of phase III is 87.5% (7/8) and the detection sensitivity of phase IV is 0 %(0/2), for all breast cancer samples, the overall sensitivity of detection is 82.98% (39/47), and the overall AUC is 0.9797, see Figure 5a-5b. The overall sensitivity has been reduced, but the sensitivity for early breast cancer is higher than the combination of top
  • the plasma samples of 22 normal people with no abnormalities in mammography and color Doppler ultrasound and 47 breast cancer patients including 3 cases of stage 0, I (12 cases in stage II, 22 cases in stage II, 8 cases in stage IV, and 2 cases in stage IV) were used for random combination test, modeling analysis was performed using logistic regression model, and 4 markers (cg23035715, cg26371731, cg04541368 and cg13973436) were further screened.
  • the data analysis method is the same as in Example 2.
  • Top1 (cg23035715) and Top1/2/3/4 (cg23035715, cg26371731, cg04541368 and cg13973436) at 98% specificity (22/22), verify the breast cancer in the set.
  • the detection sensitivity of stage I are 100% (3/3) and 100% (3/3), respectively.
  • the detection sensitivity of breast cancer stage I is 91.67% (11/12) and 91.67% (11/12), respectively.
  • Breast cancer II The detection sensitivity of stage III is 90.90% (20/22) and 90.91% (20/22), respectively.
  • the detection sensitivity of stage III is 87.5% (7/8) and 87.5% (7/8), respectively.
  • Breast cancer stage IV The detection sensitivity is 50% (1/2) and 0% (0/2).
  • the 4 biomarkers cg23035715, cg26371731, cg04541368, and cg13973436.
  • the AUC 0.9395 of the single marker cg23035715 is shown in Figure 6, and Figure 6 shows 4 of the 26 co-methylation markers (cg23035715, The combination of cg26371731, cg04541368 and cg13973436) and marker1 (cg23035715) markers in the identification of breast cancer ability, as can be seen from Figure 6, the AUC of the combination of 4 methylation markers is very close to 26 markers The combined AUC of marker1 and the marker AUC of marker1 are also 0.9395, indicating the importance of these four methylate markers, especially the marker1 marker has a strong ability to diagnose early breast cancer.
  • the detection sensitivity of Top1/2/3 and Top1/2/4 methylation marker combinations in phase 0 of the validation set is 100% (3/3), respectively And 33.33% (1/3), the detection sensitivity of phase I is 100% (12/12) and 66.67% (8/12), respectively, and the detection sensitivity of phase II is 81.82% (18/22) and 63.63% ( 14/22), the detection sensitivity of phase III is 87.5% (7/8) and 75% (6/8) respectively, and the detection sensitivity of phase IV is 50% (1/2) and 0% (0/2), respectively.
  • the overall sensitivity of the overall detection is 85.11% (40/47) and 61.7% (29/47) respectively.
  • top1/2/3 in Figure 6c represents the combination of cg23035715, cg26371731 and cg04541368
  • top1/2/4 represents the combination of 3 markers (cg23035715, cg26371731 and cg13973436), and so on.
  • top1 (SEQ ID NO.1) is combined with cg23035715, cg16304215 and cg08599259 among the random SEQ ID NO.5-26 markers and with the remaining 40 markers (SEQ ID NO.27-66) randomly selected cg01167274, cg07790615, cg23134869, and cg01832036 combinations, the detection sensitivity of phase 0 of the verification set is 100% (3/3) and 100% (3/3), respectively.
  • the detection sensitivity of phase II is 100% (12/12) and 83.33% (10/12), the detection sensitivity of phase II is 95.45% (21/22) and 95.45% (21/22), respectively, the detection sensitivity of phase III Respectively 100% (8/8) and 87.5% (7/8), the IV phase detection sensitivity is 0% (0/2) and 0% (0/2), respectively, the overall sensitivity of the overall detection is 93.61% ( 40/47) and 61.7% (29/47), see Figures 8a-8b and Figures 9a-9b.
  • top1-4 SEQ ID NO.1-4
  • SEQ ID NO.5-26 marker cg16304215
  • the detection sensitivity of phase 0 of the validation set is 100% (3/3)
  • the detection sensitivity of phase I is 91.67% (11/12)
  • the detection sensitivity of phase II is 90.91% (20/22)
  • the detection sensitivity of phase III is 87.5% (7/8)
  • the detection sensitivity of phase IV Both are 0% (0/2)
  • the overall sensitivity of the overall detection is 87.23% (41/47)
  • the AUC are 0.9739 and 0.9797, respectively, as shown in Figure 10a-10b and Figure 11a-11b.
  • cg23035715 has a good performance in the diagnosis of early breast cancer, and the combination with cg26371731, cg04541368 and cg13973436 also shows It has a diagnostic ability similar to the combination of top1-26, and cg23035715 is combined with cg23035715, cg16304215 and cg08599259 in random SEQ ID NO.5-26 markers, and with the remaining 40 markers (SEQ ID NO.27-66 ) The combination of cg01167274, cg07790615, cg23134869 and cg01832036 all showed good diagnostic capabilities.
  • top1-4 and top1-26 (SEQ ID NO.1-26) combinations are respectively combined with the remaining 40 markers (SEQ ID NO.27-66) cg22889755, cg23524195
  • the combination of cg14135814, cg15012484, cg2081748 and the combination of cg22889755, cg23524195, and cg20861607 also showed very stable diagnostic capabilities.
  • the AUC of top1/2/3/4 markers was also above 0.75, such as cg2303571, cg26371731, cg04541368 and The AUC of cg13973436 is 0.9395, 0.8020, 0.7701 and 0.7501, as shown in Table 3.1, indicating that the sites screened by this method have a very high correlation with the early diagnosis of breast cancer.
  • Table 3.1 The performance of 26 methylation marker fragments in the diagnosis of breast cancer-AUC.

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Abstract

本发明公开了一种用于乳腺癌检测的甲基化生物标记物及其组合以及应用，其选自26种血浆cfDNA甲基化修饰模式的基因组片段中的至少一种。本发明还公开了检测上述甲基化生物标记物或其组合的试剂盒。

Description

用于检测乳腺癌的甲基化生物标记物或其组合和应用 技术领域
本发明属于生物技术领域，具体涉及用于检测乳腺癌的甲基化生物标记物及其组合以及应用。
背景技术
据中国肿瘤登记年报显示：女性乳腺癌发病率在0～24岁年龄段处较低水平，25岁后逐渐上升，50～54岁年龄段达到高峰，55岁以后逐渐下降。针对乳腺癌的易感基因，欧、美国家做了大量研究，现已知的有BRCA-1、BRCA-2，还有p53、PTEN等，与这些基因突变相关的乳腺癌称为遗传性乳腺癌，占全部乳腺癌的5％～10％。“乳腺癌21基因检测”不仅提供1-5年及5年后复发风险预测，还能作为唯一多基因检测来预测雌激素受体阳性浸润性乳腺癌患者的化疗及内分泌治疗的获益程度。“乳腺癌70基因检测”也在早期乳腺癌诊断中发挥了重要作用。最新的研究成果发现72个新的常见基因变异将导致女性患乳腺癌的风险上升，这使目前已知与乳腺癌相关的基因突变数量增至177个。许多人都可能携带常见的变异基因，单个基因突变致癌的风险相对较小，但女性体内的相关变异基因越多，患乳腺癌的风险越大。目前乳腺癌基因检测手段属于一种非侵入性检测，但需要基于原来的手术(乳房肿瘤切除手术、乳房切除手术或核心穿刺活组织检查)过程中取出的组织检测基础上。
对肿瘤进行早筛和早诊，可以更大概率地发现早期癌症，从而提高其五年生存率，降低死亡率。中国乳腺癌筛查现状：乳腺癌5年生存率：原位癌100％，I期84-100％，II期76-87％，III期38-77％。中国多中心研究显示，初诊乳腺癌时，15.7％I期，44.9％II期，18.7％III期，2.4％IV期。乳腺癌影像筛查手段：1.钼靶X射线：中国女性乳腺发病年龄较轻(45-55岁)，乳腺腺体较西方女性致密，MG易遗漏，且MG对致密型乳腺病灶诊断敏感性仅为30％。2.B超：单纯MG阳性预测值为55.5％，MG联合B超阳性预测值为43.3％。3.核磁共振(MRI):MRI对微小钙化灶不敏感，且有明确禁忌症；所以，肿瘤的早筛早诊早治疗对提高五年生存率具有非常大的意义，肿瘤早筛早诊的关键是建立有效的检测模型，寻找能够作为筛查与诊断的分子标记。cfDNA(cell-free DNA)是外周血中游离的核酸小片段DNA，源自正常细胞或肿瘤细胞代谢与凋亡，包含体细胞突变和DNA甲基化等遗传信息。通过检测疾病特异性cfDNA片段，掌握疾病的发生、发展的技术，称为液体活检(Liquid Biopsy)，与传统的组织活检相比，其有着迅速、便捷、损伤性小等众多优点。2014年，来自Bert Vogelstein和Kenneth Kinzler团队的一份640例各类肿瘤的研究发现，超过75％的晚期胰腺癌、卵巢癌、结直肠癌、膀胱癌、胃食管癌、黑色素癌、肝细胞癌以及头颈癌症的患者，都能检测到ctDNA的存在。因而ctDNA是一种具有广泛适用性、敏感而特异的生物标志物，能够用于各式各样、多种不同类型癌症的临床和研究。2015年卢煜明教授通过cfDNA全基因组甲基化测序证明了液体活检替代组织活检在技术上和理论上的可行性；2017年张鹍教授团队利用ctDNA的甲基化定量描述了肿瘤负荷以及肿瘤来源的ctDNA图谱。Turner团队利用高通量测序寻找乳腺癌特异性体细胞突变位点，并监测其动态变化，证明ctDNA检测可早于CT发现肿瘤复发转移。最 新研究发现，结合其它分析物血液中的ctDNA突变能更早、更好的诊断卵巢癌，肝癌，胃癌，乳腺癌，前列腺癌，食管癌和结直肠癌这8种常见且可手术切除的癌。临床越来越多的研究表明血浆ctDNA可作为生物标志物应用在肿瘤早期诊断筛选、预测、治疗的反应，监测肿瘤大小和复发等。目前，国际上的研究方向是整合多组学/多种分子标志物、多基因/多位点来提高检测技术的灵敏度和特异性，以满足临床对检测产品的需求。
发明内容
本发明的目的之一在于提供一种可用于乳腺癌检测的甲基化生物标记物或其组合。
实现上述目的的技术方案包括如下。
一种用于乳腺癌检测的甲基化生物标记物或其组合，选自以下甲基化生物标记物中的任意至少一种：
cg23035715，cg26371731，cg04541368，cg13973436，cg16304215，cg20072171，cg08402365，cg21501525，cg22778178，cg08599259，cg25566568，cg15634980，cg07458308，cg01348584，cg14140881，cg25756435，cg00594560，cg08279008，cg09760908，cg18087672，cg14868703，cg17632299，cg18786873，cg20631750，cg25924096，cg24615528，cg22889755，cg23524195，cg20861607，cg22435300，cg25504443，cg14351528，cg25824543，cg23413809，cg24797187，cg26877715，cg01167274，cg12361223，cg04430835，cg27111970，cg22851944，cg23228540，cg26225694，cg07790615，cg14825633，cg11901043，cg24504927，cg21962423，cg14135814，cg01070209，cg14841828，cg04947764，cg01397141，cg00436496，cg01832036，cg27125093，cg21319323，cg06916239，cg11667451，cg03355998，cg19019849，，cg22009488，cg15012484，cg20817483，cg21254450和cg23134869。
在其中一些实施例中，选自以下甲基化生物标记物中的任意至少一种：
cg23035715，cg26371731，cg04541368和cg13973436。
一种用于乳腺癌检测的甲基化生物标记物组合，包括标记物cg23035715，以及选自以下标记物中的cg26371731，cg04541368和cg13973436至少一种。
在其中一些实施例中，所述用于乳腺癌检测的甲基化生物标记物组合包括标记物cg23035715，以及以下标记物中的cg26371731，cg04541368和cg13973436三种。
在其中一些实施例中，所述用于乳腺癌检测的甲基化生物标记物组合，还包括选自以下生物标记物中的至少一种：cg16304215，cg20072171，cg08402365，cg21501525，cg22778178，cg08599259，cg25566568，cg15634980，cg07458308，cg01348584，cg14140881，cg25756435，cg00594560，cg08279008，cg09760908，cg18087672，cg14868703，cg17632299，cg18786873，cg20631750，cg25924096，cg24615528，cg22889755，cg23524195，cg20861607，cg22435300，cg25504443，cg14351528，cg25824543，cg23413809，cg24797187，cg26877715，cg01167274，cg12361223，cg04430835，cg27111970，cg22851944，cg23228540，cg26225694，cg07790615，cg14825633，cg11901043，cg24504927，cg21962423，cg14135814，cg01070209，cg14841828，cg04947764，cg01397141，cg00436496，cg01832036，cg27125093，cg21319323，cg06916239，cg11667451，cg03355998，cg19019849，cg22009488，cg15012484，cg20817483，cg21254450 和cg23134869。
在其中一些实施例中，所述用于乳腺癌检测的甲基化生物标记物组合，包括标记物cg23035715，cg26371731，cg04541368，cg13973436，cg16304215，cg20072171，cg08402365，cg21501525，cg22778178，cg08599259，cg25566568，cg15634980，cg07458308，cg01348584，cg14140881，cg25756435，cg00594560，cg08279008，cg09760908，cg18087672，cg14868703，cg17632299，cg18786873，cg20631750，cg25924096，cg24615528，cg22889755，cg23524195，cg20861607，cg22435300，cg25504443，cg14351528，cg25824543，cg23413809，cg24797187，cg26877715，cg01167274，cg12361223，cg04430835，cg27111970，cg22851944，cg23228540，cg26225694，cg07790615，cg14825633，cg11901043，cg24504927，cg21962423，cg14135814，cg01070209，cg14841828，cg04947764，cg01397141，cg00436496，cg01832036，cg27125093，cg21319323，cg06916239，cg11667451，cg03355998，cg19019849，cg22009488，cg15012484，cg20817483，cg21254450和cg23134869。
在其中一些实施例中，所述标记物cg23035715的序列如SEQ ID NO.1所示、cg26371731的序列如SEQ ID NO.2所示、cg04541368的序列如SEQ ID NO.3所示、cg13973436的序列如SEQ ID NO.4所示、cg16304215的序列如SEQ ID NO.5所示、cg20072171的序列如SEQ ID NO.6所示、cg08402365的序列如SEQ ID NO.7所示、cg21501525的序列如SEQ ID NO.8所示、cg22778178序列如SEQ ID NO.9所示、cg08599259的序列如SEQ ID NO.10所示、cg25566568的序列如SEQ ID NO.11所示、cg15634980的序列如SEQ ID NO.12所示、cg07458308的序列如SEQ ID NO.13所示、cg01348584的序列如SEQ ID NO.14所示、cg14140881的序列如SEQ ID NO.15所示、cg25756435的序列如SEQ ID NO.16所示、cg00594560的序列如SEQ ID NO.17所示、cg08279008的序列如SEQ ID NO.18所示、cg09760908的序列如SEQ ID NO.19所示、cg18087672的序列如SEQ ID NO.20所示、cg14868703的序列如SEQ ID NO.21所示、cg17632299的序列如SEQ ID NO.22所示、cg18786873的序列如SEQ ID NO.23所示、cg20631750的序列如SEQ ID NO.24所示、cg25924096的序列如SEQ ID NO.25所示、cg15321298的序列如SEQ ID NO.26所示、cg22889755的序列如SEQ ID NO.27所示、cg23524195的序列如SEQ ID NO.28所示、cg20861607的序列如SEQ ID NO.29所示、cg22435300的序列如SEQ ID NO.30所示、cg25504443的序列如SEQ ID NO.31所示、cg14351528的序列如SEQ ID NO.32所示、cg25824543的序列如SEQ ID NO.33所示、cg23413809的序列如SEQ ID NO.34所示、cg24797187的序列如SEQ ID NO.35所示、cg26877715的序列如SEQ ID NO.36所示、cg01167274的序列如SEQ ID NO.37所示、cg12361223的序列如SEQ ID NO.38所示、cg04430835的序列如SEQ ID NO.39所示、cg27111970的序列如SEQ ID NO.40所示、cg22851944的序列如SEQ ID NO.41所示、cg23228540的序列如SEQ ID NO.42所示、cg26225694的序列如SEQ ID NO.43所示、cg07790615的序列如SEQ ID NO.44所示、cg14825633的序列如SEQ ID NO.45所示、cg11901043的序列如SEQ ID NO.46所示、cg24504927的序列如SEQ ID NO.47所示、cg21962423的序列如SEQ ID NO.48所示、cg14135814的序列如SEQ ID NO.49所示、cg01070209的序列如SEQ ID NO.50所示、 cg14841828的序列如SEQ ID NO.51所示、cg04947764的序列如SEQ ID NO.52所示、cg01397141的序列如SEQ ID NO.53所示、cg00436496的序列如SEQ ID NO.54所示、cg01832036的序列如SEQ ID NO.55所示、cg27125093的序列如SEQ ID NO.56所示、cg21319323的序列如SEQ ID NO.57所示、cg06916239的序列如SEQ ID NO.58所示、cg11667451的序列如SEQ ID NO.59所示、cg03355998的序列如SEQ ID NO.60所示、cg19019849的序列如SEQ ID NO.61所示、cg22009488的序列如SEQ ID NO.62所示、cg15012484的序列如SEQ ID NO.63所示、cg20817483的序列如SEQ ID NO.64所示、cg21254450的序列如SEQ ID NO.65所示、和/或cg23134869的序列如SEQ ID NO.66所示。
本发明的另一目的是涉及上述甲基化生物标记物组合在制备检测乳腺癌的试剂盒中的应用。
本发明的另一目的是提供一种检测乳腺癌的试剂盒。
一种检测乳腺癌的试剂盒，包括有检测待测样品中的上述甲基化生物标记物或其组合的甲基化状态的试剂。
在其中一些实施例中，所述乳腺癌是不同分期的乳腺癌，包括根据TNM的不同，分为0、I、II、III、IV期。
在其中一些实施例中，所述试剂盒采用的检测方法是采用焦磷酸测序法、重亚硫酸盐转化测序法、甲基化芯片法、qPCR法、数字PCR法、二代测序法、三代测序法、全基因组甲基化测序法、DNA富集检测法、简化亚硫酸氢盐测序技术、HPLC法、MassArray、甲基化特异PCR、或它们的组合所使用的试剂。
在其中一些实施例中，所述待测样品是血浆、血清或血液。
本发明公开了用于乳腺癌早期诊断的血浆基因甲基化标记物或其组合，具体地，研究获得了在乳腺癌血浆cfDNA中明显异常甲基化修饰模式的基因组片段，即cg23035715，cg26371731，cg04541368，cg13973436，cg16304215，cg20072171，cg08402365，cg21501525，cg22778178，cg08599259，cg25566568，cg15634980，cg07458308，cg01348584，cg14140881，cg25756435，cg00594560，cg08279008，cg09760908，cg18087672，cg14868703，cg17632299，cg18786873，cg20631750，cg25924096，cg24615528，cg22889755，cg23524195，cg20861607，cg22435300，cg25504443，cg14351528，cg25824543，cg23413809，cg24797187，cg26877715，cg01167274，cg12361223，cg04430835，cg27111970，cg22851944，cg23228540，cg26225694，cg07790615，cg14825633，cg11901043，cg24504927，cg21962423，cg14135814，cg01070209，cg14841828，cg04947764，cg01397141，cg00436496，cg01832036，cg27125093，cg21319323，cg06916239，cg11667451，cg03355998，cg19019849，cg22009488，cg15012484，cg20817483，cg21254450和cg23134869。通过研究这些血浆cfDNA甲基化修饰模式的基因组片段在各期乳腺癌患者及健康人群中的甲基化修饰差异，发现以上血浆基因组片段组合都能够作为乳腺癌早期诊断标记物。而且经过发明人创造性的研究发现，第一个甲基化标记物(cg23035715)或前3个甲基化标记物(cg26371731，cg04541368，cg13973436)组合均具备了与两种算法LASSO和Random Forest筛选出来重叠的26个甲基化标记物组合相近的诊断力。本发明联 合分析至少1个基因组片段上的多个甲基化胞嘧啶的共甲基化特征作为判断乳腺癌发病的生物标记物准确度远远大于其它血浆生物标记物的检测，降低假阳性和假阴性的发生。
附图说明
图1是实施例2中训练集中26个甲基化标记物组合的ROC曲线。
图2是实施例2中独立验证集中26个甲基化标记物组合的ROC曲线和乳腺癌不同分期的灵敏度、特异性。
图3是实施例2中独立验证集中LASSO筛选的42个和Random Forest筛选的50个甲基化标记物组合的ROC曲线。
图4是实施例2中独立验证集中LASSO筛选的42个(a)和Random Forest筛选的50个(b)甲基化标记物组合在不同乳腺癌分期的灵敏度、特异性。
图5是实施例2中独立验证集中SEQ ID NO.1-26标记物组合与随机挑选其它SEQ ID NO.27-66个标记物中X个的ROC曲线(a)和不同乳腺癌分期的灵敏度、特异性(b)。
图6是实施例3中独立验证集中SEQ ID NO.1或SEQ ID NO.1-4甲基化标记物组合ROC曲线。
图7是实施例3中独立验证集中SEQ ID NO.1-4甲基化标记物随机组合的不同乳腺癌分期的灵敏度和特异性。
图8是实施例3中独立验证集中SEQ ID NO.1标记物与随机挑选SEQ ID NO.5-26个标记物中X个的ROC曲线(a)和不同乳腺癌分期的灵敏度、特异性(b)。
图9是实施例3中独立验证集中SEQ ID NO.1标记物与随机挑选SEQ ID NO.27-66标记物中X个的ROC曲线(a)和不同乳腺癌分期的灵敏度、特异性(b)。
图10是实施例3中独立验证集中SEQ ID NO.1-4标记物与随机挑选SEQ ID NO.5-26个标记物中X个的ROC曲线(a)和不同乳腺癌分期的灵敏度、特异性(b)。
图11是实施例3中独立验证集中SEQ ID NO.1-4标记物与随机挑选SEQ ID NO.27-66标记物中X个的ROC曲线(a)和不同乳腺癌分期的灵敏度、特异性(b)。
具体实施方式
本发明下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。
本发明的术语“包括”和“具有”以及它们任何变形，意图在于覆盖不排他的包含。例如包含了一系列步骤的过程、方法、装置、产品或设备没有限定于已列出的步骤或模块，而是可选地还包括没有列出的步骤，或可选地还包括对于这些过程、方法、产品或设备固有的其它步骤。
在本发明中提及的“多个”是指两个或两个以上。“和/或”，描述关联对象的关联关系，表示可以存在三种关系，例如，A和/或B，可以表示：单独存在A，同时存在A和B，单独存在B这三种情况。字符“/”一般表示前后关联对象是一种“或”的关系。
在其中一些实施例中，本发明涉及筛选到的66个与乳腺癌相关的甲基化生物标记物cg23035715，cg26371731，cg04541368，cg13973436，cg16304215，cg20072171，cg08402365，cg21501525，cg22778178，cg08599259，cg25566568，cg15634980，cg07458308，cg01348584，cg14140881，cg25756435，cg00594560，cg08279008，cg09760908，cg18087672，cg14868703，cg17632299，cg18786873，cg20631750，cg25924096，cg24615528，cg22889755，cg23524195，cg20861607，cg22435300，cg25504443，cg14351528，cg25824543，cg23413809，cg24797187，cg26877715，cg01167274，cg12361223，cg04430835，cg27111970，cg22851944，cg23228540，cg26225694，cg07790615，cg14825633，cg11901043，cg24504927，cg21962423，cg14135814，cg01070209，cg14841828，cg04947764，cg01397141，cg00436496，cg01832036，cg27125093，cg21319323，cg06916239，cg11667451，cg03355998，cg19019849，cg22009488，cg15012484，cg20817483，cg21254450和cg23134869。
在其中一些实施例中，选自以下甲基化生物标记物中的任意至少一种：cg23035715，cg26371731，cg04541368和cg13973436。
本发明对上述用于预测结乳腺癌ctDNA差异性甲基化标记物(marker)的筛选方法，具体包括以下步骤：
1.根据TGCA数据筛选出来乳腺癌特异性的甲基化标记物
1.1从TCGA下载乳腺癌组织及癌旁组织的甲基化450k芯片(Illumina HumanMethylation450BeadChip)的数据；
1.2对数据进行预处理：过滤缺失值，可补全缺失值，去除批次效应，去除不稳定的CpG位点，选择乳腺癌特异性的3288个甲基化位点作为最终的标记物集；
1.3样本分组：将样本分成训练集和测试集：随机分组，保证两组样本中的病
人临床分期、年龄、随访时间等分布一致。
1.4验证3288个标记物在配对组织和血浆样本中甲基化一致，并进一步筛选、过滤确认最后为1996个甲基化标记物。
1.5 1996个显著的甲基化位点用于后面的分类器模型的构建。
首先利用TCGA筛选的3288个甲基本化标记物，进一步通过40对配对的组织和血浆来验证甲基化水平的一致性，并进一步筛选与乳腺癌诊断相关的1996个基因甲基化生物标记物。对TGCA来源筛选3288和过滤后的1996个甲基化标记物在位点数据在正常与乳腺癌样本中进行聚类分析，筛选后1996个甲基化标记物在正常血浆与乳腺癌样本存在最显著异常甲基化的位点。该1996个甲基化生物标记物，以下简称位点或者marker。再从以上1996个甲基化生物标记物经过进一步筛选,分别利用两种算法LASSO和RandomForest筛选到，其中两种算法重叠的有表1中所述的26个甲基化标记物。
66个markers甲基化率计算的特定区域的基因组序列
表1 26个甲基化标记物
Figure PCTCN2021094588-appb-000001
表2与乳腺癌相关的另外40个个甲基化标记物
Figure PCTCN2021094588-appb-000002
Figure PCTCN2021094588-appb-000003
实施例1
1.血浆cfDNA或组织提取及甲基化建库
1.1血cfDNA或组织DNA的提取。
血浆DNA提取具体操作步骤按照Life公司的MagMAX TM Cell-Free DNA Isolation Kit操作说明书进行。组织DNA的提取步骤按照QIAGEN公司的DNeasy Blood&Tissue Kit操作说明进行；
1.2转化
将提取的cfDNA(10ng)或组织DNA(50ng)进行亚硫酸氢盐转化，使DNA中未发生甲基化的胞嘧啶脱氨基转变成尿嘧啶，而甲基化的胞嘧啶保持不变，得到亚硫酸氢盐转化后的DNA，转化具体操作按照Zymo Research的EZ DNA Methylation-Lightning Kit说明书进行。
1.3末端修复
1.3.1将转化后的17ul样本加入以下试剂进行反应：
Figure PCTCN2021094588-appb-000004
1.3.2置于PCR仪中按照以下程序进行反应：
37℃ 30min
95℃ 5min
热盖 105℃
1.3.3当PCR反应第二步(95℃)达到5min时，立即将样本从PCR仪中取出，直接插入冰中，放置2min以上再进行下一步操作
1.4连接I
1.4.1配制如下反应液
Figure PCTCN2021094588-appb-000005
1.4.2置于PCR仪中按照以下程序进行反应：
37℃ 30min
95℃ 5min
10℃ hold
热盖 105℃
1.5扩增I
1.5.1配制如下反应液
Figure PCTCN2021094588-appb-000006
1.5.2置于PCR仪中按照以下程序进行反应：
Figure PCTCN2021094588-appb-000007
Figure PCTCN2021094588-appb-000008
1.6纯化I：加入166ul 1:6倍稀释的Agencourt AMPure Beads(需提前于室温平衡半小时)的对Amplification I反应后的产物进行纯化，用21ul EB进行洗脱，纯化具体步骤如下：
1.6.1取上一步反应产物并进行离心，每个样本加入166ul 1:6倍稀释的Agencourt AMPure Beads，用移液器吹打混匀。
1.6.2室温孵育5min。
1.6.3离心，置于磁力架上静置5min。
1.6.4吸去上清。
1.6.5加入200ul 80％EtOH，静置30s，吸走乙醇。
1.6.6重复步骤5)一次。
1.6.7离心，将PCR管置于磁力架上，吸走剩余乙醇。
1.6.8开盖干燥磁珠2-3min，注意不要过干。
1.6.9加入21ul EB进行洗脱，用移液器充分吹打混匀，室温静置3min。
1.6.10离心，将PCR管置于磁力架上，静置3min。
1.6.11吸取20ul上清于新的PCR管中。
1.7连接II
1.7.1配制如下反应液：
组分 体积(ul)
上一步反应体积 20
H2O 4
MSB1 Buffer 8
MSR1 Reagent 2
MSR5 Reagent 2
MSE1 Enzyme 2
MSE5 Enzyme 2
总体积 40
1.7.2置于PCR仪中按照以下程序进行反应
温度 时间 循环数
37℃ 30min 1
95℃ 5min 1
10℃ Hold 1
1.8 Indexing PCR:
1.8.1配制如下反应液：
Figure PCTCN2021094588-appb-000009
Figure PCTCN2021094588-appb-000010
1.8.2置于PCR仪中按照以下程序进行反应
Figure PCTCN2021094588-appb-000011
1.9纯化II
加入Agencourt AMPure Beads(需提前于室温平衡半小时)对Indexing PCR反应后的产物进行纯化，用41ul EB进行洗脱，纯化具体步骤如下：
1.9.1取上一步反应产物并进行离心，每个样本加入71ul未稀释的Agencourt AMPure Beads，用移液器吹打混匀.
1.9.2室温孵育5min。
1.9.3离心，置于磁力架上静置5min。
1.9.4吸去上清。
1.9.5加入200ul 80％EtOH，静置30s，吸走乙醇。
1.9.6重复步骤5)一次。
1.9.7离心，将PCR管置于磁力架上，吸走剩余乙醇。
1.9.8开盖干燥磁珠2-3min，注意不要过干。
1.9.9加入41ul EB进行洗脱，用移液器充分吹打混匀，室温静置3min。
1.9.10离心，将PCR管置于磁力架上，静置3min。
1.9.11吸取20ul上清于新的PCR管中。
1.10 Qubit定量：
1.取1ul用Qubit dsDNA HS Assay Kit对文库进行定量。
2.通过对已建库后的样本进行寡核苷酸探针捕获富集，得到特定区域的上机终文库。杂交捕获试剂盒为IDT公司的xGen Lockdown Reagents，具体按照说明书进行操作。
3.采用Illumina公司的测序仪对杂交捕获后的样本进行测序，得到测序结果。
4.数据的分析：
对测序仪的下机原始数据，进行常规的生物信息学分析处理，先通过fastp过滤低质量(QC低，长度短、太多N等)的读长(reads)，然后去除reads双端的adapter、共有序列、PolyA/T， 得到理想的插入片段序列(target区间)，使用bismark将这些reads比对hg19对应的位置后，根据UMI对reads进行去重，得到每份样本被探针捕获得到的真实reads数据(bam file)，对bam文件进行统计和分析，得到甲基化数据，用于后续的数据再分析。
本文所指的某个甲基化标记物的共甲基化率，其实为该标记物特定区域的所有甲基化组合的各自的甲基化率的和。P-vlaue所得为wilcoxn test，p<0.001为有显著性统计学意义；我们还计算了所有26种标记物的单独AUC值，AUC(area under the curve)是ROC(Receiver Operating Characteristic)曲线下的面积，用于判断该组合诊断乳腺癌的能力，AUC越接近1，表示越优。
实施例2
本实施例公开了一种用于诊断乳腺癌的甲基化特异性生物标记物组合，利用74例钼靶和彩超无异常的正常人的血浆样本以及155例乳腺癌病人的血浆样本，利用在不同组别的甲基化水平差异筛选出与乳腺癌相关的66个甲基化生物标记物，并对差异化甲基化组合模型在正常与乳腺癌血浆样本中进行独立数据集的验证，具体过程：利用LASSO和随机森进方法(Random Forest)分别在训练集和测试集进行标记物筛选和模型建立，包括了52例健康人和108例乳腺癌患者血浆cfDNA样本。用LASSO方法筛选出了42个甲基化标记物，而用随机森林法筛选出50个甲基化标记物，最终再进一步筛选出共26个甲基化标记物。最后，我们在22例健康人和47例乳腺癌患者血浆cfDNA组成的独立验证集中，对42(SEQ ID NO.1-42)、50(SEQ ID NO.1-26，SEQ ID NO.27-SEQ ID NO.66)和26(SEQ ID NO.1-26)个甲基化标记物的诊断能力分别进行了验证。
该66个甲基化生物标记物(以下简称位点或者marker)为：cg23035715，cg26371731，cg04541368，cg13973436，cg16304215，cg20072171，cg08402365，cg21501525，cg22778178，cg08599259，cg25566568，cg15634980，cg07458308，cg01348584，cg14140881，cg25756435，cg00594560，cg08279008，cg09760908，cg18087672，cg14868703，cg17632299，cg18786873，cg20631750，cg25924096，cg24615528，cg22889755，cg23524195，cg20861607，cg22435300，cg25504443，cg14351528，cg25824543，cg23413809，cg24797187，cg26877715，cg01167274，cg12361223，cg04430835，cg27111970，cg22851944，cg23228540，cg26225694，cg07790615，cg14825633，cg11901043，cg24504927，cg21962423，cg14135814，cg01070209，cg14841828，cg04947764，cg01397141，cg00436496，cg01832036，cg27125093，cg21319323，cg06916239，cg11667451，cg03355998，cg19019849，，cg22009488，cg15012484，cg20817483，cg21254450和cg23134869。具体序列见SEQ ID NO.1-66。
对52例健康人和108例乳腺癌患者的训练集中，进行以上两种算法交集的26个甲基化生物标记物(marker)的检测，在98.07％的特异性下(51/52)，0期的检测灵敏度为60％(3/5)，I期的检测灵敏度为80％(32/40)，II期的检测灵敏度为95.45％(42/44)，III期的检测灵敏度94.74％(18/19)，总体检测灵敏度为87.9％(95/108)，AUC为0.9839，结果详细见图1。
进一步，在22例钼靶和彩超无异常的正常人的组织样本以及47例乳腺癌病人的血浆样本中，对26个甲基化生物标记物(marker)进行独立验证分析，在98％的特异性下(22/22)， 0期的检测灵敏度为66.67％(2/3)，I期的检测灵敏度为75％(9/12)，II期的检测灵敏度为95％(21/22)，III期的检测灵敏度100％(8/8)和IV期的检测灵敏度100％(2/2)，对于所有的乳腺癌样本，检测的整体灵敏度为89.37％(42/47),整体的AUC为0.9816，请见图2。另外，我们也对LSSAO和RF分别筛选出来的42(SEQ ID NO.1-26和SEQ ID NO.51-66)、50(SEQ ID NO.1-SEQ ID NO.50)个甲基化生物标记物(marker)进行独立验证分析，在98％的特异性下(22/22)，0期的检测灵敏度分别为66.67％(2/3)和33.33％(1/3)，I期的检测灵敏度分别为66.67％(8/12)和58.33％(7/12)，II期的检测灵敏度分别为95.45％(21/22)和63.63％(14/22)，III期的检测灵敏度分别为100％(8/8)和75％(6/8)，IV期的检测灵敏度分别为100％(2/2)和0％(0/2)，对于所有的乳腺癌样本，检测的整体灵敏度分别为87.23％(43/47)和59.57％(28/47),整体的AUC分别为0.9709和0.9497，详细见图3和图4。
进一步验证对26个甲基化生物标记物(marker)组合的重要性，我们将26markers(SEQ ID NO.1-26)与cg22889755，cg23524195，cg20861607组合进行独立验证分析，cg22889755，cg23524195和cg20861607为随机从其它的40个甲基化标记物((SEQ ID NO.27-66))挑选的3个，在98％的特异性下(22/22)，0期的检测灵敏度为100％(3/3)，I期的检测灵敏度为83.33％(10/12)，II期的检测灵敏度为86.36％(19/22)，III期的检测灵敏度87.5％(7/8)和IV期的检测灵敏度0％(0/2)，对于所有的乳腺癌样本，检测的整体灵敏度为82.98％(39/47),整体的AUC为0.9797，请见图5a-5b。整体的灵敏度有所降低，但对于乳腺癌早期的灵敏度高于top1-26个甲基化标记物的组合。
实施例3
利用其中26个甲基化标记物(SEQ ID NO.1-26)的信息，对22例钼靶和彩超无异常的正常人以及47例乳腺癌病人血浆样本(其中，0期3例，I期12例，II期22例，III期8例，IV期2例)进行随机组合检验，利用逻辑回归模型进行建模分析，进一步筛选4个标记物(cg23035715，cg26371731，cg04541368和cg13973436)，具体的数据分析方法与实施例2一致，Top1(cg23035715)和Top1/2/3/4(cg23035715，cg26371731，cg04541368和cg13973436)在98％的特异性下(22/22)，验证集的乳腺癌0期的检测灵敏度分别为100％(3/3)和100％(3/3)，乳腺癌I期的检测灵敏度分别为91.67％(11/12)和91.67％(11/12)，乳腺癌II期的检测灵敏度分别为90.90％(20/22)和90.91％(20/22)，III期的检测灵敏度分别为87.5％(7/8)和87.5％(7/8)，乳腺癌IV期的检测灵敏度分别为50％(1/2)和0％(0/2)，对于所有的乳腺癌样本，检测的整体灵敏度分别为91.49％(43/47)87.23％(41/47)，整体的AUC分别为0.9395和0.9796，表明了这些位点组合与乳腺癌早期的诊断高度相关。结果请见图6和图7a-7b。
所述4个生物标记物：cg23035715，cg26371731，cg04541368和cg13973436，其中，单个标记物cg23035715的AUC 0.9395，见图6，且图6为26个共甲基化标记物中4个标记物(cg23035715，cg26371731，cg04541368和cg13973436)组合一起，与marker1(cg23035715)标记物在鉴别乳腺癌能力方面的比较，通过图6可看出，4个甲基化标记物组合的AUC已经很接近于26个标记物的组合的AUC，marker1的标记物AUC也有0.9395，说明这4个甲基化物标记物的重要性，特别是marker1的标记物已经具有很强的诊断早期乳腺癌的能力。
图7c-7d所示，在98％的特异性下，Top1/2/3和Top1/2/4甲基化标记物组合在验证集的0期的检测灵敏度分别为100％(3/3)和33.33％(1/3)，I期的检测灵敏度分别为100％(12/12)和66.67％(8/12)，II期的检测灵敏度分别为81.82％(18/22)和63.63％(14/22)，III期的检测灵敏度分别为87.5％(7/8)和75％(6/8)，IV期检测灵敏度分别为50％(1/2)和0％(0/2)，整体检测的整体灵敏度为分别85.11％(40/47)和61.7％(29/47)。其中，图6c中的top1/2/3代表的是cg23035715，cg26371731和cg04541368组合，而top1/2/4代表的是3个标记物(cg23035715，cg26371731和cg13973436)的组合，以此类推。
进一步验证top1单独的重要性，在98％的特异性，top1(SEQ ID NO.1)与随机SEQ ID NO.5-26标记物中的cg23035715，cg16304215和cg08599259组合以及与其它剩余40个标记物(SEQ ID NO.27-66)的随机挑先的cg01167274，cg07790615，cg23134869和cg01832036组合，验证集的0期的检测灵敏度分别为100％(3/3)和100％(3/3)，I期的检测灵敏度分别为100％(12/12)和83.33％(10/12)，II期的检测灵敏度分别为95.45％(21/22)和95.45％(21/22)，III期的检测灵敏度分别为100％(8/8)和87.5％(7/8)，IV期检测灵敏度分别为0％(0/2)和0％(0/2)，整体检测的整体灵敏度分别为93.61％(40/47)和61.7％(29/47)，见图8a-8b和图9a-9b。
进一步验证及top1/2/3/4组合的重要性，在98％的特异性，top1-4(SEQ ID NO.1-4)与随机挑选SEQ ID NO.5-26标记物中的cg16304215，cg20072171和cg08402365组合以及与其它剩余40个标记物(SEQ ID NO.27-66)的cg22889755，cg23524195，cg14135814，cg15012484和cg2081748组合，验证集的0期的检测灵敏度均为100％(3/3)，I期的检测灵敏度均为91.67％(11/12)，II期的检测灵敏度均为90.91％(20/22)，III期的检测灵敏度均为87.5％(7/8)，IV期检测灵敏度均为0％(0/2)，整体检测的整体灵敏度均为87.23％(41/47),AUC分别为0.9739和0.9797，见图10a-10b和图11a-11b。
针对上述的22例钼靶和彩超无异常的正常人以及47例乳腺癌病人血浆样本进行检测的结果，cg23035715对于早期乳腺癌的诊断有很好的表现，与cg26371731,cg04541368和cg13973436的组合也展现出与top1-26的组合相似的诊断能力，而且cg23035715与随机SEQ ID NO.5-26标记物中的cg23035715，cg16304215和cg08599259组合，以及与其它剩余40个标记物(SEQ ID NO.27-66)的cg01167274，cg07790615，cg23134869和cg01832036组合，都展现出很好的诊断能力。另外，top1-4(SEQ ID NO.1-4)以及top1-26(SEQ ID NO.1-26)个组合分别与其它剩余40个标记物(SEQ ID NO.27-66)的cg22889755，cg23524195，cg14135814，cg15012484，cg2081748的组合和cg22889755，cg23524195，cg20861607的组合，也都展现出很稳定的诊断能力，top1/2/3/4标记物的AUC也在0.75以上，如cg2303571、cg26371731,cg04541368和cg13973436的AUC为0.9395，0.8020，0.7701和0.7501，见表3.1，说明了利用该方法筛选出来的位点与乳腺癌早期的诊断有非常高的相关性。
表3.1 26个甲基化标记物片段在乳腺癌诊断中的表现-AUC。
Figure PCTCN2021094588-appb-000012
Figure PCTCN2021094588-appb-000013
Figure PCTCN2021094588-appb-000014
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (14)

  1. 一种用于乳腺癌检测的甲基化生物标记物或其组合，其特征是，选自以下甲基化生物标记物中的任意至少一种：cg23035715，cg26371731，cg04541368，cg13973436，cg16304215，cg20072171，cg08402365，cg21501525，cg22778178，cg08599259，cg25566568，cg15634980，cg07458308，cg01348584，cg14140881，cg25756435，cg00594560，cg08279008，cg09760908，cg18087672，cg14868703，cg17632299，cg18786873，cg20631750，cg25924096，cg24615528，cg22889755，cg23524195，cg20861607，cg22435300，cg25504443，cg14351528，cg25824543，cg23413809，cg24797187，cg26877715，cg01167274，cg12361223，cg04430835，cg27111970，cg22851944，cg23228540，cg26225694，cg07790615，cg14825633，cg11901043，cg24504927，cg21962423，cg14135814，cg01070209，cg14841828，cg04947764，cg01397141，cg00436496，cg01832036，cg27125093，cg21319323，cg06916239，cg11667451，cg03355998，cg19019849，cg22009488，cg15012484，cg20817483，cg21254450和cg23134869。
  2. 根据权利要求1所述的用于乳腺癌检测的甲基化生物标记物或其组合，其特征是，包括选自以下甲基化生物标记物中的任意至少一种：cg23035715，cg26371731，cg04541368和cg13973436。
  3. 根据权利要求2所述的用于乳腺癌检测的甲基化生物标记物或其组合，其特征是，包括标记物cg23035715，以及包括选自以下标记物中的cg26371731，cg04541368和cg13973436至少一种。
  4. 根据权利要求3所述的用于乳腺癌诊断的甲基化生物标记物或其组合，其特征是，包括生物标记物cg23035715，以及以下生物标记物中的cg26371731，cg04541368和cg13973436。
  5. 根据权利要求3或4所述的用于乳腺癌诊断的甲基化生物标记物或其组合，其特征是，还包括选自以下生物标记物中的至少一种：cg16304215，cg20072171，cg08402365，cg21501525，cg22778178，cg08599259，cg25566568，cg15634980，cg07458308，cg01348584，cg14140881，cg25756435，cg00594560，cg08279008，cg09760908，cg18087672，cg14868703，cg17632299，cg18786873，cg20631750，cg25924096，cg24615528。
  6. 根据权利要求3或4或5所述的用于乳腺癌诊断的甲基化生物标记物或其组合，其特征是，还包括选自以下生物标记物中的至少一种：cg22889755，cg23524195，cg20861607，cg22435300，cg25504443，cg14351528，cg25824543，cg23413809，cg24797187，cg26877715，cg01167274，cg12361223，cg04430835，cg27111970，cg22851944，cg23228540。
  7. 根据权利要求3或4或5或6所述的用于乳腺癌诊断的甲基化生物标记物或其组合，其特征是，还包括选自以下生物标记物中的至少一种：cg26225694，cg07790615，cg14825633，cg11901043，cg24504927，cg21962423，cg14135814，cg01070209，cg14841828，cg04947764，cg01397141，cg00436496，cg01832036，cg27125093， cg21319323，cg06916239，cg11667451，cg03355998，cg19019849，，cg22009488，cg15012484，cg20817483，cg21254450和cg23134869。
  8. 根据权利要求7所述的用于乳腺癌诊断的甲基化生物标记物或其组合，其特征是，包括标记物cg23035715，cg26371731，cg04541368，cg13973436，cg16304215，cg20072171，cg08402365，cg21501525，cg22778178，cg08599259，cg25566568，cg15634980，cg07458308，cg01348584，cg14140881，cg25756435，cg00594560，cg08279008，cg09760908，cg18087672，cg14868703，cg17632299，cg18786873，cg20631750，cg25924096，cg24615528，cg22889755，cg23524195，cg20861607，cg22435300，cg25504443，cg14351528，cg25824543，cg23413809，cg24797187，cg26877715，cg01167274，cg12361223，cg04430835，cg27111970，cg22851944，cg23228540，cg26225694，cg07790615，cg14825633，cg11901043，cg24504927，cg21962423，cg14135814，cg01070209，cg14841828，cg04947764，cg01397141，cg00436496，cg01832036，cg27125093，cg21319323，cg06916239，cg11667451，cg03355998，cg19019849，cg22009488，cg15012484，cg20817483，cg21254450和cg23134869。
  9. 根据权利要求1-8任一项所述的甲基化生物标记物或其组合，其特征是，所述标记物cg23035715的序列如SEQ ID NO.1所示、cg26371731的序列如SEQ ID NO.2所示、cg04541368的序列如SEQ ID NO.3所示、cg13973436的序列如SEQ ID NO.4所示、cg16304215的序列如SEQ ID NO.5所示、cg20072171的序列如SEQ ID NO.6所示、cg08402365的序列如SEQ ID NO.7所示、cg21501525的序列如SEQ ID NO.8所示、cg22778178的序列如SEQ ID NO.9所示、cg08599259的序列如SEQ ID NO.10所示、cg25566568的序列如SEQ ID NO.11所示、cg15634980的序列如SEQ ID NO.12所示、cg07458308的序列如SEQ ID NO.13所示、cg01348584的序列如SEQ ID NO.14所示、cg14140881的序列如SEQ ID NO.15所示、cg25756435的序列如SEQ ID NO.16所示、cg00594560的序列如SEQ ID NO.17所示、cg08279008的序列如SEQ ID NO.18所示、cg09760908的序列如SEQ ID NO.19所示、cg18087672的序列如SEQ ID NO.20所示、cg14868703的序列如SEQ ID NO.21所示、cg17632299的序列如SEQ ID NO.22所示、cg18786873的序列如SEQ ID NO.23所示、cg20631750的序列如SEQ ID NO.24所示、cg25924096的序列如SEQ ID NO.25所示、cg15321298的序列如SEQ ID NO.26所示、cg22889755的序列如SEQ ID NO.27所示、cg23524195的序列如SEQ ID NO.28所示、cg20861607的序列如SEQ ID NO.29所示、cg22435300的序列如SEQ ID NO.30所示、cg25504443的序列如SEQ ID NO.31所示、cg14351528的序列如SEQ ID NO.32所示、cg25824543的序列如SEQ ID NO.33所示、cg23413809的序列如SEQ ID NO.34所示、cg24797187的序列如SEQ ID NO.35所示、cg26877715的序列如SEQ ID NO.36所示、cg01167274的序列如SEQ ID NO.37所示、cg12361223的序列如SEQ ID NO.38所示、cg04430835的序列如SEQ ID NO.39所示、cg27111970的序列如SEQ ID NO.40所示、cg22851944的序列如SEQ ID NO.41所示、cg23228540的序列如SEQ ID NO.42所示、cg26225694的序列如SEQ ID NO.43所示、cg07790615的序列如SEQ ID NO.44所示、 cg14825633的序列如SEQ ID NO.45所示、cg11901043的序列如SEQ ID NO.46所示、cg24504927的序列如SEQ ID NO.47所示、cg21962423的序列如SEQ ID NO.48所示、cg14135814的序列如SEQ ID NO.49所示、cg01070209的序列如SEQ ID NO.50所示、cg14841828的序列如SEQ ID NO.51所示、cg04947764的序列如SEQ ID NO.52所示、cg01397141的序列如SEQ ID NO.53所示、cg00436496的序列如SEQ ID NO.54所示、cg01832036的序列如SEQ ID NO.55所示、cg27125093的序列如SEQ ID NO.56所示、cg21319323的序列如SEQ ID NO.57所示、cg06916239的序列如SEQ ID NO.58所示、cg11667451的序列如SEQ ID NO.59所示、cg03355998的序列如SEQ ID NO.60所示、cg19019849的序列如SEQ ID NO.61所示、cg22009488的序列如SEQ ID NO.62所示、cg15012484的序列如SEQ ID NO.63所示、cg20817483的序列如SEQ ID NO.64所示、cg21254450的序列如SEQ ID NO.65所示、和/或cg23134869的序列如SEQ ID NO.66所示。
  10. 权利要求1-9任一项所述的甲基化生物标记物或其组合在制备乳腺癌诊断试剂盒中的应用。
  11. 一种用于乳腺癌诊断的试剂盒，其特征是，包括检测待测样品中的权利要求1-9任一项所述甲基化生物标记物或其组合的甲基化状态的试剂。
  12. 根据权利要求11所述的用于乳腺癌诊断的试剂盒，其特征是，所述乳腺癌是不同分期的乳腺癌。
  13. 根据权利要求11所述的通过检测cfDNA甲基化用于乳腺癌诊断的试剂盒，其特征是，所述试剂根据采用是焦磷酸测序法、重亚硫酸盐转化测序法、甲基化芯片法、qPCR法、数字PCR法、二代测序法、三代测序法、全基因组甲基化测序法、DNA富集检测法、简化亚硫酸氢盐测序技术、HPLC法、MassArray、甲基化特异PCR、或它们的组合所使用的试剂。
  14. 根据权利要求11-13任一项所述通过检测cfDNA甲基化用于乳腺癌诊断的试剂盒，其特征是，所述待测样品是血液、血清或血浆。
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