WO2023071889A1 - 用于检测胃癌淋巴结节转移相关的甲基化生物标记物或其组合及应用 - Google Patents

用于检测胃癌淋巴结节转移相关的甲基化生物标记物或其组合及应用 Download PDF

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WO2023071889A1
WO2023071889A1 PCT/CN2022/126211 CN2022126211W WO2023071889A1 WO 2023071889 A1 WO2023071889 A1 WO 2023071889A1 CN 2022126211 W CN2022126211 W CN 2022126211W WO 2023071889 A1 WO2023071889 A1 WO 2023071889A1
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王军
阮微媚
陈志伟
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广州市基准医疗有限责任公司
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  • the invention belongs to the field of biotechnology, and in particular relates to a methylation biomarker for detecting gastric cancer lymph node metastasis or its combination and application.
  • Gastric cancer is a malignant tumor and one of the most common malignant tumors in the world. According to the 2020 global cancer statistics data, the incidence and mortality of gastric cancer rank fifth and third respectively, and the incidence rate of men is twice that of women. In China, the incidence of gastric cancer is increasing year by year, and tends to be younger. Its incidence and death rate have jumped to the second and third respectively, ranking first in malignant tumors of the digestive system, and the number of gastric cancers almost accounts for the number of gastric cancers in the world half of. Gastric cancer, as a highly aggressive and heterogeneous malignancy, remains a global health problem.
  • the traditional treatment for early gastric cancer is D1 or D2 radical resection, but the trauma of the surgery is relatively large, especially for patients without lymph node metastasis, the surgery increases the trauma but does not bring additional benefits to the patients.
  • the lymph node metastasis rate of early gastric cancer ranges from 10% to 42%, with an average of 15%. That is to say, about 80% to 85% of patients do not need lymph node dissection.
  • ECG Early gastric cancer
  • LNM lymph node metastasis
  • lymph node metastasis is between 5% and 10% in patients with early gastric cancer undergoing radical surgery, which indicates that if the status of lymph nodes under ESD can be accurately assessed, more than 90% of operations are possible avoid.
  • DNA methylation occurs in the early stages of tumors and is tissue-specific, making it one of the best options for tracking early tumor signals.
  • One of the objectives of the present invention is to provide a DNA methylation biomarker or a combination thereof for detecting gastric cancer lymph node metastasis and non-metastasis.
  • the methylated biomarkers are selected from at least one of chr12:42873140, chr8:81789921, chr8:81811441 and chr8:16884489.
  • the methylated biomarkers include a combination of chr12:42873140, chr8:81789921, chr8:81811441 and chr8:16884489.
  • the methylated biomarkers also include chr7:71801447, chr20:59828473, chr4:20254852, chr16:86542314, chr2:99439396, chr12:107713297, chr21 : 22370447, chr14: 48145305, chr16: 22825741, chr2: 210636540, chr7: 330023, chr10: 129535510, chr6: 85473377, chr5: 2749179, chr6: 118228600, chr13 :113765062, chr16:55358704, chr19:57352008, chr19:12876993 , chr20:59827184, chr17:6617301, chr7:47092638, chr12:52644274, chr5
  • the methylated biomarkers include chr12:42873140, chr8:81789921, chr8:81811441, chr5:163754234, chr8:16884489, chr10:129535510, chr2:210636540, chr4:2025485 2. Chr14: 48145305, chr6:118228600.
  • the methylated biomarkers include chr12:42873140, chr8:81789921, chr8:81811441, chr5:163754234, chr8:16884489, chr10:129535510, chr2:210636540, chr4:20254852 , chr14: 48145305, chr6:118228600.
  • the methylated biomarkers include chr12:42873140, chr8:81789921, chr8:81811441, chr5:163754234, chr8:16884489, chr10:129535510, chr2:210636540, chr4:20254852 , chr14: 48145305, chr6: 118228600, chr17: 6617301, chr5: 2749179, chr21: 22370447, chr19: 12876993, chr1: 2980508, chr16: 86542314, chr16: 22825741, chr20: 59827184, chr2: 99439396, chr8: 144790031.
  • the methylated biomarkers include chr12:42873140, chr8:81789921, chr8:81811441, chr5:163754234, chr8:16884489, chr10:129535510, chr2:210636540, chr4:20254852 , chr14: 48145305, chr6: 118228600, chr17: 6617301, chr5: 2749179, chr21: 22370447, chr19: 12876993, chr1: 2980508, chr16: 86542314, chr16: 22825741, chr20: 59827184, chr2: 99439396, chr8: 144790031, chr13: 113765062, chr6:85473377, chr7:330023, chr16:55358704, chr
  • the methylated biomarkers include chr12:42873140, chr8:81789921, chr8:81811441, chr5:163754234, chr8:16884489, chr10:129535510, chr2:210636540, chr4:20254852 , chr14: 48145305, chr6: 118228600, chr17: 6617301, chr5: 2749179, chr21: 22370447, chr19: 12876993, chr1: 2980508, chr16: 86542314, chr16: 22825741, chr20: 59827184, chr2: 99439396, chr8: 144790031, chr13: 113765062, chr6:85473377, chr7:330023, chr16:55358704, chr
  • the methylated biomarkers include chr12:42873140, chr8:81789921, chr8:81811441, chr5:163754234, chr8:16884489, chr10:129535510, chr2:210636540, chr4:20254852 , chr14: 48145305, chr6: 118228600, chr17: 6617301, chr5: 2749179, chr21: 22370447, chr19: 12876993, chr1: 2980508, chr16: 86542314, chr16: 22825741, chr20: 59827184, chr2: 99439396, chr8: 144790031, chr13: 113765062, chr6:85473377, chr7:330023, chr16:55358704, chr
  • the methylated biomarkers include chr7:71801447, chr20:59828473, chr4:20254852, chr16:86542314, chr2:99439396, chr12:107713297, chr21:22370447, chr14:4814530 5.
  • the combination of methylated biomarkers also includes the above-mentioned 60 markers other than the above-mentioned 4 kinds Any combination of substances, but also any at least one methylation biomarker in Table 1.2 of the specification of the present invention other than the above 60 kinds. Further, the methylation biomarker combinations include a total of 1366 in Table 1.2.
  • Another object of the present invention is to provide the application of the above methylation biomarkers or combinations thereof, and/or reagents for detecting their methylation levels in the preparation of detection kits for gastric cancer lymph node metastasis and non-metastasis.
  • Another object of the present invention is to provide a kit for detecting gastric cancer lymph node metastasis and non-metastasis.
  • a kit for detecting lymph node metastasis and non-metastasis of gastric cancer including a reagent for detecting any one of the above-mentioned methylated biomarkers or a combination thereof.
  • the detection adopts fluorescent quantitative PCR (qPCR), methylation-specific PCR (MSP), digital PCR (ddPCR), DNA methylation chips, targeted DNA methylation sequencing, global Genome methylation sequencing (WGBS), or DNA methylation mass spectrometry (MassArray) methods.
  • qPCR fluorescent quantitative PCR
  • MSP methylation-specific PCR
  • ddPCR digital PCR
  • DNA methylation chips DNA methylation chips
  • targeted DNA methylation sequencing targeted DNA methylation sequencing
  • WGBS global Genome methylation sequencing
  • MassArray DNA methylation mass spectrometry
  • Another object of the present invention is to provide a method for detecting gastric cancer lymph node metastasis and non-metastasis, comprising the following steps:
  • Bisulfite treatment is performed on the extracted DNA sample to obtain converted DNA;
  • Detection of methylation levels of the above methylated biomarkers or combinations thereof is performed.
  • qPCR quantitative quantitative PCR
  • MSP methylation-specific PCR
  • ddPCR digital PCR
  • DNA methylation chips targeted DNA methylation sequencing
  • WGBS genome-wide methylation Sequencing
  • MassArray DNA methylation mass spectrometry
  • the sample to be detected is a liquid biopsy sample, for example, the liquid biopsy sample is tissue, plasma, or body fluid.
  • the present invention screens out a suitable DNA methylation biomarker for detecting lymph node metastasis and non-metastasis of gastric cancer, and the DNA methylation biomarker can achieve the purpose of accurate prediction.
  • the new DNA methylation marker developed by the present invention can be used to identify lymph node metastasis of early gastric cancer, can assist in clinical accurate diagnosis and guide treatment, and has also begun to be used in technical research in laboratories.
  • Figure 1 The differential heatmap of 1366 markers in the methylation level between gastric cancer lymph node metastasis and non-metastasis in Example 1.
  • Figure 2 Heatmap of the difference in the methylation level of 60 markers in Example 1 between gastric cancer lymph node metastasis and non-metastasis.
  • Figure 3 The ROC curve of the four markers in Example 1 for the detection of gastric cancer lymph node metastasis.
  • Figure 4 ROC curves for the detection of gastric cancer lymph node metastasis by 4, 10, 20, 30, 40, 50, and 60 markers in Example 2.
  • Figure 5 In Example 4, the differential heatmap of methylation levels between gastric cancer lymph node metastasis and non-metastasis for 1366 markers of 10 gastric cancer FFPE samples.
  • the experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions recommended by the manufacturer.
  • Various commonly used chemical reagents used in the embodiment are all commercially available products.
  • nucleic acid detection refers to any method of determining the nucleotide composition of a target nucleic acid. Nucleic acid detection assays include, but are not limited to, DNA sequencing methods, probe hybridization methods.
  • sample template refers to a nucleic acid derived from a sample that is analyzed for the presence of a "target” (defined below).
  • background template is used to refer to nucleic acid other than sample template, which may or may not be present in the sample. Background templates are often unintentional. This may be the result of carryover, or it may be due to the presence of nucleic acid contaminants that were attempted to be purified from the sample. For example, nucleic acids other than those to be detected from the organism may be present as background in the test sample.
  • methylation refers to methylation of cytosine at position C5 or N4 of cytosine, position N6 of adenine or other types of nucleic acid methylation.
  • In vitro amplified DNA is usually unmethylated because in general in vitro DNA amplification methods do not preserve the methylation pattern of the amplified template.
  • unmethylated DNA or “methylated DNA” may also refer to amplified DNA in which the original template is unmethylated or methylated, respectively.
  • methylated nucleotide or “methylated nucleotide base” refers to the presence of a methyl moiety on a nucleotide base that is not present on the recognized typical nucleus. in nucleotide bases.
  • cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at the 5-position of its pyrimidine ring. Therefore, cytosine is not a methylated nucleotide, 5-methylcytosine is a methylated nucleotide.
  • thymine contains a methyl moiety at position 5 of its pyrimidine ring; however, for the purposes of this article, thymine is not considered a methylated nucleotide when present in DNA because thymine is a DNA typical nucleotide bases.
  • Methylation status can optionally be represented or indicated by a "methylation value” (eg, indicating methylation frequency, score, ratio, percentage, etc.). Methylation values can be used, for example, to quantify the amount of intact nucleic acid present after restriction digestion with a methylation-dependent restriction enzyme, or by comparing amplification profiles following a bisulfite reaction, or by comparing bisulfite treatment and Sequences of unprocessed nucleic acids are generated. Thus, a value such as a methylation value represents methylation status and thus can be used as a quantitative indicator of methylation status in multiple copies of a locus.
  • the degree of co-methylation is represented or indicated by the methylation status of more than one methylation site. In a methylated region, when the methylation status of more than one methylation site is methylation defined as co-methylation.
  • methylation assay or "methylation level or degree of methylation detection” refers to any assay for determining the methylation status of one or more CpG dinucleotide sequences within a nucleic acid sequence.
  • bisulfite reagent refers to a reagent comprising, in some embodiments, bisulfite, disulfite, hydrogen sulfite, or combinations thereof.
  • DNA treated with bisulfite reagents unmethylated cytosine nucleotides will be converted to uracil, while methylated cytosine and other bases remain unchanged, thus distinguishing, for example, CpG dinuclear Methylated and unmethylated cytidines in the nucleotide sequence.
  • Samples must meet the following requirements: 1) patients with early gastric cancer diagnosed as T1 stage; 2) no distant metastasis or family history; 3) no neoadjuvant therapy; 4) lymphoma, multiple tumors, stump carcinoma and intraepithelial carcinoma Cases with neoplastic changes were excluded; 5) At least three gastrointestinal pathology experts evaluated the tumor stage and lymph node metastasis status. 12 pairs of gastric cancer with lymph node metastasis and 11 pairs of gastric cancer with non-lymph node metastasis, a total of 46 samples, all fresh and frozen tissue samples, the clinical information of the samples is shown in Table 1.1:
  • the extracted tissue DNA (50ng) is subjected to bisulfite conversion to deaminate the unmethylated cytosine in the DNA and convert it to uracil, while the methylated cytosine remains unchanged to obtain bisulfite conversion
  • the specific operation was carried out according to the instructions of the EZ DNA Methylation-Lightning Kit of Zymo Research.
  • the hybridization capture kit is xGen Lockdown Reagents from IDT Company, and it is operated according to the instructions.
  • the sequencer of Illumina Company was used to sequence the samples captured by hybridization to obtain the sequencing results.
  • the original sequencing data was cleaned up, processed and analyzed, and the percentage of methylated cytosine at each site ( ⁇ value) was determined based on the reads; the two groups of samples were compared and analyzed, and 1366 organisms were screened Marker, the heatmap of these 1366 biomarkers (markers) in 12 pairs of gastric cancer lymph node metastasis and 11 pairs of gastric cancer non-lymph node metastasis cancer and adjacent cancer, please refer to the following figure 1, you can see These biomarkers (markers) showed significant differences in methylation levels between gastric cancer lymph node metastasis and non-metastasis.
  • Example 1 Using the 12 pairs of gastric cancer lymph node metastasis cancer and adjacent cancer and 11 pairs of gastric cancer non-lymph node metastasis cancer and adjacent cancer described in Example 1, a total of 46 samples, using the 60 biomarkers (markers) screened in Example 1 to establish Divide these 48 samples into 100 times according to the ratio of 7:3, use Random Forest to model, use Random Forest to model in the train set in each division, use this model The risk score of each sample is calculated in the test set, and the risk score is compared with the standard diagnosis to obtain the discrimination sensitivity, specificity, AUC, NPV, and PPV of the combination of methylated regions.
  • Randomly select markers based on 4marker (chr12:42873140, chr8:81789921, chr8:81811441 and chr8:16884489), respectively 4marker (chr12:42873140, chr8:81789921, chr8:81811441, chr8:168 84489), 10marker(chr12:42873140, chr8:81789921, chr8:81811441, chr5:163754234, chr8:16884489, chr10:129535510, chr2:210636540, chr4:20254852, chr14:48 145305, chr6: 118228600), 20marker (chr12: 42873140, chr8 :81789921, chr8: 81811441, chr5: 163754234, chr8: 16884489, chr10
  • biomarkers On the basis of the 60 biomarkers (markers) screened in Example 1, randomly select biomarkers from the 1366 biomarkers (markers) and 1306 biomarkers (markers) except 60 marker and 60 biomarkers (marker) combined into 100 biomarkers (marker) (chr1:2980508, chr1:9054472, chr1:53387866, chr10:15032997, chr10:129535510, chr11:460694, chr12:6729752 , chr12:42873140, chr12:52644274, chr12:107713297, chr13:50707693, chr13:113765062, chr14:48145305, chr15:21968071, chr16:22825741, chr16:3 1487303, chr16:55358704, chr16:86542314, chr17:6617301,
  • chr8 97172757, chr13: 113764582, chr3: 138665454, chr20: 61638775, chr17:7492631, chr5:178003729, chr10:49732085, chr4:1397602, chr15:58357910, chr12:101110974, chr15:89922204, chr5:37837618, chr1 9:58867745, chr5:172661905, chr5:134871183, chr3:10858345, chr18:70209165, chr2:177502678, chr5:1878770, chr17:79615509, chr1:215257074, chr12:107713303, chr11:92702977, chr16:22826055, chr5:178547 314, chr2: 5833295,
  • chr19: 29284996 chr1: 6268993, chr2: 233351145, chr16 :55358706, chr14: 24045521, chr8: 494593, chr18: 56940506, chr1: 6514750, chr19: 30021160, chr8: 109095578, chr20: 26190268, chr5: 3595961, chr10: 1 25650958, chr8: 97172744, chr1: 2980249, chr5: 76249537 , chr19:30713817, chr14:75078326, chr11:17740932, chr1:229568156, chr7:657436, chr15:89922194, chr3:126260702, chr20:54580427, chr5:1406211 29.
  • chr6 148891603, chr1: 31189195, chr20: 61637441, chr15: 68944551, chr5:178004122, chr1:12223451, chr15:22958152, chr19:4835628, chr3:123009386, chr20:52802172, chr7:9444054, chr16:896631, chr13:1 13684918, chr19:56349120, chr8:53852501, chr3:8809151, chr19:39552150, chr11:125791018, chr3:152553401, chr9:137138178, chr1:7887247, chr12:113914196, chr3:96533126, chr3:126708460, chr7:441852 30.
  • random forest (Random Forest) is used to model in the train set, and the model is used to calculate the risk score of each sample in the test set.
  • the risk score is compared with the standard diagnosis to obtain the discriminant sensitivity, specificity, and AUC of the combination of methylated regions. , NPV and PPV etc.
  • biomarkers As shown in the table below; it shows that in the case of different numbers of biomarkers (markers), the AUCs are all above 0.999, indicating that these biomarkers (markers) or combinations of biomarkers (markers) have a good ability to distinguish gastric cancer lymph nodes Ability to transfer and non-transfer;

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Abstract

本发明涉及检测胃癌淋巴结转移与非转移的甲基化生物标记物或其组合以及应用，所述甲基化生物标记物选自chr12:42873140、chr8:81789921、chr8:81811441和chr8:16884489中的至少一种。本发明筛选到合适的DNA甲基化生物标记物，用来检测胃癌淋巴结转移与非转移，所述DNA甲基化生物标记物能够实现精准预测的目的。本发明所开发的所述DNA甲基化标志物，可用于鉴别早期胃癌淋巴结转移与否，可辅助临床准确诊断和指导治疗，也可以用于实验室进行胃癌淋巴结转移的技术研究。

Description

用于检测胃癌淋巴结节转移相关的甲基化生物标记物或其组合及应用
本发明要求于2021年10月25日提交中国专利局、申请号为2021112428975的中国专利申请的优先权，其全部内容通过引用结合在本发明中。
技术领域
本发明属于生物技术领域，具体涉及用于检测胃癌淋巴结节转移相关的甲基化生物标记物或其组合及应用。
背景技术
胃癌(Gastric cancer,GC)是一种恶性肿瘤，也是世界范围内最常见的恶性肿瘤之一。根据2020年全球癌症统计数据资料显示，胃癌的发病率和死亡率分别居第五位和第三位,男性发病率是女性的两倍。在中国，胃癌发病率正逐年升高，且趋于年轻化，其发生率和死亡率分别跃居到第二位和第三位,居消化系统恶性肿瘤首位，患胃癌数量几乎占据全球胃癌数量的一半。胃癌作为一种高侵袭性的异质性恶性肿瘤，仍然是一个全球性的健康问题。
在临床上，由于胃癌发病比较隐匿，早期临床症状不明显，绝大多数胃癌患者发现时已处于进展期，错过了最佳治疗时机，5年总生存率不到30％。近年来，随着我国胃癌诊疗水平的提高，早期胃癌检出率不断提高，占所有胃癌比例的20％。与进展期胃癌对比，早期胃癌预后良好，5年生存率达90％以上，其中无淋巴结转移者5年生存率接近95％。尽管我国早期胃癌检出率远低于日本(70％)和韩国(50％)，但是基于我国人口总数和胃癌的高发病率，我国早期胃癌病例的绝对数值仍很大。早期胃癌的传统治疗方式为D1或D2根治性切除手术，但手术的创伤较大，尤其对于没有淋巴结转移的患者，手术增加了创伤却不能使患者额外获益。早期胃癌的淋巴结转移率在10％～42％，平均15％，也就是说，大约80％～85％的患者没有必要进行淋巴结清扫。随着内镜微创技术的发展，ESD已成为治疗早期胃癌的新选择，而无淋巴结转移是内镜治疗的重要前提。
早期胃癌(early gastric cancer,ECG)是肿瘤侵入黏膜层或黏膜下层，不论是否有淋巴结转移(lymph node metastasis，LNM)的一类肿瘤。目前，内镜黏膜下剥离术(endoscopic submucosal dissection，ESD)已成为治疗早期胃癌的新选择，与传统手术相比，ESD具有创伤小、术后生活质量高等优点，而无淋巴 结转移是内镜治疗的重要前提。超声内镜、计算机断层扫描(CT)、正电子发射断层扫描(PET-CT)等，对早期胃癌淋巴结状态的评估准确性有限。有研究报道，在接受根治性手术的早期胃癌患者中，淋巴结转移的发生率在5％-10％之间，这表明，如果能够准确评估ESD下淋巴结状态，那么90％以上的手术都有可能避免。DNA甲基化发生在肿瘤的早期阶段，且具有组织特异性，是追踪早期肿瘤信号的最佳选择之一。
发明内容
本发明的目的之一在于提供了一种检测胃癌淋巴结转移与非转移的DNA甲基化生物标记物或其组合。
检测胃癌淋巴结转移与非转移的甲基化生物标记物或其组合，所述甲基化生物标记物选自chr12:42873140、chr8:81789921、chr8:81811441和chr8:16884489中的至少一个。
在其中一些实施例中，所述甲基化生物标记物包括chr12:42873140、chr8:81789921、chr8:81811441和chr8:16884489的组合。
在其中一些实施例中，所述甲基化生物标记物在上述4种的基础上，还包括chr7:71801447、chr20:59828473、chr4:20254852、chr16:86542314、chr2:99439396、chr12:107713297、chr21:22370447、chr14:48145305、chr16:22825741、chr2:210636540、chr7:330023、chr10:129535510、chr6:85473377、chr5:2749179、chr6:118228600、chr13:113765062、chr16:55358704、chr19:57352008、chr19:12876993、chr20:59827184、chr17:6617301、chr7:47092638、chr12:52644274、chr5:163754234、chr1:2980508、chr20:61638536、chr1:9054472、chr10:15032997、chr11:460694、chr12:6729752、chr15:21968071、chr19:40421554、chr19:46388022、chr19:46388316、chr19:55593563、chr2:115420396、chr20:61472147、chr5:140799056、chr7:98971536、chr8:144790192、chr8:70602390、chr1:53387866、chr13:50707693、chr16:31487303、chr19:4054950、chr19:46388262、chr19:46388329、chr20:43726625、chr20:54579248、chr20:57429986、chr7:156720659、chr7:56242574、chr8:144790031、chr8:144790060、chr8:144790218、chr8:145621935中的至少一种。
在其中一些实施例中，所述甲基化生物标记物包括chr12:42873140、chr8:81789921、chr8:81811441、chr5:163754234、chr8:16884489、chr10:129535510、chr2:210636540、 chr4:20254852、chr14:48145305、chr6:118228600。
在其中一些实施例中，所述甲基化生物标记物包括chr12:42873140、chr8:81789921、chr8:81811441、chr5:163754234、chr8:16884489、chr10:129535510、chr2:210636540、chr4:20254852、chr14:48145305、chr6:118228600。
在其中一些实施例中，所述甲基化生物标记物包括chr12:42873140、chr8:81789921、chr8:81811441、chr5:163754234、chr8:16884489、chr10:129535510、chr2:210636540、chr4:20254852、chr14:48145305、chr6:118228600、chr17:6617301、chr5:2749179、chr21:22370447、chr19:12876993、chr1:2980508、chr16:86542314、chr16:22825741、chr20:59827184、chr2:99439396、chr8:144790031。
在其中一些实施例中，所述甲基化生物标记物包括chr12:42873140、chr8:81789921、chr8:81811441、chr5:163754234、chr8:16884489、chr10:129535510、chr2:210636540、chr4:20254852、chr14:48145305、chr6:118228600、chr17:6617301、chr5:2749179、chr21:22370447、chr19:12876993、chr1:2980508、chr16:86542314、chr16:22825741、chr20:59827184、chr2:99439396、chr8:144790031、chr13:113765062、chr6:85473377、chr7:330023、chr16:55358704、chr8:144790192、chr8:145621935、chr7:56242574、chr12:107713297、chr20:61638536、chr7:156720659。
在其中一些实施例中，所述甲基化生物标记物包括chr12:42873140、chr8:81789921、chr8:81811441、chr5:163754234、chr8:16884489、chr10:129535510、chr2:210636540、chr4:20254852、chr14:48145305、chr6:118228600、chr17:6617301、chr5:2749179、chr21:22370447、chr19:12876993、chr1:2980508、chr16:86542314、chr16:22825741、chr20:59827184、chr2:99439396、chr8:144790031、chr13:113765062、chr6:85473377、chr7:330023、chr16:55358704、chr8:144790192、chr8:145621935、chr7:56242574、chr12:107713297、chr20:61638536、chr7:156720659、chr8:144790060、chr15:21968071、chr7:71801447、chr1:53387866、chr7:47092638、chr20:59828473、chr19:46388022、chr11:460694、chr8:144790218、chr19:46388316。
在其中一些实施例中，所述甲基化生物标记物包括chr12:42873140、chr8:81789921、chr8:81811441、chr5:163754234、chr8:16884489、chr10:129535510、chr2:210636540、chr4:20254852、chr14:48145305、chr6:118228600、chr17:6617301、chr5:2749179、chr21:22370447、chr19:12876993、chr1:2980508、chr16:86542314、chr16:22825741、chr20:59827184、chr2:99439396、chr8:144790031、chr13:113765062、chr6:85473377、 chr7:330023、chr16:55358704、chr8:144790192、chr8:145621935、chr7:56242574、chr12:107713297、chr20:61638536、chr7:156720659、chr8:144790060、chr15:21968071、chr7:71801447、chr1:53387866、chr7:47092638、chr20:59828473、chr19:46388022、chr11:460694、chr8:144790218、chr19:46388316、chr2:115420396、chr13:50707693、chr20:57429986、chr19:46388262、chr5:140799056、chr20:61472147、chr19:57352008、chr20:54579248、chr19:40421554、chr20:43726625。
在其中一些实施例中，所述甲基化生物标记物包括chr7:71801447、chr20:59828473、chr4:20254852、chr16:86542314、chr2:99439396、chr12:107713297、chr21:22370447、chr14:48145305、chr16:22825741、chr2:210636540、chr7:330023、chr10:129535510、chr6:85473377、chr5:2749179、chr6:118228600、chr13:113765062、chr16:55358704、chr19:57352008、chr19:12876993、chr20:59827184、chr17:6617301、chr7:47092638、chr12:52644274、chr5:163754234、chr1:2980508、chr20:61638536、chr1:9054472、chr10:15032997、chr11:460694、chr12:6729752、chr15:21968071、chr19:40421554、chr19:46388022、chr19:46388316、chr19:55593563、chr2:115420396、chr20:61472147、chr5:140799056、chr7:98971536、chr8:144790192、chr8:70602390、chr1:53387866、chr13:50707693、chr16:31487303、chr19:4054950、chr19:46388262、chr19:46388329、chr20:43726625、chr20:54579248、chr20:57429986、chr7:156720659、chr7:56242574、chr8:144790031、chr8:144790060、chr8:144790218、chr8:145621935。
在其中一些实施例中，所述甲基化生物标记物组合除了包括有chr12:42873140、chr8:81789921、chr8:81811441和chr8:16884489的组合，还包括除上述4种之外的上述60种标记物的任意组合，而且还包括本发明说明书中表1.2中除上述60种之外的任意至少一种甲基化生物标记物。进一步地，所述甲基化生物标记物组合包括表1.2中共1366种。
本发明的另一目的是提供上述甲基化生物标记物或其组合，和/或检测其甲基化水平的试剂在制备胃癌淋巴结转移与非转移的检测试剂盒中的应用。
本发明的另一目的是提供一种检测胃癌淋巴结转移与非转移的试剂盒。
一种检测胃癌淋巴结转移与非转移的试剂盒，包括检测上述任意一项所述甲基化生物标记物或其组合的试剂。
在其中一些实施例中，所述检测采用应用荧光定量PCR(qPCR)、甲基化特异性PCR(MSP)、数字PCR(ddPCR)、DNA甲基化芯片、靶向DNA甲基化测序、全基因组甲基化测序(WGBS)、或DNA甲基化质谱(MassArray)的方法。
本发明的另一目的是提供一种胃癌淋巴结转移与非转移的检测方法，包括以下步骤：
对待检测样本进行DNA提取；
对提取的DNA样本进行亚硫酸氢盐处理，得到转化后的DNA；
进行上述甲基化生物标记物或其组合的甲基化水平的检测。
在其中一些实施例中，采用荧光定量PCR(qPCR)、甲基化特异性PCR(MSP)、数字PCR(ddPCR)、DNA甲基化芯片、靶向DNA甲基化测序、全基因组甲基化测序(WGBS)、DNA甲基化质谱(MassArray)方法进行检测。
在其中一些实施例中，所述待检测样本为液体活检样本，例如所述液体活检样本为组织、血浆、体液。
本发明筛选到合适的DNA甲基化生物标记物，用来检测胃癌淋巴结转移与非转移，所述DNA甲基化生物标记物能够实现精准预测的目的。本发明开发的新的所述DNA甲基化标志物，可用于鉴别早期胃癌淋巴结转移与否，可辅助临床准确诊断和指导治疗，也开始用于实验室做技术研究。
附图说明
图1：实施例1中1366marker在胃癌淋巴结转移和非转移之间的甲基化水平上的差异heatmap图。
图2：实施例1中60个marker在胃癌淋巴结转移和非转移之间的甲基化水平上的差异heatmap图。
图3：实施例1中4个marker的对于胃癌淋巴结转移的检测的ROC曲线图。
图4：实施例2中4个、10个、20个、30个、40个、50个、60个marker对于胃癌淋巴结转移的检测的ROC曲线图。
图5：实施例4中，针对10个胃癌FFPE样本的1366marker的在胃癌淋巴结转移和非转移之间的甲基化水平上的差异heatmap图。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明公开内容的理解更加透彻全面。
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。本发明所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
定义为了便于理解本技术，下面定义了一些术语和短语。
如本发明所用，术语“核酸检测”是指确定目标核酸的核苷酸组成的任何方法。核酸检测测定包括但不限于DNA测序方法、探针杂交方法。
术语“样品模板”是指来源于样品的用于分析“靶”(下文定义)的存在的核酸。相比之下，“背景模板”用于指样品模板以外的核酸，其可能存在或可能不存在于样品中。背景模板通常是无意的。这可能是遗留的结果，或者可能是由于试图从样品中纯化走的核酸污染物的存在。例如，来自生物体的待检测核酸以外的核酸可以作为测试样品的背景存在。
如本文所用，“甲基化”是指胞嘧啶位置C5或N4的胞嘧啶甲基化，腺嘌呤的N6位点或其他类型的核酸甲基化。体外扩增的DNA通常是未甲基化的，因为通常体外DNA扩增方法不能保留扩增模板的甲基化模式。然而，“未甲基化DNA”或“甲基化DNA”也可以分别指原始模板未甲基化或甲基化的扩增DNA。
因此，如本文所用，“甲基化核苷酸”或“甲基化核苷酸碱基”是指在核苷酸碱基上存在甲基部分，其中甲基部分不存在于公认的典型核苷酸碱基中。例如，胞嘧啶在其嘧啶环上不包含甲基部分，但是5-甲基胞嘧啶在其嘧啶环的5位包含甲基部分。因此，胞嘧啶不是甲基化核苷酸，5-甲基胞嘧啶是甲基化核苷酸。在另一个实例中，胸腺嘧啶在其嘧啶环的5位含有甲基部分；然而，为了本文的目的，当存在于DNA中时不认为胸腺嘧啶是甲基化核苷酸，因为胸腺嘧啶是DNA的典型核苷酸碱基。
甲基化状态可任选地由“甲基化值”表示或指示(例如，表示甲基化频率、分数、比例、百分比等)。甲基化值可以例如在用甲基化依赖性限制酶限制性消化之后定量存在的完整核酸的量，或者通过比较亚硫酸氢盐反应后的扩增谱，或者通过比较亚硫酸氢盐处理和未处理的核酸的序列来产生。因此，诸如甲基化值 的值代表甲基化状态，因此可用作基因座的多个拷贝中甲基化状态的定量指标。共甲基化程度由多于一个甲基化位点的甲基化状态表示或指示，在一段甲基化区域内，当多于一个甲基化位点的甲基化状态均为甲基化时定义为共甲基化。
术语“甲基化测定”或“甲基化水平或甲基化程度检测”是指用于确定核酸序列内的一个或多个CpG二核苷酸序列的甲基化状态的任何测定。
如本文所用，术语“亚硫酸氢盐试剂”是指在一些实施方案中包含亚硫酸氢盐(bisulfite)、亚硫酸氢盐(disulfite)、亚硫酸氢盐(hydrogen sulfite)或其组合的试剂，经过亚硫酸氢盐试剂处理的DNA，其未经过甲基化的胞嘧啶核苷酸将转化为尿嘧啶，而甲基化的胞嘧啶及其他碱基维持不变，因此可以区分例如CpG二核苷酸序列中的甲基化和未甲基化胞苷。
实施例1生物标记物(marker)筛选
1、样本来源：
样本需符合以下要求：1)诊断为T1期的早期胃癌患者；2)无远处转移或无家族史；3)未进行新辅助治疗；4)淋巴瘤、多发肿瘤、残端癌和上皮内瘤变的病例排除；5)至少由三个胃肠病理学专家评估肿瘤的分期和淋巴结转移状态等情况。12对胃癌淋巴结转移的癌与癌旁以及11对胃癌非淋巴结转移的癌与癌旁，共46例样本，均为新鲜冰冻组织样本，样本临床信息表1.1所示：
Figure PCTCN2022126211-appb-000001
Figure PCTCN2022126211-appb-000002
2、实验、数据分析及生物标记物(marker)筛选：
将上述样本，对新鲜冰冻组织或者FFPE样本进行建库，流程和步骤如下：
1、组织DNA提取及甲基化建库
1.1、组织DNA的提取。
上述胃癌组织样本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
将上述转化后的17ul样本加入以下试剂进行反应：
组分 体积(μl)
转化后样本 17
MEB1缓冲液 2
MEE2酶 1
总体积 20
置于PCR仪中按照以下程序进行反应：
37℃ 30min
95℃ 5min
热盖 105℃
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
配制如下反应液：
组分 单个用量(μl)
上一步反应产物 20
H 2O 4
MLB1缓冲液 8
MLR1试剂 2
MLR5试剂 2
MLE1酶 2
MLE5酶 2
反应混合体积 40
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
配制如下反应液
组分 单个用量(μl)
上一步反应产物 40
H2O 35
MAB2缓冲液 20
MAR1试剂 2
MAR2试剂 2
MAE3酶 1
反应混合体积 40
置于PCR仪中按照以下程序进行反应：
Figure PCTCN2022126211-appb-000003
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)的对扩增I反应后的产物进行纯化，用21μl EB进行洗脱，纯化具体步骤如下：
取上一步反应产物并进行离心，每个样本加入166μl 1:6倍稀释的Agencourt AM Pure Beads，用移液器吹打混匀。室温孵育5min。离心，置于磁力架上静置5min。吸去上清。加入200μl 80％EtOH，静置30s，吸走乙醇，重复一次后，离心，将PCR管置于磁力架上，吸走剩余乙醇，开盖干燥磁珠2-3min，注意不要过干。加入21μl EB进行洗脱，用移液器充分吹打混匀，室温静置3min。离心，将PCR管置于磁力架上，静置3min。吸取20μl上清于新的PCR管中。
1.7连接II
配制如下反应液：
组分 体积(μl)
上一步反应体积 20
H 2O 4
MSB1缓冲液 8
MSR1试剂 2
MSR5试剂 2
MSE1酶 2
MSE5酶 2
总体积 40
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(扩增产物文库构建):
配制如下反应液：
组分 体积(μl)
上一步反应体积 40
H 2O 6
2X KAPA HiFi Hot Start Ready Mix 8
I5接头引物 2
I7接头引物 2
总体积 100
置于PCR仪中按照以下程序进行反应
Figure PCTCN2022126211-appb-000004
Figure PCTCN2022126211-appb-000005
1.9纯化II
加入Agencourt AM Pure 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41μl EB进行洗脱，纯化具体步骤如下：
取上一步反应产物并进行离心，每个样本加入71μl未稀释的Agencourt AM Pure Beads，用移液器吹打混匀。室温孵育5min。离心，置于磁力架上静置5min。吸去上清。加入200μl 80％EtOH，静置30s，吸走乙醇，重复步骤一次后，离心，将PCR管置于磁力架上，吸走剩余乙醇。开盖干燥磁珠2-3min，注意不要过干。加入41μl EB进行洗脱，用移液器充分吹打混匀，室温静置3min。离心，将PCR管置于磁力架上，静置3min。吸取20μl上清于新的PCR管中。Qubit定量：取1μl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、下机数据的分析：
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。
5、对原始测序数据进行了相关清理及处理分析，并基于对reads读数来确定每一个位点甲基化胞嘧啶的百分比(β值)；对两组样本进行比较分析，筛选到1366个生物标记物(marker)，这1366个生物标记物(marker)在12对胃癌淋巴结转移的癌与癌旁和11对胃癌非淋巴结转移的癌与癌旁的heatmap图，请参见如下图1，可以看出这些生物标记物(marker)在胃癌淋巴结转移和非转移之间存在显著性的甲基化水平上的差异。
6、通过对这1366个甲基化生物标记物(marker)的基因注释信息，以及在12对胃癌淋巴结转移的癌与癌旁和11对胃癌淋巴非结转移的癌与癌旁每个甲基化生物标记物(marker)的auc如下表1.2所示，其中单生物标记物(marker)AUC大于0.6的有1342个，大于0.7有1207，大于0.8有691个，大于0.9有46个，显示这些生物标记物(marker)均具有良好的区分胃癌淋巴结转移与非转移的能力。
表1.2 1366个甲基化及其AUC值
Figure PCTCN2022126211-appb-000006
Figure PCTCN2022126211-appb-000007
Figure PCTCN2022126211-appb-000008
Figure PCTCN2022126211-appb-000009
Figure PCTCN2022126211-appb-000010
Figure PCTCN2022126211-appb-000011
Figure PCTCN2022126211-appb-000012
Figure PCTCN2022126211-appb-000013
Figure PCTCN2022126211-appb-000014
Figure PCTCN2022126211-appb-000015
Figure PCTCN2022126211-appb-000016
Figure PCTCN2022126211-appb-000017
Figure PCTCN2022126211-appb-000018
Figure PCTCN2022126211-appb-000019
Figure PCTCN2022126211-appb-000020
Figure PCTCN2022126211-appb-000021
Figure PCTCN2022126211-appb-000022
Figure PCTCN2022126211-appb-000023
Figure PCTCN2022126211-appb-000024
Figure PCTCN2022126211-appb-000025
Figure PCTCN2022126211-appb-000026
Figure PCTCN2022126211-appb-000027
Figure PCTCN2022126211-appb-000028
Figure PCTCN2022126211-appb-000029
Figure PCTCN2022126211-appb-000030
Figure PCTCN2022126211-appb-000031
Figure PCTCN2022126211-appb-000032
Figure PCTCN2022126211-appb-000033
Figure PCTCN2022126211-appb-000034
7、我们通过上述12对胃癌淋巴结转移的癌与癌旁与11对胃癌非淋巴结转移的癌与癌旁按照甲基化水平差异从大到小以及fdr从小到大排序，从1366个生物标记物(marker)里面挑选出来60个生物标记物(marker)用于后续的建模；这60个生物标记物(marker)在46个样本里面的heatmap图，请参见图2：这60个生物标记物(marker)是chr7:71801447、chr20:59828473、chr4:20254852、chr16:86542314、chr2:99439396、chr12:107713297、chr21:22370447、chr14:48145305、chr16:22825741、chr2:210636540、chr7:330023、chr10:129535510、chr6:85473377、chr5:2749179、chr6:118228600、chr8:16884489、chr13:113765062、chr8:81789921、chr16:55358704、chr8:81811441、chr19:57352008、chr19:12876993、chr20:59827184、chr17:6617301、chr7:47092638、chr12:52644274、chr12:42873140、chr5:163754234、chr1:2980508、chr20:61638536、chr1:9054472、chr10:15032997、chr11:460694、chr12:6729752、chr15:21968071、chr19:40421554、chr19:46388022、chr19:46388316、chr19:55593563、chr2:115420396、chr20:61472147、chr5:140799056、chr7:98971536、chr8:144790192、chr8:70602390、chr1:53387866、chr13:50707693、chr16:31487303、chr19:4054950、chr19:46388262、chr19:46388329、chr20:43726625、chr20:54579248、chr20:57429986、chr7:156720659、chr7:56242574、chr8:144790031、chr8:144790060、chr8:144790218、chr8:145621935；其中chr12:42873140、chr8:81789921、chr8:81811441和chr8:16884489整体表现最好，AUC分别为0.987689393939394、0.96780303030303、0.964962121212121和0.916666666666667，其AUC参见图3。
实施例2
用实施例1所述12对胃癌淋巴结转移的癌与癌旁与11对胃癌非淋巴结转移的癌与癌旁，一共46个样本，用实施例1所筛选的60个生物标记物(marker)建模，将这48个样本按照7:3的比例切分100次，利用随机森林(Random Forest)建模，每一次切分里利用随机森林(Random Forest)在train集合里面建模，用该模型在test集合里面计算每个样本的风险得分，风险得分对比标准诊断得到该甲基化区域组合的判别灵敏度、特异性、AUC、NPV以及PPV等。具体分配：在4marker(chr12:42873140、chr8:81789921、chr8:81811441和chr8:16884489)的基础上随机选择marker，分别是4marker(chr12:42873140、chr8:81789921、chr8:81811441、chr8:16884489)、10marker(chr12:42873140、chr8:81789921、chr8:81811441、chr5:163754234、chr8:16884489、chr10:129535510、chr2:210636540、chr4:20254852、chr14:48145305、chr6:118228600)、20marker(chr12:42873140、chr8:81789921、chr8:81811441、chr5:163754234、chr8:16884489、chr10:129535510、chr2:210636540、chr4:20254852、chr14:48145305、chr6:118228600、chr17:6617301、chr5:2749179、chr21:22370447、chr19:12876993、chr1:2980508、chr16:86542314、chr16:22825741、chr20:59827184、chr2:99439396、chr8:144790031)、30marker(chr12:42873140、chr8:81789921、chr8:81811441、chr5:163754234、chr8:16884489、chr10:129535510、chr2:210636540、chr4:20254852、chr14:48145305、chr6:118228600、chr17:6617301、chr5:2749179、chr21:22370447、chr19:12876993、chr1:2980508、chr16:86542314、chr16:22825741、chr20:59827184、chr2:99439396、chr8:144790031、chr13:113765062、chr6:85473377、chr7:330023、chr16:55358704、chr8:144790192、chr8:145621935、chr7:56242574、chr12:107713297、chr20:61638536、chr7:156720659)、40marker(chr12:42873140、chr8:81789921、chr8:81811441、chr5:163754234、chr8:16884489、chr10:129535510、chr2:210636540、chr4:20254852、chr14:48145305、chr6:118228600、chr17:6617301、chr5:2749179、chr21:22370447、chr19:12876993、chr1:2980508、chr16:86542314、chr16:22825741、 chr20:59827184、chr2:99439396、chr8:144790031、chr13:113765062、chr6:85473377、chr7:330023、chr16:55358704、chr8:144790192、chr8:145621935、chr7:56242574、chr12:107713297、chr20:61638536、chr7:156720659、chr8:144790060、chr15:21968071、chr7:71801447、chr1:53387866、chr7:47092638、chr20:59828473、chr19:46388022、chr11:460694、chr8:144790218、chr19:46388316)、50marker(chr12:42873140、chr8:81789921、chr8:81811441、chr5:163754234、chr8:16884489、chr10:129535510、chr2:210636540、chr4:20254852、chr14:48145305、chr6:118228600、chr17:6617301、chr5:2749179、chr21:22370447、chr19:12876993、chr1:2980508、chr16:86542314、chr16:22825741、chr20:59827184、chr2:99439396、chr8:144790031、chr13:113765062、chr6:85473377、chr7:330023、chr16:55358704、chr8:144790192、chr8:145621935、chr7:56242574、chr12:107713297、chr20:61638536、chr7:156720659、chr8:144790060、chr15:21968071、chr7:71801447、chr1:53387866、chr7:47092638、chr20:59828473、chr19:46388022、chr11:460694、chr8:144790218、chr19:46388316、chr2:115420396、chr13:50707693、chr20:57429986、chr19:46388262、chr5:140799056、chr20:61472147、chr19:57352008、chr20:54579248、chr19:40421554、chr20:43726625)、60marker的AUC图，以及在不同的marker数目下的AUC、SP、SE、ACC、PPV、NPV等如下表，从表中可以看出在不同的marker数的情况下，AUC均在0.99以上，以及SP、SE、ACC、PPV、NPV也均在0.97以上，说明这些marker或者marker的组合均具有良好的区分胃癌淋巴结转移和非转移的能力，可以作为生物标记物用于后续的研究，参见图4。
Figure PCTCN2022126211-appb-000035
Figure PCTCN2022126211-appb-000036
实施例3
在实施例1筛选到的60个生物标记物(marker)的基础上，再从1366个生物标记物(marker)里面，除开60之外的1306个生物标记物(marker)里面，随机选择生物标记物(marker)与60个生物标记物(marker)组合成100生物标记物(marker)(chr1:2980508、chr1:9054472、chr1:53387866、chr10:15032997、chr10:129535510、chr11:460694、chr12:6729752、chr12:42873140、chr12:52644274、chr12:107713297、chr13:50707693、chr13:113765062、chr14:48145305、chr15:21968071、chr16:22825741、chr16:31487303、chr16:55358704、chr16:86542314、chr17:6617301、chr19:4054950、chr19:12876993、chr19:40421554、chr19:46388022、chr19:46388262、chr19:46388316、chr19:46388329、chr19:55593563、chr19:57352008、chr2:99439396、chr2:115420396、chr2:210636540、chr20:43726625、chr20:54579248、chr20:57429986、chr20:59827184、chr20:59828473、chr20:61472147、chr20:61638536、chr21:22370447、chr4:20254852、chr5:2749179、chr5:140799056、chr5:163754234、chr6:85473377、chr6:118228600、chr7:330023、chr7:47092638、chr7:56242574、chr7:71801447、chr7:98971536、chr7:156720659、chr8:16884489、chr8:70602390、chr8:81789921、chr8:81811441、chr8:144790031、chr8:144790060、chr8:144790192、chr8:144790218、chr8:145621935、chr9:137720844、chr16:27149887、chr11:67804157、chr4:8216099、chr16:27149886、chr9:78594608、chr3:174095188、chr1:8943835、chr16:83875187、chr1:84976011、chr11:5499050、chr15:101869604、chr4:190529353、chr18:43909995、chr9:136580547、chr9:91538730、chr17:76894409、chr5:33942805、chr1:8908212、chr12:42873141、chr5:167847507、chr14:100759047、chr13:113721698、chr11:76782117、chr8:2031600、chr16:50113774、chr19:49520503、chr11:67804156、chr8:2031536、chr21:45246325、chr19:49520502、chr12:42873073、chr5:77581175、chr5:172754702、chr12:26317171、chr11:888957、 chr8:81789920、chr17:45398759、chr19:50848024、chr13:98654065)、300生物标记物(marker)(chr1:2980508、chr1:9054472、chr1:53387866、chr10:15032997、chr10:129535510、chr11:460694、chr12:6729752、chr12:42873140、chr12:52644274、chr12:107713297、chr13:50707693、chr13:113765062、chr14:48145305、chr15:21968071、chr16:22825741、chr16:31487303、chr16:55358704、chr16:86542314、chr17:6617301、chr19:4054950、chr19:12876993、chr19:40421554、chr19:46388022、chr19:46388262、chr19:46388316、chr19:46388329、chr19:55593563、chr19:57352008、chr2:99439396、chr2:115420396、chr2:210636540、chr20:43726625、chr20:54579248、chr20:57429986、chr20:59827184、chr20:59828473、chr20:61472147、chr20:61638536、chr21:22370447、chr4:20254852、chr5:2749179、chr5:140799056、chr5:163754234、chr6:85473377、chr6:118228600、chr7:330023、chr7:47092638、chr7:56242574、chr7:71801447、chr7:98971536、chr7:156720659、chr8:16884489、chr8:70602390、chr8:81789921、chr8:81811441、chr8:144790031、chr8:144790060、chr8:144790192、chr8:144790218、chr8:145621935、chr9:137720844、chr16:27149887、chr11:67804157、chr4:8216099、chr16:27149886、chr9:78594608、chr3:174095188、chr1:8943835、chr16:83875187、chr1:84976011、chr11:5499050、chr15:101869604、chr4:190529353、chr18:43909995、chr9:136580547、chr9:91538730、chr17:76894409、chr5:33942805、chr1:8908212、chr12:42873141、chr5:167847507、chr14:100759047、chr13:113721698、chr11:76782117、chr8:2031600、chr16:50113774、chr19:49520503、chr11:67804156、chr8:2031536、chr21:45246325、chr19:49520502、chr12:42873073、chr5:77581175、chr5:172754702、chr12:26317171、chr11:888957、chr8:81789920、chr17:45398759、chr19:50848024、chr13:98654065、chr10:94350680、chr18:14474510、chr18:73122598、chr19:4835629、chr19:5825819、chr8:143922789、chr5:141027225、chr1:182305083、chr16:24386374、chr1:228494790、chr11:1665309、chr11:67570640、 chr17:76894410、chr7:100823635、chr7:75803133、chr1:1913580、chr10:105824333、chr10:94350681、chr1:6269028、chr1:90189775、chr1:8943836、chr1:6269032、chr13:111143100、chr8:41166805、chr14:92859398、chr14:58041994、chr9:96716744、chr8:53852560、chr7:75803132、chr1:8908749、chr10:94310910、chr7:50778697、chr8:2216198、chr17:74585456、chr3:126708459、chr9:23821304、chr10:23460252、chr8:66555817、chr1:1912392、chr4:110348477、chr1:228482010、chr19:53861300、chr16:83875385、chr14:100126054、chr7:103969761、chr18:31286480、chr2:134024338、chr10:125732302、chr3:101843330、chr18:9722307、chr11:87940173、chr3:39543575、chr1:8908750、chr10:129191329、chr18:56940525、chr13:49794913、chr11:32355273、chr19:899568、chr17:80989930、chr5:155108270、chr7:644418、chr17:39597601、chr19:53861301、chr2:210636470、chr1:172874992、chr7:127115955、chr5:140615982、chr6:148891603、chr1:31189195、chr20:61637441、chr15:68944551、chr5:178004122、chr1:12223451、chr15:22958152、chr19:4835628、chr3:123009386、chr20:52802172、chr7:9444054、chr16:896631、chr13:113684918、chr19:56349120、chr8:53852501、chr3:8809151、chr19:39552150、chr11:125791018、chr3:152553401、chr9:137138178、chr1:7887247、chr12:113914196、chr3:96533126、chr3:126708460、chr7:44185230、chr19:58867730、chr12:78755113、chr1:85882870、chr8:63161561、chr1:90077184、chr5:170735586、chr2:119915478、chr13:28498352、chr8:41166725、chr9:131899472、chr1:6268898、chr8:70623597、chr1:12223482、chr7:25157229、chr7:50724130、chr10:13491573、chr19:40006695、chr18:76733427、chr7:50746971、chr10:125425975、chr7:50724129、chr8:87081771、chr3:48632467、chr11:1769809、chr5:140615992、chr14:75078651、chr21:46520840、chr7:50745308、chr19:1456938、chr20:61751785、chr10:13491574、chr9:91538549、chr17:17628075、chr19:50848025、chr6:167490998、chr8:109095576、chr1:7887248、 chr5:2739440、chr3:96533133、chr1:85882871、chr7:75612804、chr7:976971、chr12:54519822、chr14:24045541、chr13:49794915、chr5:1878768、chr13:53775507、chr20:62103730、chr12:888848、chr2:119915799、chr14:71714187、chr8:55370478、chr2:13614745、chr1:2560219、chr10:126290274、chr4:105716474、chr8:41166807、chr9:23821318、chr10:11761898、chr4:1397858、chr2:134024251、chr8:32410110、chr14:30018029、chr8:41166704、chr2:210636462、chr6:141398837、chr8:53852557、chr5:155108203、chr7:70597953、chr1:6268999、chr19:34112495、chr9:37787372、chr7:27204786、chr14:103989591、chr2:91777669、chr1:6269030、chr14:36973460、chr17:79770741、chr18:11149356、chr13:100548438、chr3:10858314、chr4:7338852、chr19:34972296、chr20:4230070、chr13:30494760、chr20:53092267、chr2:176931957、chr9:77113289、chr10:125732314、chr8:143546178、chr7:9444055、chr18:70209322、chr6:33656851、chr12:115109865、chr1:18963037、chr6:133562303、chr5:134386259、chr22:26166012、chr8:97172760、chr19:24154577、chr10:11761899、chr8:24858357、chr12:99288364、chr6:49481576、chr1:34642682、chr10:105037700、chr14:24045533、chr3:190104122)、500生物标记物(marker)(chr1:2980508、chr1:9054472、chr1:53387866、chr10:15032997、chr10:129535510、chr11:460694、chr12:6729752、chr12:42873140、chr12:52644274、chr12:107713297、chr13:50707693、chr13:113765062、chr14:48145305、chr15:21968071、chr16:22825741、chr16:31487303、chr16:55358704、chr16:86542314、chr17:6617301、chr19:4054950、chr19:12876993、chr19:40421554、chr19:46388022、chr19:46388262、chr19:46388316、chr19:46388329、chr19:55593563、chr19:57352008、chr2:99439396、chr2:115420396、chr2:210636540、chr20:43726625、chr20:54579248、chr20:57429986、chr20:59827184、chr20:59828473、chr20:61472147、chr20:61638536、chr21:22370447、chr4:20254852、chr5:2749179、chr5:140799056、chr5:163754234、chr6:85473377、 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chr20:44803191、chr19:38886276、chr20:57430052、chr17:81047530、chr15:58357903、chr9:138295417、chr1:79472409、chr20:21502647、chr8:144790174、chr17:65289853、chr8:144790088、chr19:58867754、chr1:6514743、chr8:143781761、chr18:77590706、chr19:46388243、chr20:21502432、chr2:5833766、chr19:58867577、chr3:72778221、chr6:50818361、chr19:58868293、chr3:147128359、chr8:144790083、chr2:242481154、chr1:34642687、chr17:48546707、chr8:144790043、chr14:103568655、chr7:71801394、chr1:247802828、chr5:170735509、chr19:10531616、chr20:26190051、chr3:126260661、chr8:53853444、chr20:43085951、chr11:420464、chr20:5297178、chr19:46388009、chr19:17518098、chr1:17186246、chr7:98246014、chr1:3567639、chr20:34894700、chr19:58868171、chr7:153585304、chr8:109095587、chr20:61472162、chr19:46387743、chr1:119522470、chr7:70597926、chr19:46388270、chr20:39317009、chr1:20669793、chr15:58357917、chr19:46388283、chr10:121578019、chr19:58868142、chr20:26190006、chr1:149137816、chr7:28855599、chr20:57089877、chr12:95942213、chr20:21087011、chr1:6269107、chr20:5297482、chr19:46388122、chr8:144790188、chr19:46388187、chr22:17628122、chr11:85645774、chr2:45156389、chr10:22623418、chr18:35144985、chr20:61472185、chr5:178004222、chr2:105472921、chr5:32712000、chr10:50887656、chr19:58867702、chr1:79472416、chr20:62004602、chr19:46388322、chr5:1882099、chr13:26303549、chr20:61638551、chr6:117086909、chr19:58868196、chr20:34188725、chr19:46388175、chr8:106332003、chr20:61472169、chr20:62662464、chr7:153585321、chr7:56242537、chr20:25062356、chr17:77789597、chr7:44185373、chr19:54466574、chr10:30404059、chr7:329671、chr8:143781736、chr19:29284957、chr20:26190246、chr8:37555581、chr16:81803746、chr2:42720247、chr8:144790211、chr7:153584277、chr1:6514724、chr7:70597908、chr3:126260652、chr20:25071624、chr19:46388040、chr17:7287324、 chr2:242481160、chr8:145559892、chr1:34642610、chr8:144790172、chr5:178004202、chr8:41166043、chr3:128764997、chr19:46388172、chr19:46388018、chr20:59886911、chr6:94128358、chr20:57089855、chr1:11752481、chr16:31487375、chr3:52324728、chr1:236850129、chr7:116963129、chr17:2953、chr1:65991433、chr1:240656236、chr1:65991381、chr20:59998372、chr17:35165761、chr10:23484127、chr18:12516125、chr19:57352012、chr19:58868198、chr17:14201981、chr8:144790035、chr8:37555635、chr19:46388256、chr17:3289601、chr18:44773144、chr6:127718818、chr5:53299、chr18:12912229、chr5:140799106、chr20:57415327、chr19:58868125、chr8:144240946、chr19:42437266、chr1:203132968、chr20:44657846、chr1:546030、chr5:1882115、chr17:1107074、chr19:46388153、chr16:696497、chr20:60243252、chr20:26190039、chr15:58357901、chr20:59827131、chr17:4663161、chr20:31555019、chr3:5137852、chr19:46388300、chr13:50707660、chr8:145107140、chr8:145106905、chr11:8103079、chr5:140503739、chr8:53853008、chr7:156720578、chr18:70209312、chr13:112712133、chr20:26190235、chr16:88683738、chr19:47951128、chr16:2737187、chr6:168502520、chr19:46388229、chr8:142413122、chr5:83679586、chr7:43152396、chr7:78685573、chr1:55464826、chr8:7275280、chr5:11903209、chr8:109095674、chr8:37555773、chr2:132057942、chr1:228246726、chr8:37555874、chr19:46388205、chr11:1981171、chr13:28367004、chr2:95540476、chr19:40421581、chr22:46343859、chr17:64978303、chr20:2539686、chr10:1260264、chr12:75601269、chr1:546102、chr1:41410055、chr1:6269070、chr14:36988160、chr13:113765054、chr16:67199738、chr20:57430002、chr16:73127619、chr7:100253853、chr20:61469892、chr11:117298903、chr20:54579254、chr20:34188727、chr8:12566064、chr17:7287326、chr20:55500336、chr8:37555586、chr20:59827155、chr20:43726604、chr20:62094777、chr8:6928714)的组合，将48个样本按照7:3的比例切分100次，利用随机森林(Random Forest)建模，每一次切分里 利用随机森林(Random Forest)在train集合里面建模，用该模型在test集合里面计算每个样本的风险得分，风险得分对比标准诊断得到该甲基化区域组合的判别灵敏度、特异性、AUC、NPV以及PPV等。
如下表所示；显示在不同的生物标记物(marker)数的情况下，AUC均在0.999以上，说明这些生物标记物(marker)或者生物标记物(marker)的组合均具有良好的区分胃癌淋巴结转移和非转移的能力；
Figure PCTCN2022126211-appb-000037
实施例4
在10个胃癌FFPE样本中，其中3个转移，7个非转移样本中，由于样本比较少，不能做建模分析，主要分析了1366生物标记物(marker)的在转移和非转移上面的heatmap图，以及筛选到的60生物标记物(marker)中的单生物标记物(marker)的AUC，heatmap及单生物标记物(marker)AUC；从heatmap图(图5)上面可以看出转移和非转移的生物标记物(marker)存在差异，60个生物标记物(marker)的单生物标记物(marker)AUC也可以看出这些生物标记物(marker)存在显著差异，其中AUC大于0.9的有3个，AUC大于0.7的有16个，即在新的样本中这些生物标记物(marker)也显示出良好的区分性能，可以作为区分胃癌转移和非转移的甲基化标记物。
marker AUC marker AUC marker AUC
chr12:52644274 0.952381 chr8:70602390 0.619048 chr19:46388329 0.666667
chr19:46388022 0.952381 chr11:460694 0.571429 chr20:59828473 0.666667
chr7:98971536 0.904762 chr12:107713297 0.571429 chr8:144790218 0.666667
chr2:210636540 0.857143 chr12:42873140 0.571429 chr1:53387866 0.619048
chr19:57352008 0.809524 chr12:6729752 0.571429 chr16:55358704 0.619048
chr8:144790031 0.809524 chr19:46388262 0.571429 chr16:86542314 0.619048
chr10:129535510 0.761905 chr19:46388316 0.571429 chr1:9054472 0.619048
chr21:22370447 0.761905 chr20:61472147 0.571429 chr2:115420396 0.619048
chr5:140799056 0.761905 chr5:163754234 0.571429 chr7:156720659 0.619048
chr5:2749179 0.761905 chr7:56242574 0.571429 chr8:145621935 0.619048
chr6:118228600 0.761905 chr8:144790060 0.571429 chr8:81811441 0.52381
chr8:144790192 0.761905 chr8:16884489 0.571429 chr13:50707693 0.52381
chr14:48145305 0.714286 chr1:2980508 0.52381 chr16:22825741 0.52381
chr15:21968071 0.714286 chr16:31487303 0.52381 chr19:4054950 0.52381
chr2:99439396 0.714286 chr19:55593563 0.52381 chr20:57429986 0.52381
chr4:20254852 0.714286 chr20:54579248 0.52381 chr6:85473377 0.52381
chr10:15032997 0.666667 chr20:59827184 0.52381 chr20:61638536 0.547619
chr13:113765062 0.666667 chr7:330023 0.52381 chr7:71801447 0.571429
chr17:6617301 0.666667 chr7:47092638 0.52381 chr20:43726625 0.571429
chr19:12876993 0.666667 chr8:81789921 0.52381 chr19:40421554 0.619048
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 (18)

  1. 检测胃癌淋巴结转移与非转移的甲基化生物标记物或其组合，其特征是，所述甲基化生物标记物选自chr12:42873140、chr8:81789921、chr8:81811441和chr8:16884489中的至少一种。
  2. 根据权利要求1所述的检测胃癌淋巴结转移与非转移的甲基化生物标记物或其组合，其特征是，所述甲基化生物标记物包括chr12:42873140、chr8:81789921、chr8:81811441和chr8:16884489的组合。
  3. 根据权利要求1或2所述的检测胃癌淋巴结转移与非转移的甲基化生物标记物或其组合，其特征是，还包括chr7:71801447、chr20:59828473、chr4:20254852、chr16:86542314、chr2:99439396、chr12:107713297、chr21:22370447、chr14:48145305、chr16:22825741、chr2:210636540、chr7:330023、chr10:129535510、chr6:85473377、chr5:2749179、chr6:118228600、chr13:113765062、chr16:55358704、chr19:57352008、chr19:12876993、chr20:59827184、chr17:6617301、chr7:47092638、chr12:52644274、chr5:163754234、chr1:2980508、chr20:61638536、chr1:9054472、chr10:15032997、chr11:460694、chr12:6729752、chr15:21968071、chr19:40421554、chr19:46388022、chr19:46388316、chr19:55593563、chr2:115420396、chr20:61472147、chr5:140799056、chr7:98971536、chr8:144790192、chr8:70602390、chr1:53387866、chr13:50707693、chr16:31487303、chr19:4054950、chr19:46388262、chr19:46388329、chr20:43726625、chr20:54579248、chr20:57429986、chr7:156720659、chr7:56242574、chr8:144790031、chr8:144790060、chr8:144790218、chr8:145621935中的至少一种。
  4. 根据权利要求3所述的检测胃癌淋巴结转移与非转移的甲基化生物标记物或其组合，其特征是，所述甲基化生物标记物包括chr12:42873140、chr8:81789921、chr8:81811441、chr5:163754234、chr8:16884489、chr10:129535510、chr2:210636540、chr4:20254852、chr14:48145305、和chr6:118228600。
  5. 根据权利要求3所述的检测胃癌淋巴结转移与非转移的甲基化生物标记物或其组合，其特征是，所述甲基化生物标记物包括chr12:42873140、chr8:81789921、chr8:81811441、chr5:163754234、chr8:16884489、chr10:129535510、 chr2:210636540、chr4:20254852、chr14:48145305、chr6:118228600、chr17:6617301、chr5:2749179、chr21:22370447、chr19:12876993、chr1:2980508、chr16:86542314、chr16:22825741、chr20:59827184、chr2:99439396、和chr8:144790031。
  6. 根据权利要求3所述的检测胃癌淋巴结转移与非转移的甲基化生物标记物或其组合，其特征是，所述甲基化生物标记物包括chr12:42873140、chr8:81789921、chr8:81811441、chr5:163754234、chr8:16884489、chr10:129535510、chr2:210636540、chr4:20254852、chr14:48145305、chr6:118228600、chr17:6617301、chr5:2749179、chr21:22370447、chr19:12876993、chr1:2980508、chr16:86542314、chr16:22825741、chr20:59827184、chr2:99439396、chr8:144790031、chr13:113765062、chr6:85473377、chr7:330023、chr16:55358704、chr8:144790192、chr8:145621935、chr7:56242574、chr12:107713297、chr20:61638536、和chr7:156720659。
  7. 根据权利要求3所述的检测胃癌淋巴结转移与非转移的甲基化生物标记物或其组合，其特征是，所述甲基化生物标记物包括chr12:42873140、chr8:81789921、chr8:81811441、chr5:163754234、chr8:16884489、chr10:129535510、chr2:210636540、chr4:20254852、chr14:48145305、chr6:118228600、chr17:6617301、chr5:2749179、chr21:22370447、chr19:12876993、chr1:2980508、chr16:86542314、chr16:22825741、chr20:59827184、chr2:99439396、chr8:144790031、chr13:113765062、chr6:85473377、chr7:330023、chr16:55358704、chr8:144790192、chr8:145621935、chr7:56242574、chr12:107713297、chr20:61638536、chr7:156720659、chr8:144790060、chr15:21968071、chr7:71801447、chr1:53387866、chr7:47092638、chr20:59828473、chr19:46388022、chr11:460694、chr8:144790218、chr19:46388316。
  8. 根据权利要求3所述的检测胃癌淋巴结转移与非转移的甲基化生物标记物或其组合，其特征是，所述甲基化生物标记物包括chr12:42873140、chr8:81789921、chr8:81811441、chr5:163754234、chr8:16884489、chr10:129535510、chr2:210636540、chr4:20254852、chr14:48145305、chr6:118228600、chr17:6617301、chr5:2749179、chr21:22370447、chr19:12876993、chr1:2980508、chr16:86542314、chr16:22825741、chr20:59827184、chr2:99439396、chr8:144790031、 chr13:113765062、chr6:85473377、chr7:330023、chr16:55358704、chr8:144790192、chr8:145621935、chr7:56242574、chr12:107713297、chr20:61638536、chr7:156720659、chr8:144790060、chr15:21968071、chr7:71801447、chr1:53387866、chr7:47092638、chr20:59828473、chr19:46388022、chr11:460694、chr8:144790218、chr19:46388316、chr2:115420396、chr13:50707693、chr20:57429986、chr19:46388262、chr5:140799056、chr20:61472147、chr19:57352008、chr20:54579248、chr19:40421554、chr20:43726625。
  9. 根据权利要求3所述的检测胃癌淋巴结转移与非转移的甲基化生物标记物或其组合，其特征是，所述甲基化生物标记物包括chr7:71801447、chr20:59828473、chr4:20254852、chr16:86542314、chr2:99439396、chr12:107713297、chr21:22370447、chr14:48145305、chr16:22825741、chr2:210636540、chr7:330023、chr10:129535510、chr6:85473377、chr5:2749179、chr6:118228600、chr13:113765062、chr16:55358704、chr19:57352008、chr19:12876993、chr20:59827184、chr17:6617301、chr7:47092638、chr12:52644274、chr5:163754234、chr1:2980508、chr20:61638536、chr1:9054472、chr10:15032997、chr11:460694、chr12:6729752、chr15:21968071、chr19:40421554、chr19:46388022、chr19:46388316、chr19:55593563、chr2:115420396、chr20:61472147、chr5:140799056、chr7:98971536、chr8:144790192、chr8:70602390、chr1:53387866、chr13:50707693、chr16:31487303、chr19:4054950、chr19:46388262、chr19:46388329、chr20:43726625、chr20:54579248、chr20:57429986、chr7:156720659、chr7:56242574、chr8:144790031、chr8:144790060、chr8:144790218、chr8:145621935。
  10. 根据权利要求2或3或9所述的检测胃癌淋巴结转移与非转移的甲基化生物标记物或其组合，其特征是，所述甲基化生物标记物组合还包括本发明说明书中表1.2中任意至少一种甲基化生物标记物。
  11. 根据权利要求3所述的检测胃癌淋巴结转移与非转移的甲基化生物标记物或其组合，其特征是，所述甲基化生物标记物组合包括本发明说明书中表1.2中1366种甲基化生物标记物。
  12. 权利要求1-11任意一项所述甲基化生物标记物或其组合，和/或检测其甲基化水平的试剂在制备胃癌淋巴结转移与非转移的检测试剂盒中的应用。
  13. 一种检测胃癌淋巴结转移与非转移的试剂盒，其特征是，包括检测权利要求1-11任意一项所述甲基化生物标记物或其组合的试剂。
  14. 根据权利要求13所述的检测胃癌淋巴结转移与非转移的试剂盒，其特征是，所述检测采用应用荧光定量PCR、甲基化特异性PCR、数字PCR、DNA甲基化芯片、靶向DNA甲基化测序、全基因组甲基化测序、或DNA甲基化质谱的方法。
  15. 一种胃癌淋巴结转移与非转移的检测方法，其特征是，包括以下步骤：
    对待检测样本进行DNA提取；
    对提取的DNA样本进行亚硫酸氢盐处理，得到转化后的DNA；
    对提取的DNA样本进行权利要求1-11任意一项所述甲基化生物标记物或其组合的甲基化水平的检测。
  16. 根据权利要求15所述的胃癌淋巴结转移与非转移的检测方法，其特征是，采用述检测采用应用荧光定量PCR、甲基化特异性PCR、数字PCR、DNA甲基化芯片、靶向DNA甲基化测序、全基因组甲基化测序、或DNA甲基化质谱的方法进行检测。
  17. 根据权利要求15或者16所述的胃癌淋巴结转移与非转移的检测方法，其特征是，所述待检测样本为液体活检样本。
  18. 根据权利要求17所述的胃癌淋巴结转移与非转移的检测方法，其特征是，所述液体活检样本为组织、血浆、体液。
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CN114317738B (zh) * 2021-10-25 2022-09-27 广州市基准医疗有限责任公司 用于检测胃癌淋巴结节转移相关的甲基化生物标记物或其组合及应用
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CN115851923A (zh) * 2022-05-25 2023-03-28 广州市基准医疗有限责任公司 用于检测结直肠癌淋巴结转移的甲基化生物标记物及其应用
CN116064806B (zh) * 2022-10-19 2023-09-22 常州国药医学检验实验室有限公司 一种评估早期胃癌淋巴结转移风险的组合物及其用途

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