WO2022188776A1 - 可用于胃癌her2伴随诊断的基因甲基化标记物或其组合和应用 - Google Patents
可用于胃癌her2伴随诊断的基因甲基化标记物或其组合和应用 Download PDFInfo
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Definitions
- the invention belongs to the field of biotechnology, and in particular relates to a gene methylation marker or a combination and application thereof that can be used for the accompanying diagnosis of gastric cancer HER2 protein overexpression or gene amplification.
- Gastric carcinoma GC is one of the most common malignant tumors. Although the incidence of gastric cancer has decreased worldwide in recent years, it is still the second most common tumor of cancer-related deaths. It is estimated that there are 930,000 new cases every year. The number of deaths exceeds 700,000, and there are large regional differences in the incidence rate, which is related to diet, lifestyle, economic status, and the prevalence of Helicobacter pylori infection. More than 2/3 of new cases and deaths occurred in developing countries, including East Asia. 42.4% (mainly China).
- HER2 human epidermal growth factor receptor 2
- HER2 protein can activate cell growth and proliferation-related signaling pathways.
- Breast cancer, gastric cancer and other tumors are closely related.
- companion diagnostics an in vitro diagnostic technology, has received increasing attention. This technology can be used to detect biomarkers and correlate with the use of specific drugs, and is an important part of the field of precise tumor diagnosis and treatment.
- the HER2 companion diagnostic test for breast cancer has been widely used in the clinical diagnosis and treatment of breast cancer patients.
- Liquid biopsy can obtain the ctDNA shed by the tumor and circulate in the blood by non-invasive blood drawing in vitro, so as to determine the gene mutation of the tumor. Therefore, the detection of HER2 gene copy number in gastric cancer patients by liquid biopsy instead of tissue biopsy will effectively alleviate the tissue sampling injury faced by gastric cancer patients, and can eliminate the problem of tissue heterogeneity through multiple blood sample collection, and improve the detection of HER2 positive gastric cancer. Accuracy and convenience.
- cfDNA cell-free DNA
- peripheral blood derived from the metabolism and apoptosis of normal cells or tumor cells, and contains genetic information such as somatic mutation and DNA methylation.
- Liquid Biopsy the technology of mastering the occurrence and development of diseases. Compared with traditional tissue biopsy, it has the advantages of rapidity, convenience, less damage, and can be passed through multiple blood Sample collection eliminates the problem of tissue heterogeneity, improves the accuracy and convenience of HER2-positive gastric cancer detection, and many other advantages.
- ctDNA is a sensitive and specific biomarker with broad applicability, which can be used for clinical and research of various types of cancers.
- Professor Lu Yuming proved the technical and theoretical feasibility of liquid biopsy to replace 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 profile In 2017, Professor Xu Ruihua's team found that by detecting the methylation level of specific sites of circulating tumor DNA (ctDNA) in a small amount (4-5ml) of peripheral blood, accurate early diagnosis of liver cancer and prediction of curative effect and prognosis can be made. Recently, more and more clinical studies have shown that plasma ctDNA methylation markers can be used as biomarkers for early diagnosis and screening of tumors, prediction, response to treatment, and monitoring tumor size and recurrence. At present, the international research direction is to integrate multi-omics/multi-molecular markers, multi-gene/multi-locus to improve the sensitivity and specificity of detection technology to meet the clinical demand for detection products.
- One of the objectives of the present invention is to provide a plasma gene methylation marker and/or a combination thereof that can be used for the companion diagnosis of HER2 protein overexpression or gene amplification in gastric cancer.
- a methylation marker or a combination thereof that can be used for the accompanying diagnosis of HER2 protein overexpression or gene amplification in gastric cancer, the methylation marker or a combination thereof comprising any one or a combination of two or more selected from the following: chr17:37879893, chr17:37881021, chr17:37886390, chr17:37893765, chr17:37860353, chr17:37843592.
- Another object of the present invention is to provide the application of the above methylation markers or combinations thereof and/or detection-related reagents in the preparation of kits for detection, screening, prognosis monitoring, and companion diagnosis of gastric cancer HER2 protein overexpression or gene amplification .
- Another object of the present invention is to provide a kit for detection, screening, prognosis monitoring, and companion diagnosis of gastric cancer HER2 protein overexpression or gene amplification.
- the kit can be used for medication guidance for accompanying diagnosis of HER2 protein overexpression or gene amplification in gastric cancer.
- Another object of the present invention is to provide a detection method for accompanying diagnosis of gastric cancer HER2, comprising the following steps: obtaining a sample to be detected;
- the detection sample is subjected to the detection of the methylation marker or a combination thereof.
- the present invention obtains genomic fragments (methylation markers chr17:37879893, chr17:37881021, chr17:37886390, chr17:37893765, chr17:37860353) that are modified by methylation in gastric cancer HER2+ and HER2- malignant patients, especially in plasma ⁇ ( ⁇ chr17:37862986, chr17:37862178,chr17:37884314,chr17:37879819,chr17:37895887,chr17:37893999,chr17:37855528,chr3:38080799,chr17:37843863,chr17:37821433,chr17:37843592 , chr17:37856695), the methylation markers provided by the present invention, for tissue and plasma, especially the combination of plasma genome fragments can be used as HER2R protein overexpression or gene amplification medication-guided companion diagnostic diagnostic markers, there are Companion
- the present invention further provides a plurality of combined markers, such as chr17:37843592, chr17:37879893 and chr17:37860353, chr17:37843592, chr17:37879893 and chr17:37860353 and chr17:37843592, chr17:37862178 for use in Detection, screening, detection, prognosis monitoring and medication guidance of patients with IHC 2+GC.
- Example 1 is a heat map of biomarkers screened by methylation level difference analysis between HER2+ gastric cancer tissue and HER2- gastric cancer tissue in Example 3.
- Figure 2 is the performance of all methylation marker models in Example 3 on the test set and validation set in tissue samples.
- Figure 3 shows the performance of the 10 methylation marker models in Example 4 on the test set and the validation set in tissue samples.
- Figure 4 is the performance of all methylation marker models in Example 6 in the plasma independent validation set.
- Figure 5 is the performance of the five methylation marker models in Example 6 in the plasma independent validation set.
- Figure 6 is the performance of the three methylation marker models in Example 6 in the plasma independent validation set.
- FIG. 7 is the performance of the two methylation marker models in Example 6 in the independent validation set of plasma.
- Figure 8 is the performance of the three combined methylation marker models in Example 7 in the plasma independent validation set.
- complementarity refers to a nucleotide (eg, 1 nucleotide) or a polynucleotide (eg, a sequence of nucleotides) related to the base pairing rules.
- sequence 5'-A-G-T-3' is complementary to the sequence 3'-T-C-A-5'.
- Complementarity can be “partial” in which only some nucleic acid bases are matched according to base pairing rules. Alternatively, there may be “complete” or “total” complementarity between nucleic acids. The degree of complementarity between nucleic acid strands affects the efficiency and strength of hybridization between nucleic acid strands. This is especially important in amplification reactions and detection methods that rely on binding between nucleic acids.
- polymerase chain reaction is used to amplify target sequences, and the method consists of the steps of introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired target sequence, followed by the presence of DNA polymerase The precise thermal cycling sequence is performed below. Both primers are complementary to the corresponding strands of the double-stranded target sequence. For amplification, the mixture is denatured and the primers are then annealed to their complementary sequences within the target molecule. After annealing, the primers are amplified with a polymerase to form a new pair of complementary strands.
- the steps of denaturation, primer annealing, and polymerase extension can be repeated multiple times (ie, denaturation, annealing, and extension constitute one "cycle”; there can be many "cycles") to obtain high concentrations of amplified fragments of the desired target sequence.
- the length of the amplified fragment of the desired target sequence is determined by the relative position of the primers with respect to each other, so this length is a controllable parameter. Due to the repetitive aspect of the method, the method is referred to as the "polymerase chain reaction" ("PCR"). Since the desired amplified fragment of the target sequence becomes the predominant sequence (in terms of concentration) in the mixture, it is said to be “PCR amplified” and is the "PCR product" or "amplicon”.
- 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.
- amplifiable nucleic acid refers to a nucleic acid that can be amplified by any amplification method. It is expected that the "amplifiable nucleic acid” will generally comprise a "sample template”.
- sample template refers to a nucleic acid derived from a sample for analysis of the presence of a "target” (defined below).
- methylation refers to the methylation of cytosine at the C5 or N4 position of cytosine, the N6 position of adenine, or other types of nucleic acid methylation.
- In vitro amplified DNA is usually unmethylated, as typically in vitro DNA amplification methods do not preserve the methylation pattern of the amplified template.
- unmethylated DNA or “methylated DNA” can also refer to amplified DNA that is unmethylated or methylated, respectively, from the original template.
- a "methylated nucleotide” or “methylated nucleotide base” refers to the presence of a methyl moiety on a nucleotide base, where the methyl moiety is not present in a typical putative 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 and 5-methylcytosine is a methylated nucleotide.
- thymine contains a methyl moiety at the 5-position of its pyrimidine ring; however, for purposes herein, thymine is not considered a methylated nucleotide when present in DNA because thymine is DNA typical nucleotide bases.
- Methylation status can optionally be represented or indicated by a "methylation value” (eg, representing methylation frequency, fraction, ratio, percentage, etc.).
- Methylation values can, for example, quantify the amount of intact nucleic acid present after restriction digestion with methylation-dependent restriction enzymes, or by comparing amplification profiles after bisulfite reaction, or by comparing bisulfite treatment and generated from untreated nucleic acid sequences.
- values such as methylation values represent methylation status and thus can be used as quantitative indicators 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 methylated is defined as co-methylation.
- bisulfite reagent refers to a reagent comprising, in some embodiments, bisulfite, disulfite, hydrogen sulfite, or a combination thereof.
- DNA treated with bisulfite reagent its unmethylated cytosine nucleotides will be converted to uracil, while methylated cytosine and other bases remain unchanged, so that for example CpG binuclear can be distinguished.
- methylation assay or “methylation level detection” refers to any assay used to determine the methylation status of one or more CpG dinucleotide sequences within a nucleic acid sequence.
- the CpG site represents a dinucleotide pair
- the base guanine (G) follows cytosine (C)
- CpG is an abbreviation for cytosine (C)-phosphate (p)-guanine (G) .
- a HER2+ patient refers to a patient with an IHC result of 3+ and a pathology report as HER2-positive;
- a HER2-patient refers to a patient with an IHC result of 0 or 1+ and a pathology report as HER2-negative.
- a "marker” refers to a condition that can be used to indicate the expression of HER2 in a subject.
- markers can be nucleic acid sequences, macromolecules, small molecules, etc., for example, nucleic acid sequences of a certain length, or nucleotides at one specific site or nucleotides at two specific sites.
- the marker provided by the present invention refers to a CpG site of a patient that can be used to detect, predict or diagnose whether a subject is a gastric cancer patient with HER2.
- gene amplification refers to a selective increase in the copy number of a specific gene (eg, HER2) while the copy number of other genes is not proportionally increased.
- the present invention detects the expression variation of HER2 gene by measuring methylation markers through methylation detection technology, and discloses the combination and application of plasma gene methylation markers for HER2 accompanying diagnosis of gastric cancer. Specifically, the study obtained genomic fragments with significantly abnormal methylation modifications in the plasma of gastric cancer HER2+ and HER2- malignant patients, namely (the site is in the genome position, and Hg19 is the reference genome)
- a methylation marker or a combination thereof that can be used for the companion diagnosis of HER2 protein overexpression or gene amplification in gastric cancer, the methylation marker or a combination thereof comprising a group selected from the following Any one or a combination of two or more: chr17:37879893, chr17:37881021, chr17:37886390, chr17:37893765, chr17:37860353, chr17:37843592.
- the methylation marker comprises: chr17:37879893.
- the methylation marker or combination thereof comprises chr17:37879893 and chr17:37860353.
- the methylation marker or a combination thereof further comprises at least one selected from chr17:37862986, chr17:37862178, chr17:37884314, chr17:37879819, chr17:37895887, chr17:37893999 , chr17:37855528, chr3:38080799, chr17:37843863, chr17:37821433, chr17:37843592, chr17:37856695, chr17:37926322.
- the combination of methylation markers includes chr17:37879893, chr17:37886390 and chr17:37893999.
- the combination of methylation markers includes chr17:37879893, chr17:37886390, chr17:37893765, chr17:37862178, and chr17:37893999.
- the methylation marker or a combination thereof further comprises at least one selected from the group consisting of: chr8:41309473, chr17:37843265, chr17:37843818, chr17:37843793, chr17:37886349, chr17: 37886390,chr17:37820452,chr17:37820496,chr17:37968912,chr17:37968904,chr17:37893804,chr17:37894352,chr17:37893005,chr17:37894334,chr17:37893813,chr17:37894439,chr17:37894258,chr17:37894288, chr17:37886321,chr17:37926309,chr17:37894165,chr17:37894469,chr17:37873266,chr17:37894233,chr17:
- the methylation marker combination comprises chr17:37879893, chr17:37862178, chr17:37862986, chr17:37894352, chr17:37820452, chr17:37893765, chr17:37886349, chr17:37856695, chr17:37856695, 37843793, chr17:37886390.
- the methylation marker combination comprises chr17:37843592, chr17:37879893, and chr17:37860353.
- the methylation marker combination comprises chr17:37843592, chr17:37893999, and chr17:37860353.
- the methylation marker combination comprises chr17:37843592, chr17:37862178.
- the above-mentioned combination of methylation markers and/or related reagents for detection thereof can be used in the preparation of a kit for detecting IHC 2+ gastric cancer.
- the methylation marker combination comprises chr17:37879893, chr17:37881021, chr17:37886390, chr17:37893765, chr17:37860353, chr17:37862986, chr17:37862178, chr17:37884314, chr17:37884314, 37879819,chr17:37895887,chr17:37893999,chr17:37855528,chr8:41309473,chr17:37843592,chr17:37843863,chr17:37843265,chr17:37843818,chr17:37843793,chr17:37886349,chr17:37886390,chr17:37820452, chr17:37820496,chr17:37968912,chr17:37968904,chr17:37893804,chr1717:37879893
- methylation markers or combinations thereof and/or detection-related reagents can be used in the preparation of kits for detection, screening, prognosis monitoring, and companion diagnosis of gastric cancer overexpression of HER2 protein or gene amplification.
- kits for detection, screening, prognosis monitoring, and companion diagnosis of gastric cancer HER2 protein overexpression or gene amplification In some embodiments of the present invention, it relates to a kit for detection, screening, prognosis monitoring, and companion diagnosis of gastric cancer HER2 protein overexpression or gene amplification.
- the kit can be used for medication guidance for accompanying diagnosis of HER2 protein overexpression or gene amplification in gastric cancer.
- kits comprise reagents for detecting, screening, prognostic monitoring, companion diagnostics, methylation levels of the aforementioned methylation markers, or a combination thereof.
- HM450K Illumina's Infinium HumanMethylation450BeadChip
- HM450K Infinium CytoSNP- 850K BeadChip
- Affymetrix any custom designed array
- Sequencing methods may include, but are not limited to: Sanger sequencing, high-throughput sequencing, pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing by ligation, sequencing by hybridization, digital gene expression (Helicos), next-generation sequencing , Single Molecule Sequencing by Synthesis (SMSS) (Helicos), Massively Parallel Sequencing, Clonal Single Molecule Arrays (Solexa/Illumina), Shotgun Sequencing, Maxim Gilbert Sequencing, Primer Walking, Using PacBio, SOLiD, Ion Torrent or Nanopore Platforms Sequencing and any other sequencing method known in the art.
- the kit adopts real-time fluorescent quantitative PCRR or other types of PCR detection methods, including the following primers and probes:
- the sample detected by the kit is tissue or plasma or gastric juice, more preferably plasma.
- ctDNA Screening markers for the diagnosis of gastric cancer based on the methylation of circulating tumor DNA (ctDNA), including the following steps:
- Step 1 Perform methylation library building, targeted hybridization capture and next-generation sequencing in advanced gastric cancer tissue samples to initially screen potential methylation marker targets related to HER2 gene expression;
- Step 2 Design and establish a corresponding methylation-specific qPCR (real-time quantitative PCR) technology platform for these targets;
- Step 3 In gastric cancer plasma samples, screen and filter these targets, and find a marker set for distinguishing HER2 negative and positive;
- Step 4 Use the marker set selected in step 6 of the above procedure to further screen markers in the plasma training set and validation set, build an algorithm model, and conduct independent data set validation;
- Step 5 Confirm the gene methylation markers and algorithm models that are finally used for the diagnosis of advanced HER2 in gastric cancer.
- This embodiment discloses the above-mentioned NGS-based detection method for differential methylation markers (markers) for the accompanying diagnosis of HER2R protein overexpression or gene amplification in gastric cancer patients, which specifically includes the following steps: 1. Tissue sample processing
- tissue samples from patients with gastric cancer After clinical surgical resection of the tissue samples from patients with gastric cancer, they were fixed with 4% neutral formalin, dehydrated with gradient alcohol, cleared with xylene, immersed in wax, embedded in sections, and finally formed FFPE samples from patients with gastric cancer.
- tissue DNA extraction was carried out according to the instructions of Qiagen's AllPre DNA/RNA FFPE Kit.
- Target BP Peak
- target BP target BP
- the extracted tissue DNA is subjected to bisulfite conversion, so that the unmethylated cytosine in the DNA is deaminated into uracil, while the methylated cytosine remains unchanged to obtain sulfite
- the specific operation of the conversion is carried out according to the instructions of Zymo Research's EZ DNA Methylation-Lightning Kit.
- the present embodiment discloses the above-mentioned qPCR-based detection method for the differential methylation marker (marker) used for the HER2 companion diagnosis of gastric cancer patients, which specifically includes the following steps:
- EDTAK2 anticoagulant vacuum blood collection tube (BD, Cat#367525) to collect 10 mL of whole blood, mix thoroughly to avoid hemolysis, and perform plasma separation processing on whole blood within 4-6 hours. 4°C, 1600g, 15min centrifugation, carefully suck the upper plasma, avoid sucking the middle buffy coat, the obtained plasma is centrifuged again at 4°C, 16000g, 10min in a high-speed centrifuge to obtain the desired sample plasma.
- reaction system single dose ⁇ L 2*ChamQ Geno-SNP probe Master Mix 10 Primer F/R+probe (single marker) 2 DEPC H 2 O 2
- This example discloses a specific biomarker for HER2 companion diagnosis of gastric cancer, using tissue samples from 44 HER2+ gastric cancer patients and 30 HER2- gastric cancer patients, using the method described in Example 1 (NGS- BASED) for methylation analysis of tissue samples.
- Biomarkers related to HER2 expression were screened out using the methylation level differences in different groups, and the locus data were clustered in normal and gastric cancer samples. The results are shown in Figure 1.
- a total of 102 sites with the most significant abnormal methylation in HER2+ and HER2- gastric cancer samples were screened (FDR ⁇ 0.01).
- the red is the hypermethylated expression of the site
- the blue is the hypomethylated expression of the site.
- the 102 methylation biomarkers (hereinafter referred to as sites or markers) are:
- the random forest model is used for modeling analysis, and the split is 6:4, and 20 repetitions are performed to obtain a model validation set AUC of 0.95, as shown in Figure 2.
- an independent validation set namely 31 HER2+ gastric cancer patient tissue samples and 42 HER2- gastric cancer patient tissue samples were used to detect and judge , using the random forest model for modeling analysis, according to the 6:4 split, and repeating 20 times, the AUC of the model validation set is as high as 0.94, and the results are shown in Figure 3. It shows that the combination of 10 methylation markers can distinguish HER2+ and HER2- tissue samples well.
- the methylation level analysis of tissue samples was carried out by using the method described in Example 2.
- the independent plasma training set that is, the plasma samples of 7 HER2+ gastric cancer patients and the plasma samples of 20 HER2- gastric cancer patients were detected and judged.
- the AUC of locus discrimination can be as high as 0.981, and the AUC of 5 marker loci (chr17:37879893, chr17:37881021, chr17:37886390, chr17:37893765, chr17:37860353) can be greater than 0.8, see Table 2 for details.
- Example 5 Using all the markers in Example 5, the random forest model was used to model the plasma samples of 7 patients with HER2+ gastric cancer and the plasma samples of 20 patients with HER22- gastric cancer. According to the detection method described in Example 2, 3 patients with HER2+ were used for modeling. It was verified with 14 HER-independent plasma samples, and the judgment was completely correct, as shown in Figure 4.
- a model was constructed using a combination of 5 markers (chr17:37879893, chr17:37886390, chr17:37893765, chr17:37862178, and chr17:37893999), and the detection method described in Example 2 was performed in 3 cases of HER2+ and 14 cases of HER2-independent
- the plasma sample set was validated, and only one case of HER2- was misjudged, see Figure 5.
- the results please See Table 7.2 and Figure 8. It shows that the combination of methylation markers can well distinguish FISH+ and FISH- tissue specimens in IHC2+ subgroup.
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Abstract
提供了一种可用于胃癌HER2伴随诊断的甲基化标记物或其组合，所述甲基化标记物或其组合包括选自以下的任意一种或两种以上的组合：chr17:37879893、chr17:37881021、chr17:37886390、chr17:37893765、chr17:37860353、chr17：37843592。还提供了上述甲基化分子标记物或其组合的检测试剂盒，以及检测方法。
Description
本发明属于生物技术领域，具体涉及可用于胃癌HER2蛋白过表达或基因扩增伴随诊断的基因甲基化标记物或其组合和应用。
胃癌(gastriccarcinoma GC)是常见的恶性肿瘤之一,近年来虽然全球范围内胃癌发病率有所下降,但其依然是癌症相关死亡的第二大常见肿瘤,据估计每年有930000新发病例,年死亡人数超过700000例,其发病率存在较大的地域差异,与饮食、生活方式、经济状况以及幽门螺旋杆菌感染流行相关,超过2/3新发病例和死亡病例发生于发展中国家,其中东亚占42.4％(主要是中国)。
胃癌现有靶向治疗手段有限，其中HER2(人表皮生长因子受体2)是肿瘤靶向治疗中的关键靶点，HER2蛋白能够激活细胞生长与增殖相关信号传导通路，该蛋白的过度表达与乳腺癌、胃癌等肿瘤的发生有密切关系。随着精准治疗时代的到来，伴随诊断这一体外诊断技术日益受到广泛关注，该技术可用于检测生物标志物并与特定药物的使用相关联，是肿瘤精准诊治领域的重要一环。针对乳腺癌的HER2伴随诊断检测已广泛应用于乳腺癌患者的临床诊治之中，而胃癌由于病变组织具有较高的异质性且传统的组织活检、免疫组织化学染色法、原位杂交检测等方法对样本采集、样本量和处理要求较高，在多次取样对患者也会造成一定伤害，且在检测实践中的一些问题也不断显现，例如胃镜活检标本HER2检测未得到普及；原位杂交检测率低下，并由此导致大多数免疫组织化学染色(IHC)2+的胃癌病例未能最终明确HER2状态；部分单位HER2阳 性率与国内外文献报道差异较大等等，因此胃癌患者的HER2检测一直是困扰胃癌临床诊疗的难题。
液态活检可通过体外无创抽血的方式获取肿瘤脱落在血液中循环的ctDNA等，以此判断肿瘤的基因突变情况。因此通过液态活检代替组织活检对胃癌患者的HER2基因拷贝数进行检测，将有效缓解胃癌患者面临的组织采样伤害，并可通过多次血液样本采集排除组织异质性问题，提高HER2阳性胃癌检测的准确率与便利性。
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、并可通过多次血液样本采集排除组织异质性问题，提高HER2阳性胃癌检测的准确率与便利性等众多优点。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。2017年徐瑞华教授团队发现，通过对少量(4～5ml)外周血中循环肿瘤DNA(ctDNA)特定位点甲基化水平的检测，可以对肝癌进行准确的早期诊断及疗效和预后预测。最新有越来越多的临床研究表明，血浆ctDNA甲基化标志物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
本发明的目的之一是提供一种可用于胃癌HER2蛋白过表达或基因扩增伴随诊断的血浆基因甲基化标记物和/或其组合。
实现上述目的的技术方案如下。
一种可用于胃癌HER2蛋白过表达或基因扩增伴随诊断的甲基化标记物或其组合，所述甲基化标记物或其组合包括选自以下的任意一种或两种以上的组合：chr 17:37879893、chr17:37881021、chr17:37886390、chr17:37893765、chr17:37860353、chr17:37843592。
本发明的另一目的是提供上述甲基化标记物或其组合和/或检测相关试剂在制备检测、筛查、预后监测、伴随诊断胃癌HER2蛋白过表达或基因扩增的试剂盒中的应用。
本发明的另一目的是提供用于检测、筛查、预后监测、伴随诊断胃癌HER2蛋白过表达或基因扩增的试剂盒。
所述试剂盒可用于伴随诊断胃癌HER2蛋白过表达或基因扩增的用药指导。
本发明的另一目的是提供一种伴随诊断胃癌HER2的检测方法，包括以下步骤：获得待检测样本；
对检测样本进行所述甲基化标记物或其组合的检测。
本发明获得了在胃癌HER2+及HER2-恶性患者甲基化修饰的基因组片段(甲基化标记物chr17:37879893、chr17:37881021、chr17:37886390、chr17:37893765、chr17:37860353)，特别是在血浆中也明显异常(还包括chr17:37862986， chr17:37862178，chr17:37884314，chr17:37879819，chr17:37895887，chr17:37893999，chr17:37855528，chr3:38080799，chr17:37843863，chr17:37821433，chr17:37843592，chr17:37856695)，本发明所提供的甲基化标记物，对于组织和血浆，特别是血浆基因组片段的组合能够作为HER2R蛋白过表达或基因扩增的用药指导的伴随诊断诊断标记物，有利于胃癌的HER2的伴随诊断、无创检测、筛查以及检测预后监测，特别是指导用药。而且，进一步地，本发明还提供了多个组合的marker，例如chr17:37843592、chr17:37879893和chr17:37860353、chr17:37843592、chr17:37879893和chr17:37860353以及chr17:37843592、chr17:37862178用于IHC 2+GC患者的检测、筛查以及检测预后监测和指导用药。
图1是实施例3中HER2+胃癌组织与HER2-胃癌组织通过甲基化水平差异分析筛选出的生物标记物热图。
图2是实施例3中所有甲基化标志物模型在组织样本中测试集和验证集上的表现。
图3是实施例4中10个甲基化标志物模型在组织样本中测试集和验证集上的表现。
图4是实施例6中所有甲基化标志物模型在血浆独立验证集里的表现。
图5是实施例6中5个甲基化标志物模型在血浆独立验证集里的表现。
图6是实施例6中3个甲基化标志物模型在血浆独立验证集里的表现。
图7是实施例6中2个甲基化标志物模型在血浆独立验证集里的表现。
图8是实施例7中三个组合的甲基化标志物模型在血浆独立验证集里的表现。
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。
在整个说明书和权利要求书中，以下术语具有与本文明确相关的含义，除非上下文另有明确规定。在本发明中使用的短语“在一个实施方案中”不一定指代相同的实施方案，尽管其可能是。此外，在本发明中使用的短语“在另一实施方案中”不一定指代不同的实施方案，尽管其可能是。因此，如下所述，可以容易地组合本发明的各个实施方案，而不脱离本发明的范围或精神。
此外，如本发明所使用的，术语“或”是包含性的“或”符号，并且等同于术语“和/或”，除非上下文另有明确规定。术语“基于”不是排他性的，并且允许基于未描述的其他因素，除非上下文另有明确规定。此外，在整个说明书中，“一个”、“一种”和“所述/该”的含义包括复数指示物。“在......中”中的含义包括“在......中”和“在......上”。
术语“互补”和“互补性”是指与碱基配对规则相关的核苷酸(例如，1个核苷酸)或多核苷酸(例如核苷酸的序列)。例如，序列5′-A-G-T-3′与序列3′-T-C-A-5′互补。互补可以是“部分的”，其中仅一些核酸碱基根据碱基配对规则进行匹配。或者，核酸之间可能存在“完全”或“总”互补。核酸链之间的互补程度影响核酸链之间杂交的效率和强度。这在扩增反应和依赖核酸之间的结合的检测方法中尤其重要。
术语“聚合酶链式反应”用于扩增靶序列,该方法由以下步骤组成：将大量过量的两种寡核苷酸引物引入到含有期望靶序列的DNA混合物中，随后在DNA聚合酶存在下进行精确的热循环顺序。两种引物与双链靶序列的相应链互补。为了进行扩增，将混合物变性，然后引物与靶分子内的其互补序列退火。退火后，用聚合酶扩增引物，形成一对新的互补链。变性、引物退火和聚合酶延伸的步骤可以重复多次(即，变性、退火和延伸构成一个“循环”；可以有许多“循环”)以获得高浓度的期望靶序列的扩增片段。期望靶序列的扩增片段的长度由引物相对于彼此的相对位置确定，因此该长度是可控参数。由于该方法的重复方面，该方法被称为“聚合酶链式反应”(“PCR”)。由于靶序列的期望扩增片段成为混合物中的主要序列(以浓度计)，所以称其被“PCR扩增”，是“PCR产物”或“扩增子”。
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“可扩增核酸”是指可以通过任何扩增方法扩增的核酸。预期“可扩增核酸”通常将包含“样品模板”。
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。
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。
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。
术语“甲基化测定”或“甲基化水平检测”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。
本发明中，CpG位点表示二核苷酸对，碱基鸟嘌呤(G)紧随胞嘧啶(C)之后，CpG是胞嘧啶(C)-磷酸(p)-鸟嘌呤(G)的缩写。
本发明中，HER2+患者指IHC结果为3+且病理报告为HER2阳性的患者；HER2-患者指IHC结果为0或1+且病理报告为HER2阴性的患者。
本发明中，“标记物”是指能够用于指示受试者HER2表达情况。这些标记物可以是核酸序列、大分子、小分子等等，例如可以是一定长度的核酸序列，也可以是一个特定位点的核苷酸或者两个特定位点的核苷酸，。根据本发明的实施例，本发明提供的标记物指的是能够用于检测、预测或者诊断受试者是否胃癌患者HER2的患者的CpG位点。
本发明中，“基因扩增”指某一个特定基因(例如HER2)的拷贝数选择性地增加而其它基因的拷贝数并未按比例增加。
本发明通过甲基化检测技术来测量甲基化标记物来检测HER2基因的表达变异情况，公开了用于胃癌HER2伴随诊断的血浆基因甲基化标记物组合及应用。具体地，研究获得了在胃癌HER2+及HER2-恶性患者血浆中明显异常甲基化修饰的基因组片段，即(位点在基因组位置，Hg19为参考基因组)
chr17:37879893，chr17:37881021，chr17:37886390，chr17:37893765，
chr17:37860353，chr17:37862986，chr17:37862178，chr17:37884314，
chr17:37879819，chr17:37895887，chr17:37893999，chr17:37855528，
chr8:41309473。
本发明的一些实施例中，提供了一种可用于胃癌HER2蛋白过表达或基因扩增伴随诊断的甲基化标记物或其组合，所述甲基化标记物或其组合包括选自 以下的任意一种或两种以上的组合：chr 17:37879893、chr17:37881021、chr17:37886390、chr17:37893765、chr17:37860353、chr17:37843592。
通过研究这些血浆甲基化修饰的基因组片段在胃癌HER2+及HER2-恶性患者人群中的甲基化修饰差异，发现该血浆基因组片段组合能够作为HER2过表达或基因扩增伴随诊断诊断标记物。进一步的研究发现5个标志物，即chr17:37879893，chr17:37886390，chr17:37893765，chr17:37862178，chr17:37893999具备与全部标志物组合基本相同的诊断力。
通过研究这些血浆甲基化修饰的基因组片段在胃癌HER2+及HER2-恶性患者人群中的甲基化修饰差异，发现该血浆基因组片段组合能够作为HER2过表达或基因扩增伴随诊断诊断标记物。进一步的研究发现前2个标志物，即chr17:37879893，chr17:37860353诊断力AUC可达0.97以上。
在其中一些实施例中，所述甲基化标记物包括有：chr17:37879893。
在其中一些实施例中，所述甲基化标记物或其组合包括有chr17:37879893和chr17:37860353。
在其中一些实施例中，所述甲基化标记物或其组合还包括选自以中的至少一种chr17:37862986，chr17:37862178，chr17:37884314，chr17:37879819，chr17:37895887，chr17:37893999，chr17:37855528，chr3:38080799，chr17:37843863，chr17:37821433，chr17:37843592，chr17:37856695，chr17:37926322。
在其中一些实施例中，所述甲基化标记物组合包括chr17:37879893，chr17:37886390和chr17:37893999。
在其中一些实施例中，所述甲基化标记物组合包括chr17:37879893，chr17:37886390，chr17:37893765，chr17:37862178，和chr17:37893999。
在其中一些实施例中，所述甲基化标记物或其组合还包括选自以中的至少一 种：chr8:41309473，chr17:37843265，chr17:37843818，chr17:37843793，chr17:37886349，chr17:37886390，chr17:37820452，chr17:37820496，chr17:37968912，chr17:37968904，chr17:37893804，chr17:37894352，chr17:37893005，chr17:37894334，chr17:37893813，chr17:37894439，chr17:37894258，chr17:37894288，chr17:37886321，chr17:37926309，chr17:37894165，chr17:37894469，chr17:37873266，chr17:37894233，chr17:37894252，chr18:19745506，chr17:37894413，chr17:37785459，chr17:37894403，chr17:37785442，chr17:37895880，chr18:19745476，chr18:19745494，chr17:37896175，chr17:37873304，chr17:37894485，chr18:19745514，chr17:37785429，chr17:37873326，chr18:19745472，chr17:37894482，chr18:19745487，chr17:37896050，chr18:19745442，chr17:37896074，chr17:37785426，chr17:37894347，chr18:19745458，chr3:38080799，chr2:27938352，chr17:37895919，chr18:19745509，chr17:37894397，chr2:27938360，chr17:37896203，chr17:37926366，chr18:19745504，chr19:36232197，chr17:37896165，chr17:37896063，chr1:26551710，chr1:26551791，chr6:167276600，chr1:26551681，chr1:26551782，chr17:38024048，chr18:19745530，chr18:19745445，chr1:28919250，chr17:37820516，chr19:50832069，chr2:157178147，chr2:157178249，chr2:27938342，chr6:167276599，chr1:28919276，chr17:37784733，chr6:167276619，chr2:157178298，chr6:43211253，chr1:46632751，chr1:26551788，chr11:94473568，chr1:46632737，chr19:50832047。
在其中一些实施例中，所述甲基化标记物组合包括chr17:37879893，chr17:37862178，chr17:37862986，chr17:37894352，chr17:37820452， chr17:37893765，chr17:37886349，chr17:37856695，chr17:37843793，chr17:37886390。
在其中一些实施例中，所述甲基化标记物组合为包括chr17:37843592、chr17:37879893和chr17:37860353。
在其中一些实施例中，所述甲基化标记物组合为包括chr17:37843592、chr17:37893999和chr17:37860353。
在其中一些实施例中，所述甲基化标记物组合为包括chr17:37843592、chr17:37862178。
上述三种组合，对HER2蛋白过表达或基因扩增的胃癌中IHC 2+的患者具有很好的检测效果。
上述甲基化标记物组合和/或检测其相关试剂可用于制备检测IHC 2+胃癌的试剂盒中。
在其中一些实施例中，所述甲基化标记物组合包括chr17:37879893，chr17:37881021，chr17:37886390，chr17:37893765，chr17:37860353，chr17:37862986，chr17:37862178，chr17:37884314，chr17:37879819，chr17:37895887，chr17:37893999，chr17:37855528，chr8:41309473，chr17:37843592，chr17:37843863，chr17:37843265，chr17:37843818，chr17:37843793，chr17:37886349，chr17:37886390，chr17:37820452，chr17:37820496，chr17:37968912，chr17:37968904，chr17:37893804，chr17:37894352，chr17:37893005，chr17:37894334，chr17:37893813，chr17:37894439，chr17:37894258，chr17:37894288，chr17:37886321，chr17:37926309，chr17:37894165，chr17:37856695，chr17:37894469，chr17:37873266，chr17:37894233，chr17:37894252，chr18:19745506， chr17:37894413，chr17:37785459，chr17:37894403，chr17:37785442，chr17:37895880，chr18:19745476，chr17:37821433，chr18:19745494，chr17:37896175，chr17:37873304，chr17:37894485，chr18:19745514，chr17:37785429，chr17:37873326，chr18:19745472，chr17:37894482，chr18:19745487，chr17:37896050，chr18:19745442，chr17:37896074，chr17:37785426，chr17:37894347，chr18:19745458，chr3:38080799，chr2:27938352，chr17:37895919，chr18:19745509，chr17:37894397，chr2:27938360，chr17:37896203，chr17:37926366，chr18:19745504，chr19:36232197，chr17:37896165，chr17:37896063，chr1:26551710，chr1:26551791，chr17:37926322，chr6:167276600，chr1:26551681，chr1:26551782，chr17:38024048，chr18:19745530，chr18:19745445，chr1:28919250，chr17:37820516，chr19:50832069，chr2:157178147，chr2:157178249，chr2:27938342，chr6:167276599，chr1:28919276，chr17:37784733，chr6:167276619，chr2:157178298，chr6:43211253，chr1:46632751，chr1:26551788，chr11:94473568，chr1:46632737，和chr19:50832047。
上述甲基化标记物或其组合和/或检测相关试剂可以应用在制备检测、筛查、预后监测、伴随诊断胃癌HER2蛋白过表达或基因扩增的试剂盒中。
本发明一些实施例中，涉及用于检测、筛查、预后监测、伴随诊断胃癌HER2蛋白过表达或基因扩增的试剂盒。
所述试剂盒可用于伴随诊断胃癌HER2蛋白过表达或基因扩增的用药指导。
在其中一些实施例中，所述试剂盒包含检测、筛查、预后监测、伴随诊断上述的甲基化标记物或其组合的甲基化水平的试剂。
在其中一些实施例中，在检测甲基化标志物时候，可采用本领域已知各种方法，包括但不限于：1)微阵列杂交技术，例如Illumina的Infinium HumanMethylation450BeadChip(HM450K)、Infinium CytoSNP-850K BeadChip或任何定制设计的阵列(Affymetrix)等；2)结合亚硫酸氢盐处理的基于测序的方法分析5-甲基胞嘧啶分布。测序方法可包括但不限于：Sanger测序、高通量测序、焦磷酸测序、合成测序、单分子测序、纳米孔测序、半导体测序、连接测序、杂交测序、数字基因表达(Helicos)、二代测序，单分子合成测序(SMSS)(Helicos)、大规模平行测序、克隆单分子阵列(Solexa/Illumina)、鸟枪测序、Maxim Gilbert测序、引物步移、使用PacBio、SOLiD、离子Torrent或纳米孔平台的测序以及本领域已知的任何其他测序方法。
在其中一些实施例中，所述试剂盒采用实时荧光定量PCRR或其他类型PCR检测方法，包括有以下引物和探针：
针对chr17:37879893的SEQ ID NO.1-SEQ ID NO.3，和/或
针对chr17:37881021的SEQ ID NO.4-SEQ ID NO.6，和/或
针对chr17:37886390的SEQ ID NO.7-SEQ ID NO.9，和/或
针对chr17:37893765的SEQ ID NO.10-SEQ ID NO.12，和/或
针对chr17:37860353的SEQ ID NO.13-SEQ ID NO.15，和/或
针对chr17:37862986的SEQ ID NO.16-SEQ ID NO.18，和/或
针对chr17:37862178的SEQ ID NO.19-SEQ ID NO.21，和/或
针对chr17:37884314的SEQ ID NO.22-SEQ ID NO.24，和/或
针对chr17:37879819的SEQ ID NO.25-SEQ ID NO.27，和/或
针对chr17:37895887的SEQ ID NO.28-SEQ ID NO.30，和/或
针对chr17:37893999的SEQ ID NO.31-SEQ ID NO.33，和/或
针对chr17:37855528的SEQ ID NO.34-SEQ ID NO.36，和/或
针对chr3:38080799的SEQ ID NO.37-SEQ ID NO.39，和/或
针对chr17:37843863的SEQ ID NO.40-SEQ ID NO.42，和/或
针对chr17:37821433的SEQ ID NO.43-SEQ ID NO.45，和/或
针对chr17:37843592的SEQ ID NO.46-SEQ ID NO.48，和/或
针对chr17:37856695的SEQ ID NO.49-SEQ ID NO.51；和/或
针对chr17:37926322的SEQ ID NO.52-SEQ ID NO.54。
在其中一些实施例中，所述试剂盒检测的样本为组织或者血浆或胃液，更优选为血浆。
基于循环肿瘤DNA(circulating tumor DNA,ctDNA)甲基化筛选胃癌诊断的标记物，包括如下步骤：
步骤一、在胃癌晚期组织样本中进行甲基化建库、靶向杂交捕获及二代测序，来初筛与HER2基因表达相关的潜在甲基化标志物靶点；
步骤二、针对这些靶点，设计并建立对应的甲基化特异性qPCR(实时荧光定量PCR)技术平台；
步骤三、在胃癌血浆样本中，对这些靶点进行筛选、过滤、并找到用于区分HER2阴性及阳性的标记物集；
步骤四、利用上术步骤六中选定的标记物集在血浆训练集和验证集中进一步筛选markers，构建算法模型，并进行独立数据集的验证；
步骤五、确认最终用于胃癌晚期HER2诊断的基因甲基化标记物及算法模型。
实施例1
本实施例公开了上述用于胃癌患者HER2R蛋白过表达或基因扩增伴随诊断差异性甲基化标记物(marker)基于NGS的检测方法，具体包括以下步骤： 1.组织样本处理
临床手术切除结胃癌患者组织样本后，采用4％的中性***固定，经过梯度酒精脱水，二甲苯透明，浸蜡，包埋切片，最终形成胃癌患者组织FFPE样本。
2.组织DNA的提取。
组织DNA提取具体操作步骤按照Qiagen公司的AllPre DNA/RNA FFPE Kit说明书进行。
3.将提取后的组织DNA进行打断，具体操作如下：
3.1取组织DNA，用EB补足体积至130ul，转移至打断管中于Covaris M220按照以下程序进行打断：
Target BP(Peak)(目的BP)200
Peak Incident Power(峰值入射功率)(W)50
Duty Factor(工作因子)20％
Cycles per Burst(脉冲周期数)200
Treatment Time(处理时间)(s)150
Temperature(温度℃)20
Sample volume(样品体积ul)130
3.2将打断完的组织DNA转移至1.5ml离心管中，用Nanodrop 2000测定打断后浓度。
4.将提取的打断后的组织DNA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。
5.对转化后的DNA进行建库，具体的操作步骤按照AnchorDx的AnchorDx EpiVisioTM Methylation Library Prep Kit(AnchorDx,Cat#A0UX00019)以及AnchorDx EpiVisioTM Indexing PCR Kit(AnchorDx,Cat#A2DX00025)说明书进行。得到测序预文库。
6.对测序预文库进行特定区域捕获，具体的操作步骤参考AnchorDx PanMet V2–Pan-cancer methylation panel(AnchorDx,Cat#A0UX00023)的说明进行，形成测序终文库。
7.采用Illumina公司的测序仪对测序终文库进行测序，得到测序结果。
8.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***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。
实施例2
本实施例公开了上述用于胃癌患者HER2伴随诊断差异性甲基化标记物(marker)基于qPCR的检测方法，具体包括以下步骤：
1.全血处理与血浆cfDNA提取
1.1利用EDTAK2抗凝真空采血管(BD，Cat#367525)采集10mL全血，充分混匀，避免出现溶血，并在4-6小时内对全血进行血浆分离处理，全血于低速离心机进行4℃，1600g，15min离心处理，小心吸取上层血浆，避免吸取中间的白膜层，所得血浆再次于高速离心机进行4℃，16000g，10min离心处理，获 得所需样品血浆。
1.2血浆cfDNA提取具体操作步骤按照Thermo Fisher公司的MagMAX TM Cell-Free DNA Isolation Kit说明书进行。
2.进行甲基化特异性qPCR，取10ng cfDNA产物，加入18μL以下试剂进行反应，每个marker pool需要3次平行反应。
| 反应体系 | 单个用量μL |
| 2*ChamQ Geno-SNP probe Master Mix | 10 |
| 引物F/R+探针(单个Marker) | 2 |
| DEPC H 2O | 2 |
3置于qPCR仪中按照以下程序进行反应：
以下为17种甲基化生物标记物的引物和探针组成：
实施例3
本实施例公开了一种用于胃癌HER2伴随诊断的特异性生物标记物，利用44例HER2+的胃癌病人组织样本以及30例HER2-胃癌病人的组织样本，利用实施例1所述方法(NGS-BASED)进行组织样本甲基化水平分析。利用在不同组别的甲基化水平差异筛选出与HER2表达相关的生物标记物，并对位点数据在正常与胃癌样本中进行聚类分析，结果请见图1。从图1可以看到，一共筛 选出102个在HER2+与HER2-胃癌样本存在最显著异常甲基化的位点(FDR<0.01)。图中，红色为位点高甲基化表达，蓝色为位点低甲基化表达。该102个甲基化生物标记物(以下简称位点或者marker)为：
chr17:37879893，chr17:37881021，chr17:37886390，chr17:37893765，chr17:37860353，chr17:37862986，chr17:37862178，chr17:37884314，chr17:37879819，chr17:37895887，chr17:37893999，chr17:37855528，chr8:41309473，chr17:37843592，chr17:37843863，chr17:37843265，chr17:37843818，chr17:37843793，chr17:37886349，chr17:37886390，chr17:37820452，chr17:37820496，chr17:37968912，chr17:37968904，chr17:37893804，chr17:37894352，chr17:37893005，chr17:37894334，chr17:37893813，chr17:37894439，chr17:37894258，chr17:37894288，chr17:37886321，chr17:37926309，chr17:37894165，chr17:37856695，chr17:37894469，chr17:37873266，chr17:37894233，chr17:37894252，chr18:19745506，chr17:37894413，chr17:37785459，chr17:37894403，chr17:37785442，chr17:37895880，chr18:19745476，chr17:37821433，chr18:19745494，chr17:37896175，chr17:37873304，chr17:37894485，chr18:19745514，chr17:37785429，chr17:37873326，chr18:19745472，chr17:37894482，chr18:19745487，chr17:37896050，chr18:19745442，chr17:37896074，chr17:37785426，chr17:37894347，chr18:19745458，chr3:38080799，chr2:27938352，chr17:37895919，chr18:19745509，chr17:37894397，chr2:27938360，chr17:37896203，chr17:37926366，chr18:19745504，chr19:36232197，chr17:37896165，chr17:37896063，chr1:26551710，chr1:26551791，chr17:37926322，chr6:167276600， chr1:26551681，chr1:26551782，chr17:38024048，chr18:19745530，chr18:19745445，chr1:28919250，chr17:37820516，chr19:50832069，chr2:157178147，chr2:157178249，chr2:27938342，chr6:167276599，chr1:28919276，chr17:37784733，chr6:167276619，chr2:157178298，chr6:43211253，chr1:46632751，chr1:26551788，chr11:94473568，chr1:46632737，chr19:50832047。
使用全部marker组合，利用随机森林模型进行建模分析，按照6：4的切分，进行20次重复，得到模型验证集AUC可达0.95，见图2。
实施例4
使用以下的10个位点的信息(表4.1，位点来自于实施例3)，对独立的验证集，即31例HER2+的胃癌病人组织样本以及42例HER2-胃癌病人的组织样本进行检测判断，利用随机森林模型进行建模分析，按照6：4的切分，进行20次重复，得到模型验证集AUC高达0.94，结果请参见图3。说明该10个甲基化标志物组合可以很好区分HER2+与HER2-的组织标本。
表4.1胃癌组织中与HER2状态相关的甲基化生物标志物模型因子
实施例5
利用实施例2所述方法进行组织样本甲基化水平分析对独立的血浆训练集，即在7例HER2+的胃癌病人血浆样本以及20例HER2-胃癌病人的血浆样本进行检测判断，单独每一个标志物位点区分AUC可以高达0.981，5个标志物位点(chr17:37879893、chr17:37881021、chr17:37886390、chr17:37893765、chr17:37860353)可AUC大于0.8，详见表2。
表2血浆HER2状态相关的甲基化生物标志物的筛选，其中AUC>0.6的标志物如下：
实施例6
使用实施例5里面全部的标志物，通过利用随机森林模型在7例HER2+的胃癌病人血浆样本以及20例HER22-胃癌病人的血浆样本进行建模，按照实施例2所述检测方法在3例HER2+和14例HER-独立血浆样本集进行验证，判断结完全正确，见图4。
使用5个标志物(chr17:37879893，chr17:37886390，chr17:37893765，chr17:37862178，和chr17:37893999)的组合构建模型，按照实施例2所述检测方法在3例HER2+和14例HER2-独立血浆样本集进行验证，仅错判一例HER2-，见图5。
按照实施例2所述检测方法，使用3个标志物(chr17:37879893，chr17:37886390和chr17:37893999)的组合构建模型，在3例HER2+和14例HER2-独立血浆样本集进行验证，仅错判一例HER2-，见图6。
按照实施例2所述检测方法，使用2个标志物(chr17:37879893，chr17:37860353)的组合构建模型，在3例HER2+和14例HER2-独立血浆样本集进行验证，仅错判两例HER2-，见图7。
实施例7
使用以下的表7.1中的7个标志物位点的信息(参见实施例2)，利用实施例2的所述方法(qPCR检测方法)进行组织样本甲基化水平分析，对独立的胃癌IHC 2+样本集，即23例IHC2+/FISH+的胃癌病人组织样本以及22例IHC2+/FISH-胃癌病人的组织样本进行检测判断，采用逻辑回归模型对7个marker进行2-3marker组合举例(参见表7.2)，repeat times＝100，2-fold交叉验证，统计平均AUC，平均AUC＞0.9marker组合较多，有多个不同组合(例如 表7.2中的三个组合)都可达到最高AUC＝0.959，结果请参见表7.2及图8。说明甲基化标志物组合可以很好区分IHC2+亚群中FISH+与FISH-的组织标本。
表7.1
表7.2
以上实验结果表明，上述marker组合在IHC 2+的胃癌人群对FISH+vs FISH-的检测，也具有很好的区分效果。
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。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 (21)
- 一种可用于胃癌HER2蛋白过表达或基因扩增伴随诊断的甲基化标记物或其组合，其特征在于，所述甲基化标记物或其组合包括选自以下标记物中的任意一种或两种及以上的组合：chr17:37879893、chr17:37881021、chr17:37886390、chr17:37893765、chr17:37860353、chr17:37843592。
- 根据权利要求1所述的可用于胃癌HER2蛋白过表达或基因扩增伴随诊断的甲基化标记物或其组合，其特征在于，所述甲基化标记物或其组合包括chr17:37879893和/或chr17:37843592标记物。
- 根据权利要求2所述的可用于胃癌HER2蛋白过表达或基因扩增伴随诊断的甲基化标记物或其组合，其特征在于，所述甲基化标记物或其组合包括标记物chr17:37879893和chr17:37860353。
- 根据权利要求1所述的可用于胃癌HER2蛋白过表达或基因扩增伴随诊断的甲基化标记物或其组合，其特征在于，所述甲基化标记物或其组合还包括选自以下的至少一种：chr17:37862986，chr17:37862178，chr17:37884314，chr17:37879819，chr17:37895887，chr17:37893999，chr17:37855528，chr3:38080799，chr17:37843863，chr17:37821433，chr17:37856695，chr17:37926322标记物。
- 根据权利要求4所述的可用于胃癌HER2蛋白过表达或基因扩增伴随诊断的甲基化标记物或其组合，其特征在于，所述甲基化标记物组合包括chr17:37879893，chr17:37886390和chr17:37893999标记物。
- 根据权利要求4所述的可用于胃癌HER2蛋白过表达或基因扩增伴随诊断的基因甲基化标记物或其组合，其特征在于，所述甲基化标记物或其组合包括chr17:37879893，chr17:37886390，chr17:37893765，chr17:37862178，和chr17:37893999标记物。
- 根据权利要求2所述的可用于胃癌HER2蛋白过表达或基因扩增伴随诊断的 甲基化标记物或其组合，其特征在于，所述甲基化标记物组合包括chr17:37843592、chr17:37879893和chr17:37860353标记物。
- 根据权利要求2所述的可用于胃癌HER2蛋白过表达或基因扩增伴随诊断的甲基化标记物或其组合，其特征在于，所述甲基化标记物组合包括chr17:37843592、chr17:37893999和chr17:37860353标记物。
- 根据权利要求2所述的可用于胃癌HER2蛋白过表达或基因扩增伴随诊断的甲基化标记物或其组合，其特征在于，所述甲基化标记物组合包括chr17:37843592、chr17:37862178标记物。
- 根据权利要求1-8任一项所述的可用于胃癌HER2蛋白过表达或基因扩增伴随诊断的甲基化标记物或其组合，其特征在于，所述甲基化标记物或其组合还包括选自以下标记物中至少一种：chr8:41309473，chr17:37843265，chr17:37843818，chr17:37843793，chr17:37886349，chr17:37886390，chr17:37820452，chr17:37820496，chr17:37968912，chr17:37968904，chr17:37893804，chr17:37894352，chr17:37893005，chr17:37894334，chr17:37893813，chr17:37894439，chr17:37894258，chr17:37894288，chr17:37886321，chr17:37926309，chr17:37894165，chr17:37894469，chr17:37873266，chr17:37894233，chr17:37894252，chr18:19745506，chr17:37894413，chr17:37785459，chr17:37894403，chr17:37785442，chr17:37895880，chr18:19745476，chr18:19745494，chr17:37896175，chr17:37873304，chr17:37894485，chr18:19745514，chr17:37785429，chr17:37873326，chr18:19745472，chr17:37894482，chr18:19745487，chr17:37896050，chr18:19745442，chr17:37896074，chr17:37785426，chr17:37894347，chr18:19745458，chr3:38080799，chr2:27938352，chr17:37895919， chr18:19745509，chr17:37894397，chr2:27938360，chr17:37896203，chr17:37926366，chr18:19745504，chr19:36232197，chr17:37896165，chr17:37896063，chr1:26551710，chr1:26551791，chr6:167276600，chr1:26551681，chr1:26551782，chr17:38024048，chr18:19745530，chr18:19745445，chr1:28919250，chr17:37820516，chr19:50832069，chr2:157178147，chr2:157178249，chr2:27938342，chr6:167276599，chr1:28919276，chr17:37784733，chr6:167276619，chr2:157178298，chr6:43211253，chr1:46632751，chr1:26551788，chr11:94473568，chr1:46632737，chr19:50832047。
- 根据权利要求10所述的可用于胃癌HER2蛋白过表达或基因扩增伴随诊断的甲基化标记物或其组合，其特征在于，所述甲基化标记物组合包括chr17:37879893，chr17:37862178,chr17:37862986，chr17:37894352，chr17:37820452，chr17:37893765，chr17:37886349，chr17:37856695，chr17:37843793，chr17:37886390标记物。
- 根据权利要求10所述的可用于胃癌HER2蛋白过表达或基因扩增伴随诊断的甲基化标记物或其组合，其特征在于，所述甲基化标记物组合包括chr17:37879893，chr17:37881021，chr17:37886390，chr17:37893765，chr17:37860353，chr17:37862986，chr17:37862178，chr17:37884314，chr17:37879819，chr17:37895887，chr17:37893999，chr17:37855528，chr8:41309473，chr17:37843592，chr17:37843863，chr17:37843265，chr17:37843818，chr17:37843793，chr17:37886349，chr17:37886390，chr17:37820452，chr17:37820496，chr17:37968912，chr17:37968904，chr17:37893804，chr17:37894352，chr17:37893005，chr17:37894334，chr17:37893813，chr17:37894439，chr17:37894258，chr17:37894288，chr17:37886321， chr17:37926309，chr17:37894165，chr17:37856695，chr17:37894469，chr17:37873266，chr17:37894233，chr17:37894252，chr18:19745506，chr17:37894413，chr17:37785459，chr17:37894403，chr17:37785442，chr17:37895880，chr18:19745476，chr17:37821433，chr18:19745494，chr17:37896175，chr17:37873304，chr17:37894485，chr18:19745514，chr17:37785429，chr17:37873326，chr18:19745472，chr17:37894482，chr18:19745487，chr17:37896050，chr18:19745442，chr17:37896074，chr17:37785426，chr17:37894347，chr18:19745458，chr3:38080799，chr2:27938352，chr17:37895919，chr18:19745509，chr17:37894397，chr2:27938360，chr17:37896203，chr17:37926366，chr18:19745504，chr19:36232197，chr17:37896165，chr17:37896063，chr1:26551710，chr1:26551791，chr17:37926322，chr6:167276600，chr1:26551681，chr1:26551782，chr17:38024048，chr18:19745530，chr18:19745445，chr1:28919250，chr17:37820516，chr19:50832069，chr2:157178147，chr2:157178249，chr2:27938342，chr6:167276599，chr1:28919276，chr17:37784733，chr6:167276619，chr2:157178298，chr6:43211253，chr1:46632751，chr1:26551788，chr11:94473568，chr1:46632737，和chr19:50832047标记物。
- 权利要求1～12任一项所述甲基化标记物或其组合和/或检测其相关试剂在制备伴随诊断胃癌HER2蛋白过表达或基因扩增的试剂盒中的应用，或权利要求7-9任一项中所述甲基化标记物组合和/或检测其相关试剂在制备检测IHC2+胃癌的试剂盒中的应用。
- 一种用于伴随诊断胃癌HER2的试剂盒，其特征在于，所述试剂盒包含检测权利要求1～12任一项所述的甲基化标记物或其组合的甲基化水平的试剂。
- 根据权利要求14所述的用于伴随诊断胃癌HER2的试剂盒，其特征在于，所 述试剂盒包括采用微阵列杂交技术，结合亚硫酸氢盐处理的基于测序方法所使用的试剂，所述测序方法包括Sanger测序、高通量测序、焦磷酸测序、合成测序、单分子测序、纳米孔测序、半导体测序、连接测序、杂交测序、数字基因表达、二代测序，单分子合成测序、大规模平行测序、克隆单分子阵列、鸟枪测序、Maxim Gilbert测序、引物步移、使用PacBio、SOLiD、离子Torrent或纳米孔平台的测序或它们的组合。
- 根据权利要求14所述的用于伴随诊断胃癌HER2的试剂盒，其特征在于，采用甲基化特异实时荧光定量PCR或其他类型PCR的检测方法，包括有以下引物和探针：针对chr17:37879893的SEQ ID NO.1-SEQ ID NO.3，和/或针对chr17:37881021的SEQ ID NO.4-SEQ ID NO.6，和/或针对chr17:37886390的SEQ ID NO.7-SEQ ID NO.9，和/或针对chr17:37893765的SEQ ID NO.10-SEQ ID NO.12，和/或针对chr17:37860353的SEQ ID NO.13-SEQ ID NO.15，和/或针对chr17:37862986的SEQ ID NO.16-SEQ ID NO.18，和/或针对chr17:37862178的SEQ ID NO.19-SEQ ID NO.21，和/或针对chr17:37884314的SEQ ID NO.22-SEQ ID NO.24，和/或针对chr17:37879819的SEQ ID NO.25-SEQ ID NO.27，和/或针对chr17:37895887的SEQ ID NO.28-SEQ ID NO.30，和/或针对chr17:37893999的SEQ ID NO.31-SEQ ID NO.33，和/或针对chr17:37855528的SEQ ID NO.34-SEQ ID NO.36，和/或针对chr3:38080799的SEQ ID NO.37-SEQ ID NO.39，和/或针对chr17:37843863的SEQ ID NO.40-SEQ ID NO.42，和/或针对chr17:37821433的SEQ ID NO.43-SEQ ID NO.45，和/或针对chr17:37843592的SEQ ID NO.46-SEQ ID NO.48，和/或针对chr17:37856695的SEQ ID NO.49-SEQ ID NO.51；和/或针对chr17:37926322的SEQ ID NO.52-SEQ ID NO.54。
- 根据权利要求14-16任一项所述的用于伴随诊断胃癌HER2的试剂盒，其特征在于，所述试剂盒检测的样本为组织，胃液或者血浆。
- 根据权利要求17所述的用于伴随诊断胃癌HER2的试剂盒，其特征在于，所述试剂盒检测的样本为血浆。
- 根据权利要求14-17任一项所述的用于伴随诊断胃癌HER2的试剂盒，其特征在于，所述试剂盒用于伴随诊断胃癌HER2蛋白过表达或基因扩增用药指导。
- 一种伴随诊断胃癌HER2的检测方法，其特征在于，包括以下步骤：获得待检测样本，对检测样本进行权利要求11～12任一项所述甲基化标记物或其组合的检测。
- 一种对IHC 2+胃癌的检测方法，其特征在于，包括以下步骤：获得待检测样本，对检测样本进行chr17:37843592、chr17:37879893和chr17:37860353的组合、或chr17:37843592、chr17:37893999和chr17:37860353的组合、或chr17:37843592、chr17:37862178的组合的检测。
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| PCT/CN2022/079736 WO2022188776A1 (zh) | 2021-03-09 | 2022-03-08 | 可用于胃癌her2伴随诊断的基因甲基化标记物或其组合和应用 |
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| CN (1) | CN115044671A (zh) |
| WO (1) | WO2022188776A1 (zh) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110269123A1 (en) * | 2010-04-30 | 2011-11-03 | The Chinese University Of Hong Kong | Marker for gastric cancer and method for detecting gastric cancer |
| WO2012070861A2 (ko) * | 2010-11-24 | 2012-05-31 | (주)지노믹트리 | 위암 진단을 위한 위암 특이적 메틸화 바이오마커 |
| CN105624279A (zh) * | 2015-01-22 | 2016-06-01 | 香港中文大学深圳研究院 | 胃癌标记物、其表达和甲基化检测方法、试剂盒及应用 |
| CN109576364A (zh) * | 2018-10-22 | 2019-04-05 | 北京交通大学 | 用于胃癌诊断、化疗和预后检测的分子标记物及试剂盒 |
-
2022
- 2022-03-08 CN CN202210220371.5A patent/CN115044671A/zh active Pending
- 2022-03-08 WO PCT/CN2022/079736 patent/WO2022188776A1/zh unknown
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110269123A1 (en) * | 2010-04-30 | 2011-11-03 | The Chinese University Of Hong Kong | Marker for gastric cancer and method for detecting gastric cancer |
| WO2012070861A2 (ko) * | 2010-11-24 | 2012-05-31 | (주)지노믹트리 | 위암 진단을 위한 위암 특이적 메틸화 바이오마커 |
| CN105624279A (zh) * | 2015-01-22 | 2016-06-01 | 香港中文大学深圳研究院 | 胃癌标记物、其表达和甲基化检测方法、试剂盒及应用 |
| CN109576364A (zh) * | 2018-10-22 | 2019-04-05 | 北京交通大学 | 用于胃癌诊断、化疗和预后检测的分子标记物及试剂盒 |
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| UESUGI NORIYUKI, SUGAI TAMOTSU, SUGIMOTO RYO, EIZUKA MAKOTO, FUJITA YASUKO, SATO AYAKA, OSAKABE MITSUMASA, ISHIDA KAZUYUKI, KOEDA : "Clinicopathological and molecular stability and methylation analyses of gastric papillary adenocarcinoma", PATHOLOGY., MODERN MEDICINE, SYDNEY., AU, vol. 49, no. 6, 1 October 2017 (2017-10-01), AU , pages 596 - 603, XP055966112, ISSN: 0031-3025, DOI: 10.1016/j.pathol.2017.07.004 * |
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