WO2022161076A1 - 用于肺结节良恶性检测的甲基化标记物或其组合及应用 - Google Patents

用于肺结节良恶性检测的甲基化标记物或其组合及应用 Download PDF

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WO2022161076A1
WO2022161076A1 PCT/CN2021/142066 CN2021142066W WO2022161076A1 WO 2022161076 A1 WO2022161076 A1 WO 2022161076A1 CN 2021142066 W CN2021142066 W CN 2021142066W WO 2022161076 A1 WO2022161076 A1 WO 2022161076A1
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benign
methylation
nodules
combination
malignant
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French (fr)
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陶锦胜
涂细祥
陈志伟
范建兵
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广州市基准医疗有限责任公司
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

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  • the invention belongs to the field of biotechnology, and in particular relates to a methylation marker or a combination and application thereof for detecting benign and malignant pulmonary nodules.
  • Lung cancer is the most common cause of cancer death worldwide, with the highest incidence of all malignancies. Due to environmental pollution, the incidence of lung cancer is increasing year by year. In my country, there are about 781,000 lung cancer patients and about 626,000 deaths each year, ranking first in cancer deaths. It is estimated that by 2025, the number of new lung cancer patients in my country will reach 1 million each year. Because most of the clinically diagnosed cases of lung cancer are in the advanced stage, the opportunity for surgical treatment is lost, and the prognosis of lung cancer is extremely poor. The 5-year survival rate of lung cancer in my country is only 16.1%.
  • Goldstraw et al found that the 5-year survival rate of early stage Ia lung cancer after surgical resection can reach more than 80%, while the 5-year survival rate of advanced stage IIIa-IV lung cancer after surgical treatment is less than 36%. Screening is the hope of improving lung cancer survival and reducing lung cancer mortality.
  • Lung cancer exists in the form of pulmonary nodules in the early stage, which is a common clinical phenomenon, including benign nodules and malignant nodules.
  • the early detection of malignant pulmonary nodules is relatively insidious. Difference.
  • there are many difficulties in the qualitative diagnosis of pulmonary nodules about 30% of the pulmonary nodules that are surgically removed are benign. Therefore, a correct evaluation of the benign and malignant pulmonary nodules will help to choose the correct treatment method. Significantly improved patient survival and improved prognosis.
  • Imaging examination is non-invasive because of its non-invasive nature. And commonly used, it mainly observes the size, volume, volume doubling time and morphological characteristics of small pulmonary nodules.
  • imaging methods mainly include chest CT, positron emission tomography-computed tomography (PET-CT) and so on. Imaging detection has obvious problems.
  • CT-CT is only effective in detecting solid nodules, and it is not satisfactory for non-solid nodules such as ground glass.
  • CT-guided percutaneous lung biopsy can be used, and the diagnostic accuracy rate is about 90%.
  • X-ray, CT or ultrasound-guided percutaneous lung biopsy is more likely to lead to serious complications such as hemorrhage, pneumothorax, and sudden death.
  • VBN is a CT-based three-dimensional imaging technique.
  • cfDNA cell-free DNA
  • cfDNA is a small fragment of free nucleic acid DNA in peripheral blood, derived from the metabolism and apoptosis of normal cells or tumor cells, and contains genetic information such as somatic mutation and DNA methylation.
  • liquid biopsy Liquid Biopsy
  • 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 Turner's team used high-throughput sequencing to find breast cancer-specific somatic mutation sites and monitor their dynamic changes, demonstrating that ctDNA detection can detect tumor recurrence and metastasis earlier than CT.
  • New research finds that combining ctDNA mutations in blood with other analytes can lead to earlier and better diagnosis of lung, ovarian, liver, stomach, breast, prostate, esophageal and colorectal cancers, which are common and surgically removed of cancer. More and more clinical studies have shown that plasma ctDNA can be used as a biomarker for early diagnosis and screening of tumors, prediction, response to treatment, and monitoring tumor size and recurrence.
  • 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 biomarkers or combinations thereof associated with benign and malignant pulmonary nodules, which can be used to diagnose benign and malignant pulmonary nodules.
  • biomarkers associated with benign and malignant lung nodules or a combination thereof including at least one of the following methylation biomarkers: cg07553761, cg22685975, cg04688351, cg07160746, cg13146465.
  • the biomarkers associated with benign and malignant lung nodules or a combination thereof further includes at least one of the following methylation biomarkers: cg20607577, cg22878622, cg17915922, cg05310764, cg22796313, cg20321153 ,cg13114497,cg08691548,cg04387597,cg15634980,cg21428324,cg18205770,cg00147160,cg15622158,cg18011916,cg03240324,cg06335867,cg2699.
  • the combination of methylation biomarkers is all 200 of the methylation biomarkers described above.
  • Another aspect of the present invention also provides the use of the above biomarkers or their combination as lung cancer-related methylation molecular markers in detecting benign and malignant pulmonary nodules, and/or lung cancer.
  • kits for detecting benign and malignant pulmonary nodules, and/or lung cancer comprising detecting the methylation level of the above DNA methylation molecular markers or a combination thereof reagent.
  • the kit can be used for the following detection platforms: including the use of PCR amplification method, fluorescence quantitative PCR method, digital PCR method, liquid chip method, first-generation sequencing method, third-generation sequencing method, second-generation sequencing method (Sanger), pyrosequencing, bisulfite sequencing, methylation chip, or a combination thereof. In some preferred embodiments, next-generation sequencing is preferred.
  • the pulmonary nodules are malignant nodules of different classifications (solid or partially solid or ground glass nodules), pulmonary nodule size, and different stages.
  • the pulmonary nodule is a partially solid or ground glass nodule.
  • the lung cancer is early stage lung cancer, preferably stage I lung cancer.
  • Another aspect of the present invention also provides a method for detecting benign and malignant pulmonary nodules, comprising the following steps:
  • the present invention finds that the combination of plasma genome fragments can be used as a diagnostic marker for pulmonary nodules by studying the differences in methylation modification of these plasma methylation-modified genome fragments in different lung nodule types, lung cancer patients of various stages, and benign nodule populations. thing. Further research found that the top 20 markers had essentially the same diagnostic power as the 100-marker combination.
  • the present invention jointly analyzes the co-methylation characteristics of multiple methylated cytosines on at least one genome segment as a biomarker for judging benign and malignant pulmonary nodules, and its accuracy is far greater than the detection of other protein plasma biomarkers , especially in the distinction of stage I lung cancer; in non-solid nodules (partial solid and ground glass nodules), the accuracy is much higher than PET-CT, which can effectively eliminate negative effects and reduce false positives. occur.
  • Figure 1 is a flow chart of the biomarker research of the present invention.
  • FIG. 2 is a schematic diagram of ROAUC of five independent markers in Example 2.
  • FIG. 2 is a schematic diagram of ROAUC of five independent markers in Example 2.
  • Figure 3 shows the performance of the 20-, 50-, 100-, and 200-marker models in Examples 2, 3, 4, and 5 on the test set.
  • the AUCs correspond to 0.81, 0.81, 0.83, and 0.82, respectively.
  • Figure 4 shows the performance of Example 4 on the independent validation set and the comparison between the model and the clinically commonly used Mayo model and VA model in the independent validation set: in the independent validation set, AUC is 0.84, AUC is higher than 0.59 of Mayo model and VA model of 0.54.
  • Figure 5 is the performance of the 100-marker model in Example 4 in early stage malignant nodules (STAGE I).
  • Figure 6 is the performance of the 100-marker model in Example 4 in 6-20mm nodules, independent validation set: AUC is 0.84, higher than Mayo model's 0.60 and VA model's 0.51.
  • Figure 7 shows the performance of the 100-marker model in Example 4 in different nodule types, independent validation sets and comparison with PET-CT.
  • the "plurality” mentioned in the present invention means two or more.
  • "And/or" which describes the association relationship of the associated objects means that there can be three kinds of relationships, for example, A and/or B, which can mean that A exists alone, A and B exist at the same time, and B exists alone.
  • the character "/" generally indicates that the associated objects are an "or" relationship.
  • At least one or a combination and application of 200 plasma gene methylation markers for the diagnosis of benign and malignant pulmonary nodules are disclosed.
  • Pulmonary nodules include different categories: solid, partially solid, or ground-glass nodules; malignant nodules are staged according to the TNM staging system.
  • Pulmonary nodules include solid or partially solid or ground-glass nodules: ground-glass density nodules refer to indistinct nodules in the lungs. The nodule density is slightly higher than that of the surrounding lung parenchyma, but the outline of the blood vessels and bronchi in the nodules is still unclear. visible.
  • a solid nodule refers to a nodule with soft tissue density in its entirety, the density is relatively uniform, and the images of blood vessels and bronchi in it are concealed. Partially solid nodules are those that contain both ground-glass and solid soft-tissue densities.
  • the staging of malignant nodular lung cancer is mainly based on the AJCC TNM international standardized staging system, which is divided into stages I-IV, and early-stage lung cancer includes stage I-II lung cancer.
  • screening markers for the diagnosis of benign and malignant pulmonary nodules based on the methylation of circulating tumor DNA includes the following steps:
  • Step 1 Lung cancer-specific methylation markers screened according to the TGCA lung cancer and paracancer methylation chip public database and the independent tumor-related methylation database;
  • Step 2 Plasma cfDNA extraction from benign and malignant pulmonary nodules, cfDNA extracted by bisulfite treatment, and methylated DNA to build a library;
  • Step 3 Benign and malignant specific panels of pulmonary nodules target hybridization to capture the methylated DNA pre-library
  • Step 4 Quantify the final library of targeted capture, and perform second-generation sequencing on the computer;
  • Step 5 Data preprocessing to obtain the real reads data (bam file) captured by the probe;
  • Step 6 methylation data analysis, screening of benign and malignant diagnostic markers (markers) of pulmonary nodules, and screening the final marker set for analysis through consistent analysis and filtering of tissue and paired plasma;
  • Step 7 Use the marker set selected in the above step 6 to further screen markers in the plasma training set and validation set, build an algorithm model, and perform independent data set validation;
  • Step 8 Confirm the gene methylation markers and algorithm models that are finally used for the diagnosis of benign and malignant pulmonary nodules.
  • Example 1 Detection method of novel ctDNA methylation markers for diagnosis of benign and malignant pulmonary nodules
  • the specific operation steps of plasma cfDNA extraction were carried out according to the operating instructions of Life's MagMAX TM Cell-Free DNA Isolation Kit.
  • the extraction steps of tissue DNA were carried out according to the DNeasy Blood&Tissue Kit operation instructions of QIAGEN company; index primer was purchased from New England Bioscience company Cat#E7600S.
  • the extracted cfDNA (10 ng) or tissue DNA (50 ng) was subjected to bisulfite conversion to deaminate unmethylated cytosine into uracil, while the methylated cytosine remained unchanged to obtain
  • the specific operation of the conversion was carried out according to the instructions of Zymo Research's EZ DNA Methylation-Lightning Kit.
  • the hybridization capture kit is xGen Lockdown Reagents from IDT Company, and the operation is carried out according to the instructions.
  • This embodiment discloses a methylation-specific biomarker for diagnosing pulmonary nodules. Based on the training set samples of 253 malignant nodules and 56 benign nodules plasma samples, the methylation described in Example 1 is used.
  • the library construction method uses the differences in methylation levels in different groups to screen out biomarkers related to malignant pulmonary nodules, and the locus data is verified in independent data sets in benign and malignant samples, and a total of 200
  • the 200 methylation biomarkers hereinafter referred to as sites or markers
  • sites or markers The 200 methylation biomarkers and their independently differentiated AUC values are shown in Table 1:
  • cg07553761, cg22685975, cg04688351, cg07160746, and cg13146465 performed the best overall, with AUC values of 0.753, 0.710, 0.715, 0.712 and 0.709, respectively, as shown in Figure 2.
  • the AUC reached 0.823
  • the accuracy was 0.838
  • the sensitivity was 90.0%
  • the specificity was 65.0 %
  • the positive predictive value PPV was 88.5%
  • the negative predictive value NPV was 68.4%, see Figure 2 and Table 2.
  • Table 2 Specific performance data for 20, 50, 100 and 200 methylation marker models
  • the AUC reached 0.83, with an accuracy of 85.0% , the sensitivity was 93.3%, the specificity was 60.0%, the PPV was 87.5%, and the NPV was 75%, see Figure 3 and Table 2.
  • the sites screened by this method have a very high correlation with the diagnosis of benign and malignant pulmonary nodules.

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Abstract

本发明涉及与肺结节良恶性相关的生物标记物及其试剂盒，其包括选自cg07553761,cg22685975,cg04688351,cg07160746,cg13146465等200种标记物中的至少一种。

Description

用于肺结节良恶性检测的甲基化标记物或其组合及应用 技术领域
本发明属于生物技术领域，具体涉及用于肺结节良恶性检测的甲基化标记物或其组合及应用。
背景技术
肺癌是全球范围内癌症死亡的最常见原因，发病率居于所有恶性肿瘤之首。由于环境的污染，肺癌的发病率逐年上升，我国每年肺癌患者约78.1万，死亡病例约62.6万，位于癌症死亡首位。预计至2025年，我国每年新发肺癌患者将达100万。由于绝大多数临床诊断肺癌病例多已为晚期，失去手术治疗机会，肺癌预后极差，我国肺癌的5年生存率仅为16.1％。Goldstraw等研究发现早期Ⅰa期肺癌经手术切除，其5年生存率可达到80％以上，而中晚期Ⅲa～Ⅳ期肺癌在手术治疗后，其5年生存率在36％以下，因此，肺癌的筛查是改善肺癌生存，降低肺癌死亡率的希望所在。
肺癌早期以肺结节形式存在，是一种临床中常见的现象，包括良性结节和恶性结节，恶性肺结节早期发现比较隐匿，如果不早期干预，其病程迅速、恶性度强、预后差。目前对肺结节的定性诊断有很多困难，在临床外科切除的肺结节中，30％左右为良性的，所以正确评价肺结节的良恶性，有助选择正确的治疗手段，且可以帮助显著提高患者的生存率改善预后。
现有肺部小结节的诊断方法有多种，但定性诊断较为困难。目前肺部小结节诊断的常用方法包括：影像学检查、经皮穿刺活组织检查、虚拟支气管镜导航系统下的活组织检查及胸腔镜手术病理组织检查：1)影像学检查因其无创性而普遍使用，其主要观察肺部小结节的大小、体积、体积倍增时间及形态特征。常用的影像学检查方法主要有胸部CT、正电子发射型计算机断层显像(positron emission tomography-computed tomography，PET-CT)等。影像学检测存在问题比较明显，例如使用CT会有过高的假阳性，可能导致过度诊断、过度治疗、医疗资源的浪费及增加受检者焦虑心理。PET-CT仅对实性结节检测效果明显，对于磨玻璃等非实性结节效果不尽如人意。2)当PET诊断的灵敏度不佳或显现出假阳性指征时，可选用CT引导下的经皮肺穿刺活组织检查，诊断准确率约为90％。但X线、CT或超声引导下经皮肺穿刺活组织检查较易导致出血、气胸、猝死等严重并发症。近年开展的VBN是一种基于CT的三维成像术，其通过图像识别建立虚拟支气管路径，引导支气管镜到达目标病灶进行活组织检查，从而达到提高肺小结节活组织微创检查阳性率的目的。但实际操作中，因为活检导致的院内感染等问题也极为普遍。
综上，筛查肺部结节及精准地进行良、恶性质的鉴别诊断是肺癌防治急需解决的重大问题。当前亟需针对此临床痛点寻找并建立有效的诊断模型以及能够作为筛查与诊断的分子标记物。
cfDNA(cell-free DNA)是外周血中游离的核酸小片段DNA，源自正常细胞或肿瘤细胞代谢与凋亡，包含体细胞突变和DNA甲基化等遗传信息。通过检测疾病特异性cfDNA片段，掌握疾病的发生、发展的技术，称为液体活检(Liquid Biopsy)，与传统的组织活检相比，其有着迅速、便捷、损伤性小等众多优点。2014年，来自Bert Vogelstein和Kenneth Kinzler团队的一份640例各类肿瘤的研究发现，超过75％的晚期胰腺癌、卵巢癌、结直肠癌、膀胱癌、胃食管癌、黑色素癌、肝细胞癌以及头颈癌症的患者，都能检测到ctDNA的存在。因而ctDNA是一种具有广泛适用性、敏感而特异的生物标志物，能够用于各式各样、多种不同类型癌症的临床和研究。2015年卢煜明教授通过cfDNA全基因组甲基化测序证明了液体活检替代组织活检在技术上和理论上的可行性；2017年张鹍教授团队利用ctDNA的甲基化定量描述了肿瘤负荷以及肿瘤来源的ctDNA图谱。Turner团队利用高通量测序寻找乳腺癌特异性体细胞突变位点，并监测其动态变化，证明ctDNA检测可早于CT发现肿瘤复发转移。最新研究发现，结合其它分析物血液中的ctDNA突变能更早、更好的诊断肺癌、卵巢癌，肝癌，胃癌，乳腺癌，前列腺癌，食管癌和结直肠癌这几种常见且可手术切除的癌。临床越来越多的研究表明血浆ctDNA可作为生物标志物应用在肿瘤早期诊断筛选、预测、治疗的反应，监测肿瘤大小和复发等。目前，国际上的研究方向是整合多组学/多种分子标志物、多基因/多位点来提高检测技术的灵敏度和特异性，以满足临床对检测产品的需求。
发明内容
本发明的目的之一在于提供了与肺结节良恶性相关的生物标记物或其组合，该生物标记物及其组合可用于诊断肺结节良恶性发病情况。
实现上述目的的技术方案如下。
与肺结节良恶性相关的生物标记物的或其组合，包括有以下至少一种甲基化生物标记物：cg07553761,cg22685975,cg04688351,cg07160746,cg13146465。
在其中一些实施例中，所述与肺结节良恶性相关的生物标记物的或其组合，还包括有以下至少一种甲基化生物标记物：cg20607577,cg22878622,cg17915922,cg05310764,cg22796313,cg20321153,cg13114497,cg08691548,cg04387597,cg15634980,cg21428324,cg18205770,cg00147160,cg15622158,cg18011916,cg03240324，cg06335867,cg26970841。
在其中一些实施例中，还包括以下甲基化生物标记物中的至少一种：cg03978375,cg24826867,cg17384889,cg18694169,cg10253847,cg09163035,cg01759136,cg21643086,cg23279117,cg12820681,cg07816637,cg17132517,cg09578794,cg00171421,cg12445832,cg23999112,cg26528551,cg11289039,cg15072319,cg16247183,cg10081761,cg10144554,cg26474549,cg11220950,cg14244013,cg09183450,cg02458065,cg21308660,cg26049726,cg17241310,cg03259243,cg24843474,cg18330573,cg05145573,cg19756358,cg01857475,cg14559388,cg11806439,cg24495585,cg14750948,cg00805193,cg11948988,cg23923820,cg05324519,cg20074395,cg25446076,cg26225694,cg13997680,cg01031101,cg09523275,cg18675097,cg00203284,cg18911817,cg06712559,cg27252696,cg17380661,cg15261247,cg10046388,cg23725149,cg05928806,cg02439789,cg24456130,cg03208426,cg10960266, cg26702958,cg22001211,cg08005730,cg06640773,cg06059409,cg21827912,cg12314713,cg06890720,cg03122669,cg16356837,cg10694030,cg01615018,cg02568274。
在其中一些实施例中，还包括以下甲基化生物标记物中的至少一种：cg00996827,cg21402748,cg03884587,cg12427163,cg18443359,cg20732876,cg14718275,cg18130044,cg09076431,cg04365202,cg21918126,cg27348152,cg16352140,cg22879098,cg07995570,cg05164926,cg24104241,cg12897502,cg15489799,cg15555898,cg23683588,cg18361098,cg17666418,cg25913016,cg11598006,cg05297854,cg11213574,cg15831346,cg03242819,cg26697286,cg26128121,cg04655953,cg22521151,cg00207921,cg20544808,cg18792131,cg16780502,cg14789818,cg20699586,cg06216683,cg07258474,cg20129806,cg22350285,cg02587648,cg27541454,cg27180750,cg24703339,cg00352681,cg15541630,cg04759874,cg19148866,cg05740609,cg22437074,cg05523911,cg17804348,cg07178825,cg23077820,cg22989843,cg05982757,cg16983211,cg15700739,cg24535439,cg00262621,cg24362661,cg07519536,cg19729116,cg12776171,cg21475076,cg07952270,cg04293152,cg26968008,cg16668728,cg04591032,cg23905164,cg26320000,cg13461447,cg23948811,cg07126167,cg14216876,cg03281661,cg27116061,cg07853668,cg06618740,cg26048630,cg00002593,cg02448922,cg06817455,cg08933227,cg00158333,cg02745847,cg15748470,cg12244647,cg27631599,cg14673618,cg00776293,cg14514924,cg12691464,cg19253333,cg13693328,cg15097000。
在其中一些实施例中，所述甲基化生物标记物或其组合为：cg07553761,cg20607577,cg22685975,cg22878622,cg17915922,cg05310764,cg22796313,cg20321153,cg13114497,cg08691548,cg04387597,cg15634980,cg21428324,cg18205770,cg00147160,cg15622158,cg18011916,cg03240324，cg06335867,和cg26970841。
在其中一个实施例中，所述甲基化生物标记物或其组合为：cg07553761,cg20607577,cg22685975,cg22878622,cg17915922,cg05310764,cg22796313,cg20321153,cg13114497,cg08691548,cg04387597,cg15634980,cg21428324,cg18205770,cg00147160,cg15622158,cg18011916,cg03240324,cg06335867,cg26970841,cg03978375,cg24826867,cg17384889,cg18694169,cg10253847,cg09163035,cg01759136,cg21643086,cg23279117,cg12820681,cg07816637,cg04688351,cg17132517,cg09578794,cg00171421,cg12445832,cg23999112,cg26528551,cg11289039,cg15072319,cg16247183,cg10081761,cg10144554,cg26474549,cg11220950,cg14244013,cg09183450,cg02458065,cg21308660,和cg07160746。
在其中一些实施例中，所述甲基化生物标记物或其组合为：cg07553761,cg20607577,cg22685975,cg22878622,cg17915922,cg05310764,cg22796313,cg20321153,cg13114497,cg08691548,cg04387597,cg15634980,cg21428324,cg18205770,cg00147160,cg15622158,cg18011916,cg03240324，cg06335867,cg26970841，cg03978375,cg24826867,cg17384889,cg18694169,cg10253847,cg09163035,cg01759136,cg21643086,cg23279117,cg12820681,cg07816637,cg04688351,cg17132517,cg09578794,cg00171421,cg12445832,cg23999112,cg26528551,cg11289039,cg15072319,cg16247183,cg10081761,cg10144554,cg26474549,cg11220950,cg14244013,cg09183450,cg02458065,cg21308660,cg07160746,cg26049726,cg13146465,cg17241310,cg03259243,cg24843474,cg18330573,cg05145573,cg19756358, cg01857475,cg14559388,cg11806439,cg24495585,cg14750948,cg00805193,cg11948988,cg23923820,cg05324519,cg20074395,cg25446076,cg26225694,cg13997680,cg01031101,cg09523275,cg18675097,cg00203284,cg18911817,cg06712559,cg27252696,cg17380661,cg15261247,cg10046388,cg23725149,cg05928806,cg02439789,cg24456130,cg03208426,cg10960266,cg26702958,cg22001211,cg08005730,cg06640773,cg06059409,cg21827912,cg12314713,cg06890720,cg03122669,cg16356837,cg10694030,cg01615018,和cg02568274。
在其中一些实施例中，所述甲基化生物标记物组合为上述全部200种所述甲基化生物标记物。
本发明的另一个方面，还提供了上述生物标记物或其组合作为肺癌相关甲基化分子标记物在检测肺结节良恶性、和/或肺癌中的应用。
本发明的另一个方面还提供了一种用于检测肺结节良恶性、和/或肺癌的试剂盒，所述试剂盒包含检测上述DNA甲基化分子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，所述试剂盒可以用于以下检测平台：包括采用PCR扩增法、荧光定量PCR法、数字PCR法、液相芯片法、一代测序法、三代测序法二代测序法(Sanger)、焦磷酸测序法、亚硫酸氢盐测序、甲基化芯片法或它们的组合所使用的试剂。在一些优选的实施例中，优选采用二代测序法。
在其中一些实施例中，所述肺结节是不同分类(实性或部分实性或磨玻璃结节)、肺结节大小、和不同分期的恶性结节。
在其中一些实施例中，所述肺结节为部分实性或磨玻璃结节。
在其中一些实施例中，所述肺癌是早期肺癌，优选为I期肺癌。
本发明的另一个方面还提供了一种肺结节良恶性的检测方法，包括以下步骤：
s1、提取待测样本的cfDNA、亚硫酸氢盐处理提取的cfDNA、甲基化DNA建库；
s2、根据上述的甲基化生物标记物或其组合进行肺结节良恶性特异性panel靶向杂交捕获甲基化预文库；
s3、靶向捕获的终文库定量，二代测序；
s4、数据预处理，获得探针捕获得到的真实数据；
s5、对上述的甲基化生物标记物或其组合甲基化数据分析。
本发明通过研究这些血浆甲基化修饰的基因组片段在不同肺结节类型及各期肺癌患者及良性结节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肺结节诊断标记物。进一步的研究发现前20个标志物具备与100个标志物组合基本相同的诊断力。本发明联合分析至少1个基因组片段上的多个甲基化胞嘧啶的共甲基化特征作为判断肺结节良恶性发病的生物标记物，其准确度远大于其它蛋白血浆生物标记物的检测，尤其在I期肺癌的区分上；在非实性结节(部分实性及磨玻璃结节)中，准确度远高于PET-CT，可有效起到排阴的效果，降低假阳性的发生。
附图说明
图1是本发明所述生物标记物研究的流程图。
图2是实施例2中五个独立标志物的ROAUC示意图。
图3是实施例2，3，4，5中20-，50-，100-，200-标志物模型在测试集上的表现，在测试集中，AUC分别对应为0.81，0.81，0.83，0.82。
图4是实施例4在独立验证集上的表现及模型和临床常用模型Mayo model及VA model在独立验证集中的对比：在独立验证集中，AUC为0.84，AUC高于Mayo model的0.59及VA model的0.54。
图5是实施例4中100-标志物模型在早期恶性结节(STAGE I)的表现。
图6是实施例4中100-标志物模型在6-20mm结节，独立验证集中的表现：AUC为0.84，高于Mayo model的0.60及VA model的0.51。
图7是实施例4中100-标志物模型在不同结节类型，独立验证集中的表现及与PET-CT的对比。
具体实施方式
本发明下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。
本发明的术语“包括”和“具有”以及它们任何变形，意图在于覆盖不排他的包含。例如包含了一系列步骤的过程、方法、装置、产品或设备没有限定于已列出的步骤或模块，而是可选地还包括没有列出的步骤，或可选地还包括对于这些过程、方法、产品或设备固有的其它步骤。
在本发明中提及的“多个”是指两个或两个以上。“和/或”，描述关联对象的关联关系，表示可以存在三种关系，例如，A和/或B，可以表示：单独存在A，同时存在A和B，单独存在B这三种情况。字符“/”一般表示前后关联对象是一种“或”的关系。
本发明的一个实施例中，公开了用于肺结节良恶性诊断的血浆基因甲基化标记物200种的至少一种或者组合及应用。具体地，发明人发现以下肺结节恶性患者血浆中明显异常甲基化修饰的基因组片段，即cg07553761,cg20607577,cg22685975,cg22878622,cg17915922,cg05310764,cg22796313,cg20321153,cg13114497,cg08691548,cg04387597,cg15634980,cg21428324,cg18205770,cg00147160,cg15622158,cg18011916,cg03240324,cg06335867,cg26970841,cg03978375,cg24826867,cg17384889,cg18694169,cg10253847,cg09163035,cg01759136,cg21643086,cg23279117,cg12820681,cg07816637,cg04688351,cg17132517,cg09578794,cg00171421,cg12445832,cg23999112,cg26528551,cg11289039,cg15072319,cg16247183,cg10081761,cg10144554,cg26474549,cg11220950,cg14244013,cg09183450,cg02458065,cg21308660,cg07160746,cg26049726,cg13146465,cg17241310,cg03259243, cg24843474,cg18330573,cg05145573,cg19756358,cg01857475,cg14559388,cg11806439,cg24495585,cg14750948,cg00805193,cg11948988,cg23923820,cg05324519,cg20074395,cg25446076,cg26225694,cg13997680,cg01031101,cg09523275,cg18675097,cg00203284,cg18911817,cg06712559,cg27252696,cg17380661,cg15261247,cg10046388,cg23725149,cg05928806,cg02439789,cg24456130,cg03208426,cg10960266,cg26702958,cg22001211,cg08005730,cg06640773,cg06059409,cg21827912,cg12314713,cg06890720,cg03122669,cg16356837,cg10694030,cg01615018,cg02568274,cg00996827,cg21402748,cg03884587,cg12427163,cg18443359,cg20732876,cg14718275,cg18130044,cg09076431,cg04365202,cg21918126,cg27348152,cg16352140,cg22879098,cg07995570,cg05164926,cg24104241,cg12897502,cg15489799,cg15555898,cg23683588,cg18361098,cg17666418,cg25913016,cg11598006,cg05297854,cg11213574,cg15831346,cg03242819,cg26697286,cg26128121,cg04655953,cg22521151,cg00207921,cg20544808,cg18792131,cg16780502,cg14789818,cg20699586,cg06216683,cg07258474,cg20129806,cg22350285,cg02587648,cg27541454,cg27180750,cg24703339,cg00352681,cg15541630,cg04759874,cg19148866,cg05740609,cg22437074,cg05523911,cg17804348,cg07178825,cg23077820,cg22989843,cg05982757,cg16983211,cg15700739,cg24535439,cg00262621,cg24362661,cg07519536,cg19729116,cg12776171,cg21475076,cg07952270,cg04293152,cg26968008,cg16668728,cg04591032,cg23905164,cg26320000,cg13461447,cg23948811,cg07126167,cg14216876,cg03281661,cg27116061,cg07853668,cg06618740,cg26048630,cg00002593,cg02448922,cg06817455,cg08933227,cg00158333,cg02745847,cg15748470,cg12244647,cg27631599,cg14673618,cg00776293,cg14514924,cg12691464,cg19253333,cg13693328,cg15097000。
针对血浆细胞游离DNA(cell-free DNA,cfDNA)中低量的肿瘤DNA(circling tumor DNA,ctDNA)样本和亚硫酸氢盐处理DNA损伤的问题，采用基准医疗的建库和杂交捕获技术：AnchorDx EpiVisio TM Methylation Library Prep Kit(AnchorDx,Cat#A0UX00019)，AnchorDx EpiVisio TM Indexing PCR Kit(AnchorDx,Cat#A2DX00025)和AnchorDx EpiVisio TM Target Enrichment Kit(AnchorDx,Cat#A0UX00031)。
肺结节包括不同的类别：实性结节、部分实性结节或磨玻璃结节；恶性结节恶性程度按TNM分期系统进行分期。
肺结节包括实性或部分实性或磨玻璃结节：磨玻璃密度结节是指肺内模糊的结节影，结节密度较周围肺实质略增加，但其内血管及支气管的轮廓尚可见。实性结节是指其内全部是软组织密度的结节，密度较均匀，其内血管及支气管影像被掩盖。部分实性结节是指其内既包含磨玻璃密度又包含实性软组织密度的结节，密度不均匀。
恶性结节肺癌分期主要依据AJCC TNM国际标准化分期系统，分为I期-IV期，早期肺癌包括I期-II期肺癌。
本发明的一个实施例中，基于循环肿瘤DNA(circulating tumor DNA,ctDNA)甲基化筛选肺结节良恶性诊断的标记物，包括如下步骤：
步骤一、根据TGCA肺癌与癌旁甲基化芯片公共数据库及自主肿瘤相关甲基化数据库筛选出来的肺癌特异性的甲基化标记物；
步骤二、肺结节良性和恶性患者血浆cfDNA提取、亚硫酸氢盐处理提取的cfDNA、甲基化DNA建库；
步骤三、肺结节良恶性特异性panel靶向杂交捕获甲基化DNA预文库；
步骤四、靶向捕获的终文库定量，上机进行二代测序；
步骤五、数据预处理，获得探针捕获得到的真实reads数据(bam file)；
步骤六、甲基化数据分析，筛选肺结节良恶性诊断标记物(markers)，通过组织与配对血浆一致的分析、过滤，筛选最终用于分析的标记物集；
步骤七、利用上述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；
步骤八、确认最终用于肺结节良恶性诊断的基因甲基化标记物及算法模型。
实施例1肺结节良恶性诊断新型ctDNA甲基化标志物检测方法
1.血浆cfDNA或组织DNA提取及甲基化建库
1.1血浆cfDNA或组织DNA的提取。
血浆cfDNA提取具体操作步骤按照Life公司的MagMAX TM Cell-Free DNA Isolation Kit操作说明书进行。组织DNA的提取步骤按照QIAGEN公司的DNeasy Blood&Tissue Kit操作说明进行；index primer购买于New England Bioscience公司Cat#E7600S。
1.2转化
将提取的cfDNA(10ng)或组织DNA(50ng)进行亚硫酸氢盐转化，使DNA中未发生甲基化的胞嘧啶脱氨基转变成尿嘧啶，而甲基化的胞嘧啶保持不变，得到亚硫酸氢盐转化后的DNA，转化具体操作按照Zymo Research的EZ DNA Methylation-Lightning Kit说明书进行。
1.3末端修复
1.3.1将转化后的17ul样本加入以下试剂进行反应：
组分 体积(ul)
转化后样本 17
MEB1 Buffer 2
MEE2 Enzyme 1
总体积 20
1.3.2置于PCR仪中按照以下程序进行反应：
37℃  30min
95℃  5min
热盖  105℃。
1.3.3当PCR反应第二步(95℃)达到5min时，立即将样本从PCR仪中取出，直接插入冰中，放置2min以上再进行下一步操作
1.4连接I
1.4.1配制如下反应液
Figure PCTCN2021142066-appb-000001
1.4.2置于PCR仪中按照以下程序进行反应：
Figure PCTCN2021142066-appb-000002
1.5扩增I
1.5.1配制如下反应液
Figure PCTCN2021142066-appb-000003
Figure PCTCN2021142066-appb-000004
1.5.2置于PCR仪中按照以下程序进行反应：
Figure PCTCN2021142066-appb-000005
1.6纯化I：加入166ul 1:6倍稀释的Agencourt AMPure Beads(需提前于室温平衡半小时)的对Amplification I反应后的产物进行纯化，用21ul EB进行洗脱，纯化具体步骤如下：
1.6.1取上一步反应产物并进行离心，每个样本加入166ul 1:6倍稀释的Agencourt AMPure Beads，用移液器吹打混匀。
1.6.2室温孵育5min。
1.6.3离心，置于磁力架上静置5min。
1.6.4吸去上清。
1.6.5加入200ul 80％EtOH，静置30s，吸走乙醇。
1.6.6重复步骤5)一次。
1.6.7离心，将PCR管置于磁力架上，吸走剩余乙醇。
1.6.8开盖干燥磁珠2-3min，注意不要过干。
1.6.9加入21ul EB进行洗脱，用移液器充分吹打混匀，室温静置3min。
1.6.10离心，将PCR管置于磁力架上，静置3min。
1.6.11吸取20ul上清于新的PCR管中。
1.7连接II
1.7.1配制如下反应液：
组分 体积(ul)
上一步反应体积 20
H 2O 4
MSB1 Buffer 8
MSR1 Reagent 2
MSR5 Reagent 2
MSE1 Enzyme 2
MSE5 Enzyme 2
总体积 40
1.7.2置于PCR仪中按照以下程序进行反应
温度 时间 循环数
37℃ 30min 1
95℃ 5min 1
10℃ Hold 1
1.8 Indexing PCR:
1.8.1配制如下反应液：
Figure PCTCN2021142066-appb-000006
1.8.2置于PCR仪中按照以下程序进行反应
Figure PCTCN2021142066-appb-000007
1.9纯化II
加入Agencourt AMPure Beads(需提前于室温平衡半小时)对Indexing PCR反应后的产物进行纯化，用41ul EB进行洗脱，纯化具体步骤如下：
1.9.1取上一步反应产物并进行离心，每个样本加入71ul未稀释的Agencourt AMPure Beads，用移液器吹打混匀.
1.9.2室温孵育5min。
1.9.3离心，置于磁力架上静置5min。
1.9.4吸去上清。
1.9.5加入200ul 80％EtOH，静置30s，吸走乙醇。
1.9.6重复步骤5)一次。
1.9.7离心，将PCR管置于磁力架上，吸走剩余乙醇。
1.9.8开盖干燥磁珠2-3min，注意不要过干。
1.9.9加入41ul EB进行洗脱，用移液器充分吹打混匀，室温静置3min。
1.9.10离心，将PCR管置于磁力架上，静置3min。
1.9.11吸取20ul上清于新的PCR管中。
1.10 Qubit定量：
取1ul用Qubit dsDNA HS Assay Kit对文库进行定量。
2.通过对已建库后的样本进行寡核苷酸探针捕获富集，得到特定区域的上机终文库。杂交捕获试剂盒为IDT公司的xGen Lockdown Reagents，具体按照说明书进行操作。
3.采用Illumina公司的测序仪对杂交捕获后的样本进行测序，得到测序结果。
4.数据的分析：
对测序仪的下机原始数据，进行常规的生物信息学分析处理，先通过fastp过滤低质量(QC低，长度短、太多N等)的读长(reads)，然后去除reads双端的adapter、共有序列、PolyA/T，得到理想的插入片段序列(target区间)，使用bismark将这些reads比对hg19对应的位置后，根据UMI对reads进行去重，得到每份样本被探针捕获得到的真实reads数据(bamfile)，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
本实施例公开了一种用于诊断肺结节的甲基化特异性生物标记物，基于训练集样本253例恶性结节和56例良性结节血浆样本，利用实施例1所述甲基化建库方法，利用在不同组别的甲基化水平差异筛选出与恶性肺结节相关的生物标记物，并对位点数据在良性与恶性样本中进行独立数据集的验证，一共筛选出200个在最显著区分良恶性的甲基化的DNA片段，该200个甲基化生物标记物(以下简称位点或者marker)及独立区分的AUC值见表1：
表1 200个甲基化标记物的具体表现数据
MARKER 位点信息 AUC值
1 cg07553761 75.35290796
2 cg20607577 69.55815923
3 cg22685975 71.05095991
4 cg22878622 68.29827781
5 cg17915922 63.79164314
6 cg05310764 69.85107284
7 cg22796313 69.85107284
8 cg20321153 56.13706945
9 cg13114497 68.43591191
10 cg08691548 68.43591191
11 cg04387597 66.20906268
12 cg15634980 68.98997741
13 cg21428324 62.39412761
14 cg18205770 66.61137775
15 cg00147160 64.51510446
16 cg15622158 63.14229249
17 cg18011916 67.80773574
18 cg03240324 69.76284585
19 cg06335867 66.5513834
20 cg26970841 66.68195935
21 cg03978375 66.68195935
22 cg24826867 66.68195935
23 cg17384889 67.65245624
24 cg18694169 67.65245624
25 cg10253847 67.65245624
26 cg09163035 56.92758329
27 cg01759136 68.53825522
28 cg21643086 68.53825522
29 cg23279117 63.58695652
30 cg12820681 61.15189159
31 cg07816637 70.10516657
32 cg04688351 71.52738566
33 cg17132517 61.00367024
34 cg09578794 53.18322981
35 cg00171421 62.79997177
36 cg12445832 52.62563523
37 cg23999112 52.62563523
38 cg26528551 62.6905703
39 cg11289039 59.79672501
40 cg15072319 64.33214286
41 cg16247183 50.53994918
42 cg10081761 67.61010728
43 cg10144554 67.62775268
44 cg26474549 63.24110672
45 cg11220950 68.09712027
46 cg14244013 68.9758611
47 cg09183450 57.31578204
48 cg02458065 59.29206663
49 cg21308660 62.45412196
50 cg07160746 71.23800113
51 cg26049726 66.32905138
52 cg13146465 70.86391869
53 cg17241310 70.86391869
54 cg03259243 67.77244495
55 cg24843474 62.535572
56 cg18330573 59.95200452
57 cg05145573 65.87380011
58 cg19756358 65.87380011
59 cg01857475 56.2852908
60 cg14559388 58.93562959
61 cg11806439 65.90909091
62 cg24495585 64.27508503
63 cg14750948 67.47247318
64 cg00805193 63.31874647
65 cg11948988 63.97515528
66 cg23923820 56.75818746
67 cg05324519 66.30434783
68 cg20074395 61.43774704
69 cg25446076 66.38198758
70 cg26225694 64.51863354
71 cg13997680 69.26171654
72 cg01031101 69.26171654
73 cg09523275 69.26171654
74 cg18675097 69.26171654
75 cg00203284 63.67165443
76 cg18911817 62.79644269
77 cg06712559 61.86476567
78 cg27252696 63.98574252
79 cg17380661 63.98574252
80 cg15261247 62.07298137
81 cg10046388 62.07298137
82 cg23725149 62.67645398
83 cg05928806 66.54079616
84 cg02439789 51.80688876
85 cg24456130 64.97741389
86 cg03208426 62.3447205
87 cg10960266 67.34189723
88 cg26702958 65.76792772
89 cg22001211 55.87238848
90 cg08005730 60.89779785
91 cg06640773 65.17504235
92 cg06059409 65.17504235
93 cg21827912 60.34373235
94 cg12314713 62.61998871
95 cg06890720 58.86857708
96 cg03122669 64.47628458
97 cg16356837 64.47628458
98 cg10694030 64.47628458
99 cg01615018 64.47628458
100 cg02568274 64.47628458
101 cg00996827 58.37803501
102 cg21402748 58.37803501
103 cg03884587 65.18210051
104 cg12427163 60.32255788
105 cg18443359 67.69833427
106 cg20732876 43.09359119
107 cg14718275 43.09359119
108 cg18130044 60.60488425
109 cg09076431 63.13523433
110 cg04365202 63.13523433
111 cg21918126 53.56789949
112 cg27348152 59.42970073
113 cg16352140 66.99251835
114 cg22879098 60.34726143
115 cg07995570 65.06916996
116 cg05164926 61.94019274
117 cg24104241 64.77625635
118 cg12897502 64.7515528
119 cg15489799 53.57142857
120 cg15555898 62.83526256
121 cg23683588 69.62168267
122 cg18361098 69.62168267
123 cg17666418 62.27766798
124 cg25913016 68.79234896
125 cg11598006 60.4743083
126 cg05297854 57.46400339
127 cg11213574 57.46400339
128 cg15831346 59.79672501
129 cg03242819 59.72744361
130 cg26697286 56.86053077
131 cg26128121 47.2473179
132 cg04655953 61.28599661
133 cg22521151 53.03500847
134 cg00207921 53.03500847
135 cg20544808 53.03500847
136 cg18792131 53.03500847
137 cg16780502 64.18690006
138 cg14789818 64.18690006
139 cg20699586 64.18690006
140 cg06216683 48.57425184
141 cg07258474 63.97515528
142 cg20129806 64.56451158
143 cg22350285 55.15598532
144 cg02587648 53.20087521
145 cg27541454 52.99160501
146 cg27180750 64.03162055
147 cg24703339 54.87718803
148 cg00352681 51.78924337
149 cg15541630 65.09034444
150 cg04759874 63.70694523
151 cg19148866 63.70694523
152 cg05740609 63.70694523
153 cg22437074 63.70694523
154 cg05523911 48.88038549
155 cg17804348 63.63636364
156 cg07178825 63.63636364
157 cg23077820 67.03133823
158 cg22989843 67.03133823
159 cg05982757 60.62605872
160 cg16983211 60.62605872
161 cg15700739 60.62605872
162 cg24535439 57.9474873
163 cg00262621 57.9474873
164 cg24362661 51.21047431
165 cg07519536 69.23348391
166 cg19729116 69.23348391
167 cg12776171 60.66840768
168 cg21475076 50.1729249
169 cg07952270 62.24943535
170 cg04293152 54.84895539
171 cg26968008 62.12944664
172 cg16668728 63.09641446
173 cg04591032 66.02202146
174 cg23905164 66.02202146
175 cg26320000 58.4133258
176 cg13461447 59.42264257
177 cg23948811 58.81474104
178 cg07126167 62.7258611
179 cg14216876 46.13565782
180 cg03281661 66.16318464
181 cg27116061 66.16318464
182 cg07853668 63.68577075
183 cg06618740 51.16106719
184 cg26048630 63.44932242
185 cg00002593 53.60671937
186 cg02448922 63.2799266
187 cg06817455 54.15725579
188 cg08933227 61.44833427
189 cg00158333 68.43591191
190 cg02745847 68.43591191
191 cg15748470 60.28020892
192 cg12244647 55.92532468
193 cg27631599 51.52103331
194 cg14673618 60.97543761
195 cg00776293 55.63946923
196 cg14514924 55.63946923
197 cg12691464 62.14356296
198 cg19253333 58.78035008
199 cg13693328 57.55575946
200 cg15097000 61.88594015
其中，cg07553761,cg22685975,cg04688351,cg07160746,cg13146465的整体表现最好，AUC值分别为0.753，0.710，0.715，0.712和0.709，见图2。
实施例3
使用所有200个甲基化标志物构建的模型进行检测验证，在60例恶性结节和20例良性结节样本的测试集中，AUC达到0.823,准确度0.838，灵敏度为90.0％，特异性为65.0％，阳性预测值PPV为88.5％，阴性预测值NPV为68.4％，见图2和表2。
在进一步100例恶性结节和40例良性结节样本的独立验证集中，准确度82.1％,灵敏度为95.0％，特异性为50.0％，PPV为82.6％，NPV为80.0％，见表2。
表2：20，50，100和200个甲基化标记物模型的具体表现数据
Figure PCTCN2021142066-appb-000008
实施例4
使用前100个甲基化标志物(表1中的1-100)构建的模型进行检测验证，在60例恶性结节和20例良性结节样本的测试集中，AUC达到0.83,准确度85.0％,灵敏度为93.3％，特异性为60.0％，PPV为87.5％，NPV为75％，见图3和表2。
在进一步100例恶性结节和40例良性结节样本的独立验证集中，AUC达到0.84,准确度80.0％,灵敏度为99.0％，特异性为32.5％，PPV为78.6％，NPV为92.9％，见图4和表2。
在1)肺癌早期(I期)样本集的验证中(Stage IA+IB，N＝90),灵敏度高达97.1％,见图5。
在2)6-20mm肺结节尺寸的验证中(N＝100),AUC达到0.84,见图6。
在3)在不同结节类别的验证中，实性结节(N＝10)灵敏度为80.0％；部分实性结节(N＝11)灵敏度为81.8％；磨玻璃结节(N＝5)灵敏度为100％，高于PET-CT的检测效果，见图7。
实施例5
使用前50个甲基化标志物((表1中的1-50)构建的模型进行检测验证，在60例恶性结节和20例良性结节样本的测试集中，AUC达到0.81,准确度80.0％,灵敏度为80.0％，特异性为80.0％，PPV为92.3％，NPV为57.1％，见图3和表2。
在进一步100例恶性结节和40例良性结节样本的独立验证集中，准确度79.3％,灵敏度为85.0％，特异性为65.0％，PPV为85.9％，NPV为63.4％，见表2。
实施例6
使用前20个甲基化生物标志物(表1中的1-20)，即cg07553761，cg20607577，cg22685975，cg22878622cg17915922,cg05310764,cg22796313,cg20321153,cg13114497,cg08691548,cg04387597,cg15634980,cg21428324,cg18205770,cg00147160,cg15622158,cg18011916,cg03240324，cg06335867,cg26970841。
在60例恶性结节和20例良性结节样本的测试集中(N＝80)，AUC达到0.81，准确度达到80.0％，灵敏度81.7％，特异性为75.0％，PPV为90.7％，NPV为57.7％，见图3和表2；在进一步100例恶性结节和40例良性结节样本的独立验证集中(N＝140)，准确度达到83.6％，灵敏度91.0％，特异性为65.0％，PPV为86.7％，NPV为74.3％，见表2。
综上可见，利用该方法筛选出来的位点与肺结节良恶性的诊断有非常高的相关性。
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 (15)

  1. 与肺结节良恶性相关的甲基化生物标记物或其组合，其特征在于，包括有以下至少一种甲基化生物标记物：cg07553761,cg22685975,cg04688351,cg07160746,cg13146465。
  2. 根据权利要求1所述的与肺结节良恶性相关的生物标记物或其组合，其特征在于，还包括有以下至少一种甲基化生物标记物：cg20607577,cg22878622,cg17915922,cg05310764,cg22796313,cg20321153,cg13114497,cg08691548,cg04387597,cg15634980,cg21428324,cg18205770,cg00147160,cg15622158,cg18011916,cg03240324，cg06335867,cg26970841。
  3. 根据权利要求1或2所述的与肺结节良恶性相关的生物标记物或其组合，其特征在于，还包括以下甲基化生物标记物中的至少一种：cg03978375,cg24826867,cg17384889,cg18694169,cg10253847,cg09163035,cg01759136,cg21643086,cg23279117,cg12820681,cg07816637,cg17132517,cg09578794,cg00171421,cg12445832,cg23999112,cg26528551,cg11289039,cg15072319,cg16247183,cg10081761,cg10144554,cg26474549,cg11220950,cg14244013,cg09183450,cg02458065,cg21308660,cg26049726,cg17241310,cg03259243,cg24843474,cg18330573,cg05145573,cg19756358,cg01857475,cg14559388,cg11806439,cg24495585,cg14750948,cg00805193,cg11948988,cg23923820,cg05324519,cg20074395,cg25446076,cg26225694,cg13997680,cg01031101,cg09523275,cg18675097,cg00203284,cg18911817,cg06712559,cg27252696,cg17380661,cg15261247,cg10046388,cg23725149,cg05928806,cg02439789,cg24456130,cg03208426,cg10960266,cg26702958,cg22001211,cg08005730,cg06640773,cg06059409,cg21827912,cg12314713,cg06890720,cg03122669,cg16356837,cg10694030,cg01615018,cg02568274。
  4. 根据权利要求1或2或3所述的与肺结节良恶性相关的生物标记物或其组合，其特征在于，还包括以下甲基化生物标记物中的至少一种：cg00996827,cg21402748,cg03884587,cg12427163,cg18443359,cg20732876,cg14718275,cg18130044,cg09076431,cg04365202,cg21918126,cg27348152,cg16352140,cg22879098,cg07995570,cg05164926,cg24104241,cg12897502,cg15489799,cg15555898,cg23683588,cg18361098,cg17666418,cg25913016,cg11598006,cg05297854,cg11213574,cg15831346,cg03242819,cg26697286,cg26128121,cg04655953,cg22521151,cg00207921,cg20544808,cg18792131,cg16780502,cg14789818,cg20699586,cg06216683,cg07258474,cg20129806,cg22350285,cg02587648,cg27541454,cg27180750,cg24703339,cg00352681,cg15541630,cg04759874,cg19148866,cg05740609,cg22437074,cg05523911,cg17804348,cg07178825,cg23077820,cg22989843,cg05982757,cg16983211,cg15700739,cg24535439,cg00262621,cg24362661,cg07519536,cg19729116,cg12776171,cg21475076,cg07952270,cg04293152,cg26968008,cg16668728,cg04591032,cg23905164,cg26320000,cg13461447,cg23948811,cg07126167,cg14216876,cg03281661,cg27116061,cg07853668,cg06618740,cg26048630,cg00002593,cg02448922,cg06817455,cg08933227,cg00158333,cg02745847,cg15748470,cg12244647,cg27631599,cg14673618,cg00776293,cg14514924,cg12691464,cg19253333,cg13693328,cg15097000。
  5. 根据权利要求2所述的与肺结节良恶性相关的生物标记物或其组合，其特征在于，所述甲基化生物标记物组合为cg07553761,cg20607577,cg22685975,cg22878622,cg17915922,cg05310764,cg22796313,cg20321153,cg13114497,cg08691548,cg04387597,cg15634980, cg21428324,cg18205770,cg00147160,cg15622158,cg18011916,cg03240324，和cg06335867,cg26970841。
  6. 根据权利要求3所述的与肺结节良恶性相关的生物标记物或其组合，其特征在于，所述甲基化生物标记物组合为cg07553761,cg20607577,cg22685975,cg22878622,cg17915922,cg05310764,cg22796313,cg20321153,cg13114497,cg08691548,cg04387597,cg15634980,cg21428324,cg18205770,cg00147160,cg15622158,cg18011916,cg03240324，cg06335867,cg26970841，cg03978375,cg24826867,cg17384889,cg18694169,cg10253847,cg09163035,cg01759136,cg21643086,cg23279117,cg12820681,cg07816637,cg04688351,cg17132517,cg09578794,cg00171421,cg12445832,cg23999112,cg26528551,cg11289039,cg15072319,cg16247183,cg10081761,cg10144554,cg26474549,cg11220950,cg14244013,cg09183450,cg02458065,cg21308660,和cg07160746。
  7. 根据权利要求3所述的与肺结节良恶性相关的生物标记物或其组合，其特征在于，所述甲基化生物标记物组合为cg07553761,cg20607577,cg22685975,cg22878622,cg17915922,cg05310764,cg22796313,cg20321153,cg13114497,cg08691548,cg04387597,cg15634980,cg21428324,cg18205770,cg00147160,cg15622158,cg18011916,cg03240324，cg06335867,cg26970841，cg03978375,cg24826867,cg17384889,cg18694169,cg10253847,cg09163035,cg01759136,cg21643086,cg23279117,cg12820681,cg07816637,cg04688351,cg17132517,cg09578794,cg00171421,cg12445832,cg23999112,cg26528551,cg11289039,cg15072319,cg16247183,cg10081761,cg10144554,cg26474549,cg11220950,cg14244013,cg09183450,cg02458065,cg21308660,cg07160746,cg26049726,cg13146465,cg17241310,cg03259243,cg24843474,cg18330573,cg05145573,cg19756358,cg01857475,cg14559388,cg11806439,cg24495585,cg14750948,cg00805193,cg11948988,cg23923820,cg05324519,cg20074395,cg25446076,cg26225694,cg13997680,cg01031101,cg09523275,cg18675097,cg00203284,cg18911817,cg06712559,cg27252696,cg17380661,cg15261247,cg10046388,cg23725149,cg05928806,cg02439789,cg24456130,cg03208426,cg10960266,cg26702958,cg22001211,cg08005730,cg06640773,cg06059409,cg21827912,cg12314713,cg06890720,cg03122669,cg16356837,cg10694030,cg01615018,和cg02568274。
  8. 根据权利要求4所述的与肺结节良恶性相关的生物标记物或其组合，其特征在于，所述甲基化生物标记物组合为全部权利要求1至权利要求4中所述甲基化生物标记物。
  9. 权利要求1-8任一项所述甲基化生物标记物或其组合作为肺癌相关甲基化分子标记物在检测肺结节良恶性、和/或肺癌检测中的应用。
  10. 一种用于检测肺结节良恶性、和/或肺癌的试剂盒，其特征在于，所述试剂盒包含检测权利要求1-8任一项所述甲基化生物标记物或其组合的甲基化水平的试剂。
  11. 根据权利要求10所述的用于检测肺结节良恶性、和/或肺癌的试剂盒，其特征在于，所述试剂盒采用以下检测平台：包括采用PCR扩增法、荧光定量PCR法、数字PCR法、液相芯片法、一代测序法、三代测序法、二代测序法、焦磷酸测序法、亚硫酸氢盐测序、甲基化芯片法或它们的组合所使用的试剂。
  12. 根据权利要求11所述的用于检测肺结节良恶性、和/或肺癌的试剂盒，其特征在于，采用二代测序法。
  13. 根据权利要求10所述的用于检测肺结节良恶性、和/或肺癌的试剂盒，其特征在于，所述肺结节包括实性或部分实性或磨玻璃结节，优选为部分实性或磨玻璃结节。
  14. 根据权利要求10所述的用于检测肺结节良恶性、和/或肺癌的试剂盒，其特征在于，所述肺癌为早期肺癌，优选为I期肺癌。
  15. 一种肺结节良恶性的检测方法，其特征在于，包括以下步骤：
    s1、提取待测样本的cfDNA、亚硫酸氢盐处理提取的cfDNA、甲基化DNA建库；
    s2、根据权利要求1-8任一项所述的甲基化生物标记物或其组合进行肺结节良恶性特异性panel靶向杂交捕获甲基化DNA预文库；
    s3、靶向捕获的终文库定量，二代测序；
    s4、数据预处理，获得探针捕获得到的真实数据；
    s5、对权利要求1-8任一项所述的甲基化生物标记物或其组合甲基化数据分析。
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