CN112899359A - Methylation marker for detecting benign and malignant lung nodules or combination and application thereof - Google Patents
Methylation marker for detecting benign and malignant lung nodules or combination and application thereof Download PDFInfo
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Abstract
The invention relates to a biomarker combination related to benign and malignant pulmonary nodules, which comprises at least one of the following components: cg07553761, cg22685975, cg04688351, cg07160746, cg13146465 and the like for 200 markers. The invention also relates to a detection and related kit of the marker combination. The 20 markers of the present invention have substantially the same diagnostic power as the 100 marker combinations. The invention jointly analyzes the co-methylation characteristics of a plurality of methylated cytosines on at least 1 genome segment as a biomarker for judging the benign and malignant onset of pulmonary nodules, and the accuracy is far higher than the detection of other protein plasma biomarkers, especially on the differentiation of stage I lung cancer; in the non-solid nodules, the accuracy is far higher than that of PET-CT, the effect of expelling yin can be effectively achieved, and the occurrence of false positive is reduced.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a methylation marker for detecting benign and malignant pulmonary nodules or a combination and application thereof.
Background
Lung cancer is the most common cause of cancer death worldwide, with morbidity at the forefront of all malignancies. Due to environmental pollution, the incidence of lung cancer is rising year by year, and in China, about 78.1 ten thousand of lung cancer patients and about 62.6 ten thousand of death cases are the first death cases of cancer every year. By 2025 years, the number of new lung cancer patients in China is estimated to be 100 ten thousand every year. As most of clinical diagnosis lung cancer cases are in the late stage, the surgery treatment opportunity is lost, the prognosis of lung cancer is extremely poor, and the 5-year survival rate of lung cancer in China is only 16.1 percent. Goldstraw and other researches find that 5-year survival rate of early-stage Ia lung cancer can reach more than 80 percent through surgical resection, and 5-year survival rate of middle-and late-stage IIIa-IV lung cancer is less than 36 percent after surgical treatment, so that the screening of lung cancer is hopeful to improve lung cancer survival and reduce lung cancer death rate.
The early stage of lung cancer exists in the form of pulmonary nodules, which are a common clinical phenomenon and comprise benign nodules and malignant nodules, the early detection of the malignant pulmonary nodules is relatively hidden, and if the early intervention is not performed, the course of the disease is rapid, the malignancy is strong, and the prognosis is poor. At present, the qualitative diagnosis of the pulmonary nodules is difficult, and about 30 percent of the clinically surgically excised pulmonary nodules are benign, so that the correct evaluation of the benign and malignant pulmonary nodules is helpful for selecting correct treatment means, and can help to significantly improve the survival rate of patients and improve prognosis.
There are many methods for diagnosing pulmonary nodules, but the qualitative diagnosis is difficult. Current methods commonly used for diagnosis of pulmonary nodules include: imaging examination, percutaneous puncture biopsy, biopsy under a virtual bronchoscope navigation system and pathological tissue examination of thoracoscope surgery: 1) imaging examinations are commonly used for their non-invasive nature, primarily to observe the size, volume doubling time, and morphological features of lung nodules. The conventional imaging examination methods include breast CT, positron emission tomography (PET-CT), and the like. Imaging tests are problematic, for example, CT may have too many false positives, which may lead to over-diagnosis, over-treatment, waste of medical resources, and increased anxiety in the subject. PET-CT only has obvious detection effect on solid nodules and has unsatisfactory effect on non-solid nodules such as ground glass. 2) When the sensitivity of PET diagnosis is poor or false positive indication appears, the percutaneous lung puncture biopsy guided by CT can be selected, and the diagnosis accuracy is about 90 percent. However, severe complications such as hemorrhage, pneumothorax, sudden death, etc. are easily caused by percutaneous lung puncture biopsy under the guidance of X rays, CT or ultrasound. The VBN developed in recent years is a three-dimensional imaging technique based on CT, which establishes a virtual bronchial path through image recognition, guides a bronchoscope to reach a target lesion for biopsy, and thus achieves the purpose of improving the positive rate of the minimally invasive biopsy of the lung nodule. However, in practice, problems such as nosocomial infection due to biopsy are also very common.
In conclusion, screening pulmonary nodules and accurately performing differential diagnosis of benign and malignant masses are important problems to be solved urgently for preventing and treating lung cancer. There is a need to find and establish an effective diagnostic model for the clinical pain point and a molecular marker capable of being used for screening and diagnosis.
cfDNA (cell-free DNA) is a small fragment of DNA free from peripheral blood, derived from normal or tumor cell metabolism and apoptosis, and contains genetic information such as somatic mutation and DNA methylation. A technology for grasping the occurrence and development of diseases by detecting disease-specific cfDNA fragments is called Liquid Biopsy (Liquid Biopsy), and compared with the traditional tissue Biopsy, the technology has the advantages of rapidness, convenience, small injury and the like. In 2014, a 640-tumor-type study from the group of Bert Vogelstein and Kenneth Kinzler found that more than 75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, melanoma, hepatocellular, and head and neck cancers detected the presence of ctDNA. ctDNA is thus a sensitive and specific biomarker with broad applicability and can be used in the clinic and research of a wide variety of different types of cancers. Lucinstead of biopsy, the technical and theoretical feasibility of liquid biopsy was demonstrated by cfDNA whole genome methylation sequencing in 2015 by professor ruyu; zhang 40525in 2017, professor team described the ctDNA profile of tumor burden and tumor origin using methylation quantification of ctDNA. The Turner team searches for breast cancer specific somatic mutation sites by using high-throughput sequencing, monitors the dynamic change of the breast cancer specific somatic mutation sites, and proves that the ctDNA detection can find the recurrence and metastasis of the tumor earlier than the CT detection. Recent studies have found that combining ctDNA mutations in blood with other analytes can provide an earlier and better diagnosis of common and surgically resectable cancers such as lung, ovarian, liver, stomach, breast, prostate, esophageal and colorectal cancers. More and more clinical researches show that plasma ctDNA can be used as a biomarker to be applied to early diagnosis, screening, prediction and treatment response of tumors, monitoring of tumor size and recurrence and the like. At present, the international research direction is to integrate multiomics/multiple molecular markers and multigenes/multiple sites to improve the sensitivity and specificity of the detection technology so as to meet the clinical requirements on detection products.
Disclosure of Invention
One of the objectives of the present invention is to provide biomarkers related to benign and malignant pulmonary nodules or combinations thereof, which can be used to diagnose the onset of benign and malignant pulmonary nodules.
The technical scheme for achieving the purpose is as follows.
A biomarker associated with benign and malignant pulmonary nodules, or a combination thereof, comprising at least one of the following methylated biomarkers: cg07553761, cg22685975, cg04688351, cg07160746 and cg 13146465.
In some embodiments, the biomarker associated with benign and malignant pulmonary nodules, or a combination thereof, further comprises 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, cg 26970841.
In some of these embodiments, at least one of the following methylated biomarkers is also included: cg03978375, cg24826867, cg17384889, cg18694169, cg10253847, cg09163035, cg01759136, cg21643086, cg23279117, cg12820681, cg07816637, cg17132517, cg09578794, cg00171421, cg12445832, cg23999112, cg26528551, cg11289039, cg15072319, cg16247183, cg10081761, cg10144554, cg 264749, cg11220950, cg 14213, cg09183450, cg02458065, cg 213243 08660, cg26049726, cg 41310, cg 59594549, cg 112459274, cg 0634063406340634064006400640065, cg 72774705, cg 774705, cg 7779,779, cg 7740779, cg 77407708, cg 7708,779, cg 7708,7780, cg 77407708,7780, cg 5977317779,7780, cg 598474, cg 057768, cg 72977768, cg 7768, cg 77317768, cg 7768, cg 72977768, cg 7768, cg 7297778, cg 05778, cg 7731778, cg 778, cg 07778, cg 7731778, cg 7795 g 200778, cg 07778, cg 778, cg 07778, cg 97778, cg 07778, cg 778, cg 97778.
In some of these embodiments, at least one of the following methylated biomarkers is also included: cg 996827, cg21402748, cg03884587, cg12427163, cg18443359, cg20732876, cg14718275, cg18130044, cg09076431, cg04365202, cg21918126, cg27348152, cg16352140, cg22879098, cg 07570, cg05164926, cg24104241, cg12897502, cg 89799, cg15555898, cg23683588, cg18361098, cg17666418, cg25913016, cg11598006, cg 11297854, cg11213574, cg 31346, cg03242819, cg 266986, cg 28121, cg04655953, cg 06980541923368, cg 33054105410541054105410541054135, cg 3305410541054135, cg 33054105410541054135, cg 329241054105410541054135, cg 054105410541054105410541054135, cg 0541054105410541054135, cg 054105410541054148, cg 2235054105410541054105410541054148, cg 223572923146, cg 2235726520, cg 2235729205410541054148, cg 2235726547, cg 2235726405410541054135, cg 2235726405410541054148, cg 22357264054105410541057, cg 223572640541054148, cg 223572640541054105410541057, cg 227346, cg 2235726405410541054105410541054105410541057, cg 227346, cg 42819, cg 2235726405410541054105410541054135, cg 223572640541054135, cg 223572640426, cg 2235726405410541054105410541054105410541054105410541054135, cg 2235726427, cg 223572640426, cg 22357264044768, cg 2227, cg 223572640426, cg 227353, cg 22435427, cg 223572640426, cg 227346, cg 2227, cg 227346, cg.
In some of these embodiments, the methylated biomarker, or combination thereof, is: cg07553761, cg20607577, cg22685975, cg22878622, cg17915922, cg05310764, cg22796313, cg20321153, cg13114497, cg08691548, cg04387597, cg15634980, cg21428324, cg18205770, cg00147160, cg15622158, cg18011916, cg03240324, cg06335867, and cg 26970841.
In one embodiment, the methylated biomarker, or combination thereof, is: cg07553761, cg20607577, cg22685975, cg22878622, cg17915922, cg05310764, cg22796313, cg20321153, cg13114497, cg08691548, cg04387597, cg15634980, cg21428324, cg18205770, cg00147160, cg15622158, cg18011916, cg 03224, cg06335867, cg26970841, cg03978375, cg24826867, cg17384889, cg18694169, cg10253847, cg09163035, cg01759136, cg 21686, cg 232232128117, cg 20620681, cg 816637, cg 04351, cg 3206517, cg 124879194, cg 72579136, cg21643086, cg 23291117, cg 2065778, cg 725778, cg 72574705, cg 725791, cg 72725791, cg 7272725791, cg 7272725751, cg 1124705, cg 72727272727272725753, cg 72727272729751, cg 1124727, cg 72727272727272725753, cg 7272725753, cg 72727272727272725753, cg 72727272729775, cg 72727272727272729760, cg 7272729775, cg 11253, cg 1124727, cg 079127, cg 074727, cg 727272.
In some of these embodiments, the methylated biomarker, or combination thereof, is: cg07553761, cg20607577, cg22685975, cg22878622, cg17915922, cg05310764, cg22796313, cg20321153, cg13114497, cg08691548, cg 04597, cg15634980, cg21428324, cg18205770, cg 001160, cg15622158, cg18011916, cg 03224, cg 063063867, cg26970841, cg03978375, cg24826867, cg 173889, cg18694169, cg10253847, cg09163035, cg01759136, cg 21621686, cg 232232117, cg 2068181, cg 816637, cg 041717292337727, cg 9277927792599720, cg 92779277317779, cg 92773177317779, cg 9277317779, cg 92773177317779, cg 924785, cg 0577317779, cg 92774785, cg 057731773177317779, cg 92774705, cg 92774785, cg 057779, cg 927759977779, cg 7759977779, cg 057779, cg 927779, cg 057779, cg 057791, cg 927791, cg 057779, cg 927791, cg 2007779, cg 927791, cg 200774785, cg 057791, cg 2007791, cg 200774785, cg 927791, cg 2007791, cg 057791, cg 2007791, cg 7297cg 2007791, cg 72977346, cg 927791, cg 2007791, cg 2007779, cg 64043297cg 2007791, cg 2007779, cg 7297cg 2007779, cg 72977346, cg 64043297cg 2007779, cg 640432977346, cg 2007779, cg 72977346, cg 2007791, cg 2007779, cg 2007791, cg 927791, cg 97cg 927791, cg 2007791, cg 7297.
In some embodiments, the combination of methylated biomarkers is all 200 of the methylated biomarkers described above.
In another aspect of the invention, the application of the above biomarkers or the combination thereof as lung cancer related methylation molecular markers in detecting benign and malignant lung nodules and/or lung cancer is also provided.
In another aspect of the present invention, a kit for detecting benign and malignant lung nodules and/or lung cancer is provided, wherein the kit comprises a reagent for detecting the methylation level of the DNA methylation molecular marker or the combination thereof.
In some of these embodiments, the kit can be used in the following assay platforms: including reagents used in PCR amplification, fluorescent quantitative PCR, digital PCR, liquid phase chip, first generation sequencing, third generation sequencing, second generation sequencing (Sanger), pyrosequencing, bisulfite sequencing, methylation chip, or combinations thereof. In some preferred embodiments, second generation sequencing is preferred.
In some of these embodiments, the lung nodules are different classifications (solid or partially solid or ground glass nodules), lung nodule sizes, and different stages of malignant nodules.
In some of these embodiments, the lung nodules are partially solid or ground glass nodules.
In some of these embodiments, the lung cancer is early stage lung cancer, preferably stage I lung cancer.
In another aspect of the present invention, there is provided a method for detecting the methylated biomarker or the combination thereof, comprising the steps of:
s1, extracting cfDNA of a sample to be detected, treating the extracted cfDNA and methylated DNA by bisulfite, and establishing a library;
s2, lung nodule benign and malignant specificity panel targeted hybridization capture methylation pre-library;
s3, final library quantification of targeted capture, second generation sequencing;
s4, preprocessing data to obtain real data obtained by probe capture;
s5, analyzing the methylation data of the above methylation biomarkers or the combination thereof.
According to the invention, the plasma genome fragment combination can be used as a lung nodule diagnostic marker by researching the methylation modification difference of the plasma methylation modified genome fragments in different lung nodule types, lung cancer patients in different stages and benign nodule groups. Further studies found that the first 20 markers had substantially the same diagnostic power as the 100 marker combinations. The invention jointly analyzes the co-methylation characteristics of a plurality of methylated cytosines on at least 1 genome segment as a biomarker for judging the benign and malignant onset of pulmonary nodules, and the accuracy is far higher than the detection of other protein plasma biomarkers, especially on the differentiation of stage I lung cancer; in non-solid nodules (partial solid and ground glass nodules), the accuracy is far higher than that of PET-CT, the negative expelling effect can be effectively achieved, and the occurrence of false positive is reduced.
Drawings
FIG. 1 is a flow chart of the biomarker study according to the present invention.
Figure 2 is a schematic representation of the ROAUC of the five independent markers in example 2.
FIG. 3 shows the performance of the 20-, 50-, 100-, and 200-marker models in examples 2, 3, 4, and 5 in the test set, where the AUC values in the test set are 0.81, 0.81, 0.83, and 0.82, respectively.
FIG. 4 is a comparison of example 4 performance and model on independent validation sets with the clinical common models Mayo model and VA model in independent validation sets: in the independent validation set, the AUC was 0.84, which is higher than 0.59 for Mayo model and 0.54 for VA model.
FIG. 5 is the representation of the 100-marker model in example 4 at early malignant nodules (STAGE I).
FIG. 6 is a representation of the 100-marker model in example 4 at 6-20mm nodules, in the independent validation set: AUC was 0.84, higher than 0.60 for Mayo model and 0.51 for VA model.
FIG. 7 is a graph of the performance of the 100-marker model in example 4 in different nodule types, independent validation set and comparison to PET-CT.
Detailed Description
The experimental procedures of the present invention, without specifying the specific conditions in the following examples, are generally carried out according to conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.
The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
In one embodiment of the invention, at least one or a combination of 200 plasma gene methylation markers for diagnosing benign and malignant pulmonary nodules and application are disclosed. Specifically, the inventors found that the genome fragments with obvious abnormal methylation modification in the plasma of the following lung nodule malignant patients, namely cg07553761, cg20607577, cg22685975, cg22878622, cg17915922, cg05310764, cg22796313, cg 20353, cg 13197, cg08691548, cg04387597, cg15634980, cg21428324, cg18205770, cg 001160, cg15622158, cg18011916, cg 03224, cg06335867, cg26970841, cg 039775, cg24826867, cg17384889, cg 1868469, cg 102847, cg09163035, cg01759136, cg 01759186, cg 43080, cg 6404320679814768, cg 924777924708, 4777924708, 470577814798, 4705774708, 470577924798, 4798, 4705774798, 4705, 4708, 4705774798, 4798, 4708 cg 924798, 4705774705, 4798, 4708, 470577598, cg 9277599777599777819793, cg 0577819727, cg 9277819777819727, cg 20077814798, 4798, cg 05774798, 4798, 4705 cg 03640432924798, 4798, cg 924798, cg 200774798, 4705, 4798, 4705, 7346, 4798, cg 64048, cg 6404774798, 4798, cg 924705, 4705, cg 924798, 4798, 4705 cg 927791, 4798, 4705, 7327, cg 64048, cg 72977791, 4798, cg 927791, 7346, cg 64048, 4705, cg 64048, cg 640480, cg 64047791, cg 640480, cg 6404327791, cg 64047791, cg 64048, cg 6404774705, cg 7297774705, cg 64047791, cg 64048, cg 640480, cg 64047791, cg 034705, cg 640480, cg 034705, cg 927791, cg 7791, cg 924705, cg 7791, cg 72977791, cg 329760, cg 640480, cg 3297cg 729760, cg 32977780, cg 3297cg 729760, cg 7297cg 3297cg 729760, cg 7297cg 729760, cg 7297cg 640480, cg 6404, cg21918126, cg27348152, cg16352140, cg22879098, cg07995570, cg05164926, cg24104241, cg12897502, cg15489799, cg15555898, cg23683588, cg18361098, cg 66418, cg25913016, cg11598006, cg05297854, cg11213574, cg 15846, cg03242819, cg26697286, cg26128121, cg04655953, cg22521151, cg00207921, cg20544808, cg 92131, cg16780502, cg14789818, cg20699586, cg 06283, cg 58474, cg 201806, cg 29806, cg 025807921, cg 337768, cg 227768, cg 227705410541054135, cg 227768, cg 22770541054105410541054135, cg 22357246, cg 224354054105410541054148, cg 223505410541054148, cg 223572357246, cg 227768, cg 227705410541054105410541054135, cg 227346, cg 223572974032057, cg 227346, cg 223572974027, cg 2235724727, cg 22357246, cg 22435427, cg 22435460, cg 22357227, cg 227746, cg 227346, cg 2243547768, cg 227346, cg 227385, cg 227346 cGC057, cg 227346 cGC05cGC05cGC05cGC057 cGC057 cGC05cGC05cGC05cGC05cGC05cGC05cGC05cGC05cGC05cGC726427 cGC05cGC726427 cGC7235 cGC72435427 cGC7227 cGC05cGC05cGC7264044727 cGC726427 cGC7264044735 cGC7235 cGC72435473817227 cGC7235 cGC7227 cGC7235 cGC7227 cGC7243cGC7243cGC7235 cGC7235 cGC7227 cGC7235 cGC72.
Targeting low amounts of tumor DNA (circulating) in plasma cell free DNA (cfDNA)tomor DNA, ctDNA) samples and bisulfite treatment of DNA damage, using standard medical banking and hybrid capture techniques: AnchorDx EpiVisioTM Methylation Library Prep Kit(AnchorDx,Cat#A0UX00019)，AnchorDx EpiVisioTMIndexing PCR Kit (AnchorDx, Cat # A2DX00025) and AnchorDx EpiVisioTM Target Enrichment Kit(AnchorDx,Cat#A0UX00031)。
Lung nodules include different categories: solid nodules, partial solid nodules, or ground glass nodules; malignant nodule malignancy was staged according to the TNM staging system.
Lung nodules include solid or partially solid or ground glass nodules: the vitreous density nodules refer to the blurred nodule shadow in the lung, and the nodule density is slightly increased compared with the surrounding lung parenchyma, but the contour of blood vessels and bronchi in the nodules is still visible. The solid nodules are all the nodules with soft tissue density, the density is uniform, and the images of blood vessels and bronchi are covered. Partially solid nodules are nodules that contain both ground glass density and solid soft tissue density within, and are not uniform in density.
The stage of the malignant nodule lung cancer is divided into stages I to IV according to an AJCC TNM international standardized stage system, and the early stage lung cancer comprises stage I to II lung cancer.
In one embodiment of the present invention, a method for screening markers for diagnosing benign and malignant lung nodules based on methylation of circulating tumor DNA (ctDNA) comprises the following steps:
firstly, screening out lung cancer specific methylation markers according to a common database of TGCA lung cancer and cancer-side methylation chips and an autonomous tumor-related methylation database;
step two, extracting the plasma cfDNA of benign and malignant lung nodule patients, treating the extracted cfDNA and methylated DNA by bisulfite, and establishing a library;
step three, capturing a methylated DNA pre-library by targeted hybridization of lung nodule benign and malignant specific panel;
quantifying the final library of the target capture, and performing second-generation sequencing on the computer;
step five, preprocessing data to obtain real reads data (bam file) acquired by probe capture;
analyzing methylation data, screening benign and malignant diagnostic markers (markers) of the lung nodules, and screening a marker set finally used for analysis through analysis and filtration of consistent tissues and paired plasma;
seventhly, further screening markers in the plasma training set and the verification set by using the marker set selected in the sixth step, constructing an algorithm model, and verifying an independent data set;
and step eight, confirming the gene methylation marker and the algorithm model which are finally used for diagnosing the benign and malignant lung nodules.
Example 1 novel ctDNA methylation marker detection method for diagnosis of benign and malignant pulmonary nodules
1. Extraction and methylation database construction of plasma cfDNA or tissue DNA
1.1 extraction of plasma cfDNA or tissue DNA.
Specific procedures for plasma cfDNA extraction were performed according to the protocol of the MagMAX TM Cell-Free DNA Isolation Kit from Life corporation. The Tissue DNA extraction step was carried out according to the DNeasy Blood & Tissue Kit protocol of QIAGEN; index primer was purchased from New England Bioscience Inc. Cat # E7600S.
1.2 transformation
The extracted cfDNA (10ng) or tissue DNA (50ng) was subjected to bisulfite conversion to deaminate unmethylated cytosine in DNA to uracil while methylated cytosine remained unchanged to obtain bisulfite converted DNA, and the specific conversion was performed according to the EZ DNA Methylation-Lighting Kit instruction of Zymo Research.
1.3 end repair
1.3.1 the 17ul sample after conversion was reacted by adding the following reagents:
| components | Volume (ul) |
| Transformed sample | 17 |
| MEB1 Buffer | 2 |
| MEE2 Enzyme | 1 |
| |
20 |
1.3.2 the reaction was carried out in a PCR apparatus according to the following procedure:
37℃ 30min
95℃ 5min
hot lid 105 ℃.
1.3.3 when the second step of the PCR reaction (95 ℃) reaches 5min, the sample is immediately taken out of the PCR instrument, directly inserted into ice, placed for more than 2min and then subjected to the next operation
1.4 connection I
1.4.1 the following reaction solutions were prepared
1.4.2 the reaction was carried out in a PCR apparatus according to the following procedure:
1.5 amplification of I
1.5.1 the following reaction solutions were prepared
1.5.2 the reaction was carried out in a PCR apparatus according to the following procedure:
1.6 purification of I: 166ul of Agencour AMPure Beads (which need to be balanced at room temperature for half an hour in advance) diluted by 6 times at a ratio of 1: 166ul are added to purify a product after the Amplification I reaction, 21ul of EB is used for elution, and the purification steps are as follows:
1.6.1 taking the reaction product in the previous step, centrifuging, adding 166ul of Agencour AMPure Beads diluted by 1:6 times into each sample, and blowing and mixing by using a pipette.
1.6.2 incubate for 5min at room temperature.
1.6.3, centrifuging, and standing for 5min on a magnetic frame.
1.6.4 the supernatant was aspirated.
1.6.5 Add 200ul 80% EtOH, let stand for 30s, and suck off the ethanol.
1.6.6 repeat step 5) once.
1.6.7 centrifugation, the PCR tube was placed on a magnetic rack and the remaining ethanol was aspirated.
1.6.8 the beads were dried for 2-3min with the lid open, taking care not to overdry.
1.6.9 adding 21ul EB for elution, fully and uniformly blowing by a pipette, and standing for 3min at room temperature.
1.6.10 centrifuging, placing the PCR tube on a magnetic frame, and standing for 3 min.
1.6.11 aspirate 20ul of supernatant into a new PCR tube.
1.7 connection II
1.7.1 the following reaction solutions were prepared:
| components | Volume (ul) |
| Reaction volume of the last step | 20 |
| H2O | 4 |
| |
8 |
| MSR1 Reagent | 2 |
| MSR5 Reagent | 2 |
| MSE1 Enzyme | 2 |
| MSE5 Enzyme | 2 |
| |
40 |
1.7.2 placing in a PCR apparatus to perform the reaction according to the following procedure
| Temperature of | Time | Number of cycles |
| 37℃ | 30min | 1 |
| 95℃ | 5min | 1 |
| 10℃ | Hold | 1 |
1.8 Indexing PCR:
1.8.1 the following reaction solutions were prepared:
1.8.2 placing in a PCR apparatus to perform the reaction according to the following procedure
1.9 purification of II
Adding Agencour AMPure Beads (needing to be balanced at room temperature for half an hour in advance) to purify a product after the exponential PCR reaction, and eluting the product by using 41ul EB, wherein the specific purification steps are as follows:
1.9.1 the reaction product from the previous step was centrifuged, and 71ul of undiluted Agencourt AMPure Beads were added to each sample and pipetted and mixed well.
1.9.2 was incubated at room temperature for 5 min.
1.9.3 centrifuging, and standing on magnetic frame for 5 min.
1.9.4 the supernatant was aspirated.
1.9.5 Add 200ul 80% EtOH, let stand for 30s, and suck off the ethanol.
1.9.6 repeat step 5) once.
1.9.7 centrifugation, the PCR tube was placed on a magnetic rack and the remaining ethanol was aspirated.
1.9.8 the beads were dried for 2-3min with the lid open, taking care not to overdry.
1.9.9 adding 41ul EB for elution, fully and uniformly blowing by a pipette, and standing for 3min at room temperature.
1.9.10 centrifuging, placing the PCR tube on a magnetic frame, and standing for 3 min.
1.9.11 aspirate 20ul of supernatant into a new PCR tube.
1.10 quant quantification:
1ul of the library was quantitated using the Qubit dsDNA HS Assay Kit.
2. And (3) carrying out oligonucleotide probe capture enrichment on the sample after the library is built to obtain the final on-computer library in a specific region. The hybridization capture kit is xGen Lockdown Reagents of IDT company, and is specifically operated according to the instruction.
3. Sequencing the sample after hybridization capture by adopting a sequencer of Illumina company to obtain a sequencing result.
4. Analysis of the data:
performing conventional bioinformatics analysis processing on off-line original data of a sequencer, filtering low-quality (low QC, short length, too much N and the like) read lengths (reads) through fastp, then removing adapters, common sequences and PolyA/T at two ends of the reads to obtain an ideal insert sequence (target interval), comparing the reads with corresponding positions of hg19 by using a bismark, removing the reads according to UMI to obtain real reads data (bamfile) obtained by capturing each sample by a probe, and counting and analyzing the bam file to obtain methylated data for subsequent data reanalysis.
Example 2
The embodiment discloses a methylation specific biomarker for diagnosing lung nodules, based on 253 malignant nodules and 56 benign nodule plasma samples in a training set sample, the methylation library building method described in embodiment 1 is utilized, the methylation level difference of different groups is utilized to screen out biomarkers related to malignant lung nodules, and the independent data sets of the site data in the benign and malignant samples are verified, so that a total of 200 methylated DNA fragments which are most obviously distinguished from benign and malignant lung nodules are screened out, wherein the 200 methylated biomarkers (hereinafter referred to as sites or markers) and the independently distinguished AUC values are shown in Table 1:
TABLE 1200 methylation marker data
Among them, cg07553761, cg22685975, cg04688351, cg07160746 and cg13146465 have the best overall performance, and AUC values are 0.753, 0.710, 0.715, 0.712 and 0.709 respectively, as shown in fig. 2.
Example 3
The detection verification is carried out by using a model constructed by all 200 methylation markers, and in the test set of 60 malignant nodule samples and 20 benign nodule samples, the AUC reaches 0.823, the accuracy is 0.838, the sensitivity is 90.0%, the specificity is 65.0%, the PPV (positive predictive value) is 88.5%, and the NPV (negative predictive value) is 68.4%, which are shown in a figure 2 and a table 2.
In an independent validation set of further 100 malignant and 40 benign nodule samples, the accuracy was 82.1%, the sensitivity was 95.0%, the specificity was 50.0%, the PPV was 82.6%, and the NPV was 80.0%, see table 2.
Table 2: data on the characterization of the 20, 50, 100 and 200 methylation marker models
Example 4
The detection using the model constructed with the first 100 methylation markers (1-100 in table 1) confirmed that in the test set of 60 malignant and 20 benign nodule samples, AUC reached 0.83, accuracy 85.0%, sensitivity 93.3%, specificity 60.0%, PPV 87.5%, and NPV 75%, see fig. 3 and table 2.
In an independent validation set of further 100 malignant and 40 benign nodule samples, AUC reached 0.84, accuracy 80.0%, sensitivity 99.0%, specificity 32.5%, PPV 78.6%, NPV 92.9%, see fig. 4 and table 2.
In 1) validation of early Stage (Stage I) lung cancer sample set (Stage IA + IB, N ═ 90), the sensitivity was as high as 97.1%, see fig. 5.
In 2) validation of lung nodule size of 6-20mm (N ═ 100), AUC reached 0.84, see fig. 6.
In 3) validation in different nodule classes, the solidity nodule (N ═ 10) sensitivity was 80.0%; partial solidity knob (N ═ 11) sensitivity was 81.8%; the sensitivity of ground glass nodules (N ═ 5) was 100% higher than the detection effect of PET-CT, see fig. 7.
Example 5
The model constructed using the first 50 methylation markers ((1-50 in table 1) was used for assay validation, and in the test set of 60 malignant and 20 benign nodule samples, AUC reached 0.81, accuracy 80.0%, sensitivity 80.0%, specificity 80.0%, PPV 92.3%, and NPV 57.1%, see fig. 3 and table 2.
In an independent validation set of further 100 malignant and 40 benign nodule samples, the accuracy was 79.3%, the sensitivity was 85.0%, the specificity was 65.0%, the PPV was 85.9%, and the NPV was 63.4%, see table 2.
Example 6
The first 20 methylation biomarkers (1-20 in table 1) were used, namely cg07553761, cg20607577, cg22685975, cg22878622cg17915922, cg05310764, cg22796313, cg20321153, cg13114497, cg08691548, cg04387597, cg15634980, cg21428324, cg18205770, cg00147160, cg15622158, cg18011916, cg 03224, cg06335867, cg 26970841.
In the test set of 60 malignant and 20 benign nodule samples (N80), AUC reached 0.81, accuracy reached 80.0%, sensitivity 81.7%, specificity 75.0%, PPV 90.7%, NPV 57.7%, see fig. 3 and table 2; in an independent validation set of further 100 malignant and 40 benign nodule samples (N140), accuracy reached 83.6%, sensitivity 91.0%, specificity 65.0%, PPV 86.7%, NPV 74.3%, see table 2.
In conclusion, the sites screened by the method have very high correlation with the diagnosis of benign and malignant lung nodules.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (15)
1. A methylation biomarker associated with benign and malignant pulmonary nodules, or a combination thereof, comprising at least one of the following methylation biomarkers: cg07553761, cg22685975, cg04688351, cg07160746 and cg 13146465.
2. The biomarker or the combination thereof for benign and malignant lung nodules according to claim 1, further comprising 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, cg 26970841.
3. The biomarker or the combination thereof associated with benign and malignant lung nodules according to claim 1 or 2, further comprising at least one of the following methylation biomarkers: cg03978375, cg24826867, cg17384889, cg18694169, cg10253847, cg09163035, cg01759136, cg21643086, cg23279117, cg12820681, cg07816637, cg17132517, cg09578794, cg00171421, cg12445832, cg23999112, cg26528551, cg11289039, cg15072319, cg16247183, cg10081761, cg10144554, cg 264749, cg11220950, cg 14213, cg09183450, cg02458065, cg 213243 08660, cg26049726, cg 41310, cg 59594549, cg 112459274, cg 0634063406340634064006400640065, cg 72774705, cg 774705, cg 7779,779, cg 7740779, cg 77407708, cg 7708,779, cg 7708,7780, cg 77407708,7780, cg 5977317779,7780, cg 598474, cg 057768, cg 72977768, cg 7768, cg 77317768, cg 7768, cg 72977768, cg 7768, cg 7297778, cg 05778, cg 7731778, cg 778, cg 07778, cg 7731778, cg 7795 g 200778, cg 07778, cg 778, cg 07778, cg 97778, cg 07778, cg 778, cg 97778.
4. The biomarker or the combination thereof associated with benign and malignant pulmonary nodules of claim 1, 2 or 3, further comprising at least one of the following methylation biomarkers: cg 996827, cg21402748, cg03884587, cg12427163, cg18443359, cg20732876, cg14718275, cg18130044, cg09076431, cg04365202, cg21918126, cg27348152, cg16352140, cg22879098, cg 07570, cg05164926, cg24104241, cg12897502, cg 89799, cg15555898, cg23683588, cg18361098, cg17666418, cg25913016, cg11598006, cg 11297854, cg11213574, cg 31346, cg03242819, cg 266986, cg 28121, cg04655953, cg 06980541923368, cg 33054105410541054105410541054135, cg 3305410541054135, cg 33054105410541054135, cg 329241054105410541054135, cg 054105410541054105410541054135, cg 0541054105410541054135, cg 054105410541054148, cg 2235054105410541054105410541054148, cg 223572923146, cg 2235726520, cg 2235729205410541054148, cg 2235726547, cg 2235726405410541054135, cg 2235726405410541054148, cg 22357264054105410541057, cg 223572640541054148, cg 223572640541054105410541057, cg 227346, cg 2235726405410541054105410541054105410541057, cg 227346, cg 42819, cg 2235726405410541054105410541054135, cg 223572640541054135, cg 223572640426, cg 2235726405410541054105410541054105410541054105410541054135, cg 2235726427, cg 223572640426, cg 22357264044768, cg 2227, cg 223572640426, cg 227353, cg 22435427, cg 223572640426, cg 227346, cg 2227, cg 227346, cg.
5. The biomarker or combination thereof associated with the malignancy and well-being of a lung nodule of claim 2, wherein the methylation biomarker combination is cg07553761, cg20607577, cg22685975, cg22878622, cg17915922, cg05310764, cg22796313, cg20321153, cg13114497, cg08691548, cg04387597, cg15634980, cg21428324, cg18205770, cg00147160, cg15622158, cg18011916, cg03240324, and cg06335867, cg 2697080841.
6. The biomarker or the combination thereof associated with benign and malignant lung nodules according to claim 3, wherein the methylated biomarker combination is cg07553761, cg20607577, cg22685975, cg22878622, cg17915922, cg05310764, cg22796313, cg20321153, cg13114497, cg08691548, cg 04597, cg15634980, cg21428324, cg18205770, cg00147160, cg 15658, cg18011916, cg03240324, cg06335867, cg 269708970841, cg 979797979797978375, cg 828282827, cg 84889, cg 11294169, cg 53847, cg 5384035, cg 017563035, cg 019136, cg 0686, cg 1247980, cg 72794397204797, cg 729740924727, cg 72974705, cg 724705, cd 724705, cg 724794, cg 724705 and cg 724705.
7. The biomarker related to benign and malignant lung nodules or the combination thereof according to claim 3, wherein the methylation biomarker combination is cg07553761, cg20607577, cg22685975, cg22878622, cg17915922, cg05310764, cg22796313, cg20321153, cg13114497, cg08691548, cg 04597, cg15634980, cg21428324, cg18205770, cg00147160, cg 15658, cg 18016, cg03240324, cg06335867, cg 97089708970841, cg 97979797979797979775, cg 2482827, cg 84889, cg 10294169, cg 53847, cg 53035 035, cg 017556379792819726, 01077927792479, cg 779277927720, cg 7792779277924798, cg 057727, cg 779277927731977727, cg 77317727, cg 924798, 4798, 4708, 4798, 4394, 43977780, 4795, cg 7780, cg 778, cg 97778, cg 778, cg 977780, cg 97778, cg 778, cg 773195, cg 773194, cg 778, cg 97773194, cg 97773195, cg 97778, cg 778, cg 97778, cg 773195 g, cg 97778, cg 97773195 g, cg 97778, cg 97773195 g, cg 97778, cg 977780, cg 97778, cg 977780, cg 977775, cg 97773194, cg 97778, cg 977775, cg 97cg 97778, cg 97773195, cg 97778, cg 97cg.
8. The biomarker or the combination thereof associated with the benign and malignant pulmonary nodules according to claim 4, wherein the combination of the methylated biomarkers is the methylated biomarker described in all of claims 1 to 4.
9. Use of the methylated biomarkers of any one of claims 1 to 8 or a combination thereof as lung cancer associated methylated molecular markers for the detection of benign and malignant lung nodules and/or lung cancer detection.
10. A kit for detecting benign and malignant lung nodules and/or lung cancer, the kit comprising reagents for detecting the methylation level of the methylated biomarker or combination thereof of any one of claims 1 to 8.
11. The kit for detecting benign and malignant lung nodules and/or lung cancer according to claim 10, wherein said kit employs the following detection platforms: comprises reagents used by a PCR amplification method, a fluorescent quantitative PCR method, a digital PCR method, a liquid phase chip method, a first generation sequencing method, a third generation sequencing method, a second generation sequencing method, a pyrosequencing method, a bisulfite sequencing method, a methylation chip method or a combination thereof.
12. The kit for detecting benign or malignant lung nodules and/or lung cancer according to claim 11, wherein a second generation sequencing method is used.
13. The kit for detecting benign or malignant lung nodules and/or lung cancer according to claim 10, wherein said lung nodules comprise solid or partially solid or ground glass nodules, preferably partially solid or ground glass nodules.
14. The kit for detecting benign and malignant lung nodules and/or lung cancer according to claim 10, wherein said lung cancer is early stage lung cancer, preferably stage I lung cancer.
15. The method for detecting a methylated biomarker or combination thereof according to any one of claims 1 to 8, comprising the steps of:
s1, extracting cfDNA of a sample to be detected, treating the extracted cfDNA and methylated DNA by bisulfite, and establishing a library;
s2, lung nodule benign and malignant specificity panel targeted hybridization capture methylated DNA pre-library;
s3, final library quantification of targeted capture, second generation sequencing;
s4, preprocessing data to obtain real data obtained by probe capture;
s5, methylation data analysis of the methylated biomarker of any one of claims 1 to 8, or a combination thereof.
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