CN112899359B - Methylation marker for benign and malignant lung nodule detection or combination and application thereof - Google Patents
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Abstract
The present invention relates to a biomarker combination associated with benign and malignant lung nodules comprising at least one of the following: cg07553761, cg22685975, cg04688351, cg07160746, cg13146465 and the like. The invention also relates to a detection and related kit of the marker combination. The 20 markers of the invention possess substantially the same diagnostic power as the 100 marker combinations. The invention combines the analysis of the co-methylation characteristics of a plurality of methylated cytosines on at least 1 genome segment as a biomarker for judging benign and malignant diseases of lung nodules, and has the accuracy far greater than that of detection of other protein plasma biomarkers, especially in the differentiation of phase I lung cancer; in the non-solid nodule, the accuracy is far higher than that of PET-CT, the effect of discharging 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 lung nodule or a combination and application thereof.
Background
Lung cancer is the most common cause of cancer death worldwide, with the incidence leading to all malignant tumors. Due to environmental pollution, the incidence rate of lung cancer rises year by year, and about 78.1 ten thousand of lung cancer patients and about 62.6 ten thousand of death cases are in the first death of cancer in China every year. By 2025, new lung cancer patients in China are expected to reach 100 ten thousand per year. As most clinical diagnosis cases of lung cancer are advanced, the chance of surgical treatment is lost, the prognosis of lung cancer is extremely bad, and the survival rate of lung cancer in China in 5 years is only 16.1 percent. Goldstraw et al have found that early stage Ia lung cancer is surgically removed, its 5-year survival rate can reach more than 80%, and intermediate stage IIIa-IV lung cancer has a 5-year survival rate of less than 36% after surgical treatment, so that screening of lung cancer is a hope for improving lung cancer survival and reducing lung cancer mortality.
Lung cancer exists in early stage in the form of lung nodules, which is a common phenomenon in clinic, including benign nodules and malignant nodules, and early detection of malignant lung nodules is more hidden, and if early intervention is not performed, the disease course is rapid, the malignancy is strong, and the prognosis is poor. At present, qualitative diagnosis of lung nodules is difficult, and about 30% of lung nodules which are clinically and surgically resected are benign, so that the benign and malignant lung nodules are correctly evaluated, correct treatment means are selected, and the survival rate of patients can be remarkably improved, and prognosis is improved.
The existing diagnosis methods of the pulmonary nodules are various, but the qualitative diagnosis is difficult. Current common methods for pulmonary nodule diagnosis include: imaging examination, percutaneous puncture biopsy, biopsy under a virtual bronchoscope navigation system and thoracoscopic surgery pathological tissue examination: 1) Imaging examinations are commonly used for their non-invasive nature, primarily to observe the size, volume doubling time and morphological features of the pulmonary nodules. The commonly used imaging examination methods mainly include chest CT, positron emission computed tomography (positron emission tomography-computed tomography, PET-CT), etc. Imaging tests are problematic, for example, in that use of CT has excessively high false positives, which can lead to overdiagnosis, overdherapy, waste of medical resources, and increased anxiety in the subject. PET-CT has obvious detection effect only on solid nodules, and has unsatisfactory effect on glass grinding and other non-solid nodules. 2) Percutaneous pulmonary biopsy under CT guidance can be selected when the sensitivity of PET diagnosis is poor or false positive indication is displayed, and the diagnosis accuracy is about 90%. However, X-ray, CT or ultrasonic guided percutaneous pulmonary puncture biopsy is more likely to cause serious complications such as bleeding, pneumothorax, sudden death and the like. VBN developed in recent years is a CT-based three-dimensional imaging procedure, which establishes a virtual bronchial path through image recognition, and guides a bronchoscope to reach a target focus for biopsy, thereby achieving the purpose of improving the positive rate of minimally invasive biopsy of a lung nodule. However, in practice, problems such as nosocomial infections due to biopsies are also very common.
In conclusion, screening lung nodules and accurately performing differential diagnosis of benign and malignant masses are important problems to be solved urgently for lung cancer prevention and treatment. There is a need to find and build an effective diagnostic model for this clinical pain spot and to be able to serve as a molecular marker for screening and diagnosis.
cfDNA (cell-free DNA) is a small fragment of free nucleic acid DNA in peripheral blood derived from normal or tumor cell metabolism and apoptosis, and contains genetic information such as somatic mutation and DNA methylation. By detecting the disease specific cfDNA fragment, the technology for grasping the occurrence and development of the disease is called Liquid Biopsy (Liquid Biopsy), and compared with the traditional tissue Biopsy, the method has the advantages of rapidness, convenience, small injury and the like. In 2014, a 640-case study of various tumors from the group of Bert Vogelstein and Kenneth kenzler found that the presence of ctDNA was detected in more than 75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, melanoma, hepatocellular, and head and neck cancers. ctDNA is thus a widely applicable, sensitive and specific biomarker that can be used in the clinical and research of a wide variety of different types of cancer. 2015, lu teaches that liquid biopsy replacement tissue biopsies demonstrate technical and theoretical feasibility by cfDNA whole genome methylation sequencing; professor team 2017 Zhang described tumor burden and tumor derived ctDNA profile quantitatively using methylation of ctDNA. The Turner team uses high throughput sequencing to find breast cancer specific somatic mutation sites and monitors dynamic changes thereof, which proves that ctDNA detection can be earlier than CT detection of tumor recurrence and metastasis. Recent studies have found that ctDNA mutations in blood in combination with other analytes can provide an early and better diagnosis of lung, ovarian, liver, stomach, breast, prostate, esophageal and colorectal cancer, several common and surgically resectable cancers. More and more clinical researches show that the plasma ctDNA can be used as a biomarker for early diagnosis screening, prediction, treatment response of tumors, tumor size and recurrence monitoring and the like. At present, the international research direction is to integrate multiple groups of chemical/multiple molecular markers and multiple genes/multiple sites to improve the sensitivity and specificity of the detection technology so as to meet the clinical demands on detection products.
Disclosure of Invention
It is an object of the present invention to provide biomarkers associated with benign and malignant lung nodules, or combinations thereof, which can be used to diagnose the benign and malignant lung nodule condition.
The technical scheme for achieving the purpose is as follows.
A biomarker, or a combination thereof, associated with benign and malignant lung nodules comprising at least one of the following methylation biomarkers: cg07553761, cg22685975, cg04688351, cg07160746, cg13146465.
In some embodiments, the biomarker, or combination thereof, associated with benign and malignant lung nodules 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, cg26970841.
In some of these embodiments, at least one of the following methylation biomarkers is also included: cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, a first, cg, a second, cg, a first, cg, cg, cg, cg, cg, cg, cg, cg.
In some of these embodiments, at least one of the following methylation biomarkers is also included: the present invention relates to a method for producing a solid-state catalyst, which comprises the steps of (a) preparing a solid-state catalyst from a solid-state catalyst, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg.
In some of these embodiments, the methylation 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 cg26970841.
In one embodiment, the methylation biomarker, or combination thereof, is: the method includes the steps of a cell, g, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, and cg.
In some of these embodiments, the methylation biomarker, or combination thereof, is: cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, the method comprises the steps of cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, and cg.
In some of these embodiments, the methylation biomarkers are combined to all 200 of the methylation biomarkers described above.
In another aspect, the invention also provides the application of the biomarker or the combination thereof as a methylation molecular marker related to lung cancer in detecting benign and malignant lung nodules and/or lung cancer.
In another aspect, the invention also provides a kit for detecting benign and malignant lung nodules, and/or lung cancer, the kit comprising reagents for detecting the methylation level of the above DNA methylation molecular marker or a combination thereof.
In some of these embodiments, the kit may be used in the following assay platform: including reagents used in PCR amplification, fluorescent quantitative PCR, digital PCR, liquid chip, first generation sequencing, third generation sequencing, second generation sequencing (Sanger), pyrosequencing, bisulfite sequencing, methylation chip, or combinations thereof. In some preferred embodiments, a second generation sequencing method is preferred.
In some of these embodiments, the lung nodules are malignant nodules of different classifications (solid or partially solid or ground glass), lung nodule sizes, and different stages.
In some of these embodiments, the lung nodule is a partially solid or ground glass nodule.
In some of these embodiments, the lung cancer is early stage lung cancer, preferably stage I lung cancer.
In another aspect, the present invention also provides a method for detecting the methylation biomarker or the methylation biomarker combination, comprising the following steps:
s1, extracting cfDNA of a sample to be detected, and constructing a library by bisulphite treatment of the extracted cfDNA and methylated DNA;
s2, capturing methylation pre-library by lung nodule benign and malignant specific panel targeting hybridization;
s3, quantifying a final library captured in a targeted manner, and sequencing in the second generation;
s4, preprocessing data to obtain real data captured by a probe;
s5, methylation data analysis of the methylation biomarker or the methylation biomarker combination.
The invention discovers that the plasma genome fragment combination can be used as a lung nodule diagnostic marker by researching methylation modification differences of the plasma methylation modified genome fragments in lung nodule types, lung cancer patients in each stage and benign nodule populations. Further studies found that the first 20 markers had substantially the same diagnostic power as the 100 marker combinations. The invention combines the analysis of the co-methylation characteristics of a plurality of methylated cytosines on at least 1 genome segment as a biomarker for judging benign and malignant diseases of lung nodules, and has the accuracy far greater than that of detection of other protein plasma biomarkers, especially in the differentiation of phase I lung cancer; in non-solid nodules (partially solid and ground glass nodules), the accuracy is far higher than that of PET-CT, the effect of discharging yin can be effectively achieved, and the occurrence of false positive is reduced.
Drawings
FIG. 1 is a flow chart of a biomarker study according to the present invention.
Fig. 2 is a schematic representation of the ROAUC of five independent markers in example 2.
FIG. 3 shows the performance of the 20-,50-,100-, 200-marker models of examples 2,3,4,5 on test sets where AUC corresponds to 0.81,0.81,0.83,0.82, respectively.
FIG. 4 is a comparison of the performance of example 4 on independent validation sets and the model and the clinically 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 of the Mayo model and 0.54 of the VA model.
FIG. 5 is the performance of the 100-marker model in example 4 at early malignant nodules (STAGE I).
FIG. 6 is the performance of the 100-marker model of example 4 in 6-20mm nodules, independent validation set: AUC is 0.84, higher than that of the Mayo model 0.60 and VA model 0.51.
FIG. 7 is a graph of performance and comparison with PET-CT for the 100-marker model of example 4 at different nodule types in independent validation sets.
Detailed Description
The experimental procedure of the present invention, in which no specific conditions are noted in the following examples, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. The various chemicals commonly 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 a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.
In the present invention, the term "plurality" means two or more. "and/or", describes an association relationship of an association object, and indicates that there may be three relationships, for example, a and/or B, and may indicate: a exists alone, A and B exist together, and B exists alone. The character "/" generally indicates that the context-dependent object is an "or" relationship.
In one embodiment of the invention, at least one or a combination and use of 200 plasma gene methylation markers for diagnosis of benign and malignant lung nodules is disclosed. Specifically, the inventors found that genomic fragments that are significantly modified by aberrant methylation in the plasma of patients with malignant lung nodules, i.e., cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, the present invention relates to a method for producing a solid-state catalyst, which comprises the steps of (a) preparing a solid-state catalyst from a solid-state catalyst, the present invention relates to a method for producing a solid-state catalyst, which comprises the steps of (a) preparing a solid-state catalyst from a solid-state catalyst, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg.
Aiming at the problems of low-amount tumor DNA (ctDNA) samples and bisulphite treatment DNA damage in plasma cell free DNA (cfDNA), the stock building and hybrid capture technology of standard medical treatment is adopted: anchorDx EpiVisio TM Methylation Library Prep Kit(AnchorDx,Cat#A0UX00019)，AnchorDx EpiVisio TM Indexing PCR Kit (Anchor Dx, cat#A2DX00025) and AnchorDx EpiVisio TM Target Enrichment Kit(AnchorDx,Cat#A0UX00031)。
Lung nodules include different categories: solid nodules, partially solid nodules or ground glass nodules; malignant nodule malignancy is staged according to the TNM staging system.
Pulmonary nodules include solid or partially solid or ground glass nodules: ground glass density nodules refer to blurred sarcoidosis in the lungs, which has a slightly increased density compared to the surrounding lung parenchyma, but the contours of the internal vessels and bronchi remain visible. Solid nodules are nodules of soft tissue density throughout, which are of relatively uniform density and in which the vessel and bronchus images are masked. Partially solid nodules refer to nodules that include both ground glass density and solid soft tissue density therein that are non-uniform in density.
The stage of malignant nodular lung cancer is divided into stages I-IV according to an AJCC TNM international standardized stage system, and early lung cancer comprises stage I-II lung cancer.
In one embodiment of the invention, a marker for diagnosis of benign and malignant lung nodules is selected based on methylation of circulating tumor DNA (circulating tumor DNA, ctDNA), comprising the steps of:
step one, screening out lung cancer specific methylation markers according to a TGCA lung cancer and paracancestor methylation chip public database and an autonomous tumor related methylation database;
step two, extracting cfDNA of plasma of benign and malignant patients of lung nodules, and constructing libraries of cfDNA and methylated DNA extracted by bisulfite treatment;
capturing a methylation DNA pre-library through lung nodule benign and malignant specific panel targeting hybridization;
step four, quantifying a final library captured in a targeting way, and performing second generation sequencing on the machine;
step five, data preprocessing, namely obtaining real reads data (bam file) obtained by capturing by a probe;
step six, methylation data analysis, screening lung nodule benign and malignant diagnosis markers (markers), and screening a marker set finally used for analysis through analysis and filtration of tissues consistent with paired plasma;
step seven, further screening markers in the plasma training set and the verification set by utilizing the marker set selected in the step six, constructing an algorithm model, and verifying an independent data set;
and step eight, confirming a gene methylation marker and an algorithm model which are finally used for diagnosing benign and malignant lung nodules.
Example 1 detection method of novel ctDNA methylation marker for diagnosis of benign and malignant lung nodule
1. Extraction and methylation library establishment of cfDNA (cfDNA) or tissue DNA of blood plasma
1.1 extraction of plasma cfDNA or tissue DNA.
The specific procedure for plasma cfDNA extraction was performed according to the MagMAX TM Cell-Free DNA Isolation Kit protocol from Life Corp. The Tissue DNA extraction step is carried out according to DNeasy Blood & Tissue Kit operation instructions of QIAGEN company; index primer was purchased from New England Bioscience company cat#E7600S.
1.2 conversion
The extracted cfDNA (10 ng) or tissue DNA (50 ng) was subjected to bisulfite conversion to convert unmethylated cytosines in the DNA to uracil, while methylated cytosines remained unchanged, resulting in bisulfite converted DNA, and the conversion was performed according to the EZ DNA Methylation-lighting Kit instructions of Zymo Research.
1.3 terminal repair
1.3.1 the converted 17ul sample was reacted with the following reagents:
| component (A) | Volume (ul) |
| Post-conversion samples | 17 |
| MEB1 Buffer | 2 |
| MEE2 Enzyme | 1 |
| Total volume of | 20 |
1.3.2 reaction was carried out in a PCR apparatus according to the following procedure:
37℃ 30min
95℃ 5min
the lid was heated to 105 ℃.
1.3.3 when the second step of PCR reaction (95 ℃ C.) reaches 5min, immediately taking out the sample from the PCR instrument, directly inserting into ice, standing for more than 2min, and performing the next step
1.4 connection I
1.4.1 preparation of the following reaction solution
1.4.2 placed in a PCR apparatus and reacted according to the following procedure:
1.5 amplification I
1.5.1 preparation of the following reaction solution
1.5.2 placed in a PCR apparatus and reacted according to the following procedure:
1.6 purification I: the product after Amplification I reaction was purified by adding 166ul 1:6 fold dilution Agencourt AMPure Beads (half an hour prior to room temperature equilibration) and eluted with 21ul EB, the purification steps were as follows:
1.6.1 the reaction product of the previous step was taken and centrifuged, and each sample was added to 166ul of Agencourt AMPure Beads diluted 1:6 times and mixed by pipetting.
1.6.2 incubation for 5min at room temperature.
Centrifuging at 1.6.3, and standing on a magnetic rack for 5min.
1.6.4 the supernatant was aspirated.
1.6.5 200ul of 80% EtOH are added, left to stand for 30s and the ethanol is taken away.
1.6.6 repeating step 5) once.
1.6.7 the PCR tube was placed on a magnetic rack and the remaining ethanol was aspirated.
1.6.8 the beads were left open and dried for 2-3min, taking care not to overdry.
1.6.9 adding 21ul EB, eluting, stirring with a pipette, and standing at room temperature for 3min.
1.6.10 centrifuging, placing the PCR tube on a magnetic rack, and standing for 3min.
1.6.11 20ul of supernatant was pipetted into a new PCR tube.
1.7 connection II
1.7.1 the following reaction liquid was prepared:
| component (A) | Volume (ul) |
| Reaction volume of the last step | 20 |
| H 2 O | 4 |
| |
8 |
| MSR1 Reagent | 2 |
| MSR5 Reagent | 2 |
| MSE1 Enzyme | 2 |
| MSE5 Enzyme | 2 |
| Total volume of | 40 |
1.7.2 placing in a PCR apparatus and performing the reaction according to the following procedure
| Temperature (temperature) | Time | Cycle number |
| 37℃ | 30min | 1 |
| 95℃ | 5min | 1 |
| 10℃ | Hold | 1 |
1.8 Indexing PCR:
1.8.1 the following reaction liquid was prepared:
1.8.2 placing in a PCR apparatus and performing a reaction according to the following procedure
1.9 purification II
The product after the Indexing PCR reaction was purified by adding Agencourt AMPure Beads (half an hour prior to equilibration at room temperature), eluting with 41ul EB, and the purification steps were as follows:
1.9.1 the reaction product of the previous step was centrifuged and 71ul of undiluted Agencourt AMPure Beads was added to each sample and mixed by pipetting.
1.9.2 incubated at room temperature for 5min.
1.9.3 centrifuging, and standing on a magnetic rack for 5min.
1.9.4 the supernatant was aspirated.
1.9.5 200ul of 80% EtOH are added, left to stand for 30s and the ethanol is taken away.
1.9.6 the procedure of step 5) is repeated once.
1.9.7 the PCR tube was placed on a magnetic rack and the remaining ethanol was aspirated.
1.9.8 the beads were left open and dried for 2-3min, taking care not to overdry.
1.9.9 adding 41ul EB, eluting, stirring with a pipette, and standing at room temperature for 3min.
1.9.10 centrifuging, placing the PCR tube on a magnetic rack, and standing for 3min.
1.9.11 20ul of supernatant was pipetted into a new PCR tube.
1.10 Quantitative Qubit:
1ul of the library was quantified using Qubit dsDNA HS Assay Kit.
2. And (3) carrying out oligonucleotide probe capturing enrichment on the samples after library establishment to obtain the on-machine final library in the specific area. The hybridization capture kit was xGen Lockdown Reagents from IDT company, and was specifically prepared according to the instructions.
3. And sequencing the sample after hybridization capture by using a sequencer of Illumina company to obtain a sequencing result.
4. Analysis of data:
performing conventional bioinformatics analysis on the original data of the sequencer, filtering low-quality reads (reads) through fastp, removing adapters, consensus sequences and PolyA/T at the two ends of the reads to obtain ideal insert sequences (target intervals), comparing the reads with positions corresponding to hg19 by using bismark, performing de-duplication on the reads according to UMI to obtain real reads data (bamfile) obtained by capturing each sample by a probe, and performing statistics and analysis on the bam file to obtain methylation data for subsequent data re-analysis.
Example 2
This example discloses a methylation specific biomarker for diagnosing lung nodules, based on 253 cases of malignant nodules and 56 cases of benign nodule plasma samples of training set samples, using the methylation library building method described in example 1, using methylation level differences in different groups to screen out biomarkers related to malignant lung nodules, and performing independent dataset verification on bit point data in benign and malignant samples, wherein 200 methylated DNA fragments most notably distinguishing benign and malignant are screened out, and the 200 methylation biomarkers (hereinafter referred to as sites or markers) and AUC values of independent distinction are shown in table 1:
TABLE 1 characterization data for 200 methylation markers
Of these, cg07553761, cg22685975, cg04688351, cg07160746, cg13146465 performed best overall with AUC values of 0.753,0.710,0.715,0.712 and 0.709, respectively, see fig. 2.
Example 3
The test was performed using all 200 methylation marker-constructed models, with AUC reaching 0.823, accuracy 0.838, sensitivity 90.0%, specificity 65.0%, positive predictive value PPV 88.5%, negative predictive value NPV 68.4% in the test set of 60 malignant and 20 benign nodule samples, see fig. 2 and table 2.
In a further independent validation set of 100 malignant nodules and 40 benign nodule samples, accuracy 82.1%, sensitivity 95.0%, specificity 50.0%, PPV 82.6%, NPV 80.0%, see table 2.
Table 2:20 Characterization data for 50, 100 and 200 methylation marker models
Example 4
The test using the model constructed with the first 100 methylation markers (1-100 in Table 1) demonstrated an AUC of 0.83, an accuracy of 85.0%, a sensitivity of 93.3%, a specificity of 60.0%, a PPV of 87.5% and an NPV of 75% in the test set of 60 malignant and 20 benign nodule samples, see FIG. 3 and Table 2.
In a further independent validation set of 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 lung cancer early (Stage I) sample sets (Stage ia+ib, n=90), the sensitivity was as high as 97.1%, see fig. 5.
In 2) validation of the 6-20mm lung nodule size (n=100), AUC reached 0.84, see fig. 6.
In 3) validation of different nodule categories, the sensitivity of solid nodules (n=10) was 80.0%; the sensitivity of the partially solid nodule (n=11) is 81.8%; the sensitivity of the ground glass nodules (n=5) was 100% higher than the detection effect of PET-CT, see fig. 7.
Example 5
The test using the model constructed with the first 50 methylation markers ((1-50 in table 1) demonstrated AUC of 0.81, accuracy of 80.0%, sensitivity of 80.0%, specificity of 80.0%, PPV of 92.3%, NPV of 57.1% in the test set of 60 malignant and 20 benign nodule samples, see fig. 3 and table 2.
In a further independent validation set of 100 malignant nodules and 40 benign nodule samples, accuracy 79.3%, sensitivity 85.0%, specificity 65.0%, PPV 85.9%, NPV 63.4%, see table 2.
Example 6
The first 20 methylation biomarkers (1-20 in Table 1) were used, i.e., cg07553761, cg20607577, cg22685975, cg22878622cg17915922, cg05310764, cg22796313, cg20321153, cg13114497, cg08691548, cg04387597, cg15634980, cg21428324, cg18205770, cg00147160, cg15622158, cg18011916, cg03240324, cg06335867, cg26970841.
In the test set of 60 malignant nodules and 20 benign nodule samples (n=80), 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 a further independent validation set of 100 malignant nodules and 40 benign nodule samples (n=140), the accuracy reached 83.6%, the sensitivity 91.0%, the specificity 65.0%, PPV 86.7%, and NPV 74.3%, see table 2.
Taken together, the sites screened by the method have very high correlation with diagnosis of benign and malignant lung nodules.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (4)
1. Use of an agent for detecting the methylation level of a methylation biomarker combination in plasma for the manufacture of a product for detecting benign and malignant lung nodules, characterized in that the methylation biomarker combination is cg07553761, cg20607577, cg22685975, cg22878622, cg17915922, cg05310764, cg22796313, cg20321153, cg13114497, cg08691548, cg04387597, cg15634980, cg21428324, cg18205770, cg00147160, cg15622158, cg18011916, cg03240324, cg06335867, and cg26970841.
2. Use of a reagent for detecting the methylation level of a methylation biomarker combination in plasma for the manufacture of a product for detecting benign and malignant lung nodules, characterized in that the methylation biomarker combination is cg, cg, cg, the method includes the steps of a cell, g, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, and cg.
3. Use of a reagent for detecting the methylation level of a combination of methylation biomarkers in plasma for the manufacture of a product for detecting benign and malignant pulmonary nodules, characterized in that, the methylation biomarker combination is a cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg.
4. Use of a reagent for detecting the methylation level of a methylation biomarker combination in plasma for the manufacture of a product for detecting benign and malignant lung nodules, characterized in that the methylation biomarker combination is cg, cg, cg, cg, the present invention relates to a method for producing a solid-state catalyst, which comprises the steps of (a) preparing a solid-state catalyst from a solid-state catalyst, the present invention relates to a method for producing a solid-state imaging device, comprising the steps of (1) performing a process for producing a solid-state imaging device, the present invention relates to a method for producing a solid-state catalyst, which comprises the steps of (a) preparing a solid-state catalyst from a solid-state catalyst, the method includes the steps of a cell, g, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, cg, and cg.
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