Method, combination and kit for detecting malignant pulmonary nodules and lung cancer

Abstract

The invention provides a minimally invasive method for determining whether pulmonary nodules are malignant or not by measuring methylation levels of different gene combinations such as CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781. The disclosed methods also enable the detection of lung cancer. The invention also discloses a polynucleotide and a kit which can be used for measuring the methylation level of CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781.

Description

[0001] The present invention provides a minimally invasive method for determining whether a lung nodule is malignant by measuring the methylation level of a gene combination (including CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781). The disclosed method is also capable of detecting lung cancer. The present invention also discloses polynucleotides and kits that can be used to measure the methylation level of CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781. summary

[0002] Lung cancer is the leading cause of cancer-related deaths worldwide. Currently, there is no effective minimally invasive detection method for clinical differentiation of benign and malignant lung nodules. If malignant lung nodules can be identified when they are small, then this method can also be used for early detection of lung cancer. Computed tomography (CT) is commonly used for the detection of lung cancer. However, CT examinations have a high false positive rate and also require doctors to be well-trained and experienced to use CT to identify and distinguish malignant from benign lung nodules. In addition, high-risk populations in developing countries do not have easy access to CT and cannot afford the high cost of CT.

[0003] Other methods, such as detecting gene mutations and whole genome sequencing, have also been used for early detection of malignant pulmonary nodules and lung cancer. However, the results and effects of these existing methods are not satisfactory. A recent study showed that the sensitivity of detecting stage I lung cancer using existing methods is less than 22% (Klein et al., 2021). In addition, the second-generation sequencing in existing methods usually requires a test package covering a large number of genes, which greatly increases the cost of testing. Furthermore, many existing methods are invasive and require lung tissue samples for testing. Therefore, there is an urgent need for a minimally invasive detection method with high accuracy and specificity and low cost for early detection of malignant pulmonary nodules and lung cancer in the clinic.

[0004] The present invention provides a minimally invasive method for determining whether a pulmonary nodule is malignant by measuring the methylation level of a combination of genes selected from the CDO1, PTGER4 and HOXA9 genes in a sample of a subject. These methods can also be used to detect whether a subject has at least one malignant pulmonary nodule and / or lung cancer. The present invention also provides polynucleotides and kits for measuring the methylation levels of CDO1, PTGER4 and HOXA9.

[0005] DNA methylation is considered to be a promising marker for early detection of cancer due to its stable and inherited properties. Abnormal DNA methylation can lead to the dysregulation of multiple genes at various stages of lung cancer initiation, growth and metastasis. The present invention identifies six specific methylated genes in subjects with malignant lung nodules compared to normal lung tissue. High methylation of any combination of at least two of the six genes disclosed by the present invention indicates the presence of malignant lung nodules and enables early diagnosis of lung cancer. In some embodiments, the combined methylation level of CDO1 and PTGER4 is sufficient to distinguish between malignant lung nodules and benign lung nodules. The low-cost and minimally invasive detection method provided by the present invention is suitable for the identification of malignant lung nodules and the diagnosis of lung cancer.

[0006] In one aspect, the present invention provides a method comprising (a) measuring the methylation levels of at least two genes in a sample from a subject, wherein the two genes are selected from CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781, and (b) determining at least whether the two genes are hypermethylated, wherein detection of at least hypermethylation of the two genes indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0007] In one aspect, the present invention provides a method comprising (a) measuring the methylation level of at least two genes in a sample from a subject, wherein the two genes are selected from CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781, (b) at least determining whether the two genes are hypermethylated, (c) when hypermethylation of the two genes is detected, diagnosing the subject with at least one malignant pulmonary nodule and / or lung cancer, and (d) administering at least one effective amount of lung cancer treatment to the subject diagnosed with at least one malignant pulmonary nodule and / or lung cancer.

[0008] In some embodiments, the sample is a blood sample, a sputum sample, a bronchial washing sample, a bronchial brushing sample, a urine sample, or a saliva sample. In some embodiments, the sample is a blood sample.

[0009] In some embodiments, the method further comprises, prior to measuring, collecting a blood sample from the subject, separating plasma from the blood sample, and extracting DNA from the separated plasma.

[0010] In some embodiments, the lung cancer treatment is selected from surgery, chemotherapy, radiation therapy, immunotherapy, and targeted drug therapy.

[0011] In some embodiments, the methylation level is measured by (a) converting unmethylated cytosine in the DNA in step (c) to uracil while leaving the methylated cytosine unchanged, and (b) measuring the level of conversion of unmethylated cytosine to uracil.

[0012] In some embodiments, unmethylated cytosine in DNA is converted to uracil by bisulfite treatment or enzymatic treatment.

[0013] In some embodiments, methylation levels are measured by real-time polymerase chain reaction (PCR), sequencing, or microarray.

[0014] In some embodiments, the PCR is methylation-specific real-time fluorescent quantitative PCR.

[0015] In some embodiments, the methylation-specific real-time fluorescence quantitative PCR uses a forward primer comprising SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2, and a probe of SEQ ID NO: 3.

[0016] In some embodiments, the methylation-specific real-time fluorescence quantitative PCR uses a forward primer comprising SEQ ID NO:4, a reverse primer of SEQ ID NO:5, and a probe of SEQ ID NO:6.

[0017] In some embodiments, the methylation-specific real-time fluorescence quantitative PCR uses a forward primer comprising SEQ ID NO:7, a reverse primer of SEQ ID NO:8, and a probe of SEQ ID NO:9.

[0018] In some embodiments, the methylation-specific real-time fluorescence quantitative PCR uses a forward primer comprising SEQ ID NO:10, a reverse primer of SEQ ID NO:11, and a probe of SEQ ID NO:12.

[0019] In some embodiments, the methylation-specific real-time fluorescence quantitative PCR uses a forward primer comprising SEQ ID NO:13, a reverse primer of SEQ ID NO:14, and a probe of SEQ ID NO:15.

[0020] In some embodiments, the methylation-specific real-time fluorescent quantitative PCR uses a forward primer comprising SEQ ID NO:16, a reverse primer of SEQ ID NO:17, and a probe of SEQ ID NO:18.

[0021] In some embodiments, methylation levels are measured by methylation-specific high-resolution melting, pyrosequencing, nanopore long-read technology, or methylation-specific restriction endonuclease digestion.

[0022] In some embodiments, the lung cancer is non-small cell lung cancer or small cell lung cancer.

[0023] In some embodiments, the subject has been previously determined to have at least one pulmonary nodule.

[0024] In some embodiments, the subject is from a population at high risk for lung cancer.

[0025] In one aspect, the present invention provides a polynucleotide having any one of SEQ ID NOs: 1-18.

[0026] In one aspect, the present invention provides a kit for performing CDO1 methylation-specific real-time fluorescence quantitative PCR ("CDO1 kit"), comprising a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2, and a probe of SEQ ID NO: 3. In some embodiments, the kit further comprises bisulfite.

[0027] In one aspect, the present invention provides a kit for performing PTGER4 methylation-specific real-time fluorescence quantitative PCR ("PTGER4 kit"), comprising a forward primer of SEQ ID NO: 4, a reverse primer of SEQ ID NO: 5, and a probe of SEQ ID NO: 6. In some embodiments, the kit further comprises bisulfite.

[0028] In one aspect, the present invention provides a kit for performing methylation-specific real-time fluorescence quantitative PCR on HOXA9 ("HOXA9 kit"), comprising a forward primer of SEQ ID NO: 7, a reverse primer of SEQ ID NO: 8, and a probe of SEQ ID NO: 9. In some embodiments, the kit further comprises bisulfite.

[0029] In one aspect, the present invention provides a kit for performing SHOX2 gene methylation-specific real-time fluorescence quantitative PCR (hereinafter referred to as "SHOX2 kit"), comprising a forward primer of SEQ ID NO: 10, a reverse primer of SEQ ID NO: 11, and a probe of SEQ ID NO: 12. In some embodiments, the kit further comprises bisulfite.

[0030] In one aspect, the present invention provides a kit for performing SP9 gene methylation-specific real-time fluorescence quantitative PCR (hereinafter referred to as "SP9 kit"), comprising a forward primer of SEQ ID NO: 13, a reverse primer of SEQ ID NO: 14, and a probe of SEQ ID NO: 15. In some embodiments, the kit further comprises bisulfite.

[0031] In one aspect, the present invention provides a kit for performing methylation-specific real-time fluorescence quantitative PCR on the ZNF781 gene (hereinafter referred to as "ZNF781 kit"), comprising a forward primer of SEQ ID NO: 16, a reverse primer of SEQ ID NO: 17, and a probe of SEQ ID NO: 18. In some embodiments, the kit further comprises bisulfite.

[0032] In one aspect, the present invention provides a kit for determining whether at least one pulmonary nodule found in a subject is malignant, the kit comprising bisulfite and reagents for performing methylation-specific real-time fluorescence quantitative PCR on at least two genes selected from CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781, wherein the reagents for performing methylation-specific real-time fluorescence quantitative PCR on CDO1 comprise a CDO1 forward primer, a CDO1 reverse primer and a CDO1 probe, wherein the reagents for performing methylation-specific real-time fluorescence quantitative PCR on PTGER4 comprise a PTGER4 forward primer, a PTGER4 reverse primer and a PTGER4 probe. needle, wherein the reagents for performing methylation-specific real-time fluorescence quantitative PCR on HOXA9 include a HOXA9 forward primer, a HOXA9 reverse primer and a HOXA9 probe, wherein the reagents for performing methylation-specific real-time fluorescence quantitative PCR on SHOX2 include a SHOX2 forward primer, a SHOX2 reverse primer and a SHOX2 probe, wherein the reagents for performing methylation-specific real-time fluorescence quantitative PCR on SP9 include an SP9 forward primer, an SP9 reverse primer and an SP9 probe, wherein the reagents for performing methylation-specific real-time fluorescence quantitative PCR on ZNF781 include a ZNF781 forward primer, a ZNF781 reverse primer and a ZNF781 probe.

[0033] In some embodiments, the kit further comprises at least two kits selected from the CDO1 kit, PTGER4 kit, HOXA9 kit, SHOX2 kit, SP9 kit and ZNF781 kit disclosed in the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a receiver operating characteristic curve describing the performance of using CDO1 gene methylation level to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0035] Figure 2is a receiver operating characteristic curve describing the performance of using PTGER4 gene methylation level to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0036] Figure 3 is a receiver operating characteristic curve describing the performance of using HOXA9 gene methylation levels to differentiate malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0037] Figure 4 is a receiver operating characteristic curve describing the performance of using the methylation levels of the CDO1 gene and the PTGER4 gene to distinguish malignant pulmonary nodules from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0038] Figure 5 is a receiver operating characteristic curve describing the performance of using the methylation levels of the CDO1 gene and the HOXA9 gene to distinguish malignant pulmonary nodules from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0039] Figure 6 is a receiver operating characteristic curve describing the performance of using the methylation levels of the PTGER4 gene and the HOXA9 gene to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0040] Figure 7 is a receiver operating characteristic curve describing the performance of using the methylation levels of the CDO1 gene, PTGER4 gene, and HOXA9 gene to distinguish malignant pulmonary nodules from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0041] Figure 8 is a receiver operating characteristic curve describing the performance of using SHOX2 gene methylation levels to differentiate malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0042] Fig. 9 is a receiver operating characteristic curve describing the performance of using SP9 gene methylation level to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0043] Fig.10is a receiver operating characteristic curve describing the performance of using ZNF781 gene methylation level to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0044] Fig.11 is a receiver operating characteristic curve describing the performance of using the methylation levels of the SHOX2 gene and the SP9 gene to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0045] Fig.12 is a receiver operating characteristic curve describing the performance of using the methylation levels of the SHOX2 gene and the PTGER4 gene to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0046] Fig.13 is a receiver operating characteristic curve describing the performance of using the methylation levels of the SHOX2 gene and the CDO1 gene to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0047] Fig.14 is a receiver operating characteristic curve describing the performance of using the methylation levels of the SHOX2 gene and the ZNF781 gene to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0048] Fig.15 is a receiver operating characteristic curve describing the performance of using SP9 gene methylation level and PTGER to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0049] Fig.16 is a receiver operating characteristic curve describing the performance of using the methylation levels of the SP9 gene and the CDO1 gene to distinguish malignant pulmonary nodules from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0050] Fig.17 is a receiver operating characteristic curve describing the performance of using the methylation levels of the SP9 gene and the ZNF781 gene to distinguish malignant pulmonary nodules from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0051] Fig.18is a receiver operating characteristic curve describing the performance of using the methylation levels of the ZNF781 gene and the PTGER4 gene to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0052] Fig.19 is a receiver operating characteristic curve describing the performance of using the methylation levels of the ZNF781 gene and the CDO1 gene to distinguish malignant pulmonary nodules from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0053] Fig. 20 is a receiver operating characteristic curve describing the performance of using the methylation levels of the ZNF781 gene, CDO1 gene, and SHOX2 gene to distinguish malignant pulmonary nodules from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0054] Fig.21 is a receiver operating characteristic curve describing the performance of using the methylation levels of SHOX2, SP9, and PTGER4 genes to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0055] Fig. 22 is a receiver operating characteristic curve describing the performance of using the methylation levels of SHOX2, SP9, and CDO11 genes to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0056] Fig.23 is a receiver operating characteristic curve describing the performance of using methylation levels of SHOX2, SP9, and ZNF781 genes to differentiate malignant from benign pulmonary nodules. The y-axis reports sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0057] Fig.24 is a receiver operating characteristic curve describing the performance of using the methylation levels of SHOX2, PTGER4, and CDO1 genes to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0058] Fig.25 is a receiver operating characteristic curve describing the performance of using the methylation levels of SHOX2, PTGER4, and ZNF781 genes to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0059] Fig.26 is a receiver operating characteristic curve describing the performance of using the methylation levels of the SP9 gene, PTGER4 gene, and CDO1 gene to distinguish malignant pulmonary nodules from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0060] Fig. 27 is a receiver operating characteristic curve describing the performance of using the methylation levels of the SP9 gene, PTGER4 gene, and ZNF781 gene to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0061] Fig.28 is a receiver operating characteristic curve describing the performance of using the methylation levels of PTGER4, CDO1, and ZNF781 genes to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0062] Fig.29 is a receiver operating characteristic curve describing the performance of using the methylation levels of SHOX2, SP9, PTGER4, and CDO1 genes to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0063] Fig.30 is a receiver operating characteristic curve describing the performance of using the methylation levels of SHOX2, SP9, PTGER4, and ZNF781 genes to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0064] Fig.31 is a receiver operating characteristic curve describing the performance of using the methylation levels of the SP9 gene, PTGER4 gene, CDO1 gene, and ZNF781 gene to distinguish malignant pulmonary nodules from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0065] Fig.32 is a receiver operating characteristic curve describing the performance of using the methylation levels of the SP9 gene, PTGER4 gene, CDO1 gene, ZNF781 gene, and SHOX2 gene to distinguish malignant pulmonary nodules from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0066] Fig.33is a receiver operating characteristic curve describing the performance of using the methylation levels of the SP9 gene, CDO1 gene, and ZNF781 gene to distinguish malignant pulmonary nodules from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0067] Fig.34 is a receiver operating characteristic curve describing the performance of using the methylation levels of SHOX2, SP9, CDO1, and ZNF781 genes to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0068] Fig.35 is a receiver operating characteristic curve describing the performance of using the methylation levels of SHOX2, PTGER4, CDO1, and ZNF781 genes to distinguish malignant from benign pulmonary nodules. The y-axis reports the sensitivity and the x-axis reports 1-specificity. Connect the diagonal lines.

[0069] Fig.36 is the gene sequence of the CDO1 gene, with the CpG sites highlighted.

[0070] Fig.37 is the gene sequence of the PTGER4 gene, with the CpG sites highlighted.

[0071] Fig.38 is the gene sequence of the HOXA9 gene, with the CpG sites highlighted.

[0072] Fig.39 is the gene sequence of the SHOX2 gene, with the CpG sites highlighted.

[0073] Fig.40 is the gene sequence of the SP9 gene, with the CpG sites highlighted.

[0074] Fig.41 is the gene sequence of the ZNF781 gene, with the CpG sites highlighted. Detailed description definition

[0075] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Therefore, the terms defined herein will be more fully described by reference to the entire specification.

[0076] As used herein, singular terms such as "a," "an," and plural terms such as "the" are used unless the context clearly indicates otherwise.

[0077] As used herein, "and / or" refers to any and all possible combinations of one or more related items listed, and "or" is interpreted as an alternative combination. In addition, the present invention also contemplates that in some embodiments disclosed in the present invention, any feature or combination of features described herein may be excluded or omitted.

[0078] Unless the context requires otherwise, the terms "comprises," "comprising," "containing" or similar terms are intended to mean a non-exclusive inclusion, so that the list of elements or features includes not only the elements stated or listed, but may also include other elements or features that are not listed or described.

[0079] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation.

[0080] It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described as these may vary depending on the context in which they are used by one skilled in the art.

[0081] As used herein, "nodule" refers to an abnormal growth or mass of cells. A pulmonary nodule refers to a nodule that forms in the lungs.

[0082] As used herein, "malignant" refers to the presence of cancer cells that are capable of spreading to other parts of the body or invading nearby tissues and destroying them.

[0083] As used herein, "minimally invasive" surgery refers to surgery that does not involve an incision in the body. In some embodiments, the minimally invasive method includes puncture; in some embodiments, the minimally invasive method is bronchial washing or bronchial brushing. For the purposes of this application, the minimally invasive method includes collecting a blood sample, a sputum sample, a urine sample, a bronchial washing sample, a bronchial brushing sample, or a saliva sample from a subject.

[0084] As used herein, "subject" refers to any animal, including but not limited to humans, non-human primates, rodents, etc., to which the methods of the present invention are applicable. Generally, the terms "subject" and "patient" are used interchangeably herein when referring to human subjects.

[0085] For the purposes of this application, "methylation level" refers to the number of 5mC bases contained in a gene. As used herein, if the target gene extracted from the subject sample has more 5mC than the corresponding gene from the subject sample of non-malignant pulmonary nodules, the target gene is "hypermethylated". In some embodiments, the methylation level is measured by methylation-specific real-time fluorescence quantitative PCR, and if ΔCt≤15.5 (ΔCt=candidate gene Ct-reference gene Ct), the target gene is "hypermethylated". For more information about methylation-specific real-time fluorescence quantitative PCR, please refer to the following sections of the present invention.

[0086] As used herein, "lung cancer" includes all types of cancers that begin in the lungs. Typical lung cancers include, but are not limited to, adenocarcinoma, large cell carcinoma, squamous cell carcinoma, small cell carcinoma, combined small cell carcinoma, and mesothelioma.

[0087] As used herein, "reference gene" refers to a gene present in all cells for the purpose of internal reaction control. "Reference gene" can be used to normalize the quantity and quality of the starting nucleic acid and the differences in PCR amplification. In general, the expression level of the reference gene does not differ significantly between different tissues and experiments (Radonic et al., 2004). In some embodiments, the reference gene is selected from basic metabolic genes (called housekeeping genes, HKGs), which, by definition, are involved in processes necessary for cell survival and are expressed at a stable, non-regulated constant level (Thellin et al., 1999). DNA methylation

[0088] DNA methylation is an epigenetic mechanism that regulates gene expression and cell differentiation. In mammals, DNA methylation is essential for normal development and is associated with many key processes, including genomic imprinting, X chromosome inactivation, transposable element repression, aging, and carcinogenesis.

[0089] It was found that natural enzymatic DNA methylation occurs on two bases, cytosine and adenine. The modified bases are 5-methylcytosine (5mC), N4-methylcytidine (4mC), and N6-methyladenine (6mA). Among them, 6mA and 4mC are limited to prokaryotes and some eukaryotes. In mammals, 5mC is the main form of DNA methylation, which mainly occurs in cytosine phosphate guanine (CpG) dinucleotides, and usually cytosine on both strands is methylated. CpG sites or CG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in a linear sequence of bases in the 5' to 3' direction. The mammalian genome has a particularly high level of CpG methylation. Although there are some tissue-specific differences, about 70-80% of CpGs are methylated. Although most CpGs are methylated, densely clustered CpG regions, namely CpG islands (CGIs), usually lack methylation. Many CGIs exist near gene promoters, and about two-thirds of genes have CGIs in their promoters. Methylation of promoter CGIs can cause long-term transcriptional repression of related genes.

[0090] In mammals, 5mC methylation is catalyzed by a family of DNA methyltransferases (DNMTs), whose members transfer a methyl group from S-adenosylmethionine to the fifth carbon of a cytosine residue, forming 5mC. DNMT3a and DNMT3b are methyltransferases that establish DNA methylation patterns early in development. DNMT3L is a protein that is homologous to the other DNMT3s but is catalytically inactive. Instead, DNMT3L assists the methyltransferases by enhancing their ability to bind to DNA and stimulating their activity. DNMT1 is considered a maintenance methyltransferase responsible for copying DNA methylation patterns to daughter strands during DNA replication. Malignant pulmonary nodules and lung cancer

[0091] Lung cancer is the most common cause of cancer-related death worldwide, causing more than 1 million deaths each year. Patients with advanced lung cancer usually have a poor prognosis. Malignant lung nodules usually indicate the early stages of lung cancer. Therefore, distinguishing between benign and malignant lung nodules is very important for early diagnosis of lung cancer and formulating appropriate treatment plans.

[0092] Over the past few decades, studies have shown that changes in DNA methylation patterns can distinguish cancer cells from normal cells. Abnormal DNA methylation leads to dysregulation of various genes and occurs at various stages of lung cancer initiation, growth, and metastasis. It is reported that changes in DNA methylation patterns occur in a developmental stage and tissue-specific manner and are often associated with the development of cancer. During carcinogenesis, both alleles of tumor suppressor genes need to be inactivated by genomic changes, such as chromosomal deletions or loss-of-function mutations in gene coding regions. As an alternative mechanism, hypermethylation of CpG islands spanning the promoter region of tumor suppressor genes leading to transcriptional silencing is a common and important process in carcinogenesis. Since hypermethylation usually leads to inactivation of gene expression, this epigenetic change is considered to be a key mechanism for long-term silencing of tumor suppressor genes (Esteller, 2002; Wajed et al., 2001). As research progresses, people are increasingly aware of the importance of promoter methylation in the functional inactivation of lung cancer suppressor genes. It is estimated that in lung cancer, 0.5% to 3% of all genes carrying CpG islands may be silenced by DNA methylation (Costello et al., 2000).

[0093] The present invention provides a minimally invasive method for determining whether at least one pulmonary nodule found in a subject is malignant by measuring the DNA methylation level of a gene combination. Since malignant pulmonary nodules are cancerous and can develop into lung cancer, the method provided herein can also be used to detect lung cancer. The method disclosed herein also provides useful guidance for doctors to determine the appropriate treatment plan for subjects with at least one malignant pulmonary nodule. Free DNA

[0094] Cell-free DNA (cfDNA) is a DNA fragment that can be isolated from mammalian serum or plasma. In 1948, Mandel and Metais first reported the presence of extracellular nucleic acids in circulation (Mandel et al., 1948). In 1989, Stroun et al. showed that circulating DNA in cancer patients exhibited some characteristics of tumor DNA, such as decreased stability of DNA chains (Stroun et al., 1989). In some embodiments, cfDNA extracted from a blood sample is used in the methods disclosed herein; in some embodiments, cfDNA extracted from a urine sample is used in the methods disclosed herein; in some embodiments, cfDNA extracted from a saliva sample is used in the methods disclosed herein.

[0095] Because phagocytes can effectively remove defective cells from the circulation, cfDNA concentrations in healthy individuals are generally low, generally between 0 and 100 ng / mL (Elshimali et al., 2013). In cancer patients, cfDNA is released in increased amounts from necrotic and apoptotic tumor cells, but its levels vary widely, ranging from 0 to >1000 ng / mL (Schwarzenbach et al., 2011; Fleischhacker et al., 2007). Because molecular alterations can be detected in cfDNA, analysis of cfDNA isolated from plasma or serum provides a minimally invasive approach to studying epigenetic changes in cancer patients. For example, high levels of TSG promoter methylation have been found in cfDNA from lung cancer patients, indicating that specific epigenetic changes detected in tumor cell genomic DNA can also be found in patient serum / plasma (Fujiwara et al., 2005; Sigalotti et al., 2019). method

[0096] In one aspect, the present invention provides a method comprising (a) measuring the methylation levels of at least two genes in a sample from a subject, wherein the genes are selected from CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781, and (b) determining whether the at least two genes are hypermethylated, wherein detection of hypermethylation of the at least two genes indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0097] In some embodiments, the method comprises (a) collecting a sample from a subject; (b) extracting DNA from the sample; (c) measuring the methylation level of at least two genes in the extracted DNA, wherein the at least two genes are selected from CDO1 (SEQ ID NO: 19), PTGER4 (SEQ ID NO: 20), HOXA9 (SEQ ID NO: 21), SHOX2 (SEQ ID NO: 22), SP9 (SEQ ID NO: 23) and ZNF781 (SEQ ID NO: 23); and (d) determining whether the at least two genes are hypermethylated, wherein detection of hypermethylation of the at least two genes indicates that the subject has at least one malignant pulmonary nodule and / or lung cancer. In some embodiments, the sample is a blood sample, a sputum sample, a bronchial washing fluid sample, a bronchial brushing sample, a urine sample or a saliva sample. In some embodiments, the sample is a blood sample.

[0098] In some embodiments, the method comprises (a) collecting a blood sample from a subject; (b) separating plasma from the blood sample; (c) extracting DNA from the separated plasma; (d) measuring the methylation levels of at least two genes in the extracted DNA, wherein the genes are selected from CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781; and (e) determining whether the at least two genes are hypermethylated, wherein detection of hypermethylation of the at least two genes indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0099] In some embodiments, the methylation levels of CDO1 and PTGER4 are measured, and detection of hypermethylation of CDO1 and PTGER4 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0100] In some embodiments, the methylation levels of CDO1 and HOXA9 are measured, and detection of hypermethylation of CDO1 and HOXA9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0101] In some embodiments, the methylation levels of CDO1 and SHOX2 are measured, and detection of hypermethylation of CDO1 and SHOX2 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0102] In some embodiments, the methylation levels of CDO1 and SP9 are measured, and detecting high methylation of CDO1 and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0103] In some embodiments, the methylation levels of CDO1 and ZNF781 are measured, and detection of hypermethylation of CDO1 and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0104] In some embodiments, the methylation levels of PTGER4 and HOXA9 are measured, and detection of hypermethylation of PTGER4 and HOXA9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0105] In some embodiments, the methylation levels of PTGER4 and SHOX2 are measured, and detecting hypermethylation of PTGER4 and SHOX2 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0106] In some embodiments, the methylation levels of PTGER4 and SP9 are measured, and detection of high methylation of PTGER4 and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0107] In some embodiments, the methylation levels of PTGER4 and ZNF781 are measured, and detecting hypermethylation of PTGER4 and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0108] In some embodiments, the methylation levels of HOXA9 and SHOX2 are measured, and detecting hypermethylation of HOXA9 and SHOX2 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0109] In some embodiments, the methylation levels of HOXA9 and SP9 are measured, and detection of hypermethylation of HOXA9 and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0110] In some embodiments, the methylation levels of HOXA9 and ZNF781 are measured, and detecting hypermethylation of HOXA9 and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0111] In some embodiments, the methylation levels of SHOX2 and SP9 are measured, and detecting hypermethylation of both SHOX2 and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0112] In some embodiments, the methylation levels of SHOX2 and ZNF781 are measured, and detecting hypermethylation of SHOX2 and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0113] In some embodiments, the methylation levels of SP9 and ZNF781 are measured, and detecting high methylation of SP9 and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0114] In some embodiments, the methylation levels of CDO1, PTGER4, and HOXA9 are measured, and detecting hypermethylation of all of CDO1, PTGER4, and HOXA9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0115] In some embodiments, the methylation levels of CDO1, PTGER4, and SHOX2 are measured, and detection of hypermethylation of all of CDO1, PTGER4, and SHOX2 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0116] In some embodiments, the methylation levels of CDO1, PTGER4, and SP9 are measured, and detecting hypermethylation of all of CDO1, PTGER4, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0117] In some embodiments, the methylation levels of CDO1, PTGER4, and ZNF781 are measured, and detecting hypermethylation of all of CDO1, PTGER4, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0118] In some embodiments, the methylation levels of CDO1, HOXA9, and SHOX2 are measured, and detection of hypermethylation of all of CDO1, HOXA9, and SHOX2 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0119] In some embodiments, the methylation levels of CDO1, HOXA9, and SP9 are measured, and detection of hypermethylation of all of CDO1, HOXA9, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0120] In some embodiments, the methylation levels of CDO1, HOXA9, and ZNF781 are measured, and detection of hypermethylation of all of CDO1, HOXA9, and ZnF771 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0121] In some embodiments, the methylation levels of CDO1, SHOX2, and SP9 are measured, and detection of hypermethylation of all of CDO1, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0122] In some embodiments, the methylation levels of CDO1, SHOX2, and ZNF781 are measured, and detection of hypermethylation of all of CDO1, SHOX2, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0123] In some embodiments, the methylation levels of CDO1, SP9, and ZNF781 are measured, and detection of hypermethylation of all of CDO1, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0124] In some embodiments, the methylation levels of PTGER4, HOXA9, and SHOX2 are measured, and detection of hypermethylation of all of PTGER4, HOXA9, and SHOX2 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0125] In some embodiments, the methylation levels of PTGER4, HOXA9, and SP9 are measured, and detection of hypermethylation of all of PTGER4, HOXA9, and SP4 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0126] In some embodiments, the methylation levels of PTGER4, HOXA9, and ZNF781 are measured, and detection of hypermethylation of all of PTGER4, HOXA9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0127] In some embodiments, the methylation levels of PTGER4, SHOX2, and SP9 are measured, and detection of hypermethylation in all of PTGER4, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0128] In some embodiments, the methylation levels of PTGER4, SHOX2, and ZNF781 are measured, and detection of hypermethylation of all of PTGER4, SHOX2, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0129] In some embodiments, the methylation levels of PTGER4, SP9, and ZNF781 are measured, and detection of hypermethylation of all of PTGER4, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0130] In some embodiments, the methylation levels of HOXA9, SHOX2, and SP9 are measured, and detection of hypermethylation of all of HOXA9, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0131] In some embodiments, the methylation levels of HOXA9, SHOX2, and ZNF781 are measured, and detection of hypermethylation of all of HOXA9, SHOX2, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0132] In some embodiments, the methylation levels of HOXA9, SP9, and ZNF781 are measured, and detection of hypermethylation of all of HOXA9, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0133] In some embodiments, the methylation levels of SHOX2, SP9, and ZNF781 are measured, and detection of hypermethylation of all of SHOX2, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0134] In some embodiments, the methylation levels of CDO1, PTGER4, HOXA9, and SHOX2 are measured, and detecting hypermethylation of all of CDO1, PTGER4, HOXA9, and SHOX2 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0135] In some embodiments, the methylation levels of CDO1, PTGER4, HOXA9, and SP9 are measured, and detecting hypermethylation of all of CDO1, PTGER4, HOXA9, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0136] In some embodiments, the methylation levels of CDO1, PTGER4, HOXA9, and ZNF781 are measured, and detecting hypermethylation of all of CDO1, PTGER4, HOXA9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0137] In some embodiments, the methylation levels of CDO1, PTGER4, SHOX2, and SP9 are measured, and detecting hypermethylation of all of CDO1, PTGER4, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0138] In some embodiments, the methylation levels of CDO1, PTGER4, SHOX2, and ZNF781 are measured, and detecting hypermethylation of all of CDO1, PTGER4, SHOX2, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0139] In some embodiments, the methylation levels of CDO1, PTGER4, SP9, and ZNF781 are measured, and detecting hypermethylation of all of CDO1, PTGER4, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0140] In some embodiments, the methylation levels of CDO1, HOXA9, SHOX2, and SP9 are measured, and detection of hypermethylation in all of CDO1, HOXA9, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0141] In some embodiments, the methylation levels of CDO1, HOXA9, SHOX2, and ZNF781 are measured, and detection of hypermethylation of all of CDO1, HOXA9, SHOX2, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0142] In some embodiments, the methylation levels of CDO1, HOXA9, SP9, and ZNF781 are measured, and detecting hypermethylation of all of CDO1, HOXA9, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0143] In some embodiments, the methylation levels of CDO1, SHOX2, SP9, and ZNF781 are measured, and detecting hypermethylation of all of CDO1, SHOX2, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0144] In some embodiments, the methylation levels of PTGER4, HOXA9, SHOX2, and SP9 are measured, and detecting hypermethylation in all of PTGER4, HOXA9, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0145] In some embodiments, the methylation levels of PTGER4, HOXA9, SHOX2, and ZNF781 are measured, and detecting hypermethylation of all of PTGER4, HOXA9, SHOX2, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0146] In some embodiments, the methylation levels of PTGER4, HOXA9, SP9, and ZNF781 are measured, and detecting hypermethylation of all of PTGER4, HOXA9, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0147] In some embodiments, the methylation levels of PTGER4, SHOX2, SP9, and ZNF781 are measured, and detecting hypermethylation of all of PTGER4, SHOX2, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0148] In some embodiments, the methylation levels of HOXA9, SHOX2, SP9, and ZNF781 are measured, and detection of hypermethylation of all of HOXA9, SHOX2, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0149] In some embodiments, the methylation levels of CDO1, PTGER4, HOXA9, SHOX2, and SP9 are measured, and detecting hypermethylation in all of CDO1, PTGER4, HOXA9, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0150] In some embodiments, the methylation levels of CDO1, PTGER4, HOXA9, SHOX2, and ZNF781 are measured, and detecting hypermethylation of all of CDO1, PTGER4, HOXA9, SHOX2, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0151] In some embodiments, the methylation levels of CDO1, PTGER4, HOXA9, ZNF781 and SP9 are measured, and detecting hypermethylation of all of CDO1, PTGER4, HOXA9, ZNF781 and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0152] In some embodiments, the methylation levels of CDO1, PTGER4, ZNF781, SHOX2, and SP9 are measured, and detecting hypermethylation of all of CDO1, PTGER4, ZNF781, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0153] In some embodiments, the methylation levels of CDO1, ZNF781, HOXA9, SHOX2, and SP9 are measured, and detecting hypermethylation of all of CDO1, ZNF781, HOXA9, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0154] In some embodiments, the methylation levels of ZNF781, PTGER4, HOXA9, SHOX2, and SP9 are measured, and detecting hypermethylation in all of ZNF781, PTGER4, HOXA9, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0155] In some embodiments, the methylation levels of CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781 are measured, and detecting hypermethylation of all of CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0156] In some embodiments, the sample is a blood sample, a sputum sample, a bronchial washing sample, a bronchial brushing sample, a urine sample, or a saliva sample. In some embodiments, the sample is a blood sample.

[0157] Blood samples can be collected using any suitable method known in the art. Exemplary methods for collecting blood samples include, but are not limited to, venipuncture sampling, arterial sampling, and finger sampling. One skilled in the art will be able to determine the appropriate conditions and amount of blood to be collected for the blood tests disclosed herein.

[0158] Plasma can be collected using any suitable method known in the art. An exemplary method for collecting plasma is to use blood centrifugation. The centrifuge applies centrifugal force to the blood collection tube by high-speed rotation, and separates the various components in the blood by different densities. Therefore, the various components of the blood can be divided into different layers for easy separation. In some embodiments, one or more anticoagulants (e.g., EDTA, citric acid-glucose) are added to the blood sample before centrifugation. Those skilled in the art can select suitable methods and conditions to separate plasma from whole blood samples. DNA can be extracted from plasma using any suitable method known in the art. There are a variety of commercial kits that can be used to extract DNA from plasma, such as but not limited to QIAampCirculating Nucleic Acid Kit, QIAamp DNA Blood kit, and chemagic cfDNA 2k kit H24. Those skilled in the art can select methods and conditions suitable for extracting DNA from plasma.

[0159] Sputum samples can be collected using any suitable method known in the art. Exemplary methods for collecting sputum samples include, but are not limited to, spontaneous sputum sampling, sputum induction, bronchoscopy, and gastric lavage. In spontaneous sputum sampling, the subject coughs the sputum into a sterile container. In sputum induction, the subject inhales a saline mist that can cause a deep cough, and then coughs the sputum into a sterile container. In bronchoscopy, a bronchoscope is passed directly into the lungs through the subject's mouth or nose, and sputum or lung tissue is obtained. During gastric lavage, a tube is inserted through the subject's mouth or nose and sent into the stomach to obtain a gastric secretion sample containing sputum obtained by swallowing sputum into the stomach. DNA can be extracted from sputum samples using any suitable method known in the art. There are a variety of commercial kits for extracting DNA from sputum, such as, but not limited to, NorgenBioteck's Sputum DNA Isolation Kit (Catalog No. 46200) and Qiagen's DNeasy blood and tissue kits. Those skilled in the art can select methods and conditions suitable for extracting DNA from sputum.

[0160] Bronchial washing is a procedure to extract cells from the inside of the airways leading to the lungs. A bronchoscope is inserted into the lungs through the nose or mouth. The airway surface is washed with a mild saline solution to collect cells, which are then viewed under a microscope. A person skilled in the art will be able to select the appropriate method and conditions for collecting a bronchial washing sample.

[0161] A bronchial brushing is a procedure in which a bronchoscope is inserted through the nose or mouth into the lungs. A small brush is then used to collect cells from the airways. These cells are then viewed under a microscope. A bronchial brushing is also called a bronchial brush biopsy. A person skilled in the art will be able to select the appropriate method and conditions for collecting a bronchial brushing sample.

[0162] DNA can be extracted from bronchial washing fluid or bronchial brushing samples using any suitable method known in the art. There are a variety of commercial kits available for extracting DNA from samples collected from bronchial washing fluid and bronchial brushing samples, such as, but not limited to, Takara's Nucleospin Tissue Kit and Qiagen's DNeasy blood and tissue kits. Those skilled in the art will be able to select methods and conditions suitable for extracting DNA from bronchial washing fluid and bronchial brushing samples.

[0163] Urine samples can be collected using any suitable method known in the art. Exemplary methods for collecting urine samples include, but are not limited to, sterile urine bags, catheterization (CATH), suprapubic aspiration (SPA), or clean capture (CC). Those skilled in the art can select methods and conditions suitable for collecting urine samples. DNA can be extracted from urine samples using any suitable method known in the art. There are a variety of commercial kits for extracting DNA from urine, such as, but not limited to, Zymo Research's Quick-DNA Urine Kit, Abcam's DNA Isolation Kit-Urine (ab156899), and NorgenBiotek's Urine DNA isolation kit. Those skilled in the art can select methods and conditions suitable for extracting DNA from urine.

[0164] Saliva samples can be collected using any suitable method known in the art. In some embodiments, saliva samples are collected using a passive saliva method or an oral swab method. One skilled in the art will be able to select methods and conditions suitable for collecting saliva samples. DNA can be extracted from saliva samples using any suitable method known in the art. There are a variety of commercial kits for extracting DNA from saliva, such as, but not limited to, MagMAX Saliva gDNA Isolation Kit from Thermo Fisher, ELISA Kit from Norgen Biotek, and ELISA Kit from Norgen Biotek. DNA Saliva Kit and Saliva DNA Isolation Kit (Cat. No. RU45400). A person skilled in the art can select a method and conditions suitable for extracting DNA from saliva.

[0165] Gene methylation levels can be measured by any suitable method known in the art and disclosed herein. Methods for measuring gene methylation levels will be discussed in detail in the next section.

[0166] In one aspect, the present invention provides a method comprising (a) measuring the methylation level of at least two genes in a blood sample from a subject, wherein the two genes are selected from CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781; (b) determining whether the at least two genes are hypermethylated; (c) when hypermethylation of the at least two genes is detected, diagnosing the subject with at least one malignant pulmonary nodule and / or lung cancer; (d) administering an effective amount of at least one lung cancer treatment to the subject diagnosed with at least one malignant pulmonary nodule.

[0167] In some embodiments, the method comprises (a) collecting a sample from a subject; (b) extracting DNA from the sample; (c) measuring the methylation level of at least two genes in the extracted DNA, wherein the at least two genes are selected from CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781; (d) determining whether the at least two genes are hypermethylated; (e) when hypermethylation of the at least two genes is detected, diagnosing the subject with at least one malignant pulmonary nodule and / or lung cancer; (f) administering an effective amount of at least one lung cancer treatment to the subject diagnosed with at least one malignant pulmonary nodule.

[0168] In some embodiments, the sample is a blood sample, a sputum sample, a bronchial washing sample, a bronchial brushing sample, a urine sample, or a saliva sample. In some embodiments, the sample is a blood sample.

[0169] In some embodiments, the method comprises (a) collecting a blood sample from a subject; (b) separating plasma from the blood sample; (c) extracting DNA from the separated plasma; (d) measuring the methylation level of at least two genes in the extracted DNA, wherein the at least two genes are selected from CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781; (e) determining whether the at least two genes are hypermethylated; (f) when hypermethylation of the at least two genes is detected, diagnosing the subject with at least one malignant pulmonary nodule and / or lung cancer; (g) administering an effective amount of at least one lung cancer treatment to the subject diagnosed with having at least one malignant pulmonary nodule.

[0170] In some embodiments, the methylation levels of CDO1 and PTGER4 are measured, and detecting hypermethylation of CDO1 and PTGER4 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0171] In some embodiments, the methylation levels of CDO1 and HOXA9 are measured, and detecting hypermethylation of CDO1 and HOXA9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0172] In some embodiments, the methylation levels of CDO1 and SHOX2 are measured, and detecting high methylation of CDO1 and SHOX2 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0173] In some embodiments, the methylation levels of CDO1 and SP9 are measured, and detecting high methylation of CDO1 and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0174] In some embodiments, the methylation levels of CDO1 and ZNF781 are measured, and detection of high methylation of CDO1 and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0175] In some embodiments, the methylation levels of PTGER4 and HOXA9 are measured, and detecting hypermethylation of PTGER4 and HOXA9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0176] In some embodiments, the methylation levels of PTGER4 and SHOX2 are measured, and detecting hypermethylation of PTGER4 and SHOX2 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0177] In some embodiments, the methylation levels of PTGER4 and SP9 are measured, and detecting hypermethylation of PTGER4 and SP7 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0178] In some embodiments, the methylation levels of PTGER4 and ZNF781 are measured, and detecting hypermethylation of PTGER4 and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0179] In some embodiments, the methylation levels of HOXA9 and SHOX2 are measured, and detecting hypermethylation of HOXA9 and SHOX2 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0180] In some embodiments, the methylation levels of HOXA9 and SP9 are measured, and detecting high methylation of HOXA9 and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0181] In some embodiments, the methylation levels of HOXA9 and ZNF781 are measured, and detecting hypermethylation of HOXA9 and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0182] In some embodiments, the methylation levels of SHOX2 and SP9 are measured, and detecting hypermethylation of SHOX2 and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0183] In some embodiments, the methylation levels of SHOX2 and ZNF781 are measured, and detecting hypermethylation of SHOX2 and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0184] In some embodiments, the methylation levels of SP9 and ZNF781 are measured, and detecting high methylation of SP9 and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0185] In some embodiments, the methylation levels of CDO1, PTGER4, and HOXA9 are measured, and detecting hypermethylation of all of CDO1, PTGER4, and HOXA9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0186] In some embodiments, the methylation levels of CDO1, PTGER4, and SHOX2 are measured, and detecting hypermethylation of all of CDO1, PTGER4, and SHOX2 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0187] In some embodiments, the methylation levels of CDO1, PTGER4, and SP9 are measured, and detecting hypermethylation of all of CDO1, PTGER4, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0188] In some embodiments, the methylation levels of CDO1, PTGER4, and ZNF781 are measured, and detecting hypermethylation of all of CDO1, PTGER4, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0189] In some embodiments, the methylation levels of CDO1, HOXA9, and SHOX2 are measured, and detection of hypermethylation of all of CDO1, HOXA9, and SHOX2 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0190] In some embodiments, the methylation levels of CDO1, HOXA9, and SP9 are measured, and detection of hypermethylation of all of CDO1, HOXA9, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0191] In some embodiments, the methylation levels of CDO1, HOXA9, and ZNF781 are measured, and detection of hypermethylation of all of CDO1, HOXA9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0192] In some embodiments, methylation levels of CDO1, SHOX2, and SP9 are measured, and detection of hypermethylation in all of CDO1, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0193] In some embodiments, the methylation levels of CDO1, SHOX2, and ZNF781 are measured, and detecting hypermethylation of all of CDO1, SHOX2, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0194] In some embodiments, the methylation levels of CDO1, SP9, and ZNF781 are measured, and detection of high methylation of all of CDO1, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0195] In some embodiments, the methylation levels of PTGER4, HOXA9, and SHOX2 are measured, and detecting hypermethylation of all of PTGER4, HOXA9, and SHOX2 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0196] In some embodiments, the methylation levels of PTGER4, HOXA9, and SP9 are measured, and detecting hypermethylation of all of PTGER4, HOXA9, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0197] In some embodiments, the methylation levels of PTGER4, HOXA9, and ZNF781 are measured, and detecting hypermethylation of all of PTGER4, HOXA9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0198] In some embodiments, the methylation levels of PTGER4, SHOX2, and SP9 are measured, and detection of hypermethylation of all of PTGER4, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0199] In some embodiments, the methylation levels of PTGER4, SHOX2, and ZNF781 are measured, and detection of hypermethylation of all of PTGER4, SHOX2, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0200] In some embodiments, the methylation levels of PTGER4, SP9, and ZNF781 are measured, and detecting hypermethylation of all of PTGER4, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0201] In some embodiments, the methylation levels of HOXA9, SHOX2, and SP9 are measured, and detecting hypermethylation in all of HOXA9, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0202] In some embodiments, the methylation levels of HOXA9, SHOX2, and ZNF781 are measured, and detecting hypermethylation of all of HOXA9, SHOX2, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0203] In some embodiments, the methylation levels of HOXA9, SP9, and ZNF781 are measured, and detecting hypermethylation of all of HOXA9, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0204] In some embodiments, the methylation levels of SHOX2, SP9, and ZNF781 are measured, and detecting hypermethylation of all of SHOX2, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0205] In some embodiments, the methylation levels of CDO1, PTGER4, HOXA9, and SHOX2 are measured, and detecting hypermethylation of all of CDO1, PTGER4, HOXA9, and SHOX2 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0206] In some embodiments, the methylation levels of CDO1, PTGER4, HOXA9, and SP9 are measured, and detecting hypermethylation of all of CDO1, PTGER4, HOXA9, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0207] In some embodiments, the methylation levels of CDO1, PTGER4, HOXA9, and ZNF781 are measured, and detecting hypermethylation of all of CDO1, PTGER4, HOXA9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0208] In some embodiments, the methylation levels of CDO1, PTGER4, SHOX2, and SP9 are measured, and detecting hypermethylation of all of CDO1, PTGER4, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0209] In some embodiments, the methylation levels of CDO1, PTGER4, SHOX2, and ZNF781 are measured, and detecting hypermethylation of all of CDO1, PTGER4, SHOX2, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0210] In some embodiments, the methylation levels of CDO1, PTGER4, SP9, and ZNF781 are measured, and detecting high methylation of all of CDO1, PTGER4, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0211] In some embodiments, the methylation levels of CDO1, HOXA9, SHOX2, and SP9 are measured, and detecting hypermethylation of all of CDO1, HOXA9, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0212] In some embodiments, the methylation levels of CDO1, HOXA9, SHOX2, and ZNF781 are measured, and detecting hypermethylation of all of CDO1, HOXA9, SHOX2, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0213] In some embodiments, the methylation levels of CDO1, HOXA9, SP9, and ZNF781 are measured, and detecting hypermethylation of all of CDO1, HOXA9, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0214] In some embodiments, the methylation levels of CDO1, SHOX2, SP9, and ZNF781 are measured, and detecting hypermethylation of all of CDO1, SHOX2, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0215] In some embodiments, the methylation levels of PTGER4, HOXA9, SHOX2, and SP9 are measured, and detecting hypermethylation in all of PTGER4, HOXA9, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0216] In some embodiments, the methylation levels of PTGER4, HOXA9, SHOX2, and ZNF781 are measured, and detecting hypermethylation of all of PTGER4, HOXA9, SHOX2, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0217] In some embodiments, the methylation levels of PTGER4, HOXA9, SP9, and ZNF781 are measured, and detecting hypermethylation of all of PTGER4, HOXA9, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0218] In some embodiments, the methylation levels of PTGER4, SHOX2, SP9, and ZNF781 are measured, and detecting hypermethylation of all of PTGER4, SHOX2, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0219] In some embodiments, the methylation levels of HOXA9, SHOX2, SP9, and ZNF781 are measured, and detecting hypermethylation of all of HOXA9, SHOX2, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0220] In some embodiments, the methylation levels of CDO1, PTGER4, HOXA9, SHOX2, and SP9 are measured, and detecting hypermethylation in all of CDO1, PTGER4, HOXA9, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0221] In some embodiments, the methylation levels of CDO1, PTGER4, HOXA9, SHOX2, and ZNF781 are measured, and detecting hypermethylation of all of CDO1, PTGER4, HOXA9, SHOX2, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0222] In some embodiments, measuring the methylation levels of CDO1, PTGER4, HOXA9, ZNF781, and SP9, and detecting hypermethylation of all of CDO1, PTGER4, HOXA9, ZNF781, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0223] In some embodiments, the methylation levels of CDO1, PTGER4, ZNF781, SHOX2, and SP9 are measured, and detecting hypermethylation of all of CDO1, PTGER4, ZNF781, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0224] In some embodiments, the methylation levels of CDO1, ZNF781, HOXA9, SHOX2, and SP9 are measured, and detecting hypermethylation in all of CDO1, ZNF781, HOXA9, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0225] In some embodiments, the methylation levels of ZNF781, PTGER4, HOXA9, SHOX2, and SP9 are measured, and detection of hypermethylation of all of ZNF781, PTGER4, HOXA9, SHOX2, and SP9 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0226] In some embodiments, measuring the methylation levels of CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781, and detecting hypermethylation of all of CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0227] In some embodiments, the sample is a blood sample, a sputum sample, a bronchial washing sample, a bronchial brushing sample, a urine sample, or a saliva sample. In some embodiments, the sample is a blood sample.

[0228] In some embodiments, the lung cancer treatment method is any treatment method known in the art or disclosed herein. In some embodiments, the lung cancer treatment method is selected from surgery, chemotherapy, radiation therapy, immunotherapy and targeted drug therapy.

[0229] Lung cancer treatment surgeries include, but are not limited to, therapeutic surgery to remove cancerous tumors from the body; preventive surgery to remove tissue that does not contain cancer cells but may develop into malignant nodules or tumors; diagnostic surgery to remove tissue samples for testing and evaluation to help confirm the diagnosis; staging surgery to reveal the extent of cancer development in the body or the extent of physical disease; tumor resection surgery to remove a portion (although not all) of a cancerous tumor or tumor growth; palliative surgery to relieve discomfort or correct problems caused by other cancer treatments; supportive surgery, used in conjunction with other cancer treatments to help them work effectively; restorative surgery to help subjects restore their appearance or function of body parts after other cancer treatments or surgery. A person skilled in the art will be able to use the methods disclosed herein to select the appropriate type of surgery for the treatment of subjects who have been diagnosed with carrying malignant lung nodules or lung cancer.

[0230] Chemotherapy is a cancer treatment method that uses one or more anticancer drugs (chemotherapeutic drugs) as part of a standardized cancer treatment regimen, with a curative or palliative effect. Chemotherapy is the application of chemicals or drugs to kill cancer cells, and its effects are systemic. Based on their mechanism of action, they are divided into different categories, including: a) alkylating agents that damage DNA; b) antimetabolites that replace the normal structural units of RNA and DNA; c) antibiotics that interfere with DNA replication enzymes; d) topoisomerase inhibitors, which inhibit topoisomerase I or II (the enzyme involved in unwinding DNA during replication and transcription); e) mitotic inhibitors that inhibit mitosis and cell division; f) corticosteroids, which are used to treat cancer and reduce the side effects of other drugs. The chemotherapeutic drugs used in the treatment of lung cancer include but are not limited to Xeloda, Avastin, Tarceva, cyclophosphamide, paclitaxel, docetaxel, Gemcitabine, Elbitol, Alimta, Navibin, Platinum, Tresal, Ethylthiol, Iressa, Nesso, Platinum AQ, Photophorin, Onxol, Cisplatin, Taxane, Gefitinib, Gemcitabine, Erlotinib, Doxorubicin, Belotecan, Bendamustine, Picolatine and Palifenil. Those skilled in the art can use the method disclosed herein to select a suitable chemotherapy regimen to treat a subject determined to carry a malignant pulmonary nodule or lung cancer.

[0231] Radiation therapy is the application of high-energy radiation to kill cancer cells, shrink tumors, or slow their growth. High doses of radiation therapy kill cancer cells or slow their growth by damaging their DNA. Cancer cells with DNA that is damaged beyond repair stop dividing and die. When the damaged cells die, they are broken down and removed by the body. There are two forms of radiation therapy: external and internal. External radiation therapy uses a machine that directs high-energy rays or beams from outside the body to the tumor or tumor growth. Types of external radiation therapy include, but are not limited to, three-dimensional conformal radiation therapy (3D-CRT), image-guided radiation therapy (IGRT), intensity-modulated radiation therapy (IMRT), helical tomotherapy, photon beam radiation therapy, proton beam radiation therapy, intraoperative radiation therapy (IORT), and stereotactic radiosurgery. Internal radiation therapy is a treatment in which a radiation source is placed inside the body. The radiation source can be solid or liquid. Internal radiation therapy using a solid radiation source is called brachytherapy, and pellets, strips, or capsules containing the radiation source are placed inside or near the tumor or growth in the subject's body. Internal radiation therapy using a liquid radioactive source is called systemic therapy, which means that the treatment spreads through the blood to tissues throughout the body to find and kill cancer cells. A person skilled in the art can use the methods disclosed herein to select appropriate radiation therapy for a subject who has been determined to carry malignant lung nodules or lung cancer. Targeted drug therapy is the use of drugs or other substances to block the growth and spread of cancer by interfering with specific molecular targets involved in the growth, progression and spread of cancer. Targeted drug therapy is also known as "molecular targeted drug therapy", "molecular targeted drugs", "precision medicine" or other similar names. Many different targeted drug therapies have been approved for cancer treatment, including but not limited to hormone therapy, signal transduction inhibitors, apoptosis inducers, angiogenesis inhibitors, immunotherapy and gene therapy.

[0232] Hormone therapy can slow or stop the growth of hormone-sensitive tumors. Hormone therapy works by stopping the body from producing hormones or interfering with the effects of hormones. In some embodiments, estrogen and progesterone can be used to treat female patients with non-small cell lung cancer (Katcoff et al., 2014).

[0233] Signal transduction inhibitors can block the activity of molecules involved in signal transduction. Signal transduction is the process by which cells respond to signals from their environment. During this process, once a cell receives a specific signal, the signal is transmitted within the cell through a series of biochemical reactions, ultimately producing an appropriate response. In some cancers, malignant cells are stimulated to divide continuously without being stimulated by external growth factors. Signal transduction inhibitors can interfere with this inappropriate signal transduction. In some embodiments, targeted signal pathways include but are not limited to EGFR, VEGF, IGF, PI3K / Akt, TRAIL, HSP, HDAC, and EML4 / ALK pathways. In some embodiments, signal transduction inhibitors used for the treatment of lung cancer are BIBW2992, wandatinib, sunitinib, sorafenib, motesanib, bevacizumab in combination with erlotinib, filgemax, CI-994, or cyclizumab.

[0234] Apoptosis inducers are agents that cause cancer cells to undergo a controlled cell death process called apoptosis. Apoptosis is a method used by the body to eliminate unwanted or abnormal cells, but cancer cells have strategies to avoid apoptosis. Apoptosis inducers can bypass these cancer cell strategies to avoid apoptosis and cause cancer cell death. Studies have shown that cysteine-binding [6]-tocopherol (M2), AZD6244, and Viburnum grandiflorum extract can induce apoptosis in lung cancer cells (Meng et al., 2010; Warin et al., 2014; Han et al., 2020).

[0235] Angiogenesis inhibitors can prevent new blood vessels from growing toward a tumor or malignancy (a process called tumor angiogenesis). Because blood provides the oxygen and nutrients needed for tumors to continue to grow, blood supply is necessary for tumors to grow beyond a certain size. Treatments that interfere with angiogenesis may prevent tumor growth. Some targeted therapies that inhibit angiogenesis interfere with the effects of vascular endothelial growth factor (VEGF, a substance that stimulates the formation of new blood vessels). Other angiogenesis inhibitors target other molecules that stimulate the growth of new blood vessels. In some embodiments, the angiogenesis inhibitor used in the methods described herein is bevacizumab or ramucirumab.

[0236] Immunotherapies can trigger the immune system to destroy cancer cells. Some immunotherapies are monoclonal antibodies that recognize specific molecules on the surface of cancer cells, and the binding of the monoclonal antibody to the target molecule can cause the immune system to destroy cells expressing the target molecule. Other monoclonal antibodies bind to certain immune cells and can help these cells better kill cancer cells. Some monoclonal antibodies are protein inhibitors produced by certain types of immune system cells (such as T cells and some cancer cells). These proteins help prevent the immune response from being too strong and sometimes prevent T cells from killing cancer cells. When these proteins are blocked, T cells can kill cancer cells. Examples of such proteins found on T cells or cancer cells include PD-1 / PD-L1 and CTLA-4 / B7-1 / B7.2. In some embodiments, the monoclonal antibodies used in the methods described herein are nivolumab, pembrolizumab, or atezolizumab (Doroshow 2019). Cancer vaccines are another type of immunotherapy because they fight cancer by boosting the immune system. In some embodiments, the cancer vaccine used in the methods described herein is CIMAvaxEGF.

[0237] In some embodiments, lung cancer treatment methods involve gene therapy. Gene therapy is achieved by changing or manipulating gene expression or changing the biological properties of living cells. In some embodiments, p53 gene therapy or TUSC2 gene therapy is used in the methods described herein.

[0238] Molecular targets for lung cancer treatment include, but are not limited to, the following genes: KRAS, EGFR, ALK, ROS1, BRAF, RET, MET, and NTRK. Targeted drug therapies currently available for lung cancer include, but are not limited to, bevacizumab, ramucirumab, sotolacib, erlotinib, afatinib, gefitinib-osimertinib, dacomitinib, osimertinib, amivanta monoclonal antibody, mobotinib, nesitutumab, crizotinib, citinib, alectinib, britatinib, lorlatinib, entinib, dabrafenib, trametinib, selpatinib, plasitinib, capmatinib, tepotinib, and larotrectinib. Those skilled in the art are able to select appropriate targeted drug therapies for treating subjects who have been determined to carry malignant pulmonary nodules or lung cancer using the methods described herein.

[0239] In some embodiments, the above lung cancer treatments are used in combination. For example, in some embodiments, radiation therapy is given before surgery to reduce the size of the tumor so that it can be removed surgically and is less likely to recur. In some embodiments, a combination of the chemotherapy drug topotecan and the targeted drug berzosertib is used to treat small cell lung cancer.

[0240] In some embodiments, the subject has been previously determined to have at least one pulmonary nodule. In some embodiments, the subject has not been previously determined to have at least one pulmonary nodule.

[0241] In some embodiments, the subject is from the general population. As used herein, the general population for the purposes of the present invention refers to the entire population, including the lung nodule population and the population without lung nodules. In some embodiments, the subject is from a high-risk population for lung cancer. As used herein, a high-risk population for lung cancer refers to a population determined to have a higher likelihood of lung cancer (there are many reasons why a person is more likely to have lung cancer). In some embodiments, because someone has at least one lung nodule, it is determined that the possibility of lung cancer is higher. In some embodiments, because someone smokes, or has a family history of lung cancer, or is frequently exposed to high levels of radon, or has received chest radiotherapy, it is determined that the possibility of lung cancer is higher. DNA methylation level measurement

[0242] Gene methylation levels can be measured by any suitable method known in the art and disclosed herein. "Recent Advances in DNA Methylation Analysis Methods" provides an exemplary method for measuring DNA methylation levels (Khodadadi et al., 2021).

[0243] In some embodiments, the level of DNA methylation is measured by (1) converting unmethylated cytosine to uracil while retaining methylated cytidine as cytosine; and (2) measuring the level of conversion of unmethylated cytosine to uracil.

[0244] In some embodiments, unmethylated cytosine is converted to uracil by bisulfite treatment. Bisulfite conversion is a three-step reaction in which cytosine is first converted to sulfonated cytosine at low pH and high temperature, then deaminated to sulfonated uracil, and finally converted to uracil in an alkaline desulfurization process. Subsequent PCR amplification converts uracil to thymine. In the case of 5mC, the deamination step is nearly two orders of magnitude slower than that of cytosine. Therefore, after bisulfite conversion, 5mC remains unchanged and is amplified to cytosine by PCR. Since double-stranded DNA (dsDNA) protects C from deamination, DNA generally needs to be denatured and purified prior to conversion. There are a variety of commercial bisulfite conversion kits, including but not limited to Epigentek's Bisulflash TM DNA Modification Kit (Cat. No. P-1026), Bisulflash TMDNA Bisulfite Conversion Easy Kit (Cat. No. P-1054), Diagenode's Premium Bisulfite Kit (Cat. No. C02030030), Sigma-Aldrich's DNA Modification Kit (Cat. No. MOD50), EZ DNA Methylation-GoldT from Zymo Research M Kit (Cat. No. D5005), EZ DNA Methylation-Lightning TM Kit (Cat. No. D5030), Fast Bisulfite Conversion Kit (Cat. No. ab1127127) of the company, InnuCONVERT Bisulfite Basic Kit (Cat. No. 845-IC-1000008) of Analytic Jena, and Qiagen Fast DNA Bisulfite Kit (Cat. No. 59824), Bisulfite Kit (Qiagen, 59110), CpGenome from Merck Millipore TM TurboBisulfite Modification Kit (Cat. No. S7847) and Methyleasy from Human Genetic Signatures TM Xceed (Cat. No. ME001) (Knit et al., 2018). Other available methods include, for example, Wang et al., "An improved bisulfite conversion method for detecting DNA methylation (Epigenomics 2017, 9955-969)".

[0245] In some embodiments, unmethylated cytosine in DNA is converted to uracil by an enzyme-based method. In some embodiments, unmethylated cytosine in DNA is converted to uracil using a commercially available NEBNext Enzymatic Methylation Sequence Kit. NEBNext Enzymatic Methylation Sequence (EM-seq) is a method for identifying 5mC and 5hmC. The efficient enzymatic conversion in this method minimizes damage to DNA, and through the provided NEBNext UltraTMII library preparation reagents, a high-quality library is established, and excellent detection of 5mC and 5hmC can be achieved from fewer sequencing reads. Gentle treatment of DNA through the steps in the EM-seq workflow minimizes damage to DNA. Therefore, EM-seq-converted DNA is more complete, resulting in a higher percentage of longer insertions in the library. This can achieve longer sequencing reads, thereby improving the confidence of mapping and potentially reducing the sequencing cost per base (depending on the instrument and other sequencing reaction details). In addition, gentle treatment in the EM-seq workflow can also maintain high-quality DNA libraries, which can be effectively amplified, resulting in higher library yields with fewer PCR cycles. Importantly, these higher yields are not due to the presence of PCR duplicates, which may be particularly evident at low input amounts. In fact, EM-seq libraries consistently show low levels of duplicates across a range of input amounts. While sufficient library yield is required for successful sequencing, the quality of the library is also a factor. High-quality libraries are representative of the original sample, as evidenced by uniform coverage of GC spectra. EM-seq libraries show consistent GC coverage, significantly reduced damage to DNA, and are representative of the original sample.

[0246] In some embodiments, only 10 ng of DNA, NEBNext Ultra II reagents, and optimized EM-seq adapters are used to prepare the library. TET2 then oxidizes 5-mC and 5-hmC to prevent them from being deaminated by APOBEC in the next step. Instead, unmodified cytosine is deaminated to uracil. The library is then amplified using the NEBNext master mix recipe of Q5U (an improved version of Q5 High-Fidelity DNA Polymerase).

[0247] In some embodiments, the conversion product is detected and the conversion level is measured, i.e., indicating the methylation level. In some embodiments, the methylation level after conversion is measured by real-time polymerase chain reaction. Real-time fluorescence PCR (qPCR) monitors the amplification of the targeted DNA molecule during PCR (i.e., in real time) rather than at the end thereof. qPCR includes quantitative PCR (real-time quantitative PCR) and semi-quantitative PCR (real-time semi-quantitative PCR). Two common methods for detecting PCR products in real-time PCR are: (1) non-specific fluorescent dyes inserted into any double-stranded DNA; (2) sequence-specific DNA probes composed of oligonucleotides labeled with fluorescent reporters, which only allow detection after the probe hybridizes with its complementary sequence.

[0248] The use of nonspecific fluorescent dyes involves DNA binding dyes that bind to double-stranded (ds) DNA produced during the PCR reaction, increasing the fluorescence quantum yield of the dye. Therefore, the increase in DNA products during the PCR reaction will increase the fluorescence intensity measured in each cycle. In real-time PCR using double-stranded DNA dyes, double-stranded DNA fluorescent dyes are added, the reaction is then carried out in a real-time PCR instrument, and the fluorescence intensity is measured with a detector after each cycle; the dye only fluoresces when it binds to double-stranded DNA (i.e., PCR products). The advantages of this method are: (1) only one pair of primers is required for amplification, thereby reducing costs; and (2) by using different types of dyes, multiple target sequences can be monitored in a tube. However, double-stranded DNA dyes (such as SYBR Green) can bind to all double-stranded DNA PCR products, including nonspecific PCR products (such as primer dimers), which may interfere with or prevent accurate monitoring of the intended target sequence.

[0249] Unlike nonspecific fluorescent dyes, fluorescent reporter probes only detect DNA containing sequences complementary to the probe. Therefore, the use of fluorescent reporter probes significantly increases the specificity of PCR and enables the use of the technique even in the presence of other double-stranded DNA. The use of fluorescent reporter probes of different colors allows for multiplexed analysis to monitor multiple target sequences in the same PCR tube. The specificity of fluorescent reporter probes also prevents measurement interference caused by primer dimers, which are potential undesirable byproducts of PCR. The method relies on a DNA-based probe with a fluorescent reporter at one end and a fluorescent quencher at the other end. Since the fluorescent reporter of the primer dimer is very close to the fluorescent quencher, its fluorescence cannot be detected; the 5′ to 3′ exonuclease activity of Taq DNA polymerase breaks the probe, destroying the proximity of the reporter quencher, allowing unquenched fluorescence emission and can be detected after laser excitation. Therefore, in each PCR cycle, the increase in the product targeted by the reporter probe leads to a proportional increase in fluorescence through the breakdown of the probe and the release of the reporter.

[0250] Real-time PCR can quantify nucleic acids by two common methods: absolute quantification and relative quantification. Absolute quantification gives the exact number of target DNA molecules by comparing with DNA standards using a calibration curve. Relative quantification determines the fold difference in target gene expression based on an internal reference gene. Because relative quantification does not require a calibration curve and only compares the amount of the studied gene with the amount of the control reference gene, relative quantification is easier to perform than absolute quantification. The use of one or more reference genes enables those skilled in the art to correct for nonspecific variations, such as differences in the quantity and quality of the nucleic acids used, as this may affect the efficiency of reverse transcription and thus the efficiency of the entire PCR process.

[0251] In some embodiments, the real-time PCR (qPCR) used to measure the methylation level after conversion is a methylation-specific real-time quantitative PCR, also referred to as methylation-specific qPCR, MS-qPCR or qMSP. If the primers and / or probes used in the qPCR have methylation discrimination capabilities, the qPCR is methylation-specific. In some embodiments, "methylation discrimination capabilities" are achieved by designing primers and / or probes to bind to the converted methylated sites or the converted non-methylated sites. In some embodiments, the methylated sites of interest are CpG sites. Figure 36-41 CpG sites in CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781 are highlighted, where one or more of the CpG sites are methylation sites of interest.

[0252] In some embodiments, the method for measuring the methylation level after conversion is MethyLight. Methylight is a highly sensitive assay that can detect methylated alleles in the presence of 10,000-fold excess of non-methylated alleles. In some embodiments, MethyLight includes amplifying bisulfite-converted DNA using site-specific PCR primers, which are flanked by oligonucleotide probes with 5' fluorescent reporter dyes and 3' quenchers. In some embodiments, the 5' fluorescent reporter dye is 6-carboxyfluorescein (6-FAM), 5-carboxyfluorescein (5-FAM), 5'-dichlorodimethoxyfluorescein (JOE), hexagonal dyes, VIC dyes, Cy5, Cy3, TAMRA or TET dyes. In some embodiments, the 3' quencher is Black Hole Quenchers, Iowa Black FQ, Iowa Black RQ, Dabsyl, Qxl or internal ZENQuencher. The 5′ to 3′ nuclease activity of Taq DNA polymerase cleaves the probe and releases the reporter, whose fluorescence can be detected by a laser detector on an ABI Prism 7700 Sequence Detection System (Perkin Elmer, Foster City, CA). An example of the MethyLight assay is provided in MethyLight: A High-Throughput Assay for Measuring DNA Methylation (Eads et al., 2000).

[0253] In some embodiments, ΔCt is used. As used herein, "Ct" refers to the cycle number, which is defined as the number of cycles required for the fluorescent signal to exceed the threshold value (ie, exceed the background level). The Ct value is inversely proportional to the amount of target nucleic acid in the sample (ie, the lower the Ct value, the higher the target nucleic acid content in the sample). The initial template amount can be derived by the Ct value. A Ct value of <29 is considered a strong positive reaction, indicating that the sample contains abundant target nucleic acid; a Ct value between 30-37 is considered a medium positive reaction, indicating a moderate amount of target nucleic acid; a Ct value between 38-40 is considered a weak reaction, indicating that the number of target nucleic acids is small. ΔCt is calculated by subtracting the Ct value of the reference gene from the Ct value of the candidate gene. The most commonly used reference genes in qPCR include, but are not limited to, β-actin (ACTB), diacylglycerol-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), and hypoxanthine guanine phosphoribosyltransferase (HPRT1).

[0254] In some embodiments, the primers and probes used in the qPCR analysis are designed to specifically anneal to the methylation sites of interest. In some embodiments, the probes are designed not to cover the methylation sites. In some embodiments, the probes are designed to cover the methylation sites, providing a greater degree of methylation recognition capability.

[0255] In some embodiments, DNA methylation is measured by methylation-specific qPCR according to the MethyLight protocol, and the target gene is determined to be hypermethylated if ΔCt≤15.5.

[0256] In some embodiments, methylation-specific qPCR uses a forward primer comprising SEQ ID NO:1, a reverse primer of SEQ ID NO:2, and a probe of SEQ ID NO:3.

[0257] In some embodiments, methylation-specific qPCR uses a forward primer comprising SEQ ID NO:4, a reverse primer of SEQ ID NO:5, and a probe of SEQ ID NO:6.

[0258] In some embodiments, methylation-specific qPCR uses a forward primer comprising SEQ ID NO:7, a reverse primer of SEQ ID NO:8, and a probe of SEQ ID NO:9.

[0259] In some embodiments, methylation-specific qPCR uses a forward primer comprising SEQ ID NO:10, a reverse primer of SEQ ID NO:11, and a probe of SEQ ID NO:12.

[0260] In some embodiments, methylation-specific qPCR uses a forward primer comprising SEQ ID NO:13, a reverse primer of SEQ ID NO:14, and a probe of SEQ ID NO:15.

[0261] In some embodiments, methylation-specific qPCR uses a forward primer of SEQ ID NO:16, a reverse primer of SEQ ID NO:17, and a probe of SEQ ID NO:18.

[0262] In one aspect, the invention provides a sequence of any one of SEQ ID NOs: 1-18. In some embodiments, SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 are used as forward primers, reverse primers, and probes, respectively, in methylation-specific qPCR; in some embodiments, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 are used as forward primers, reverse primers, and probes, respectively, in methylation-specific qPCR; in some embodiments, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are used as forward primers, reverse primers, and probes, respectively, in methylation-specific qPCR; in some embodiments, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 are used as forward primers, reverse primers, and probes, respectively, in methylation-specific qPCR; in some embodiments, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 are used as forward primers, reverse primers, and probes, respectively, in methylation-specific qPCR; in some embodiments, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 are used as forward primers, reverse primers, and probes, respectively, in methylation-specific qPCR.

[0263] In one aspect, the present invention provides a kit for performing CDO1 gene methylation-specific qPCR (hereinafter referred to as "CDO1 kit"), comprising a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2, and a probe of SEQ ID NO: 3. In some embodiments, the kit further comprises bisulfite.

[0264] In one aspect, the present invention provides a kit for performing PTGER4 gene methylation-specific qPCR (hereinafter referred to as "PTGER4 kit"), comprising a forward primer of SEQ ID NO: 4, a reverse primer of SEQ ID NO: 5, and a probe of SEQ ID NO: 6. In some embodiments, the kit further comprises bisulfite.

[0265] In one aspect, the present invention provides a kit for performing HOXA9 gene methylation-specific qPCR (hereinafter referred to as "HOXA9 kit"), comprising a forward primer of SEQ ID NO: 7, a reverse primer of SEQ ID NO: 8, and a probe of SEQ ID NO: 9. In some embodiments, the kit further comprises bisulfite.

[0266] In one aspect, the present invention provides a kit for performing SHOX2 gene methylation-specific qPCR (hereinafter referred to as "SHOX2 kit"), comprising a forward primer of SEQ ID NO: 10, a reverse primer of SEQ ID NO: 11, and a probe of SEQ ID NO: 12. In some embodiments, the kit further comprises bisulfite.

[0267] In one aspect, the present invention provides a kit for performing SP9 gene methylation-specific qPCR (hereinafter referred to as "SP9 kit"), comprising a forward primer of SEQ ID NO: 13, a reverse primer of SEQ ID NO: 14, and a probe of SEQ ID NO: 15. In some embodiments, the kit further comprises bisulfite.

[0268] In one aspect, the present invention provides a kit for performing ZNF781 gene methylation-specific qPCR (hereinafter referred to as "ZNF781 kit"), comprising a forward primer of SEQ ID NO: 16, a reverse primer of SEQ ID NO: 17, and a probe of SEQ ID NO: 18. In some embodiments, the kit further comprises bisulfite.

[0269] In one aspect, the present invention provides a kit for determining whether at least one pulmonary nodule found in a subject is malignant, comprising bisulfite and reagents for performing methylation-specific real-time fluorescence quantitative PCR on at least two genes selected from CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781, wherein the reagents for performing methylation-specific real-time fluorescence quantitative PCR on CDO1 comprise a CDO1 forward primer, a CDO1 reverse primer and a CDO1 probe; the reagents for performing methylation-specific real-time fluorescence quantitative PCR on PTGER4 comprise a PTGER4 forward primer, a PTGER4 reverse primer and a CDO1 probe; 4 reverse primer and PTGER4 probe; reagents for methylation-specific real-time fluorescence quantitative PCR of HOXA9 include HOXA9 forward primer, HOXA9 reverse primer and HOXA9 probe; reagents for methylation-specific real-time fluorescence quantitative PCR of SHOX2 include SHOX2 forward primer, SHOX2 reverse primer and SHOX2 probe; reagents for methylation-specific real-time fluorescence quantitative PCR of SP9 include SP9 forward primer, SP9 reverse primer and SP9 probe; reagents for methylation-specific real-time fluorescence quantitative PCR of ZNF781 include ZNF781 forward primer, ZNF781 reverse primer and ZNF781 probe. In some embodiments, the kit further includes at least two kits selected from CDO1 kit, PTGER4 kit, HOXA9 kit, SHOX2 kit, SP9 kit and ZNF781 kit, as described herein.

[0270] In addition, after methylation-nonspecific PCR amplification, sequence differences caused by various DNA methylation patterns after conversion can also be revealed by other methods. In some embodiments, the method for measuring the methylation level after conversion is methylation-specific high-resolution melting (MS-HRM). MS-HRM analysis is based on the different melting temperatures (Tm) between CG pairs (3H bonds) and AT pairs (2H bonds). Melting analysis is performed after methylation-nonspecific PCR amplification. The temperature gradually increases, and the PCR products of non-methylated alleles containing AT pairs dissociate at lower temperatures and are detected by fluorescent mutations when the inserted dyes (such as SYBR-Green, Eva-Green and SYTO9) are released from double-stranded DNA. PCR amplifications of 100bp or less usually use such analysis methods to avoid the formation of secondary structures that may interfere with the analysis. Wojdacz et al. describe such methods and primer design in "Methylation-sensitive high-resolution melting". Compared with other conversion-based methods, this method is relatively fast and can distinguish between fully or partially methylated sites and non-methylated sites, so it is particularly useful when only a small part of the site in the analysis sample is methylated. However, this method is generally not sensitive enough when analyzing single CpGs. However, if the unknown sample is compared to a standard with a known methylated to unmethylated ratio, the method is semi-quantitative. Overall, MS-HRM provides a rapid and relatively economical method for assessing methylation rates.

[0271] In some embodiments, the method used to measure the methylation level after conversion is pyrosequencing. Bisulfite pyrosequencing is a PCR amplification with a biotinylated primer after bisulfite conversion, the amplicon is fixed on streptavidin beads and hybridized with a sequencing primer, and then sequenced. This method does not provide information about allele-specific methylation, but rather provides the average methylation level of the two alleles in a quantitative manner. The main advantage of this technology is that it can generate background-free chromatograms where the methylation percentage is accurately calculated as C / (C+T). EpigenDx provides commercial gene-specific methylation pyrosequencing analysis.

[0272] DNA methylation levels can also be measured without converting unmethylated cytosine to uracil in advance. In some embodiments, the method for measuring methylation levels is nanopore long read technology. Nanopore long read technology can sequence DNA molecules containing methylation modifications in their native state without the use of enzymes or chemical treatments. When DNA molecules pass through the nanopore, methylated bases can produce unique signals for detection and can be distinguished from unmodified DNA bases. For more information on nanopore technology and its application in DNA methylation measurement, please refer to "Detection of DNA cytosine methylation using nanopore sequencing" (Simpson, JT et al., 2017), "Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA" (Laszlo, AH et al., 2013), "Mapping DNA methylation using high-throughput nanopore sequencing" (Rand, AC et al., 2017).

[0273] In certain embodiments, DNA methylation levels are measured by methylation-specific restriction endonuclease digestion. Several site-specific methylation-dependent restriction endonucleases are available, such as the nuclease HpaI, which can digest CCGG sequences when not methylated. In contrast, the MspI enzyme that cuts DNA at the CCGG site is not affected by DNA methylation. Other methylation-specific restriction endonucleases include, but are not limited to, HpaII, AatII, and ClaI. After digestion with one or more methylation-specific restriction endonucleases, the position of the DNA methylation site is revealed by sequencing analysis, qPCR analysis using primers for the cleavage site, or electrophoresis analysis of the digestion product. Qiagen's EpiTect-Methyl II PCR array system is a commercial system for DNA methylation analysis based on digestion (Kurdyukove et al., 2016; Zo et al., 2009; Jiang et al., 2012). Example

[0274] The present invention can be further described by the following non-limiting examples, wherein, where appropriate, standard techniques known to those skilled in the art and techniques similar to those described in these examples can be used. It should be understood that those skilled in the art will envision other embodiments consistent with the disclosure provided herein. Example 1: Whole genome array for identification of candidate genes for detection of malignant pulmonary nodules

[0275] Whole genome 850K Illumina methylation array analysis was performed using blood from patients with malignant and benign pulmonary nodules. Genomic DNA was extracted from the blood using the QIAamp circulating nucleic acid kit (Qiagen, Germany) and treated with bisulfite conversion using the EZ-DNA methylation lightning kit (Zymo, USA). The Infinium Methylation EPIC BeadChip (850K) (Illumina, USA) was used to determine candidate differentially methylated sites. The methylation status of the probe was expressed as a β value, and CpG sites containing β ≥ 0.20 and an adjusted p value ≤ 0.05 were defined as differentially methylated sites. result

[0276] The following candidate genes were identified from genome-wide analysis of clinical samples: CDO1, NEUROD1, NEUROG1, ONECUT2, PITX2, SFRP2, SLC38A4, TFAP2A, GDNF, SFRP5, CCND2, HOXA1, SIM1, GALR1, PTGER4, LOC643719, ADCY4, C1ORF114, OTX1, TFAP2B, DMRTA2, TBX15, SOX1, EVX2, SOX17, SHOX2, VIPR2, TRIM58, BCL2, CDX2, TWIST1, DPP6, TBX4, CMTM2, LBXCOR, AJAP1, HOXA9, RASSF1A, MARCH 11. ZNF781, RPPH1, ZIC4, TAC1, HOXA7, PRRX1, OPCML, CDKN2A, SIX3, DLX4, TMEFF2, RUNX3, GATA3, HOXA11, RAR β, PRDM14, HOXB4, HOXA10, PENK, SP9, DLEC1, MT1G, GRIN2B, CDH13, RARRES1, ITGA4, CRABP1, SEZ6L.

[0277] CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781 were identified as the most promising candidates for distinguishing malignant from benign pulmonary nodules. Example 2: Detection of malignant pulmonary nodules by methylation levels of CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781 Blood samples were collected from 179 patients with lung cancer and 71 patients with benign pulmonary nodules, and plasma was separated by centrifugation. DNA was extracted from the plasma samples and converted to uracil after bisulfite treatment. The methylation status of CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781 genes was measured by methylation-specific real-time fluorescence quantitative PCR. The primer and probe sequences used for qPCR are shown in Table 3. Methylation-specific real-time fluorescence quantitative PCR (q-MSP) was used to detect and verify specific methylation sites. All samples were tested in duplicate three times. For some samples where no methylation was detected, Ct=60 was used to calculate, and the difference between the Ct of each methylation repeat detection and the average Ct of β-actin (ACTB) was calculated, ΔCt and 2 -ΔCt , calculate mean2 according to the following formula -ΔCt : result The results of qPCR analysis are shown in Table 1. Figure 1-3 and Figure 8-10 The data showed that CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781 genes were able to distinguish between malignant and benign pulmonary nodules.

[0281] Table 1 Specificity and sensitivity of each biomarker in blood CDO1 PTGER4 HOXA9 SHOX2 SP9 ZNF781 Sensitivity (%) 46.6 39.4 26.5 33.8 60.0 61.5 Specificity (%) 90.3 94.9 80 89.1 54.0 63.5 Example 3: Detection of malignant pulmonary nodules by methylation levels of at least two genes selected from CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781

[0282] The data collected in Example 2 were further analyzed to determine whether detecting methylation of the combination of CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781 genes improves the ability to distinguish between malignant and benign pulmonary nodules. The area under the receiver operating characteristic curve (ROC) was calculated using 2 -ΔCt Ct values ​​used to evaluate the diagnostic performance of specific genes selected. All data analyses were performed using GraphPad Prism. result

[0283] Further analysis is shown in Table 2. Figure 4-7 and Figure 11-35 The analysis showed that the combination of multiple genes produced better sensitivity and specificity compared with a single gene. The analysis further showed that the combination of CDO1 and PTGER4 had the best sensitivity and specificity in distinguishing benign from malignant pulmonary nodules. Adding HOXA9 to the combination of CDO1 and PTGER4 did not improve sensitivity and specificity. Table 2 Results of combined analysis of at least two biomarkers in blood. 1: SHOX2; 2: SP9; 3: PTGER4; 4: CDO1; 5: ZNF781; 6.HOXA9 Table 3 Primer and probe sequences

Claims

1. A method comprising: (a) measuring the methylation level of at least two genes in a sample from a subject, wherein the at least two genes are selected from CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781; (b) determining whether the at least two genes are hypermethylated, wherein detecting the hypermethylation of the at least two genes indicates that the subject has at least one malignant lung nodule and / or lung cancer.

2. A method comprising: (a) measuring the methylation level of at least two genes in a sample from a subject, wherein the at least two genes are selected from CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781; (b) determining whether at least two genes are hypermethylated; (c) when hypermethylation of at least two genes is detected, diagnosing the subject with at least one malignant lung nodule and / or lung cancer; (d) administering an effective amount of at least one lung cancer treatment to a subject diagnosed with at least one malignant lung nodule and / or lung cancer.

3. The method of claim 2, wherein the lung cancer treatment is selected from surgery, chemotherapy, radiation therapy, immunotherapy and targeted drug therapy.

4. The method according to any one of claims 1 to 3, wherein the sample is a blood sample, a sputum sample, a bronchial washing fluid sample, a bronchial brushing sample, a urine sample or a saliva sample.

5. The method according to any one of claims 1 to 4, wherein the methylation level is measured by: (1) converting the unmethylated cytosine in the DNA of step (c) into uracil, while retaining the methylated cytosine as cytosine; (2) Measure the level of conversion of unmethylated cytosine to uracil. The method according to claim 5 , wherein the unmethylated cytosine in the DNA is converted into uracil by bisulfite treatment or enzyme treatment.

7. The method according to any one of claims 5 and 6, wherein the measuring is performed by real-time polymerase chain reaction (PCR), sequencing or microarray methods.

8. The method according to claim 7, wherein the real-time polymerase chain reaction (PCR) is methylation-specific real-time fluorescent quantitative PCR. 9 . The method according to claim 8 , wherein the reagents used in the methylation-specific real-time fluorescence quantitative PCR comprise a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2, and a probe of SEQ ID NO:

3. 10 . The method according to claim 8 , wherein the reagents used in the methylation-specific real-time fluorescence quantitative PCR include a forward primer of SEQ ID NO: 4, a reverse primer of SEQ ID NO: 5, and a probe of SEQ ID NO:

6. 11 . The method according to claim 8 , wherein the reagents used in the methylation-specific real-time fluorescence quantitative PCR comprise a forward primer of SEQ ID NO: 7, a reverse primer of SEQ ID NO: 8, and a probe of SEQ ID NO:

9. 12 . The method according to claim 8 , wherein the reagents used in the methylation-specific real-time fluorescence quantitative PCR include a forward primer of SEQ ID NO: 10, a reverse primer of SEQ ID NO: 11, and a probe of SEQ ID NO:

12. 13 . The method according to claim 8 , wherein the reagents used in the methylation-specific real-time fluorescence quantitative PCR comprise a forward primer of SEQ ID NO: 13, a reverse primer of SEQ ID NO: 14, and a probe of SEQ ID NO:

15. 14 . The method according to claim 8 , wherein the methylation-specific real-time fluorescence quantitative PCR uses reagents comprising a forward primer of SEQ ID NO: 16, a reverse primer of SEQ ID NO: 17, and a probe of SEQ ID NO:

18.

15. The method of any one of claims 1-4, wherein the methylation level is measured by methylation-specific high-resolution melting, pyrosequencing, nanopore long-read technology, or methylation-specific restriction endonuclease digestion.

16. The method according to any one of claims 1 to 15, wherein the lung cancer is non-small cell lung cancer or small cell lung cancer.

17. The method of any one of claims 1-16, wherein the subject has been previously determined to have at least one pulmonary nodule.

18. The method according to any one of claims 1 to 17, wherein the subject belongs to a high-risk population for lung cancer.

19. A polynucleotide having any one of SEQ ID NOs: 1-18.

20. A kit for performing methylation-specific real-time fluorescent quantitative PCR on CDO1, comprising a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2 and a probe of SEQ ID NO:

3.

21. A kit for performing methylation-specific real-time fluorescence quantitative PCR on PTGER4, comprising a forward primer of SEQ ID NO: 4, a reverse primer of SEQ ID NO: 5 and a probe of SEQ ID NO:

6.

22. A kit for performing methylation-specific real-time fluorescent quantitative PCR on HOXA9, comprising a forward primer of SEQ ID NO: 7, a reverse primer of SEQ ID NO: 8 and a probe of SEQ ID NO:

9.

23. A kit for performing methylation-specific real-time fluorescent quantitative PCR on SHOX2, comprising a forward primer of SEQ ID NO: 10, a reverse primer of SEQ ID NO: 11 and a probe of SEQ ID NO:

12.

24. A kit for performing methylation-specific real-time fluorescent quantitative PCR on SP9, comprising a forward primer of SEQ ID NO: 13, a reverse primer of SEQ ID NO: 14 and a probe of SEQ ID NO:

15.

25. A kit for performing methylation-specific real-time fluorescent quantitative PCR on ZNF781, comprising a forward primer of SEQ ID NO: 16, a reverse primer of SEQ ID NO: 17 and a probe of SEQ ID NO:

18.

26. The kit according to any one of claims 20-25, further comprising bisulfite.

27. A kit for determining whether at least one pulmonary nodule found in a subject is malignant, comprising bisulfite and reagents for performing methylation-specific real-time fluorescence quantitative PCR on at least two genes selected from CDO1, PTGER4, HOXA9, SHOX2, SP9 and ZNF781. The reagents used for methylation-specific real-time fluorescence quantitative PCR of CDO1 include CDO1 forward primer, CDO1 reverse primer and CDO1 probe; the reagents used for methylation-specific real-time fluorescence quantitative PCR of PTGER4 include PTGER4 forward primer, PTGER 4 reverse primer and PTGER4 probe; the reagents used for methylation-specific real-time fluorescence quantitative PCR of HOXA9 include HOXA9 forward primer, HOXA9 reverse primer and HOXA9 probe; the reagents used for methylation-specific real-time fluorescence quantitative PCR of SHOX2 include SHOX2 forward primer, SHOX2 reverse primer and SHOX2 probe; the reagents used for methylation-specific real-time fluorescence quantitative PCR of SP9 include SP9 forward primer, SP9 reverse primer and SP9 probe; the reagents used for methylation-specific real-time fluorescence quantitative PCR of ZNF781 include ZNF781 forward primer, ZNF781 reverse primer and ZNF781 probe.

28. The kit of claim 25 further comprising at least two of the kits of claims 20-25.

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