Methods, Compositions, and Kits for Detecting Malignant Lung Nodules and Lung Cancer

Abstract

The present disclosure provides a minimally invasive method for determining whether a pulmonary nodule is malignant by measuring the methylation levels of a combination of genes including CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781. The disclosed method can also detect lung cancer. The present disclosure also discloses polynucleotides and kits that can be used in measuring the methylation levels of CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781.

Description

Technical Field

[0001] The present disclosure relates to a minimally invasive method for identifying malignant pulmonary nodules by measuring the methylation levels of a combination of genes including CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781 in a sample of interest. The disclosed method can also detect lung cancer. The present disclosure also relates to polynucleotides and kits for use in measuring the methylation levels of CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781.

Summary of the Invention

[0002] Lung cancer is a major global cause of cancer-related death. Effectively and efficiently distinguishing malignant pulmonary nodules from benign pulmonary nodules using a minimally invasive method is a major unmet clinical need. If such a distinction can be made when the malignant pulmonary nodule is small, such a 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 has a high false positive rate. In addition, to identify and distinguish malignant pulmonary nodules from benign pulmonary nodules using CT, a physician needs to be well-trained and experienced. Moreover, CT is not easily accessible and is not affordable for high-risk populations in developing countries.

[0003] Several other approaches, including detection of gene mutations and whole-genome sequencing, have been studied for the early detection of malignant pulmonary nodules and lung cancer. However, the results and effects of these existing methods are not satisfactory. Recent studies have demonstrated that the sensitivity for the detection of stage I lung cancer using existing methods is less than 22% (Klein et al., 2021). In addition, existing methods require large panels of next-generation sequencing, which significantly increases the test cost. Furthermore, many existing methods are invasive and require lung tissue samples for the test. Therefore, there is a need for a highly accurate, specific, cost-effective, and minimally invasive test for the early detection of malignant pulmonary nodules and lung cancer.

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

[0005] DNA methylation is a promising marker for early cancer detection due to its stability and heritability. Aberrant DNA methylation leads to dysregulation of various genes and occurs at all stages of lung cancer, including initiation, proliferation, and metastasis. The present disclosure has identified six genes that are differentially methylated in subjects with malignant lung nodules compared to normal lung tissue. The present disclosure has shown that detection of hypermethylation of a combination of at least two of the six genes disclosed herein indicates the presence of a malignant lung nodule and enables early diagnosis of lung cancer. In some embodiments, the methylation level of the combination of CDO1 and PTGER4 is sufficient to distinguish malignant lung nodules from benign nodules. The cost-effective and minimally invasive method provided herein is suitable for both distinguishing malignant lung nodules and diagnosing lung cancer.

[0006] In one aspect, the present disclosure provides a method comprising: (a) measuring the methylation levels of at least two genes in a sample of a subject, wherein the at least two 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.

[0007] In one aspect, the present disclosure provides a method comprising: (a) measuring the methylation levels of at least two genes in a sample of 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; (c) diagnosing that the subject has at least one malignant pulmonary nodule and / or lung cancer if hypermethylation of the at least two genes is detected; and (d) administering to the subject diagnosed with having at least one malignant pulmonary nodule and / or lung cancer an effective amount of at least one lung cancer treatment.

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

[0009] In some embodiments, the method further comprises, prior to the 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, radiotherapy, immunotherapy, and targeted drug therapy.

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

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

[0013] In some embodiments, the measurement is performed by real-time polymerase chain reaction (PCR), sequencing, or microarray.

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

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

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

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

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

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

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

[0021] In some embodiments, the methylation level is measured by methylation-specific high-resolution melting, pyrosequencing, nanopore long-read technology, or methylation-specific restriction enzyme 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 lung nodule.

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

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

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

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

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

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

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

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

[0032] In one aspect, the present disclosure provides a kit for determining whether at least one lung nodule found in a subject is malignant, the kit comprising bisulfite and reagents for performing methylation-specific quantitative real-time PCR on at least two genes selected from the group consisting of CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781, wherein the reagent for performing methylation-specific quantitative real-time PCR on CDO1 comprises a CDO1 forward primer, a CDO1 reverse primer, and a CDO1 probe; the reagent for performing methylation-specific quantitative real-time PCR on PTGER4 comprises a PTGER4 forward primer, a PTGER4 reverse primer, and a PTGER4 probe; the reagent for performing methylation-specific quantitative real-time PCR on HOXA9 comprises a HOXA9 forward primer, a HOXA9 reverse primer, and a HOXA9 probe; the reagent for performing methylation-specific quantitative real-time PCR on SHOX2 comprises a SHOX2 forward primer, a SHOX2 reverse primer, and a SHOX2 probe; the reagent for performing methylation-specific quantitative real-time PCR on SP9 comprises a SP9 forward primer, a SP9 reverse primer, and a SP9 probe; and the reagent for performing methylation-specific quantitative real-time PCR on ZNF781 comprises a ZNF781 forward primer, a ZNF781 reverse primer, and a ZNF781 probe.

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

[0034]

Figure 1

[0035]

Figure 2

[0036]

Figure 3

[0037]

Figure 4

[0038]

Figure 5

[0039]

Figure 6

[0040]

Figure 7

[0041]

Figure 8

[0042]

Figure 9

[0043]

Figure 10

[0044]

Figure 11

[0045]

Figure 12

[0046]

Figure 13

[0047]

Figure 14

[0048]

Figure 15

[0049]

Figure 16

[0050]

Figure 17

[0051]

Figure 18

[0052]

Figure 19

[0053]

Figure 20

[0054]

Figure 21

[0055]

Figure 22

[0056]

Figure 23

[0057]

Figure 24

[0058]

Figure 25

[0059]

Figure 26

[0060]

Figure 27

[0061]

Figure 28

[0062]

Figure 29

[0063]

Figure 30

[0064]

Figure 31

[0065]

Figure 32

[0066]

Figure 33

[0067]

Figure 34

[0068]

Figure 35

[0069]

Figure 36

[0070]

Figure 37

[0071]

Figure 38

[0072]

Figure 39

[0073]

Figure 40

[0074]

Figure 41

Mode for Carrying Out the Invention

[0075] [Definition] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings generally understood by those skilled in the art. Accordingly, the terms defined herein are more fully explained by reference to the entire specification.

[0076] As used herein, the terms "a", "an", and "the" in the singular form include plural references unless the context clearly dictates otherwise.

[0077] As used herein, "and / or" means any and all possible combinations of one or more of the associated listed items and includes the case where, when interpreted alternatively ("or"), there is no combination. Further, in the present disclosure, it is also contemplated that in some embodiments of the disclosure, any feature or combination of features described herein can be excluded or omitted.

[0078] Unless the context otherwise requires, the terms "comprise", "comprises", and "comprising", or similar terms, are intended to mean non-exclusive inclusion, such that a list of recited elements or features does not include only the recited elements but may include other elements or features not recited or listed.

[0079] Unless otherwise specifically stated, nucleic acids are described from left to right in a 5' to 3' orientation.

[0080] It should be understood that this disclosure is not limited to the specific methodologies, protocols, and reagents described, as these may vary depending on the context in which those skilled in the art use them.

[0081] As used herein, "nodule" refers to an abnormal growth or mass of cells. Lung nodules, also called pulmonary nodules, are nodules formed in the lungs.

[0082] As used herein, "malignant" or "malignant lesion" refers to the presence of cancerous cells that have the ability to spread to other parts of the body or invade nearby tissues and destroy them.

[0083] As used herein, "minimally invasive" procedures refer to procedures that do not involve incisions into the body. In some embodiments, minimally invasive procedures include needle punctures. In some embodiments, the minimally invasive procedure is bronchial lavage or bronchial brushing. For the purposes of this application, minimally invasive methods include obtaining a blood sample, sputum sample, urine sample, sample obtained from bronchial lavage, sample obtained from bronchial brushing, or 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 disclosure are applied. Typically, the terms "subject" and "patient" are used interchangeably herein with respect to human subjects.

[0085] For the purposes of this application, "methylation level" refers to the number of 5mC bases contained within a gene. As used herein, a target gene extracted from a sample of a subject is "hypermethylated" if it has more 5mC than the corresponding gene in a sample from a subject that does not have a malignant lung nodule. In some embodiments where the methylation level is measured by methylation-specific qPCR, the target gene is hypermethylated if ΔCt ≤ 15.5, where ΔCt is calculated as Ct of the candidate gene - Ct of the reference gene. More information regarding methylation-specific qPCR is provided in a later section of this disclosure.

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

[0087] As used herein, "reference gene" refers to a gene that is present in all cells and is used for the purpose of internal reaction control so as to be able to standardize the amount and quality of the starting nucleic acid and the differences in PCR amplification. Generally speaking, the expression level of a reference gene does not significantly vary between the tissues being analyzed and the experimental situations (Radonic et al., 2004). In some embodiments, the reference gene is selected from housekeeping genes (HKGs), which by definition are involved in processes essential for cell survival and are expressed at a stable, unregulated, constant level (Thellin et al 1999).

[0088] [DNA methylation] DNA methylation is an epigenetic mechanism that controls gene expression and cell differentiation. In mammals, DNA methylation is essential for normal development and is associated with several important processes, including genomic imprinting, X-chromosome inactivation, suppression of transposable elements, aging, and carcinogenesis.

[0089] DNA methylation by natural enzymes has been found to occur at two nucleobases, cytosine and adenine. The modified bases are 5-methylcytosine (5mC), N 4 -methylcytosine (4mC), and N 6-Methyladenine (6mA). The latter (6mA and 4mC) is limited to prokaryotes and certain eukaryotes. In mammals, 5mC is the dominant form of DNA methylation, which mainly occurs in the context of cytosine-phosphate-guanine (CpG) dinucleotides where cytosines on both strands are usually methylated. A CpG site or CG site is a DNA region where a guanine nucleotide follows a cytosine nucleotide in a linear sequence of bases in the 5' to 3' direction. The mammalian genome exhibits particularly high levels of CpG methylation. Although there are some tissue-specific differences, approximately 70 - 80% of CpGs are methylated. While most CpGs are methylated, densely clustered CpG regions known as CpG islands (CGIs) are often not methylated. Many CGIs are found near gene promoters, and approximately two-thirds of genes have a CGI in their promoter. Methylation of promoter CGIs causes long-term transcriptional repression of the associated gene.

[0090] In mammals, 5mC methylation is catalyzed by the DNA methyltransferase (Dnmt) family, which transfers a methyl group from S-adenosylmethionine to the 5th carbon of the cytosine residue to form 5mC. DNMT3a and DNMT3b are de novo methyltransferases that set the DNA methylation pattern early in development. DNMT3L is a protein that is homologous to other DNMT3s but has no catalytic activity. Instead, DNMT3L assists de novo methyltransferases by enhancing its ability to bind to DNA and stimulating its activity. DNMT1 is a proposed maintenance methyltransferase that is responsible for copying the DNA methylation pattern to the daughter strand during DNA replication.

[0091] [Malignant pulmonary nodules and lung cancer] Lung cancer is the most common cause of cancer-related deaths worldwide, with over 1 million deaths occurring annually. Patients presenting with advanced lung cancer usually have a poor prognosis. Malignant pulmonary nodules usually indicate the early stage of lung cancer. Therefore, differentiating between malignant and benign pulmonary nodules is very important for the early detection of lung cancer and the formulation of a treatment plan suitable for the patient.

[0092] Studies over the past few decades have shown that changes in DNA methylation patterns can distinguish cancer cells from normal cells. Aberrant DNA methylation causes dysregulation of various genes and occurs at all stages of lung cancer, including initiation, proliferation, and metastasis. Changes in DNA methylation patterns occur in a tissue-specific manner during the development stage and are often associated with the onset of cancer. During the carcinogenesis process, both alleles of tumor suppressor genes need to be inactivated by genomic changes such as chromosomal deletions or loss-of-function mutations in the coding regions of genes. As an alternative mechanism, transcriptional silencing due to hypermethylation of CpG islands spanning the promoter regions of tumor suppressor genes is a common and important process in carcinogenesis. Since hypermethylation generally leads to the inactivation of gene expression, this epigenetic change is considered an important mechanism for the long-term silencing of tumor suppressor genes (Esteller, 2002; Wajed et al., 2001). The importance of promoter methylation in the functional inactivation of lung cancer suppressor genes is increasingly being recognized. In lung cancer, it is estimated that 0.5% to 3% of all genes with CpG islands may be silenced by DNA methylation (Costello et al., 2000).

[0093] The present disclosure provides a minimally invasive method for determining whether at least one lung nodule found in a subject is malignant by measuring the DNA methylation levels of gene combinations. Since malignant lung nodules are cancerous and may progress to lung cancer, the method provided here can also be used for the detection of lung cancer. The method disclosed here also provides useful guidance to physicians in determining an appropriate treatment plan for a subject having at least one malignant lung nodule.

[0094] [Extracellular circulating DNA] Extracellular circulating DNA (cfDNA) is a DNA fragment that can be isolated from mammalian serum or plasma. The existence of extracellular nucleic acids in circulation was first reported by Mandel and Metais in 1948 (Mandel et al., 1948). In 1989, Stroun et al. demonstrated that the DNA circulating in the body of cancer patients exhibits some specific characteristics of tumor DNA, such as a decrease in strand stability (Stroun et al., 1989). In some embodiments, cfDNA extracted from a blood sample is used in the method disclosed herein. In some embodiments, cfDNA extracted from a urine sample is used in the method disclosed herein. In some embodiments, cfDNA extracted from a saliva sample is used in the method disclosed herein.

[0095] In healthy individuals, the concentration of cfDNA is generally low, usually in the range of 0 to 100 ng / mL, because defective cells are efficiently removed from the circulation by phagocytes (Elshimali et al., 2013). In cancer patients, the amount of cfDNA released by necrotic and apoptotic tumor cells increases, but the levels can vary widely in the range of 0 to >1000 ng / mL (Schwarzenbach et al., 2011, Fleischhacker et al., 2007). Since molecular changes can be detected in cfDNA, analyzing cfDNA isolated from plasma or serum provides a minimally invasive approach for studying epigenetic changes in cancer patients. For example, it has been shown that the methylation level of the TSG promoter is high in the cfDNA of lung cancer patients, and it has been demonstrated that specific epigenetic changes detected in the genomic DNA of tumor cells may also be found in the serum / plasma of patients (Fujiwara et al., 2005, Sigalotti et al., 2019).

[0096] [Method] In one aspect, the present disclosure provides a method comprising: (a) measuring the methylation levels 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; 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 pulmonary 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 levels 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: 24); 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 sample collected from bronchial lavage, a sample collected from bronchial brushing, 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 at least two 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 pulmonary nodule and / or lung cancer.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0114] In some embodiments, the methylation levels of CDO1, PTGER4, and HOXA9 are measured, and detection of all of the hypermethylation 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 all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 all of the hypermethylation 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 all of the hypermethylation 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 all of the hypermethylation of CDO1, HOXA9, and ZNF781 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 all of the hypermethylation 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 all of the hypermethylation 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 all of the hypermethylation 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 all of the hypermethylation 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 all of the hypermethylation of PTGER4, HOXA9, and SP9 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 all hypermethylation 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 all hypermethylation 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 all hypermethylation 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 all hypermethylation 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 all hypermethylation 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 all hypermethylation 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 all of the hypermethylation 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 all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 all of the hypermethylation 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 all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all hypermethylation 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, sputum sample, sample collected from bronchial lavage, sample collected from bronchial brushing, urine sample, or saliva sample. In some embodiments, the sample is a blood sample.

[0157] The blood sample can be collected by any suitable method known in the art. Exemplary methods for collecting a blood sample include, but are not limited to, venipuncture sampling, arterial sampling, and fingertip puncture sampling. One of ordinary skill in the art will understand that the appropriate conditions and amounts of blood to be collected for the purposes of the blood tests disclosed herein can be determined.

[0158] Plasma can be collected by any suitable method known in the art. An exemplary method of collecting plasma is blood centrifugation. A blood separation centrifuge functions by rotating a blood sample (in a collection tube) at high speed. Due to the high-speed rotation, a rotational force is applied to the blood collection tube, which is called centrifugal force. When the blood collection tube rotates within the blood separation centrifuge, the various components of the blood in the sample are separated according to their density and quantity by the centrifugal force. Thus, the various components of the blood can be separated into different layers to facilitate separation. In some embodiments, one or more anticoagulants (e.g., EDTA, citrate dextrose) are added to the blood sample before centrifugation. Those skilled in the art will understand that they can select suitable conditions for separating plasma from a whole blood sample. DNA can be extracted from plasma using any suitable method known in the art. Kits for extracting DNA from plasma are commercially available, including but not limited to the QIAamp Circulating Nucleic Acid Kit, QIAamp DNA Blood Kit, and chemagic cfDNA 2k Kit H24. Those skilled in the art will understand that they can select suitable methods and conditions 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 expectorates sputum into a sterile container. In sputum induction, the subject inhales a saline mist that may cause a deep cough and then expectorates sputum into a sterile container. In bronchoscopy, a bronchoscope is passed directly from the subject's mouth or nose into the lungs to remove sputum or lung tissue. In gastric lavage, a tube is inserted through the subject's mouth or nose into the stomach, and a sample of gastric secretions containing sputum that has been coughed up and swallowed is collected. DNA can be extracted from sputum samples using any suitable method known in the art. Kits for extracting DNA from sputum are commercially available, for example, but not limited to, the Norgen Bioteck Sputum DNA Isolation Kit (Catalog No. 46200) and the Qiagen DNeasy Blood and Tissue Kit. Those skilled in the art will understand that they can select suitable methods and conditions for extracting DNA from sputum.

[0160] Bronchoalveolar lavage is a procedure for collecting cells from the inner surface of the airways leading to the lungs. A bronchoscope is inserted from the nose or mouth into the lungs. Cells are collected by washing the surface of the airways with a weak saline solution and then observed under a microscope. Those skilled in the art will understand that they can select suitable methods and conditions for collecting liquid and / or cell samples from bronchoalveolar lavage.

[0161] Bronchial brushing is a procedure in which a bronchoscope is inserted from the nose or mouth into the lungs. Then, a small brush is used to remove cells from the airways. These cells are then observed under a microscope. Bronchial brushing is also called bronchial brush biopsy. Those skilled in the art will understand that they can select suitable methods and conditions for collecting liquid and / or cell samples from bronchial brushing.

[0162] DNA can be extracted from samples collected from bronchial lavage or bronchial brushing using any suitable method known in the art. Kits for extracting DNA from samples collected from bronchial lavage or bronchial brushing are commercially available in plurality, including but not limited to Takara's Nucleospin Tissue Kit and DNeasy Blood and Tissue Kit. It will be understood by those skilled in the art that they can select a method and conditions suitable for extracting DNA from samples collected from bronchial lavage or bronchial brushing.

[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, urethral catheterization (CATH), suprapubic aspiration (SPA), or clean catch (CC). It will be understood by those skilled in the art that they can select a method and conditions suitable for collecting urine samples. DNA can be extracted from urine samples using any suitable method known in the art. Kits for extracting DNA from urine are commercially available in plurality, for example, including but not limited to Zymo Research's Quick-DNA Urine Kit, Abcam's DNA Isolation Kit-Urine (ab156899), Norgen Biotek's Urine DNA Isolation Kit. It will be understood by those skilled in the art that they can select a method 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 passive drooling or oral swabbing methods. Those skilled in the art will understand that they can select methods and conditions suitable for collecting saliva samples. DNA can be extracted from saliva samples using any suitable method known in the art. For example, but not limited to, there are multiple commercially available kits for extracting DNA from saliva, such as Thermo Fisher's MagMAX Saliva gDNA Isolation Kit, Oragene® DNA Saliva Kit, and Norgen Biotek's Saliva DNA Isolation Kit (Cat. RU45400). Those skilled in the art will understand that they can select methods 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. The methods for measuring gene methylation levels will be discussed in detail in a later section.

[0166] In one aspect, the present disclosure provides a method comprising: (a) measuring the methylation levels of at least two genes in a blood 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; (c) diagnosing the subject as having at least one malignant lung nodule and / or lung cancer if hypermethylation of the at least two genes is detected; and (d) administering an effective amount of at least one lung cancer therapeutic agent to a subject diagnosed as having at least one malignant lung 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 levels 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; and (d) determining whether the at least two genes are hypermethylated; (e) diagnosing the subject as having at least one malignant pulmonary nodule and / or lung cancer if hypermethylation of the at least two genes is detected; and (d) administering an effective amount of at least one lung cancer therapeutic agent to a subject diagnosed as having at least one malignant pulmonary nodule.

[0168] In some embodiments, the sample is a blood sample, a sputum sample, a sample collected from bronchial lavage, a sample collected from bronchial brushing, 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 levels 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) diagnosing the subject as having at least one malignant pulmonary nodule and / or lung cancer if hypermethylation of the at least two genes is detected; and (g) administering an effective amount of at least one lung cancer therapeutic agent to a subject diagnosed as having at least one malignant pulmonary nodule.

[0170] In some embodiments, the methylation levels of CDO1 and PTGER4 are measured, and detection of hypermethylation of both 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 detection of hypermethylation of both 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 detection of hypermethylation of both 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 detection of hypermethylation of both 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 hypermethylation of both 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 detection of hypermethylation of both 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 detection of hypermethylation of both 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 detection of hypermethylation of both PTGER4 and SP9 indicates that the subject has at least one malignant pulmonary nodule and / or lung cancer.

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

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

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

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

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

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

[0184] In some embodiments, the methylation levels of SP9 and ZNF781 are measured, and detection of hypermethylation of both 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 detection of 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 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.

[0187] In some embodiments, the methylation levels of CDO1, PTGER4, and SP9 are measured, and detection of 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 detection of 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 all hypermethylation of CDO1, HOXA9, and ZNF781 indicates that the subject has at least one malignant lung nodule and / or lung cancer.

[0192] In some embodiments, the methylation levels of CDO1, SHOX2, and SP9 are measured, and detection of all hypermethylation 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 detection of all hypermethylation 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 all hypermethylation 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 detection of all hypermethylation 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 detection of all hypermethylation 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 detection of all of the hypermethylation 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 all of the hypermethylation 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 all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all hypermethylation of PTGER4, HOXA9, SHOX2, and SP9 indicates that the subject has at least one malignant pulmonary nodule and / or lung cancer.

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

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

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

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

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

[0221] In some embodiments, the methylation levels of CDO1, PTGER4, HOXA9, SHOX2, and ZNF781 are measured, and detection of all of the hypermethylation 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, the methylation levels of CDO1, PTGER4, HOXA9, ZNF781, and SP9 are measured, and detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 detection of all of the hypermethylation 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 all of the hypermethylation 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, the methylation levels of CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781 are measured, and detection of all hypermethylation 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, sputum sample, sample collected from bronchial lavage, sample collected from bronchial brushing, urine sample, or saliva sample. In some embodiments, the sample is a blood sample.

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

[0229] Surgery for lung cancer treatment includes, but is not limited to, curative surgery to remove a cancerous tumor or growth from the body; preventive surgery to remove tissue that does not contain cancerous cells but may grow into a malignant nodule or tumor; diagnostic surgery to remove a tissue sample for tests and evaluations useful for confirming a diagnosis; staging diagnostic surgery to clarify the extent of the cancer or the extent of the disease in the body; debulking surgery to remove part but not all of a cancerous tumor or growth; palliative surgery used to relieve discomfort caused by other cancer treatments or to correct other problems; supportive surgery used to act effectively in combination with other cancer treatments; and reconstructive surgery used to restore the appearance or function of a body part of the subject after other cancer treatments or other surgeries. One of ordinary skill in the art will understand that the methods disclosed herein can be used to select the type of surgery appropriate for the treatment of lung cancer in a subject determined to have a malignant lung nodule.

[0230] Chemotherapy is a type of cancer treatment that uses one or more anti-cancer drugs (chemotherapeutic agents) as part of a standardized cancer treatment regimen. Chemotherapy may be carried out for curative or palliative purposes. Chemotherapy involves applying chemicals or drugs to kill cancer cells, and its effects are systemic. There are several different classes of anti-cancer drugs based on their mechanism of action, including the following: a) alkylating agents that damage DNA; b) antimetabolites that replace the normal building blocks of RNA and DNA; c) antibiotics that interfere with enzymes involved in DNA replication; d) topoisomerase inhibitors that inhibit either topoisomerase I or II, enzymes involved in the unwinding of DNA during replication and transcription; e) mitotic inhibitors that inhibit mitosis and cell division; and f) corticosteroids used to treat cancer and reduce the side effects of other drugs. Chemotherapeutic agents used in the treatment of lung cancer include, but are not limited to, Xeloda, Avastin, Tarceva, Cytoxan, Taxol, Taxotere, Gemzar, Arimidex, Alimta, Navelbine, Platinol, Trexall, Ethyol, Iressa, Neosar, Platinol-AQ, Photofrin, Onxol, Cisplatin, Taxane, Gefitinib, Gemcitabine, Erlotinib, Amrubicin, Belotecan, Bendamustine, Picoplatin, and Parifosfamide. It will be understood by those skilled in the art that the methods disclosed herein can be used to select a chemotherapy regimen suitable for the treatment of lung cancer in a subject determined to have malignant pulmonary nodules.

[0231] Radiation therapy irradiates high-energy radiation to kill cancerous cells and shrink tumors or growths. High-dose radiation therapy kills cancer cells or damages their DNA to slow down their growth. Cancer cells with irreparably damaged DNA stop dividing and die. When damaged cells die, they are broken down and removed from the body. Radiation therapy can be performed externally or internally. External radiation therapy uses a machine that irradiates high-energy radiation or beams from outside the body to the tumor or 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 method that places a radiation source inside the body. The radiation source can be solid or liquid. Internal radiation therapy with a solid source is called brachytherapy, and a seed, ribbon, or capsule containing the radiation source is placed inside or near the tumor or growth in the body of the subject. Internal radiation therapy with a liquid source is called systemic therapy. Systemic means that the therapeutic rays travel through the blood to tissues in the body, search for cancer cells, and kill them. Those skilled in the art will understand that the methods disclosed herein can be used to select a radiation therapy suitable for the treatment of lung cancer in a subject determined to have malignant pulmonary nodules. Targeted drug therapy uses drugs or other substances to prevent the growth and spread of cancer by interfering with specific molecular targets involved in cancer growth, progression, and spread. Targeted drug therapy is also called "molecular targeted drug therapy", "molecular targeted drug", "precision medicine", or other similar names. Many different targeted drug therapies, including, but not limited to, hormone therapy, signal transduction inhibitors, apoptosis inducers, angiogenesis inhibitors, immunotherapy, and gene therapy, have been approved for cancer treatment.

[0232] Hormone therapy slows or stops the growth of hormone-sensitive tumors or growth areas that require certain hormones to grow. Hormone therapy works by preventing the production of hormones in the body or interfering with the action of hormones. In some embodiments, estrogen and progesterone are used to treat female patients with non-small cell lung cancer (Katcoff et al., 2014).

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

[0234] Apoptosis inducers cause a process of controlled cell death called apoptosis in cancer cells. Apoptosis is a way the body uses to eliminate unwanted or abnormal cells, but cancer cells have strategies to avoid apoptosis. Apoptosis inducers can bypass these strategies and cause cancer cell death. Studies have shown that cysteine conjugate [6]-gingerol (M2), AZD6244, and extracts of the plant Viburnum grandiflorum can induce apoptosis in lung cancer cells (Meng et al., 2010; Warin et al., 2014; Han et al., 2020).

[0235] An angiogenesis inhibitor blocks the growth of new blood vessels (a process called tumor angiogenesis) to a tumor or malignant growth site. Since blood provides the oxygen and nutrients required for the continued growth of a tumor, a blood supply is necessary for a tumor to grow beyond a certain size. Treatments that interfere with angiogenesis may prevent tumor growth. Some targeted therapies that inhibit angiogenesis interfere with the action of vascular endothelial growth factor (VEGF), a substance that stimulates new blood vessel formation. 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] Immunotherapy triggers the immune system to destroy cancer cells. Some immunotherapy agents are monoclonal antibodies that recognize specific molecules on the surface of cancer cells. When a monoclonal antibody binds to a target molecule, the immune system destroys the cells expressing that target molecule. Other monoclonal antibodies bind to certain immune cells and help these cells kill cancer cells more effectively. Some monoclonal antibodies are checkpoint inhibitors. Checkpoints are proteins produced by some types of immune system cells, such as T cells, and some cancer cells. These checkpoints help prevent the immune response from becoming too strong and may prevent T cells from killing cancer cells. When these checkpoints are blocked, T cells can kill cancer cells. Examples of checkpoint 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 antibody used in the methods described herein is nivolumab, pembrolizumab, or atezolizumab (Doroshow 2019). Cancer vaccines are another type of immunotherapy because they function to strengthen the immune system to fight cancer. In some embodiments, the cancer vaccine used in the methods described herein is CIMAvax-EGF.

[0237] In some embodiments, the treatment includes gene therapy. Gene therapy modifies or manipulates the expression of genes for therapeutic purposes or alters 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. Currently available targeted drug therapy agents for lung cancer include, but are not limited to, bevacizumab, ramucirumab, sotorasib, erlotinib, afatinib, gefitinib, osimertinib, dacomitinib, osimertinib, amivantamab, mobocertinib, necitumumab, crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, entrectinib, dabrafenib, trametinib, selpercatinib, pralsetinib, capmatinib, tempotinib, and larotrectinib. Those skilled in the art will understand that the methods described herein can be used to select a targeted drug therapy suitable for the treatment of lung cancer in a subject determined to have a malignant pulmonary nodule.

[0239] In some embodiments, the lung cancer treatments discussed above are used in combination. For example, in some embodiments, radiation therapy is administered before surgery to reduce the size of the cancer so that it can be removed by surgery and the likelihood of recurrence is reduced. In some embodiments, a combination of the chemotherapy drug topotecan and the targeted drug belzocertib is used in combination for the treatment of 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 a population. As used herein, the population refers to the entire population including both people with and without lung nodules for the purposes of the present invention. In some embodiments, the subject is from a population at high risk of developing lung cancer. As used herein, a population at high risk of developing lung cancer refers to a group of people who have been determined to have a high likelihood of developing lung cancer. There are numerous reasons for a person to be determined to have a high likelihood of developing lung cancer. In some embodiments, a person is determined to have a high likelihood of developing lung cancer because they have at least one lung nodule. In some embodiments, a person is determined to have a high likelihood of developing lung cancer because they smoke, have a family history of lung cancer, have been exposed to high levels of radon, or have received radiation therapy to the chest, among other reasons.

[0242] [Measurement of DNA methylation level] Gene methylation levels can be measured by any suitable method known in the art and disclosed herein. Exemplary methods for measuring DNA methylation levels are provided in Khodadadi et al., Current Advances in DNA Methylation Analysis Methods, Biomed Res Int. 2021.

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

[0244] In some embodiments, unmethylated cytosine is converted to uracil by bisulfite treatment. Bisulfite conversion is a three-step reaction where, at low pH and high temperature, cytosine is first converted to sulfonated cytosine, then deaminated to sulfonated uracil, and finally converted to uracil in an alkaline desulfonation process. Subsequent PCR amplification converts uracil to thymine. In the case of 5mC, the deamination step is nearly two orders of magnitude slower than in the case of cytosine. Thus, after bisulfite conversion, 5mC remains unchanged and is amplified as cytosine by PCR. Since double-stranded DNA (dsDNA) protects C from deamination, the DNA typically needs to be denatured and purified before conversion. Without limitation, Bisulflash TM DNA Modification Kit (Epigentek, P-1026), Bisulflash TM DNA Bisulfite Conversion Easy Kit (Epigentek, P-1054), Premium Bisulfite Kit (Diagenode, C02030030), Imprint® DNA Modification Kit (Sigma-Aldrich, MOD50), EZ DNA Methylation-Gold TM Kit (Zymo Research, D5005), EZ DNA Methylation-Lightning TM Kit (Zymo Research, D5030), Fast Bisulfite Conversion Kit (Abcam®, ab1127127), InnuCONVERT Bisulfite Basic Kit (Analytic Jena, 845-IC-1000008), Epitect® Fast DNA Bisulfite Kit (Qiagen, 59824), Epitect® Bisulfite Kit (Qiagen, 59110), CpGenome TM Turbo Bisulfite Modification Kit (Merck Millipore, S7847), and Methyleasy TMThere are several commercially available kits for bisulfite conversion, including Xceed (Human Genetic Signatures, ME001) (Knit, etc., 2018). Other available methods include, for example, the method described in Wang et al., A modified bisulfite conversion method for the detection of DNA methylation. Epigenomics 2017, 9, 955 - 969.

[0245] In some embodiments, unmethylated cytosines in DNA are converted to uracil by an enzyme-based approach. In some embodiments, unmethylated cytosines in DNA are converted to uracil using the commercially available NEBNext Enzymatic Methyl-seq kit. NEBNext Enzymatic Methyl-seq (EM-seq) is a method for discriminating 5mC and 5hmC. The highly effective enzymatic conversion in this method minimizes damage to DNA and generates high-quality libraries that enable excellent detection of 5mC and 5hmC from fewer sequencing reads using the accompanying NEBNext UltraTM II library preparation workflow reagents. Since DNA is gently processed in the steps of the EM-seq workflow, damage to DNA is minimized. As a result, the DNA converted by EM-seq remains more intact, and libraries with a high proportion of long inserts are generated. This results in longer sequencing reads, increased mapping reliability, and potentially lower per-base sequencing costs depending on the instrument and other details of the sequencing reaction. In addition, the gentle processing in the EM-seq workflow allows for the maintenance of high-quality DNA libraries, which can be amplified efficiently and the number of PCR cycles can be reduced to increase library yield. Importantly, these high yields are not due to the presence of PCR duplicates, which can be prominent especially when the input amount is low. In fact, EM-seq libraries consistently show low levels of duplication across a range of input amounts. Sufficient library yield is required for successful sequencing, but library quality is also an important factor. High-quality libraries have a uniform representation of the original sample, including uniform coverage across the GC spectrum. EM-seq libraries show uniform GC coverage, indicating no damage to the DNA and that the libraries are representative of the original sample.

[0246] In some embodiments, libraries are prepared using as little as 10 ng of input DNA, the accompanying NEBNext Ultra II reagents, and optimized EM-seq adapters. Next, TET2 oxidizes 5-mC and 5-hmC, protecting them from deamination by APOBEC in the next step. In contrast, unmodified cytosine is deaminated to uracil. Next, the library is amplified using a NEBNext master mix formulation of Q5U (a modified version of Q5 High-Fidelity DNA polymerase).

[0247] In some embodiments, the conversion products are then detected and a conversion level indicative of the level of methylation is measured. In some embodiments, measurement of the methylation level after conversion is performed by real-time polymerase chain reaction. Real-time PCR (qPCR) monitors the amplification of target DNA molecules during PCR (i.e., in real-time), rather than at the end of PCR. Real-time PCR (qPCR) includes both quantitative PCR (quantitative real-time PCR) and semi-quantitative PCR (semi-quantitative real-time PCR). Two common methods for detecting PCR products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate into any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides labeled with a fluorescent reporter, where detection is only possible after hybridization of the probe to its complementary sequence.

[0248] The use of a non-specific fluorescent dye involves a DNA-binding dye that binds to double-stranded (ds) DNA generated during PCR, increasing the fluorescence quantum yield of the dye. Thus, the increase in DNA product during PCR leads to an increase in fluorescence intensity measured at each cycle. In real-time PCR using a dsDNA dye, a fluorescent dsDNA dye is added and the reaction is prepared as usual. Next, the reaction is run on a real-time PCR instrument, and the fluorescence intensity is measured with a detector after each cycle; the dye fluoresces only when bound to dsDNA (i.e., the PCR product). The advantages of this method are that the cost is reduced because only a pair of primers is required to perform the amplification; and multiple target sequences can be monitored in a tube by using different types of dyes. However, dsDNA dyes such as SYBR Green bind to all dsDNA PCR products, including non-specific PCR products (such as primer dimers). This may interfere with or prevent accurate monitoring of the intended target sequence.

[0249] Unlike non-specific fluorescent dyes, fluorescent reporter probes detect only DNA containing sequences complementary to the probe. Thus, the use of reporter probes significantly improves specificity and enables this approach to be carried out even in the presence of other dsDNA. By using differently colored labels, fluorescent probes can be used in multiplex assays to monitor several target sequences within the same tube. The specificity of the fluorescent reporter probe also prevents interference in measurements caused by primer dimers, which are undesirable potential by-products of PCR. This method relies on DNA-based probes with a fluorescent reporter at one end of the probe and a fluorescent quencher at the opposite end. Since the reporter is in close proximity to the quencher, its fluorescence is not detected; the degradation of the probe by the 5'-to-3' exonuclease activity of Taq polymerase disrupts the proximity of the reporter-quencher, resulting in the emission of unquenched fluorescence that can be detected after excitation by a laser. Thus, as the product targeted by the reporter probe increases in each PCR cycle, the degradation of the probe and the release of the reporter cause a proportional increase in fluorescence.

[0250] Real-time PCR can be used to quantify nucleic acids by two general methods: relative quantification and absolute quantification. In absolute quantification, an accurate number of target DNA molecules is obtained by comparing with a DNA standard using a calibration curve. Relative quantification is based on an internal reference gene to determine the fold difference in the expression of the target gene. Relative quantification is easier to perform than absolute quantification because it does not require a calibration curve as it compares the amount of the gene of interest to the amount of a control reference gene. By using one or more reference genes, practitioners can correct for non-specific variations such as differences in the amount and quality of the nucleic acids used, which can potentially affect the efficiency of reverse transcription and thus the overall PCR process.

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

[0252] In some embodiments, the method used to measure the methylation level after conversion is MethyLight. MethyLight is a sensitive assay that can detect methylated alleles in the presence of 10,000-fold excess of unmethylated alleles. In some embodiments, MethyLight involves amplifying bisulfite-converted DNA using locus-specific PCR primers that flank an oligonucleotide probe having a 5' fluorescent reporter dye and a 3' quencher. In some embodiments, the 5' fluorescent reporter dye is 6-carboxyfluorescein (6-FAM), 5-carboxyfluorescein (5-FAM), 5'-dichlorodimethoxy-fluorescein (JOE), HEX dye, VIC dye, Cy5, Cy3, TAMRA, or TET dye. In some embodiments, the 3' quencher is a Black Hole Quencher, Iowa Black FQ, Iowa Black RQ, Dabcyl, Qxl, or an internal ZEN quencher. The probe is cleaved by the 5'-to-3' nuclease activity of Taq DNA polymerase, releasing the reporter, the fluorescence of which can be detected by, for example, a laser detector of an ABI Prism 7700 sequence detection system (Perkin-Elmer, Foster City, CA). An exemplary protocol for MethyLight is provided in Eads et al., MethyLight: a high-throughput assay to measure DNA methylation, Nucleic Acids Res. 2000.

[0253] In some embodiments, the methylation level of a gene is quantified using ΔCt. As used herein, "Ct" refers to the cycle threshold and is defined as the number of cycles required for the fluorescence signal to exceed the threshold (i.e., exceed the background level). The Ct level is inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Ct level, the higher the amount of target nucleic acid in the sample). The initial template amount can be derived from its Ct. When the Ct is less than 29, it is considered a strong positive reaction indicating an abundance of target nucleic acid in the sample. When the Ct is between 30 and 37, it is considered a positive reaction indicating a moderate amount of target nucleic acid. When the Ct is between 38 and 40, it is considered a weak reaction indicating a minimal amount of target nucleic acid. The ΔCt score is calculated by subtracting the Ct of the candidate gene from the Ct of the reference gene. Genes most commonly applied as references in qPCR include, but are not limited to, beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-glucuronidase (GUSB), and hypoxanthine-guanine phosphoribosyltransferase (HPRT1).

[0254] In some embodiments, the primers and probes used in the qPCR assay are designed to specifically anneal to the methylation site of interest. In some embodiments, the probe is designed not to cover the methylation site. In some embodiments, the probe is designed to cover the methylation site, providing a higher degree of methylation discrimination ability.

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

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

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

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

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

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

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

[0262] In one aspect, the present disclosure provides any one of the sequences of SEQ ID NO: 1 to 18. In some embodiments, in methylation-specific qPCR, SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 are used as forward primer, reverse primer, and probe, respectively. In some embodiments, in methylation-specific qPCR, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 are used as forward primer, reverse primer, and probe, respectively. In some embodiments, in methylation-specific qPCR, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are used as forward primer, reverse primer, and probe, respectively. In some embodiments, in methylation-specific qPCR, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 are used as forward primer, reverse primer, and probe, respectively. In some embodiments, in methylation-specific qPCR, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 are used as forward primer, reverse primer, and probe, respectively. In some embodiments, in methylation-specific qPCR, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 are used as forward primer, reverse primer, and probe, respectively.

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

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

[0265] In one aspect, the present disclosure provides a kit (hereinafter referred to as the "HOXA9 kit") for performing methylation-specific qPCR of the HOXA9 gene, 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 disclosure provides a kit (hereinafter referred to as the "SHOX2 kit") for performing methylation-specific qPCR of the SHOX2 gene, 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 disclosure provides a kit (hereinafter referred to as the "SP9 kit") for performing methylation-specific qPCR of the SP9 gene, 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 disclosure provides a kit (hereinafter referred to as the "ZNF781 kit") for performing methylation-specific qPCR of the ZNF781 gene, 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 disclosure provides a kit for determining whether at least one lung nodule found in a subject is malignant, the kit comprising bisulfite and reagents for performing methylation-specific quantitative real-time PCR of at least two genes selected from the group consisting of CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781, wherein the reagents for performing methylation-specific quantitative real-time PCR of CDO1 comprise a CDO1 forward primer, a CDO1 reverse primer, and a CDO1 probe; the reagents for performing methylation-specific quantitative real-time PCR of PTGER4 comprise a PTGER4 forward primer, a PTGER4 reverse primer, and a PTGER4 probe; the reagents for performing methylation-specific quantitative real-time PCR of HOXA9 comprise a HOXA9 forward primer, a HOXA9 reverse primer, and a HOXA9 probe; the reagents for performing methylation-specific quantitative real-time PCR of SHOX2 comprise a SHOX2 forward primer, a SHOX2 reverse primer, and a SHOX2 probe; the reagents for performing methylation-specific quantitative real-time PCR of SP9 comprise a SP9 forward primer, a SP9 reverse primer, and a SP9 probe; and the reagents for performing methylation-specific quantitative real-time PCR of ZNF781 comprise a ZNF781 forward primer, a ZNF781 reverse primer, and a ZNF781 probe. In some embodiments, the kit further comprises at least two of the kits selected from the CDO1 kit, PTGER4 kit, HOXA9 kit, SHOX2 kit, SP9 kit, and ZNF781 kit described herein.

[0270] In addition, sequence differences resulting from the various DNA methylation patterns after conversion can also be revealed by other methods after methylation-independent PCR amplification. In some embodiments, the method used to measure 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 C-G (3H bonds) and A-T (2H bonds) pairs. The melting analysis is performed after methylation-independent PCR amplification. The temperature is gradually increased, and PCR products from unmethylated alleles containing A-T pairs dissociate at a lower temperature and are detected by a sharp decrease in fluorescence when an intercalating dye (e.g., SYBR Green, Eva Green, SYTO9) is released from the dsDNA. To avoid the formation of secondary structures that may interfere with the analysis, PCR amplicons of 100 bp or less are typically used in such assays. Such protocols, including primer design, are described in Wojdacz et al., Methylation-sensitive high-resolution melting, Nat Protoc. 2008. This method is relatively rapid compared to other conversion-based methods and can distinguish between fully or partially methylated sites and unmethylated sites, making it particularly useful when only a small fraction of the analyzed sample loci are methylated. On the other hand, this method is generally not sensitive enough to analyze only a single CpG. Nevertheless, when comparing an unknown sample to a standard with a known methylation-to-unmethylation ratio, this approach is semi-quantitative. Overall, MS-HRM provides a fast and relatively cost-effective estimate of the methylation rate.

[0271] In some embodiments, the method used to measure the level of methylation after conversion is pyrosequencing. Bisulfite pyrosequencing utilizes bisulfite conversion, followed by PCR amplification with a single biotinylated primer, immobilization of the amplicon on streptavidin beads, hybridization with a sequencing primer, and subsequent sequencing. With this method, information regarding allele-specific methylation is not obtained, but the average methylation level of both alleles is obtained in a quantitative form. The main advantage of this technique is that a background-free chromatogram is generated from which the methylation ratio is accurately calculated as C / (C+T). EpigenDx provides a commercially available pyrosequencing analysis of gene-specific methylation.

[0272] It is also possible to measure the DNA methylation level without first converting unmethylated cytosine to uracil. In some embodiments, the method used to measure the methylation level is nanopore long-read technology. DNA molecules containing methylation modifications can be sequenced in their native state without enzymatic or chemical treatment using such nanopore long-read technology. Methylation modifications can be detected by the unique signals generated by such modified bases when the DNA molecule passes through the nanopore and can be distinguished from unmodified DNA bases. More information regarding nanopore technology and its use in DNA methylation measurements is provided in Simpson, J.T. et al., Detecting DNA cytosine methylation using nanopore sequencing, Nat. Methods 2017, Laszlo, A.H. et al., Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA, Proc. Natl Acad. Sci. USA 2013, and Rand, A.C. et al., Mapping DNA methylation with high-throughput nanopore sequencing, Nat. Methods 2017.

[0273] In some embodiments, DNA methylation levels are measured by methylation-specific restriction enzyme digestion. Several site-specific methylation-dependent restriction enzymes are available. For example, the endonuclease HpaI can digest the CCGG sequence, but only when it is not methylated. In contrast, the MspI enzyme, which also cleaves DNA at the CCGG site, is not affected by DNA methylation. Other methylation-specific restriction enzymes include, but are not limited to, HpaII, AatII, and ClaI. After digestion with one or more methylation-specific restriction enzymes, the location of DNA methylation sites is revealed by sequencing analysis, qPCR analysis using primers targeting the cleavage sites, or electrophoretic analysis of the digestion products. Qiagen's EpiTect Methyl II PCR Array System is a commercially available system for digestion-based DNA methylation analysis (Kurdyukove et al., 2016; Zuo et al., 2009; Jiang et al., 2012).

Example

[0274] The present disclosure can be further illustrated by the following non-limiting examples, in which standard techniques known to those skilled in the art and techniques similar to those described in these examples can be used where appropriate. It is understood that those skilled in the art will envision additional embodiments consistent with the disclosure provided herein.

[0275] Example 1: Whole Genome Array for Identifying Candidate Genes for Malignant Lung Nodule Detection Genome-wide 850K Illumina methylation array analysis was performed using blood from patients with malignant and benign lung nodules. Genomic DNA was extracted from blood using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany) and processed with the Zymo (USA) bisulfite conversion kit (EZ DNA Methylation-Lightning Kit) according to the manufacturer's protocol. Candidate differentially methylated sites were identified using the Infinium Methylation EPIC BeadChip (850K) (Illumina, USA). The methylation status of the probes was represented as β values. CpG sites with β ≥ 0.20 and adjusted p-value ≤ 0.05 were defined as differentially methylated sites.

[0276] [Results] From the genome-wide analysis of clinical samples, the following candidates were identified: 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, MARCH11, ZNF781, RPPH1, ZIC4, TAC1, HOXA7, PRRX1, OPCML, CDKN2A, SIX3, DLX4, TMEFF2, RUNX3, GATA3, HOXA11, RARβ, 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 differentiating malignant from benign lung nodules.

[0278] Example 2: Detection of Malignant Pulmonary Nodules by Methylation Levels of CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781 Blood samples were collected from 179 lung cancer patients and 71 patients with benign pulmonary nodules, and plasma was separated by centrifugation. DNA was extracted from the plasma samples and treated with bisulfite to convert unmethylated cytosine to uracil. The methylation status of the CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781 genes was measured by methylation-specific quantitative real-time PCR. The primer and probe sequences used for qPCR are shown in Table 3.

[0279] Quantitative real-time methylation-specific PCR (q-MSP) was used to detect and verify specific methylation sites. All samples were run in triplicate. Since methylation was not detected in some of the sample replicates, 60 Ct was used to create an almost zero value for each methylation detection replicate and compared with the average Ct of β-actin (ACTB). The formula for calculating the average value was as follows: -△Ct Since methylation was not detected in some of the sample replicates, 60 Ct was used to create an almost zero value for each methylation detection replicate and compared with the average Ct of β-actin (ACTB). -△Ct The formula for calculating the average value was as follows: TIFF2025519538000001.tif13170

[0280] [Results] The results of the qPCR analysis are shown in Table 1, Figures 1-3, and Figures 8-10. It was revealed from the data that each of the CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781 genes could distinguish malignant pulmonary nodules from benign pulmonary nodules.

[0281] TIFF2025519538000002.tif31170

[0282] Example 3: Detection of Malignant Pulmonary Nodules by Methylation Levels of Combinations of at Least Two Genes Selected from CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781 The data collected in Example 2 was further analyzed to determine whether the detection of methylation in the combination of CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781 genes improves the ability to distinguish malignant lung nodules from benign lung nodules. The area under the receiver operating characteristic curve (ROC) was calculated as the accuracy index using 2 -△Ct values. All data analyses were performed using GraphPad Prism.

[0283] [Results] Further analyses are shown in Table 2, Figures 4-7, and Figures 11-35. The analyses showed that the combination resulted in better sensitivity and specificity than the individual genes alone. The analyses further revealed that the combination of CDO1 and PTGER4 resulted in the best sensitivity and specificity in distinguishing malignant lung nodules from benign lung nodules. Adding HOXA9 to the combination of CDO1 and PTGER4 did not improve the sensitivity and specificity. TIFF2025519538000003.tif222170TIFF2025519538000004.tif141170

Claims

Claim 1 (a) measuring the methylation levels of at least two genes in a sample of interest, 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 detection of hypermethylation of the at least two genes indicates that the subject has at least one malignant pulmonary nodule and / or lung cancer; A method comprising the steps above. Claim 2 (a) measuring the methylation levels of at least two genes in a sample of interest, 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; (c) diagnosing that the subject has at least one malignant pulmonary nodule and / or lung cancer if hypermethylation of the at least two genes is detected; and (d) administering to the subject diagnosed with having at least one malignant pulmonary nodule and / or lung cancer an effective amount of at least one lung cancer treatment. A method comprising the steps above. Claim 3 The method according to claim 2, wherein the lung cancer treatment is selected from surgery, chemotherapy, radiotherapy, immunotherapy, and targeted drug therapy. Claim 4 The method according to any one of claims 1 to 3, wherein the sample is a blood sample, sputum sample, sample collected from bronchial lavage, sample collected from bronchial brushing, urine sample, or saliva sample. Claim 5 The methylation level is measured by (1) converting unmethylated cytosine in the DNA of step (c) to uracil while leaving methylated cytosine as cytosine; (2) measuring the level of conversion of unmethylated cytosine to uracil. The method according to any one of claims 1 to 4. Claim 6 The method according to claim 5, wherein unmethylated cytosine in the DNA is converted to uracil by bisulfite treatment or enzymatic treatment. Claim 7 The method according to any one of claims 5 and 6, wherein the measurement is performed by real-time polymerase chain reaction (PCR), sequencing, or microarray. Claim 8 The method according to claim 7, wherein the PCR is methylation-specific quantitative real-time PCR.

9. The method according to claim 8, wherein the methylation-specific quantitative real-time PCR uses a forward primer comprising SEQ ID NO: 1, a reverse primer comprising SEQ ID NO: 2, and a probe comprising SEQ ID NO:

3.

10. The method according to claim 8, wherein the methylation-specific quantitative real-time PCR uses a forward primer comprising SEQ ID NO: 4, a reverse primer comprising SEQ ID NO: 5, and a probe comprising SEQ ID NO:

6.

11. The method according to claim 8, wherein the methylation-specific quantitative real-time PCR uses a forward primer comprising SEQ ID NO: 7, a reverse primer comprising SEQ ID NO: 8, and a probe comprising SEQ ID NO:

9.

12. The method according to claim 8, wherein the methylation-specific quantitative real-time PCR uses a forward primer comprising SEQ ID NO: 10, a reverse primer comprising SEQ ID NO: 11, and a probe comprising SEQ ID NO:

12.

13. The method according to claim 8, wherein the methylation-specific quantitative real-time PCR uses a forward primer comprising SEQ ID NO: 13, a reverse primer comprising SEQ ID NO: 14, and a probe comprising SEQ ID NO:

15.

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

18.

15. The method according to any one of claims 1 to 4, wherein the methylation level is measured by methylation-specific high-resolution melting, pyrosequencing, nanopore long-read technology, or methylation-specific restriction enzyme 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 according to any one of claims 1 to 16, wherein the subject has been determined to have had at least one lung nodule previously.

18. The method according to any one of claims 1 to 17, wherein the subject is from a population at high risk of developing lung cancer.

19. A polynucleotide having any one of the sequences of SEQ ID NOs: 1 to 18.

20. A kit for performing methylation-specific quantitative real-time PCR of 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 quantitative real-time PCR of 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 quantitative real-time PCR of 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 quantitative real-time PCR of 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 quantitative real-time PCR of 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 quantitative real-time PCR of 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 to 25, further comprising bisulfite.

27. A kit for determining whether at least one lung nodule found in a subject is malignant, comprising bisulfite and reagents for performing methylation-specific quantitative real-time PCR of at least two genes selected from the group consisting of CDO1, PTGER4, HOXA9, SHOX2, SP9, and ZNF781, The reagent for performing methylation-specific quantitative real-time PCR of CDO1 comprises a CDO1 forward primer, a CDO1 reverse primer, and a CDO1 probe, The reagent for performing methylation-specific quantitative real-time PCR of PTGER4 comprises a PTGER4 forward primer, a PTGER4 reverse primer, and a PTGER4 probe, Reagents for performing methylation-specific quantitative real-time PCR of HOXA9 include a HOXA9 forward primer, a HOXA9 reverse primer, and a HOXA9 probe. Reagents for performing methylation-specific quantitative real-time PCR of SHOX2 include a SHOX2 forward primer, a SHOX2 reverse primer, and a SHOX2 probe. Reagents for performing methylation-specific quantitative real-time PCR of SP9 include a SP9 forward primer, a SP9 reverse primer, and a SP9 probe. A kit for performing methylation-specific quantitative real-time PCR of ZNF781 includes a ZNF781 forward primer, a ZNF781 reverse primer, and a ZNF781 probe.

28. The kit according to claim 27, further comprising at least two of the kits according to claims 20 to 25.

Images