Method for Detecting Lung Cancer

Abstract

The present invention relates to a method for providing information for the diagnosis of lung cancer, a method for diagnosing lung cancer, a composition for diagnosing lung cancer, and a kit containing the same. More specifically, the present invention relates to a method for providing information for the diagnosis of lung cancer, a composition for diagnosing lung cancer, and a kit containing the same, using primers that specifically amplify a plurality of methylated lung cancer marker genes.

Description

Technical Field

[0001] The present invention relates to a method for providing information for the diagnosis of lung cancer, a method for diagnosing lung cancer, a composition for diagnosing lung cancer, and a kit containing the same. More specifically, the present invention relates to a method for providing information for the diagnosis of lung cancer, a composition for diagnosing lung cancer, and a kit containing the same, using primers that specifically amplify a plurality of methylated lung cancer marker genes.

Background Art

[0002] In addition to A, C, G, and T, the genomic DNA of mammalian cells has a fifth base, which is 5-methylcytosine (5-mC) with a methyl group attached to the fifth carbon of the cytosine ring. 5-mC always attaches only to the C of the CG dinucleotide (5'-mCG-3'), and such CG is often denoted as CpG. Most of the C in CpG is methylated with a methyl group. Such methylation of CpG suppresses the expression of repetitive sequences such as alu and transposons in the genome and is the site where epigenetic changes most frequently occur in mammalian cells. Such 5-mC of CpG is naturally deaminated and changed to T, whereby CpG in the mammalian genome shows only a frequency of 1%, which is much lower than the frequency (1 / 4 x 1 / 4 = 6.25%) at which it should normally appear.

[0003] Among CpGs, there are some that appear exceptionally densely, and these are called CpG sites (CpG islands). A CpG site refers to a site that is 0.2 - 3 kb in length, where the distribution percentages of C and G bases exceed 50%, and the distribution percentage of CpG is highly concentrated at 3.75% or more. Approximately 45,000 CpG sites appear in the entire human genome, and they are particularly concentrated in promoter sites that regulate gene expression. In fact, CpG sites appear in the promoters of important genes (housekeeping genes) that account for approximately half of human genes (Cross, S. et al., Curr. Opin. Gene Develop., 5:309, 1995). Abnormal DNA methylation mainly occurs in the 5' regulatory region of the gene and is known to reduce the expression of the gene.

[0004] On the other hand, in the somatic cells of healthy individuals, the CpG islands in these important gene promoter sites are not methylated, but the genes imprinted not to be expressed during development and the genes on the inactivated X chromosome are methylated.

[0005] During the carcinogenesis process, methylation appears in the promoter CpG island, and the expression of the gene is impaired. In particular, when methylation occurs in the CpG island, which is the expression regulatory site of tumor suppressor genes that regulate the cell cycle and cell death, repair DNA, are involved in cell adhesion and cell - cell cooperation, and suppress invasion and metastasis, this blocks the expression and function of these genes, similar to mutations in the coding sequence, and as a result, promotes the occurrence and progression of cancer. In addition, partial methylation may also appear in CpG islands due to aging.

[0006] Methylation of the regulatory region of tumor-related genes is an important indicator of cancer. Therefore, it can be used in many aspects such as cancer diagnosis and early diagnosis, prediction of carcinogenic risk, prediction of cancer prognosis, follow-up after treatment, and prediction of response to anti-cancer therapy. In fact, attempts have been actively made in recent years to investigate the promoter methylation of tumor-related genes in blood, sputum, saliva, feces, urine, etc. and use them in the diagnosis and treatment of various cancers (Ahlquist, D.A. et al., Gastroenterol., 119:1219, 2000).

[0007] Under such a technical background, the inventors of the present application confirmed that the methylation of the marker gene of lung cancer can be detected with a high detection limit and accuracy by using primers that specifically amplify multiple methylated marker genes of lung cancer, and thus completed the present invention.

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a method for providing information for the diagnosis of lung cancer through primers that specifically amplify multiple methylated marker genes of lung cancer.

[0009] An object of the present invention is to provide a method for diagnosing lung cancer through primers that specifically amplify multiple methylated marker genes of lung cancer.

[0010] An object of the present invention is to provide a composition for diagnosing lung cancer through primers that specifically amplify multiple methylated marker genes of lung cancer.

[0011] An object of the present invention is to provide a kit for diagnosing lung cancer containing the above composition.

Means for Solving the Problems

[0012] To achieve the above object, the present invention relates to a method for providing information for the diagnosis of lung cancer, comprising the following steps: (a) treating a sample with one or more reagents that modify the methylated PCDHGA12 (Protocadherin Gamma Subfamily A, 12) gene and CDO1 (Cysteine Dioxygenase Type 1) gene, and the unmethylated PCDHGA12 gene and CDO1 gene to be different from each other; and (b) treating with primers that specifically amplify the methylated PCDHGA12 gene and CDO1 gene.

[0013] The present invention also provides a composition for the diagnosis of lung cancer, comprising one or more reagents that modify the methylated PCDHGA12 (Protocadherin Gamma Subfamily A, 12) gene and CDO1 (Cysteine Dioxygenase Type 1) gene, and the unmethylated PCDHGA12 gene and CDO1 gene to be different from each other; and primers that specifically amplify the methylated PCDHGA12 gene and CDO1 gene.

[0014] The present invention also provides a kit for the diagnosis of lung cancer, comprising the above composition.

Brief Description of the Drawings

[0015]

Figure 1

[0016]

Figure 2

[0017]

Figure 3

Mode for Carrying Out the Invention

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the relevant technical field.

[0019] The present invention relates to a method for detecting the methylation of the lung cancer-specific methylated gene CDO1 for the detection of lung cancer. More specifically, by measuring the methylation of a gene specifically methylated in lung cancer cells, information related to the diagnosis of lung cancer is provided. Also, when combined with the methylation of the lung cancer-specific methylated gene PCDHGA12, it is provided that the detection ability of lung cancer is improved.

[0020] The inventors of the present application have confirmed that, through primers that specifically amplify multiple methylated lung cancer marker genes, the sensitivity and specificity of methylated DNA detection can be improved compared to the methylation detection degree based on a single lung cancer marker gene. In a specific example according to the present invention, by detecting the methylation of the PCDHGA12 (Protocadherin Gamma Subfamily A, 12) gene and the CDO1 (Cysteine Dioxygenase Type 1) gene compared to the methylation detection of the SDC2 gene, the sensitivity and specificity for the diagnosis of lung cancer are improved, and it is confirmed that it is highly useful for the diagnosis of lung cancer.

[0021] Accordingly, in one aspect, the present invention relates to a method for providing information for the diagnosis of lung cancer, comprising the following steps: (a) treating a sample with one or more reagents that modify the methylated PCDHGA12 (Protocadherin Gamma Subfamily A, 12) gene and the CDO1 (Cysteine Dioxygenase Type 1) gene, and the non-methylated PCDHGA12 gene and the CDO1 gene to be different from each other; and (b) treating with primers that specifically amplify the methylated PCDHGA12 gene and the CDO1 gene.

[0022] Accordingly, in one aspect, the present invention relates to a method for diagnosing lung cancer, comprising the following steps: (a) treating a sample with one or more reagents that modify the methylated PCDHGA12 (Protocadherin Gamma Subfamily A, 12) gene and the CDO1 (Cysteine Dioxygenase Type 1) gene, and the non-methylated PCDHGA12 gene and the CDO1 gene to be different from each other; and (b) treating with primers that specifically amplify the methylated PCDHGA12 gene and the CDO1 gene.

[0023] In the present invention, step (a) is a step of treating a sample with one or more reagents that modify the methylated PCDHGA12 gene and the CDO1 gene, and the non-methylated PCDHGA12 gene and the CDO1 gene to be different from each other.

[0024] The "methylation" in the present invention means that a methyl group is attached to the 5th carbon of the cytosine base ring and is modified to 5-methylcytosine (5-mC). 5-methylcytosine is always attached only to the C of the CG dinucleotide (5'-mCG-3'), and such CG is often denoted as CpG. Such methylation of CpG suppresses the expression of base sequences repeated in the genome such as alu and transposons, and is the site where epigenetic changes most frequently appear in mammalian cells. Such 5-mC of CpG is naturally deaminated and changed to T, and thus CpG in the mammalian genome shows only a frequency of 1%, which is much lower than the frequency (1 / 4 x 1 / 4 = 6.25%) at which it should normally appear.

[0025] There are some that appear exceptionally densely among CpGs, which are called CpG islands. A CpG island refers to a site that is 0.2 - 3 kb in length, the distribution percentage of C and G bases exceeds 50%, and the distribution percentage of CpG is highly concentrated at 3.75% or more. Approximately 45,000 CpG islands appear in the entire human genome, and they appear particularly concentrated in promoter sites that regulate gene expression. In fact, CpG islands appear in the promoters of important genes (housekeeping genes) that account for about half of human genes.

[0026] The nucleic acid isolated from the specimen is obtained from the biological sample of the specimen. When it is desired to diagnose lung cancer or the progression stage of lung cancer, it is necessary to isolate nucleic acid from lung tissue by scraping or biopsy. Such samples can be obtained by various medical procedures known in the art.

[0027] The degree of methylation of the nucleic acid of the sample obtained from the specimen is measured by comparing it with the same nucleic acid portion of a specimen without abnormal cell proliferation in lung tissue. Hypermethylation means that there are alleles methylated in one or more nucleic acids. When examining the same nucleic acid, specimens without abnormal cell proliferation in lung tissue do not show methylated alleles.

[0028] "Normal" cells refer to cells that do not show abnormal cell morphology or changes in cytological properties. "Tumor" cells refer to cancer cells, and "non-tumor" cells refer to cells that are part of diseased tissue but are determined not to be in the tumor site.

[0029] According to the present invention, it is possible to determine the methylation level of one or more nucleic acids isolated from a specimen and early diagnose cell proliferative abnormalities in the lung tissue of the specimen. The methylation level of the one or more nucleic acids can be characterized by comparison with the methylation state of one or more nucleic acids isolated from a specimen without cell proliferative abnormalities in the lung tissue. The nucleic acid is preferably a CpG-containing nucleic acid such as a CpG island.

[0030] According to the present invention, it is possible to diagnose the predisposition of cell proliferative abnormalities in the lung tissue of a specimen, including determining the methylation of one or more nucleic acids isolated from the specimen. The methylation level of the one or more nucleic acids can be characterized by comparison with the methylation state of one or more nucleic acids isolated from a specimen without a predisposition to cell proliferative abnormalities.

[0031] "Predisposition" means the property of being prone to the cell proliferative abnormality. A specimen with a predisposition refers to a specimen that does not yet have a cell proliferative abnormality but has an increased tendency for the presence of a cell proliferative abnormality.

[0032] The presence of CpG methylation in the target DNA can be an indicator of a disease. For example, it is possible to measure the CpG methylation at any one site in the promoter, 5' untranslated region, and intron of the target DNA.

[0033] CpG-containing genes are usually DNA. However, the method of the present invention can be applied to a sample containing, for example, DNA or RNA containing DNA and mRNA, where the DNA or RNA may be single-stranded or double-stranded, or a sample containing a DNA-RNA hybrid.

[0034] Nucleic acid mixtures can also be used. The "multiplex" used in the present invention includes all cases where there are multiple specific nucleic acid sequence sites detected within one gene and cases where multiple target DNAs are included in one tube (single reactor). The specific nucleic acid sequences to be detected may be fractions of large molecules and may initially exist in the form of separated molecules in which the specific sequences constitute the entire nucleic acid sequence. The nucleic acid sequences do not have to be nucleic acids in pure form, and the nucleic acids may be a small fraction within a complex mixture such as one containing the entire human DNA.

[0035] Specifically, the present invention is for detecting the methylation of multiple target DNAs in a sample within a single reactor, and the sample can contain multiple multiplex target DNAs. The target DNAs can be used without limitation as long as they are genes that affect the occurrence or progression of lung cancer when abnormally methylated and their expression is suppressed, not just control group genes.

[0036] In the present invention, the sample can be characterized by being derived from a human body. For example, the sample can use lung cancer tissue, cells, feces, urine, blood, serum or plasma.

[0037] One or more reagents for modifying the methylated DNA and non-methylated DNA to be different from each other can be used without limitation as long as they can distinguish between non-methylated cytosine bases and methylated cytosine bases. For example, they may be bisulfite, hydrogen sulfite, disulfite and combinations thereof, but are not limited thereto. Specifically, with these reagents, methylated cytosine bases are not converted, and non-methylated cytosine bases can be converted to bases other than uracil or cytosine.

[0038] In the present invention, step (b) is a step of treating primers that specifically amplify the methylated PCDHGA12 gene and CDO1 gene.

[0039] The primers can be used simultaneously in pairs, for example, a forward primer and a reverse primer for PCR. Further, the 3'-end of the primer is designed such that a methylated "C" of the CpG site is necessarily located, and it has the characteristic of high specificity for methylation detection. The forward primer that specifically amplifies the methylated PCDHGA12 gene can include, for example, the sequence of SEQ ID NO: 1. The reverse primer can include, for example, the sequence of SEQ ID NO: 2. The primer that specifically amplifies the methylated PCDHGA12 gene can include the primer pair of SEQ ID NO: 1 and 2.

[0040] The forward primer that specifically amplifies the methylated CDO1 gene can include, for example, the sequence of SEQ ID NO: 4. The reverse primer can include, for example, the sequence of SEQ ID NO: 5. The primer that specifically amplifies the methylated CDO1 gene can include the primer pair of SEQ ID NO: 4 and 5.

[0041] The present invention can further include, in the step (b), a step of treating probes that can hybridize complementarily to the methylated PCDHGA12 gene and the methylated CDO1 gene specifically amplified by the primers, respectively.

[0042] In the hybridization reaction, the conditions used to achieve a strict specific level vary depending on the nature of the nucleic acid to be hybridized. For example, the length of the nucleic acid site to be hybridized, the degree of homology, the nucleotide sequence composition (e.g., GC / AT composition ratio), and the nucleic acid type (e.g., RNA, DNA), etc. are considered for selecting the hybridization conditions. An additional consideration is whether the nucleic acid is immobilized on, for example, a filter or the like.

[0043] Examples of conditions that are carried out very strictly are as follows: 2X SSC / 0.1% SDS at room temperature (hybridization condition); 0.2X SSC / 0.1% SDS at room temperature (low stringency condition); 0.2X SSC / 0.1% SDS at 42 °C (condition with medium stringency); 0.1X SSC at 68 °C (condition with high stringency). The washing process can be carried out using one of these conditions, for example, the condition with high stringency, or each of the above conditions can be used, and each of the above-described conditions can be repeated for 10 to 15 minutes each in the described order, either all or part of them. However, as described above, the optimal conditions vary depending on the specific hybridization reaction involved and can be determined through experiments. Generally, conditions with high stringency are used for the hybridization of important probes.

[0044] Probes that can hybridize complementarily to the amplified methylated CDO1 gene or methylated PCDHGA12 gene can include, for example, the sequences of SEQ ID NO: 3 or 6. Probes that can hybridize complementarily to the amplified methylated CDO1 gene can include the sequence of SEQ ID NO: 3. Probes that can hybridize complementarily to the amplified methylated PCDHGA12 gene can include the sequence of SEQ ID NO: 6.

[0045] In some cases, the probe can be labeled so as to be detectable, for example, labeled with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelate, or an enzyme. Appropriately labeling a probe as described above is a technique widely known in the art and can be carried out through ordinary methods.

[0046] The amount of the amplification product can be detected by a fluorescence signal. There are methods such as the intercalating method using a reagent (intercalator) that binds to the double-stranded DNA of the amplification product to which the probe is bound and shows fluorescence, and a method using an oligonucleotide labeled with a fluorescent substance at the 5'-end and a quencher at the 3'-end.

[0047] The amplification according to the present invention can be performed through real-time quantitative amplification, for example, real-time polymerase chain reaction (Real-Time PCR). In the real-time polymerase chain reaction, the amount of the PCR amplification product can be detected by a fluorescence signal. As the real-time polymerase chain reaction progresses, the intensity of the fluorescence signal increases according to the increasing amount of polynucleotide, and an amplification profile curve showing the intensity of the fluorescence signal according to the number of amplification cycles is obtained.

[0048] The amplification profile curve is generally divided into a baseline region where a background fluorescence signal that does not substantially reflect the amount of polynucleotide appears, an exponential region where an increase in the fluorescence signal appears with an increase in the amount of polynucleotide product, and a plateau region where the PCR reaction reaches a saturated state and no increase in the fluorescence signal intensity appears.

[0049] Usually, the point at which the baseline region shifts to the exponential region, that is, the fluorescence signal intensity when the amount of the PCR amplification product reaches a detectable amount by fluorescence, is defined as the threshold, and the number of amplification cycles corresponding to the threshold in the amplification profile curve is defined as the threshold cycle (Ct) value.

[0050] By measuring the Ct value, analyzing a standard curve whose concentration is determined based on the Ct (threshold cycle) value for a standard substance, and confirming the concentration of the amplified gene, the methylation specificity and / or specificity can be determined.

[0051] In one embodiment, the methylation detection can be performed by a method selected from the group consisting of PCR, methylation specific PCR, real time methylation specific PCR, PCR using a methylation DNA specific binding protein, PCR using a methylation DNA specific binding antibody, quantitative PCR, gene chip, sequencing, sequencing by synthesis, and sequencing by ligation.

[0052] (1) Methylation specific PCR: When detected by the methylation specific PCR, when cytosine at the 5'-CpG'-3 site is methylated after bisulfite treatment, it remains as cytosine, and when it is unmethylated, it changes to uracil. Therefore, primers corresponding to the sites where the 5'-CpG-3' base sequence exists can be prepared for the base sequence converted after bisulfite treatment. When PCR is performed using the primers, if it is methylated, a PCR product is prepared from the one using the primer corresponding to the methylated base sequence, and the presence or absence of methylation can be confirmed by agarose gel electrophoresis. Here, the methylation detection probe may be a probe having a TaqMan, Molecular Beacon, self-reporting function, or an energy transfer labeling function, and is not limited thereto.

[0053] (2) Real-time methylation-specific PCR: The real-time methylation-specific PCR is a method that converts the methylation-specific PCR method into a real-time measurement method. After treating genomic DNA with bisulfite, PCR primers corresponding to the methylated case are designed, and real-time PCR is performed using these primers. At this time, there are two methods of detection: one is to use a TaqMan probe complementary to the amplified nucleotide sequence for detection, and the other is to use Sybergreen for detection. Therefore, real-time methylation-specific PCR can selectively perform quantitative analysis only on methylated DNA. At this time, a standard curve is created using an in vitro methylated DNA sample, and a gene without a 5'-CpG-3' sequence in the nucleotide sequence is amplified together as a negative control group for standardization, and the degree of methylation can be quantitatively analyzed.

[0054] (3) PCR or quantitative PCR and DNA chip using methylated DNA-specific binding protein: In the PCR or DNA chip method using the methylated DNA-specific binding protein, when a protein that specifically binds only to methylated DNA is mixed with DNA, the protein specifically binds only to methylated DNA, so that only methylated DNA can be selectively separated.

[0055] Also, the presence or absence of methylation can be measured by the quantitative PCR method. The methylated DNA separated by the methylated DNA-specific binding protein is labeled with a fluorescent dye and hybridized to a DNA chip on which complementary probes are accumulated, so that the presence or absence of methylation can be measured.

[0056] (4) Detection of differential methylation - bisulfite sequencing method: Other methods for detecting nucleic acids containing methylated CpG include contacting a sample containing the nucleic acid with an agent that modifies unmethylated cytosine, and amplifying the CpG - containing nucleic acid of the sample using CpG - specific oligonucleotide primers. Here, the oligonucleotide primers can be characterized by distinguishing modified methylated and unmethylated nucleic acids and detecting methylated nucleic acids. The amplification step is optional, preferred but not essential. The method relies on a PCR reaction that distinguishes modified (e.g., chemically modified) methylated and unmethylated DNA.

[0057] (5) Bisulfite sequencing method: Other methods for detecting nucleic acids containing methylated CpG include contacting a sample containing the nucleic acid with an agent that modifies unmethylated cytosine, and amplifying the CpG - containing nucleic acid of the sample using methylation - independent oligonucleotide primers. Here, the oligonucleotide primers can be characterized by not distinguishing modified methylated and unmethylated nucleic acids and amplifying the nucleic acid. The amplified product is sequenced by the Sanger method using sequencing primers or is described in connection with bisulfite sequencing for the detection of methylated nucleic acids by next generation sequencing method.

[0058] (6) Here, the next-generation sequencing method can be characterized by performing sequencing by synthesis and sequencing by ligation methods. The feature of this method is that instead of creating bacterial clones, single DNA fragments are spatially separated and clonally amplified in situ for sequencing. At this time, since hundreds of thousands of fragments are read simultaneously, it is sometimes called the massively parallel sequencing method.

[0059] Basically, it is a sequencing by synthesis method, and a method of obtaining signals while sequentially attaching mono- or dinucleotides is used. The pyrosequencing, ion torrent, and Solexa methods correspond to this.

[0060] NGS devices based on sequence determination by synthesis include the 454 platform from Roche, the HiSeq platform from Illumina, the Ion PGM platform from Life Technology, and finally the PacBio platform from Pacific BioSciences. 454 and Ion PGM use emulsion PCR with the clonal amplification method, and HiSeq uses Bridge amplification. The sequence determination method by synthesis reads the sequence by detecting phosphate, hydrogen ions, or pre-attached fluorescence generated when DNA is synthesized while sequentially attaching one nucleotide at a time. In the method of detecting the sequence, 454 uses the pyrosequencing method using phosphate, and Ion PGM uses hydrogen ion detection. HiSeq and PacBio detect fluorescence to decode the sequence.

[0061] Sequencing by ligation is a sequencing technique that uses DNA ligase and is a technique for confirming nucleotides at specific positions in a DNA base sequence. Unlike most sequencing techniques that use polymerase, it does not use polymerase and takes advantage of the fact that DNA ligase does not ligate mismatched sequences. The SOLiD system corresponds to this. In this technique, two bases are read at intervals, but since it is repeated independently 5 times through primer reset, ultimately each base is read twice in duplicate to improve accuracy.

[0062] In the case of sequencing by ligation, among the dinucleotide primer sets prepared in 16 combinations, the dinucleotide primer corresponding to the base sequence is sequentially ligated, and the combination of these ligations is finally analyzed to complete the base sequence of the DNA.

[0063] Here, the next-generation sequencing method can be characterized by being performed by a method of sequencing by synthesis or sequencing by ligation. Here, the methylated DNA-specific binding protein is not limited to MBD2bt, and the antibody is not limited to a 5'-methyl-cytosine antibody.

[0064] Regarding the primer used in the present invention, when a reagent such as bisulfite is treated according to the step (a), if the cytosine at the 5'-CpG'-3 site is methylated, it remains as cytosine, and if it is not methylated, it changes to uracil. Therefore, a primer corresponding to the site where the 5'-CpG-3' base sequence exists can be prepared for the base sequence converted after treatment with a reagent such as bisulfite.

[0065] The primer can be prepared to have "substantially" complementarity with each strand of the gene locus to be amplified. This means that under the conditions for performing the polymerization reaction, the primer has sufficient complementarity to hybridize with the corresponding nucleic acid strand.

[0066] In another aspect, the present invention relates to a diagnostic composition for lung cancer comprising one or more reagents for modifying the methylated PCDHGA12 gene and CDO1 gene and the non-methylated PCDHGA12 gene and CDO1 gene to be different from each other; and primers specifically amplifying the methylated PCDHGA12 gene and the methylated CDO1 gene.

[0067] Since the components included in the composition according to the present invention overlap with the components described above, the description thereof is similarly applicable.

[0068] The present invention also relates, in another aspect, to a kit for detecting methylation of target DNA containing the above composition.

[0069] In one embodiment, the kit can include a compartmentalized carrier means for receiving a sample, a container containing a reagent, and a container containing primers for each of the methylated PCDHGA12 gene and the methylated CDO1 gene. Optionally, the kit can further include a container containing a probe for detecting each of the amplification products of the methylated PCDHGA12 gene and the methylated CDO1 gene.

[0070] The carrier means is suitable for containing one or more containers such as bottles and tubes, and each container contains independent components used in the method of the present invention. In the specification of the present invention, those having ordinary knowledge in the art can easily dispense the necessary preparations in the container.

[0071] Hereinafter, the present invention will be described in more detail through examples. It will be apparent to those having ordinary knowledge in the art that these examples are solely for illustrating the present invention and should not be construed as limiting the scope of the present invention by these examples.

[0072] Example 1. Verification of Primers and Probes for Detecting PCDHGA12 and CDO1 Methylated Genes A primer set for multiplex polymerase chain reaction (multiplex PCR) was prepared to simultaneously detect methylation of the PCDHGA12 and CDO1 genes. Using MethPrimer (http: / / www.urogene.org / cgi-bin / methprimer / methprimer.cgi), methylation-specific oligonucleotide primer and probe sets were designed for the bisulfite-converted PCDHGA12 and CDO1 gene sequences (Table 1). The PCDHGA12 gene was designed based on the nucleotide sequence of exon 1, and the CDO1 gene was designed based on the nucleotide sequence of the promoter region. In addition, oligonucleotide primers and probes specific for the control gene COL2A1 were designed (Table 1).

[0073]

Table 1

[0074] To confirm the methylation-specific detection ability of the designed primers and probes for multiplex PCR of the PCDHGA12 and CDO1 genes, methylation and unmethylated control DNAs (EpiTect PCR control DNA set (Qiagen, cat. no. 59695)) were used and verified by quantitative methylation-specific real-time polymerase chain reaction (Quantitative methylation-specific real time PCR (qMSP)). At this time, the COL2A1 gene was used as the internal target gene.

[0075] A total of 25 μl of PCR reaction solution (bisulfite-converted template human DNA, 20 ng / 5 μl; Qplex Master Mix (CellSafe, Korea), 10 μl; methylation-specific PCR primers (IDT, USA), 1 μl each (1 - 10 pmole / μl); methylation-specific TaqMan probes (IDT, USA), 1 μl each (2.5 - 5 pmole / μl); D.W. 1 μl) was prepared and subjected to PCR reaction with the bisulfite-converted template human DNA (Table 2). The PCR conditions were as follows: after treatment at 95°C for 5 minutes, 15 seconds at 95°C and 45 seconds at 72°C were performed 5 times in total, followed by 15 seconds at 95°C and 45 seconds at 58°C for a total of 35 times in the qMSP stage. The presence or absence of amplification of the PCR products of qMSP was confirmed in real time using a CFX96 (Bio-Rad) device (Figure 1).

[0076]

Table 2

[0077] As shown in Figure 1, in methylated DNA, both the PCDHGA12 and CDO1 genes and the control gene COL2A1 were amplified. In unmethylated DNA, it was confirmed that the PCDHGA12 and CDO1 genes were not amplified and only the control gene COL2A1 was amplified, thus confirming that the methylation-specific primers for the PCDHGA12 and CDO1 genes functioned properly.

[0078] Example 2. Confirmation of increased detection performance of lung cancer by methylation combination of CDO1 and PCDHGA12 genes in bronchial lavage fluid DNA To confirm whether the detection ability of lung cancer is improved when the CDO1 gene is combined with the methylation of the PCDHGA12 gene (Korean Registered Patent No. 10-1106727), a conventional methylation biomarker for early diagnosis of lung cancer, methylation of the PCDHGA12 and CDO1 genes was measured in bronchial lavage fluid DNA collected through bronchoscopy from 38 patients with benign lung diseases (Kyungyang University Hospital) and 73 patients with lung cancer (Kyungyang University Hospital), a total of 111 people (Figure 2). Methylation measurement of the PCDHGA12 and CDO1 genes for these specimens used the qMSP method described in Example 2.

[0079] The PCR instrument used was CFX96 (Bio-Rad), and bisulfite conversion used the EZ Methylation Gold Kit (Catalog No. D5006, Zymo Research, USA).

[0080] The methylation level of each gene was shown by the 35-dCt (PCDHGA12 or CDO1 Ct - COL2A1 Ct) (Chung W et al., Cancer Epidemiol Biomarkers Prev., 2011;20(7):1483-1491) method. The larger the 35-dCt value, the higher the methylation level. To evaluate the performance of each gene for the diagnosis of lung cancer, Receiver Operating Characteristic (ROC) analysis was used with the MedCalc program (MedCalc software, Belgium) to set the optimal cutoff for positive determination and calculate sensitivity and specificity.

[0081] When measuring the methylation of the CDO1 gene using the bronchial lavage fluid DNA that had been converted by bisulfite treatment, the methylation positive cutoff was calculated to be 27.5. Only 1 out of 38 patients with benign lung diseases showed positive methylation, with a specificity of 97.4%. Among lung cancer patients, 59 out of 73 showed positive methylation, and the sensitivity was confirmed to be 80.8%. When combining the methylation of the PCDHGA12 gene, 3 out of 38 patients with benign lung diseases showed positive methylation, and the specificity was 92.1% with no significant decrease (P = 0.615, Fisher's exact test). The sensitivity was that 68 out of 73 lung cancer patients showed positive methylation. Compared with using CDO1 alone, 9 more lung cancer patients showed positive results, and a total of 68 out of 73 (68 / 73) showed a significantly increased sensitivity of 93.2% (P < 0.047, Fisher's exact test) (Table 3). Therefore, it was confirmed that when combining the methylation of the PCDHGA12 gene with the methylation of the CDO1 gene, the detection ability of lung cancer increased significantly.

Table 3

Industrial Applicability

[0082] The present invention can improve the detection ability of lung cancer compared to the method of detecting with a single marker gene by detecting methylation with high detection sensitivity through primers that specifically amplify multiple methylated lung cancer marker genes, and it can enable accurate and rapid diagnosis of lung cancer, which is useful.

[0083] As described in detail the specific parts of the content of the present invention above, it will be clear to those with ordinary knowledge in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention thereby. Therefore, it can be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

**Claim 1** A method for providing information for the diagnosis of lung cancer, comprising the following steps: (a) treating a sample with one or more reagents that modify the methylated PCDHGA12 (Protocadherin Gamma Subfamily A, 12) gene and the CDO1 (Cysteine Dioxygenase Type 1) gene, and the non-methylated PCDHGA12 gene and the CDO1 gene to be different from each other; and (b) treating with primers that specifically amplify the methylated PCDHGA12 gene and the CDO1 gene. **Claim 2** The method according to claim 1, wherein the reagent is bisulfite, hydrogen sulfite, disulfite or a combination thereof. **Claim 3** The method according to claim 1, wherein at least one cytosine base is converted to a base different from uracil or cytosine by the reagent treatment. **Claim 4** The method according to claim 1, wherein the primers that specifically amplify the methylated CDO1 gene comprise the primer pair of SEQ ID NOs: 4 and 5. **Claim 5** The method according to claim 1, wherein the primers that specifically amplify the methylated PCDHGA12 gene comprise the primer pair of SEQ ID NOs: 1 and 2. **Claim 6** (c) The method according to claim 1, further comprising treating with a probe that can specifically hybridize to the methylated PCDHGA12 gene and the methylated CDO1 gene specifically amplified by the primers in step (b). **Claim 7** The method according to claim 6, wherein the probe that can specifically hybridize to the amplified methylated CDO1 gene comprises the sequence of SEQ ID NO:

6. **Claim 8** The method according to claim 6, wherein the probe that can specifically hybridize to the amplified methylated PCDHGA12 gene comprises the sequence of SEQ ID NO:

3. **Claim 9** The methylation detection is performed by a method selected from the group consisting of PCR, methylation specific PCR, real time methylation specific PCR, PCR using a methylation DNA specific binding protein, PCR using a methylation DNA specific binding antibody, quantitative PCR, gene chip, sequencing, sequencing by synthesis, and sequencing by ligation, according to the method of claim 1.

10. The method according to claim 6, characterized in that a substance that binds to the probe and exhibits fluorescence is detected to detect methylation.

11. One or more reagents for modifying the methylated PCDHGA12 (Protocadherin Gamma Subfamily A, 12) gene and CDO1 (Cysteine Dioxygenase Type 1) gene, the non-methylated PCDHGA12 gene and CDO1 gene to be different from each other; and A composition for diagnosing lung cancer comprising primers that specifically amplify the methylated PCDHGA12 gene and CDO1 gene.

12. The composition according to claim 11, characterized in that the reagent is bisulfite, hydrogen sulfite, disulfite or a combination thereof.

13. The composition according to claim 11, characterized in that at least one cytosine base is converted to uracil or a base different from cytosine by the reagent treatment.

14. The composition according to claim 11, characterized in that the primers that specifically amplify the methylated CDO1 gene comprise the primer pair of SEQ ID NOs: 4 and 5.

15. The composition according to claim 11, characterized in that the primers that specifically amplify the methylated PCDHGA12 gene comprise the primer pair of SEQ ID NOs: 1 and 2.

16. The composition according to claim 11, further comprising probes that can hybridize complementarily to the methylated PCDHGA12 gene and CDO1 gene specifically amplified by the primers.

17. The composition according to claim 16, characterized in that the probe that can hybridize complementarily to the amplified methylated CDO1 gene comprises the sequence of SEQ ID NO:

6.

18. The composition according to claim 16, wherein the probe capable of hybridizing complementarily to the amplified methylated PCDHGA12 gene comprises the sequence of SEQ ID NO:

3.

19. A diagnostic kit for lung cancer comprising the composition according to any one of claims 11 to 18.

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