Lung cancer-specific biomarkers and composition for diagnosing or predicting prognosis of lung cancer by using same

Abstract

The present invention relates to novel use of a gene as a lung cancer-specific methylation biomarker and, specifically, to: a biomarker comprising one or more genes selected from the group consisting of HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, and GATA5; and lung cancer diagnosis using the detection of a methylation level of the biomarker. The present invention can effectively contribute to early screening and definitive diagnosis of lung cancer, prediction of prognosis after surgery or treatment, and reduction of unnecessary invasive examinations, and can greatly improve the accuracy of differential diagnosis of benign lesions and malignant lesions, which are difficult to distinguish by LDCT alone, and thus is highly likely to be used as a precision medicine-based screening and diagnostic platform for high-risk lung cancer groups.

Description

Lung cancer-specific biomarkers and compositions for lung cancer diagnosis or prognosis prediction using the same

[0001] The present invention relates to lung cancer-specific methylation biomarkers and their uses, specifically to epigenetic biomarkers comprising one or more genes selected from the group consisting of HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, and GATA5, a composition for diagnosing lung cancer comprising detecting the methylation level of said biomarkers, a kit, and a method for providing information for diagnosing lung cancer.

[0002]

[0003] Lung cancer remains a serious public health issue and is one of the leading causes of cancer-related deaths worldwide. Current diagnostic methods rely on invasive procedures, and due to a lack of efficient means for early detection, the disease is often diagnosed at a terminal stage, resulting in poor treatment outcomes. Low-Dose Computed Tomography (LDCT) is known to be a useful method for the early detection of lung lesions, including nodules, and is utilized in national lung cancer screening programs targeting high-risk groups; however, it has limitations in effectively distinguishing between malignant and benign lesions, leading to the disadvantage of frequent unnecessary invasive examinations. In fact, while a significant number of patients undergoing LDCT show suspicious nodules or masses, the rate of final diagnosis for lung cancer among them is very low, at less than 1%. Therefore, there is a need for the development of diagnostic methods that can differentiate between malignant and benign lung diseases more accurately, reliably, and simultaneously less invasively.

[0004] One promising approach to overcome these diagnostic limitations is the use of bronchoalveolar lavage fluid (BALF). BALF is a relatively easily obtainable biological fluid that contains cellular components from the bronchial and alveolar spaces, which reflect the tumor microenvironment, and is therefore considered a suitable biological resource for detecting lung cancer-related biomarkers. BALF collection can be performed repeatedly using a minimally invasive method, offering the advantage of securing valid and sufficient specimens for lung cancer diagnosis. In particular, due to its proximity to tumor tissue, it is expected to have higher sensitivity for biomarker detection compared to other body fluids such as plasma, pleural fluid, and urine.

[0005] Epigenetic changes, particularly DNA methylation patterns, are attracting attention as promising biomarkers for various types of cancer, including lung cancer. Recent studies have reported that DNA methylation patterns in BALF can be effectively utilized for the diagnosis of lung cancer, but in actual clinical practice, there is a problem where sensitivity and specificity are limited due to the low proportion of cancer cell-derived DNA in BALF.

[0006] To overcome these limitations and effectively differentiate lung cancer from benign diseases at an early stage, the inventors selected lung cancer-specific exosomal DNA biomarkers derived from differential methylation regions (DMRs) within major tumor suppressor genes known to be associated with the early stages of cancer development. Exosomes are considered a highly useful resource for detecting cancer-specific biomarkers, particularly DNA methylation patterns, as they directly contain biomolecules derived from cancer cells, such as DNA, RNA, and proteins, and play an important role in intercellular signaling involved in the progression and metastasis of cancer. Furthermore, according to several studies, BALF contains a high concentration of pure DNA derived from cancer cells, exhibiting higher sensitivity and specificity than existing fluid-based diagnostic methods, and can be expected to achieve accuracy approaching that of standard tissue-based diagnostic methods.

[0007] DNA methylation primarily occurs in the cytosines of CpG islands within the promoter regions of specific genes. It disrupts the binding of transcription factors, thereby blocking gene expression (gene silencing). This serves as a major mechanism by which gene function is lost in vivo, even without mutations in the gene's base sequence. Furthermore, it is interpreted as a cause of the loss of function in numerous tumor suppressor genes in human cancer. It has been confirmed that abnormal methylation / demethylation in promoter CpG islands leads to hypermethylation of tumor suppressor genes, DNA repair genes, and cell cycle regulatory genes in various cancers, resulting in the blockage of their expression. The inventors used a PNA (peptide nucleic acid) probe and a methylation specific restriction enzyme (MSRE) to detect DNA methylation of lung cancer-specific biomarkers without treatment with bisulfite.

[0008] Meanwhile, smoking history, a risk factor closely associated with the development of lung cancer, is considered a key variable in defining high-risk groups for lung cancer and assisting in clinical judgment. Accordingly, the inventors further improved the accuracy of diagnosis and the reliability of prognosis prediction by integrating the subjects' smoking history (e.g., current smoking status, total smoking amount, duration of abstinence, etc.) with the results of methylation biomarker analysis.

[0009]

[0010] (비특허문헌 0001) Bade B.C., Dela Cruz C.S. Lung Cancer 2020: Epidemiology, Etiology, and Prevention. Clin. Chest Med. 2020;41:1-24. doi: 10.1016 / j.ccm.2019.10.001.

[0011] (비특허문헌 0002) Leiter A., Veluswamy R.R., Wisnivesky J.P. The global burden of lung cancer: Current status and future trends. Nat. Rev. Clin. Oncol. 2023;20:624-639. doi: 10.1038 / s41571-023-00798-3.

[0012] (비특허문헌 0003) Chen H., Ma Y., Xu J., Wang W., Lu H., Quan C., Yang F., Lu Y., Wu H., Qiu M. Circulating microbiome DNA as biomarkers for early diagnosis and recurrence of lung cancer. Cell Rep. Med. 2024;5:101499. doi: 10.1016 / j.xcrm.2024.101499

[0013] (비특허문헌 0004) Wang X., Tian L., Lu J., Ng I.O. Exosomes and cancer-Diagnostic and prognostic biomarkers and therapeutic vehicle. Oncogenesis. 2022;11:54. doi: 10.1038 / s41389-022-00431-5.

[0014] (Non-patent literature 0005) Hong Y., Kim WJ DNA Methylation Markers in Lung Cancer. Curr. Genom. 2021;22:79-87. doi: 10.2174 / 1389202921999201013164110.

[0015] (Non-patent document 0006) Lu H., Lin D. Diagnostic value of exfoliated tumor cells combined with DNA methylation in bronchoalveolar lavage fluid for lung cancer. Medicine. 2023;102:e34955. doi: 10.1097 / MD.0000000000034955.

[0016]

[0017] The information described above in the background section is intended solely to enhance understanding of the background of the present invention and may not include information that forms prior art already known to those skilled in the art to which the present invention belongs.

[0018]

[0019] Summary of the Invention

[0020] The objective of the present invention is to provide a lung cancer-specific biomarker comprising a specific gene, a composition for diagnosing lung cancer using the measurement of the methylation level thereof, and a kit for diagnosing lung cancer comprising said composition.

[0021] Another objective of the present invention is to provide a method for providing information for lung cancer diagnosis and a lung cancer diagnosis method using methylated lung cancer-specific biomarker genes.

[0022]

[0023] To achieve the above objective, the present invention provides a biomarker composition for diagnosing lung cancer comprising one or more genes selected from the group consisting of HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, and GATA5.

[0024] The present invention also provides a composition for diagnosing lung cancer comprising a preparation for measuring the methylation level of a CpG island of one or more genes selected from the group consisting of HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, and GATA5.

[0025] The present invention also provides a use for a preparation for measuring the methylation level of a CpG island of one or more genes selected from the group consisting of HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, and GATA5, to be used in the preparation of a composition for diagnosing lung cancer.

[0026] The present invention also provides a lung cancer diagnostic kit comprising the above composition.

[0027] The present invention also provides a method for providing information for lung cancer diagnosis and a method for diagnosing lung cancer, comprising the step of measuring the methylation level of a CpG island of one or more genes selected from the group consisting of HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, and GATA5 in nucleic acids isolated from a clinical specimen.

[0028]

[0029] Figure 1 is a workflow for lung cancer diagnosis, prognosis prediction, and screening through the present invention.

[0030] Figure 2 shows the results of the Percent Methylated Ratio (PMR) value analysis for the primary screening of lung cancer-specific biomarkers.

[0031] Figure 3 shows the workflow of the lung cancer detection kit (Epi-TOP™ mLUNG assay).

[0032]

[0033] Detailed Description of the Invention and Preferred Embodiments

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0035]

[0036] In one embodiment of the present invention, as a result of research on the early screening, accurate diagnosis, and prognosis prediction of lung cancer, an algorithm was developed utilizing the methylation levels of promoter CpG sites of specific genes in exosome DNA within BALF and the patient's smoking history information, and it was confirmed that using this, it is possible to measure and diagnose lung cancer risk with high sensitivity and specificity.

[0037]

[0038] Accordingly, in one aspect, the present invention relates to a biomarker composition for diagnosing lung cancer comprising one or more genes selected from the group consisting of HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, and GATA5.

[0039] In the present invention, the biomarker composition may be characterized by including methylated CpG within the CpG island of the gene.

[0040] In this specification, the term “biomarker” generally refers to an indicator capable of detecting changes within the body using organic biomolecules such as proteins, nucleic acids (DNA and mRNA, etc.), and metabolites (lipids, glycolipids, glycoproteins, sugars, etc.). Specifically, it refers to a marker that can distinguish between normal and pathological states in the case of specific diseases or cancer, predict treatment responses, and be objectively measurable. Depending on their application, biomarkers include diagnostic markers for diagnosing the presence or absence of disease, target markers for confirming the existence of drug targets, predictive markers for distinguishing between responders and non-responders to specific drugs, surrogate markers for monitoring drug therapeutic effects, and prognostic biomarkers for indicating the prognosis of a disease.

[0041]

[0042] In another aspect, the present invention relates to a composition for diagnosing lung cancer comprising a preparation for measuring the methylation level of a CpG island of one or more genes selected from the group consisting of HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, and GATA5.

[0043] In the present invention, the diagnostic composition may further comprise a preparation for quantitative analysis of the ACTB (human β-actin) gene as an internal control, preferably a preparation for amplifying or detecting a CpG-free region of the ACTB gene.

[0044]

[0045] In the present invention, the agent for measuring the methylation level may be characterized by comprising a primer capable of amplifying a fragment containing methylated CpG, or a probe capable of specifically binding to methylated CpG.

[0046] In this specification, the term “primer” means a short nucleic acid sequence having a short free 3’ end hydroxyl group, capable of forming base pairs with a complementary template, and functioning as a starting point for template strand replication. The primer can initiate DNA synthesis in the presence of a reagent for a polymerization reaction (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer solution and temperature.

[0047] The primers of the present invention may preferably be designed according to the sequence of a specific CpG site to be analyzed for methylation status, and may be, for example, a primer or primer pair capable of specifically amplifying a fragment containing a methylated CpG island of each gene, and a primer or primer pair capable of specifically amplifying a fragment containing an unmethylated CpG island of each gene.

[0048] In this specification, the term "probe" refers to a single-stranded nucleic acid molecule capable of hybridization that includes a sequence substantially complementary to a target nucleic acid sequence. More specifically, as a nucleic acid strand partially or completely complementary to the target nucleic acid sequence, it may be an oligonucleotide capable of binding in a base-specific manner. The probe comprises any previously known nucleic acid derivative capable of complementary binding, including oligonucleotides, Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA), TaqMan DNA probes, and combinations thereof.

[0049] In one embodiment of the present invention, the probe may be a PNA (Peptide Nucleic Acid) combined with a reporter and a quencher.

[0050] In some cases, primers or probes are labeled so as to be detectable, and may be labeled, for example, with radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelates, or enzymes. Suitablely labeling such primers or probes is a technique widely known in the art and can be performed through conventional methods.

[0051] The amount of the amplification product can be detected by a fluorescent signal. Examples include the intercalating method, which uses an intercalator that binds to the double helix DNA of the amplification product bound to a primer or probe and exhibits fluorescence, and the method which uses an oligonucleotide labeled with a fluorescent substance at the 5' end or a quencher at the 3' end.

[0052] In the present invention, the primer may be characterized by comprising one or more sequences selected from the group consisting of SEQ ID NOs 1 to 18, and the probe may be characterized by comprising one or more sequences selected from the group consisting of SEQ ID NOs 19 to 27.

[0053] In the present invention, the primers may be used simultaneously, for example, by forming forward and reverse primers in pairs. In one embodiment of the present invention, the forward primer that specifically amplifies the methylated HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, or GATA5 gene may each include the sequence of SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, or 15, and the reverse primer may each include the sequence of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, or 16.

[0054] Preferably, the primer for specifically amplifying the methylated HOXA9 gene may include the primer pair of SEQ ID NOs 1 and 2, the primer for specifically amplifying the methylated HOXD3 gene may include the primer pair of SEQ ID NOs 3 and 4, the primer for specifically amplifying the methylated NID2 gene may include the primer pair of SEQ ID NOs 5 and 6, the primer for specifically amplifying the methylated NPTX2 gene may include the primer pair of SEQ ID NOs 7 and 8, the primer for specifically amplifying the methylated SEPT9 gene may include the primer pair of SEQ ID NOs 9 and 10, the primer for specifically amplifying the methylated PCDH17 gene may include the primer pair of SEQ ID NOs 11 and 12, the primer for specifically amplifying the methylated GATA5 gene may include the primer pair of SEQ ID NOs 15 and 16, and the primer for specifically amplifying the methylated RUNX3 gene may include the primer pair of SEQ ID NOs 13 and 14.

[0055] The primers that specifically amplify the ACTB gene may include the primer pair of SEQ ID NOs 17 and 18.

[0056] In the present invention, the diagnostic composition may further comprise at least one of Hinp1I, HpaII, and AciI, which are methylation-specific restriction enzymes. The methylation-specific restriction enzymes can cleave the gene promoter region by distinguishing the degree of methylation.

[0057]

[0058] In this specification, the term “methylation” refers to the attachment of a methyl group to a base constituting a gene. Preferably, as used herein, methylation refers to methylation occurring in the cytosine of a specific CpG island of a specific gene.

[0059] In the present invention, methylation refers to the modification of a cytosine base ring into 5-methylcytosine (5-mC) by attaching a methyl group to the 5th carbon. 5-methylcytosine always occupies only the C of a CG dinucleotide (5'-mCG-3'), and such a CG is commonly denoted as CpG. Methylation of this CpG inhibits the expression of repetitive nucleotide sequences within the genome, such as alu or transposons, and is the site where extragenetic changes most frequently occur in mammalian cells. The 5-mC of this CpG naturally undergoes deamination to change to T; consequently, CpGs within the mammalian genome exhibit a frequency of only 1%, which is much lower than the normal frequency (1 / 4 x 1 / 4 = 6.25%).

[0060] Among CpGs, there are some that appear in exceptionally high concentrations, known as CpG islands. A CpG island refers to a region with a length of 0.2 to 3 kb where the distribution percentage of C and G bases exceeds 50% and the distribution percentage of CpGs is highly concentrated at 3.75% or higher. Approximately 45,000 CpG islands appear throughout the entire human genome, and they are particularly concentrated in promoter regions that regulate gene expression. In fact, CpG islands are found in the promoters of housekeeping genes, which account for about half of all human genes.

[0061]

[0062] In this specification, the term "diagnosis" means confirming the existence or characteristics of a pathological condition. For the purposes of the present invention, diagnosis means determining whether lung cancer has developed and may include predicting the prognosis.

[0063] In this specification, the term "prognosis" refers to a prediction regarding the progression of the disease or recovery, and signifies a forecast or preliminary evaluation. For the purposes of the present invention, prognosis refers to determining the success of treatment, survival, recurrence, metastasis, drug responsiveness, resistance, etc., in the subject after treatment for lung cancer. That is, it refers to an expectation regarding medical outcomes (e.g., long-term survival potential, disease-free survival rate, etc.), and includes a positive prognosis or a negative prognosis; the negative prognosis includes disease progression or mortality such as recurrence, tumor growth, metastasis, or drug resistance, while the positive prognosis includes disease remission such as a disease-free state, or disease improvement or stabilization such as tumor regression.

[0064] The term "prediction" in this specification means to estimate or guess in advance regarding medical outcomes, and for the purposes of the present invention, it may mean to estimate in advance the course of a patient diagnosed with lung cancer (disease progression, improvement, recurrence of cancer, tumor growth, drug resistance).

[0065]

[0066] In another aspect, the present invention relates to a lung cancer diagnostic kit comprising the above-mentioned diagnostic composition.

[0067] In one embodiment of the present invention, the kit may include a partitioned carrier means for holding a sample, a container containing a reagent, a preparation for measuring the methylation level of CpG islands of the HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, or GATA5 gene, and a container containing a preparation for quantitative analysis of the ACTB gene. In some cases, the kit may each include a container containing a preparation for measuring the methylation level of CpG islands of the HOXA9, HOXD3, NID2, or NPTX2 gene and a preparation for quantitative analysis of the ACTB gene, and a container containing a preparation for measuring the methylation level of CpG islands of the SEPT9, PCDH17, RUNX3, or GATA5 gene and a preparation for quantitative analysis of the ACTB gene.

[0068] The above-mentioned carrier means is suitable for containing one or more containers, such as bottles or tubes, and each container contains independent components used in the method of the present invention. In the specification of the present invention, a person skilled in the art can easily dispense the necessary formulations in the containers.

[0069]

[0070] In another aspect, the present invention relates to a method for diagnosing lung cancer and a method for diagnosing lung cancer, comprising the steps of: (a) measuring the methylation level of a CpG island of one or more genes selected from the group consisting of HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, and GATA5 in nucleic acids isolated from a clinical specimen; and (b) comparing the measured methylation level with the methylation level of a normal group and predicting lung cancer if hypermethylated.

[0071] In the present invention, the above step (a) may be characterized by including the following step:

[0072] (i) a step of treating nucleic acids isolated from clinical specimens with methylation-specific restriction enzymes such as Hinp1I, HpaII, or AciI;

[0073] (ii) a step of amplifying and hybridizing the methylation site by treating with a primer capable of amplifying a fragment containing a methylated CpG of the gene and a probe capable of specifically binding to the methylated CpG; and

[0074] (iii) A step of determining the degree of methylation by analyzing the amplification curve of the hybridized reactants.

[0075] In the present invention, step (iii) may include a step of amplifying a methylation-specific region of the gene or gene using a pair of forward primers and reverse primers capable of amplifying a specific region of the gene or gene, and hybridizing the generated amplification product with a probe, and step (iv) may include a step of analyzing the degree of methylation of the methylation-specific region of the gene or gene by analyzing the amplification curve of the product generated by hybridization.

[0076] In the present invention, the measurement of the methylation level may be characterized by being performed by one or more methods selected from the group consisting of Polymerase Chain Reaction (PCR), real-time PCR, methylation specific PCR, real-time methylation specific PCR, PCR using a methylation DNA-specific binding protein, quantitative PCR, digital PCR, isothermal amplification method, DNA chip, FISH (Fluorescence in situ hybridization), and NGS (Next Generation Sequencing).

[0077] In the present invention, the measurement of the methylation level may be characterized by being performed by comparing the Percent Methylated Ratio (PMR) values ​​of the HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, or GATA5 genes of standard DNA and a clinical sample, and calculating the Methylation Score (MS).

[0078] Here, the PMR value is the methylation ratio of the HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, or GATA5 genes of standard DNA or clinical specimens, derived by the following formula, and

[0079]

[0080] (Here, X is the Ct (Cycle threshold) of the ACTB gene, which is the internal control, and Y is the Ct of the HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, or GATA5 gene);

[0081] The above methylation score may be characterized as being assigned as MS as listed in the table below.

[0082]

[0083] In the present invention, the method may further include the step of calculating a risk score (RS) by integrating a smoking score (SS) calculated based on the patient's smoking history into the methylation score, and determining the patient as a high-risk group for lung cancer if the lung cancer score (LCS) calculated based on the risk score is 0.5 or higher.

[0084] Smoking Score (SS) = 0 (if no smoking history or previous smoking history (less than 10 pack-years)) or 1 (if currently smoking or previous smoking history (10 pack-years or more);

[0085]

[0086] In the present invention, the pack year is a unit indicating lifetime smoking history, and means daily smoking amount (pack) x smoking period (year).

[0087] In the present invention, the clinical specimen may be characterized as being tissue, cell, blood, serum, plasma, urine, bronchoalveolar lavage fluid, or extracellular vesicle derived from a patient suspected of having lung cancer or a subject of diagnosis, but is not limited thereto.

[0088] In the present invention, the clinical specimen may be a bronchoalveolar lavage fluid (BALF) from a patient suspected of having lung cancer based on low-dose chest computed tomography (LDCT) results, and the nucleic acid may be an exosomal DNA isolated from the bronchoalveolar lavage fluid, but is not limited thereto.

[0089] In the present invention, the "normal group" may refer to a general individual that has not developed lung cancer, a non-lung cancer patient group, a non-patient group, etc.

[0090]

[0091] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0092]

[0093] Example 1: Screening of Methylation Biomarkers for Lung Cancer Diagnosis and Prognosis Prediction

[0094] To select lung cancer-specific biomarkers, genes reported to be differentially methylated between normal individuals and cancer patient groups were primarily selected based on prior research and open source data. The selected genes are KCNA4, REPRIMO, RNF, SLC-19, CYP, MINT25, ADAM23, ESR1, TIMP3, DAPK, RASSF1A, RUNX3, CDKN2A, SHOX2, SEPTIN9, PTGER4, MTHFR, CALCA, HIST1H4F, APC, MLH1, SFRP, VIM, ALX, NEW, GATA, RAR-β, SCGB3A1, p14ARF, ATM, CST6, FHIT, CDKN2B, WT1, HOXA9, HOXD3, GSTP2, TWIST-R2, POU4F2, PCDH17, NID2, ZNF154, ADAMTS, BMP3, NPTX, BNC1, C13ORF18, CD1D, HAND2, PTGDR, POU4F3, MAGI2, There are a total of 61 genes, including ADCY, MME, 14-3-3-γ, HS3ST2, NIS, PTEN, FASSF10, TSHR, MGMT, etc.

[0095] To finally select lung cancer-specific methylated biomarkers among the above genes, the methylation rates of CpG island regions of each gene promoter were compared and analyzed using DNA derived from extracellular vesicles (EVs) isolated from bronchoalveolar lavage fluid (BALF) of patients with non-cancer lung disease and lung cancer.

[0096] The nucleotide sequences of the promoter regions of each gene were analyzed to design primers capable of selectively amplifying the corresponding regions, and peptide nucleic acid (PNA) probes were constructed to specifically bind to methylated sequences. A composition comprising the above primers (or primer pairs) and PNA probes was utilized as a composition for biomarker detection experiments for lung cancer screening, and the composition included methylation-specific restriction enzymes Hinp1I, HpaII, AciI, and Taq polymerase. EV-derived DNA isolated from bronchoalveolar lavage fluid of healthy individuals and lung cancer patients was used as the experimental samples.

[0097] The experiment was performed using Real-Time Polymerase Chain Reaction (Real-Time PCR), and the Cycle Threshold (ΔCt) and Percent Methylated Ratio (PMR) were calculated based on standard methylated and non-methylated DNA (Table 1). Based on these criteria, the degree of methylation of each target gene was analyzed according to Tables 2 and 3 below.

[0098]

[0099]

[0100]

[0101] To accurately distinguish BALF exosome DNA samples derived from healthy individuals and lung cancer patients, the PMR threshold with the highest clinical accuracy was established for each marker. Subsequently, the lung cancer detection performance for each marker was evaluated, and as a result, 12 biomarkers with a lung cancer detection accuracy of 70% or higher were finally selected as lung cancer-specific biomarkers (Fig. 1). The list of selected biomarkers is presented in Table 4.

[0102]

[0103] Example 2: Selection of Final Combination of Methylation Biomarkers for Lung Cancer Diagnosis and Prognosis Prediction

[0104] Based on the 12 biomarkers selected in Example 1, various multiple combinations were formed, and clinical performance analysis was performed for each combination. As a result, the biomarker combination that showed the best diagnostic performance in lung cancer detection was selected as the final combination (Table 5).

[0105]

[0106] The clinical performance of the eight finally selected biomarker combinations is shown in Table 6 below, and they demonstrated high sensitivity and specificity for lung cancer overall and by stage.

[0107]

[0108] Example 3: Preparation of a gene methylation kit for lung cancer diagnosis and prognosis prediction

[0109] The eight lung cancer-specific methylation biomarkers listed in Table 5 above were divided into four sets each to form two sets (Set A, Set B), and the human β-actin (ACTB) gene was added as an internal control to finally produce a lung cancer detection kit (Epi-TOP™ mLUNG assay) with a composition of '4 types of methylation biomarkers + ACTB' for each set. The kit composition is as presented in Tables 7 to 9 below.

[0110]

[0111]

[0112]

[0113]

[0114] Example 4: Development of a Lung Cancer Risk Analysis Algorithm

[0115] Based on the results of analyzing a total of 100 additional samples from healthy individuals and lung cancer patients using the lung cancer detection kit (Epi-TOP™ mLUNG assay) of Example 3, a lung cancer risk analysis algorithm capable of achieving optimal clinical performance was developed as follows:

[0116] 1) Calculation of PMR values ​​for each biomarker: The PMR calculation method is performed according to Table 2 above.

[0117] 2) A methylation score (MS) was assigned based on the PMR values ​​of each biomarker relative to the Control DNA. The scoring criteria are presented in Table 10.

[0118]

[0119]

[0120]

[0121] 3) Calculation of Smoking Score (SS) based on smoking history: A smoking score is assigned as shown in Table 11 below, based on the individual patient's smoking history information.

[0122]

[0123] 4) The Risk Score (RS) and Lung Cancer Score (LCS) for each patient were calculated based on the formula presented in Table 12 below to classify the final results.

[0124]

[0125] Example 5: Verification of the analytical performance of the lung cancer detection kit (Epi-TOP™ mLUNG assay)

[0126] To evaluate the analytical sensitivity of the lung cancer detection kit (Epi-TOP™ mLUNG assay) of Example 3, gDNA mixed samples were prepared by mixing unmethylated DNA (methylation ratio approx. 0%) and fully methylated DNA (methylation ratio approx. 100%) in various ratios (Table 13).

[0127]

[0128] As a result of analyzing the mixed DNA, it was confirmed that the kit can detect at least 1.25% of methylated DNA based on a pre-set methylation threshold (Threshold, Table 14), and the minimum detectable methylation level for each target biomarker was found to be in the range of 0.31% to 1.25% (Table 15).

[0129]

[0130]

[0131] Example 6: Verification of Clinical Performance of the Lung Cancer Detection Kit (Epi-TOP™ mLUNG Assay)

[0132] To evaluate the clinical performance of the lung cancer detection kit (Epi-TOP™ mLUNG assay) of Example 3, 22 BALF-derived DNA samples were used from patients who were finally diagnosed with non-cancer lung disease among those who underwent pathological confirmation testing due to suspected lung cancer based on low-dose chest computed tomography (LDCT) results, and 36 BALF-derived DNA samples were used from patients confirmed to have lung cancer. When the results of this analysis were compared with the confirmed diagnosis results, the kit demonstrated clinical performance with a sensitivity of 94.44% and a specificity of 100.00% (Table 16).

[0133]

[0134] According to the present invention, by utilizing the methylation status of exosome DNA within BALF together with the patient's smoking history information, it is possible to effectively contribute to the early screening and definitive diagnosis of lung cancer, the prediction of prognosis after surgery or treatment, and the reduction of unnecessary invasive examinations. In addition, since it can significantly improve the accuracy of differential diagnosis between benign and malignant lesions that are difficult to distinguish with LDCT alone, it has very high potential to be utilized as a precision medicine-based screening diagnostic platform for high-risk groups for lung cancer.

[0135]

[0136] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0137]

[0138] I have attached the electronic file.

Claims

1. A biomarker composition for diagnosing lung cancer comprising one or more genes selected from the group consisting of HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, and GATA5.

2. A biomarker composition according to claim 1, characterized by including methylated CpG within a CpG island of the gene.

3. A composition for diagnosing lung cancer comprising a preparation for measuring the methylation level of CpG islands of one or more genes selected from the group consisting of HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, and GATA5.

4. A composition according to claim 3, further comprising a preparation for amplifying or detecting a CpG-free region of the ACTB (human β-actin) gene, which serves as an internal control, for measuring the methylation level of the CpG islands of the gene.

5. A composition according to claim 3, wherein the agent for measuring the methylation level comprises a primer capable of amplifying a fragment containing methylated CpG, or a probe capable of specifically binding to CpG.

6. A composition according to claim 5, wherein the primer comprises one or more sequences selected from the group consisting of SEQ ID NOs 1 to 18, and the probe comprises one or more sequences selected from the group consisting of SEQ ID NOs 19 to 27.

7. A composition according to claim 4, further comprising at least one of the methylation-specific restriction enzymes Hinp1I, HpaII, and AciI.

8. A lung cancer diagnostic kit comprising the composition of claim 2.

9. Method for providing information for lung cancer diagnosis including the following steps: (a) a step of measuring the methylation level of CpG islands of one or more genes selected from the group consisting of HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, and GATA5 in nucleic acids isolated from clinical specimens; and (b) A step of predicting lung cancer if the measured methylation level is hypermethylated by comparing it with the methylation level of a normal group.

10. A method according to claim 9, wherein the above step (a) comprises the following step: (i) a step of treating nucleic acids isolated from clinical specimens with methylation-specific restriction enzymes such as Hinp1I, HpaII, or AciI; (ii) a step of amplifying and hybridizing the methylation site by treating with a primer capable of amplifying a fragment containing a methylated CpG of the gene and a probe capable of specifically binding to the methylated CpG; and (iii) A step of determining the degree of methylation by analyzing the amplification curve of the hybridized reactants.

11. A method according to claim 9, wherein the measurement of the methylation level is performed by one or more methods selected from the group consisting of Polymerase Chain Reaction (PCR), real-time PCR, methylation specific PCR, real-time methylation specific PCR, PCR using methylation DNA-specific binding proteins, quantitative PCR, digital PCR, isothermal amplification, DNA chip, FISH (Fluorescence in situ hybridization), and NGS (Next Generation Sequencing).

12. A method according to claim 9, characterized in that the measurement of the methylation level is performed by comparing the Percent Methylated Ratio (PMR) values ​​of the HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, or GATA5 genes of a standard DNA and a clinical sample, and calculating the Methylation Score (MS): Here, the PMR value is the methylation ratio of the HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, or GATA5 genes of standard DNA or clinical specimens, derived by the following formula, and (Here, X is the Ct (Cycle threshold) of the ACTB gene, which is the internal control, and Y is the Ct of the HOXA9, HOXD3, NID2, NPTX2, SEPT9, PCDH17, RUNX3, or GATA5 gene); The above methylation scores were assigned as MS as listed in the table below:

13. A method according to claim 12, further comprising the step of calculating a Risk Score (RS) by integrating a Smoking Score (SS) calculated based on the patient's smoking history with the methylation score, and determining the patient as a high-risk group for lung cancer if the Lung Cancer Score (LCS) calculated based on the Risk Score is 0.5 or higher: Smoking Score (SS) = 0 (no smoking history or previous smoking history (less than 10 pack-years)) or 1 (current smoking or previous smoking history (10 pack-years or more); 14. A method according to claim 9, characterized in that the clinical specimen is tissue, cell, blood, serum, plasma, urine, bronchoalveolar lavage fluid, or extracellular vesicle derived from a patient suspected of having lung cancer or a subject of diagnosis.

15. A method according to claim 9, wherein the clinical specimen is bronchoalveolar lavage fluid (BALF) from a patient suspected of having lung cancer based on low-dose chest computed tomography (LDCT) results, and the nucleic acid is exosomal DNA isolated from the bronchoalveolar lavage fluid.

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