Biomarkers for predicting the possibility of gastric cancer development and their uses

Abstract

The present invention relates to a biomarker for predicting the possibility of gastric cancer development and its use, and by measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2 or the protein expressed therefrom, the possibility of gastric cancer development including metachronous gastric cancer can be predicted. The composition for predicting the possibility of gastric cancer development according to the present invention not only has high accuracy in predicting the development of gastric cancer, but also enables the prediction of gastric cancer by a single measurement method by using multiplex reverse transcription polymerase chain reaction (multi-plex PCR) in one tissue specimen, so it has a high utilization rate. In addition, since it includes a method for quantifying basal gastritis by a quantitative method capable of predicting not only metachronous gastric cancer but also the development of gastric cancer, it provides a method for predicting the risk of gastric cancer development in normal people with a family history or risk factors of gastric cancer. In addition, by predicting the development of metachronous gastric cancer in patients with early gastric cancer treated by endoscopic submucosal dissection, there is an effect that the prognosis of the patient can be predicted.

Description

Technical Field

[0001] The present invention relates to a biomarker for predicting the possibility of gastric cancer development and its use.

[0002] This application claims priority based on Korean Patent Application No. 10-2022-0067245 filed on May 31, 2022, and Korean Patent Application No. 10-2022-0118936 filed on September 20, 2022, and all the contents disclosed in the specifications and drawings of the applications are incorporated herein by reference.

[0003] (Blank)

Background Art

[0004] Regarding gastric cancer, not only surgical procedures and adjuvant chemotherapies to assist them, but also early diagnosis methods have made many advancements. However, gastric cancer ranks second globally as a cause of cancer death regardless of gender and remains one of the main causes of death. However, the exact mechanism underlying the most important gastric cancer development and tumor progression has not yet been clarified. In particular, it is known that the reason for the highest gastric cancer incidence rate in Korea worldwide is due to the progression of a large number of cases of basal gastritis.

[0005] Metachronous Gastric Cancer (MGC) means the case where it is discovered 12 months after being diagnosed with gastric cancer. Specifically, as an example, it can be cited as gastric cancer newly occurring at other sites other than the original treatment site during follow-up observation after treating gastric cancer by endoscopic resection.

[0006] In patients with early gastric cancer treated by endoscopic submucosal dissection (ESD), the probability of metachronous gastric cancer occurring in normal gastric mucosa other than the site where the completely cured gastric cancer was resected is extremely high, with an incidence rate of about 3% - 5% per year, which is approximately 100 times the incidence risk of normal people. Therefore, predicting the occurrence of such metachronous gastric cancer is very important for predicting the prognosis of patients.

[0007] However, after endoscopic resection of gastric cancer, since there are no predictive factors for the occurrence of metachronous gastric cancer, all patients will undergo endoscopic examination once a year throughout their lives, but in fact, there are many follow-up losses in the middle.

[0008] In conventional endoscopy, basal gastritis (intestinal metaplasia, atrophic gastritis) is used as a predictive factor for future gastric cancer occurrence, but the inter-observer or intra-observer variability is intense. To overcome this, the OLGA (Operative Link on Gastritis Assessment) and OLGIM (Operative Link on Gastritis Assessment based on Intestinal Metaplasia) stages were developed. However, this requires at least 5 pairs of tissue biopsies endoscopically, with a total of 10 endoscopic tissue specimens needed. As a result, there is a problem that the risk of complications such as bleeding increases and it is hardly used in actual clinical practice.

[0009] Regarding recurrence-specific markers for gastric cancer, they are disclosed in Korean Patent Registration Publication No. 10 - 2253304, etc., but the biomarkers used for the diagnosis or prediction of metachronous gastric cancer in the present invention have not been disclosed.

[0010] (Blank) Summary of the Invention Problems to be Solved by the Invention

[0011] Under the above background, the present inventor confirmed that when performing endoscopic submucosal dissection for early gastric cancer, the expression levels of CDK1 and KDF1 measured in normal mucosa other than gastric cancer can be used to predict the occurrence of metachronous gastric cancer in the future. After confirming the correlation between the expression of CREB5 and AKT2 isoform genes and the OLGIM stage, it was confirmed that through a single gene expression measurement in one tissue specimen, it is possible to predict not only the occurrence of metachronous gastric cancer but also the occurrence of gastric cancer in normal individuals, leading to the completion of the present invention.

[0012] Through the expression patterns of the CDK1, KDF1, CREB5, and AKT2 isoform genes of the present invention, for patients who have undergone endoscopic submucosal dissection for early gastric cancer, not only can an individualized approach to observing the future occurrence of metachronous gastric cancer be achieved through risk stratification, but it is also a quantitative method that can predict the occurrence of gastric cancer in normal individuals, and can also predict the occurrence of gastric cancer in normal individuals with a family history of gastric cancer or risk factors.

[0013] Therefore, an object of the present invention is to provide a composition for predicting the possibility of gastric cancer onset, which comprises a preparation for measuring the level of mRNA of any one or more genes selected from the group consisting of CREB5 and AKT2 or the protein expressed therefrom.

[0014] Another object of the present invention is to provide a kit for predicting the possibility of gastric cancer onset, which comprises the above composition.

[0015] Another object of the present invention is to provide a composition for predicting the possibility of metachronous gastric cancer onset or a composition for predicting the prognosis of patients with early gastric cancer treated by endoscopic submucosal dissection, which comprises a preparation for measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2 or the protein expressed therefrom.

[0016] Still another object of the present invention is to provide a method for providing information for predicting the possibility of gastric cancer development, which includes measuring the level of mRNA of any one or more genes selected from the group consisting of CREB5 and AKT2, or the protein expressed therefrom.

[0017] Still another object of the present invention is to provide a method for providing information for predicting the possibility of metachronous gastric cancer development, which includes measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2, or the protein expressed therefrom, or a method for providing information for predicting the prognosis after endoscopic submucosal dissection treatment in patients with early gastric cancer.

[0018] (Blank)

Means for Solving the Problems

[0019] In order to achieve the above object, the present invention provides a composition for predicting the possibility of gastric cancer development, which includes a preparation for measuring the level of mRNA of any one or more genes selected from the group consisting of CREB5 and AKT2, or the protein expressed therefrom.

[0020] As one embodiment of the present invention, the gastric cancer may include, but is not limited to, metachronous gastric cancer.

[0021] As another embodiment of the present invention, the composition may further include, but is not limited to, a preparation for measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1 and KDF1, or the protein expressed therefrom.

[0022] As still another embodiment of the present invention, the preparation for measuring the mRNA level of the gene may be, but is not limited to, a primer or a probe that specifically binds to the gene or mRNA.

[0023] As yet another embodiment of the present invention, the agent for measuring the level of the protein may be, but is not limited to, an antibody or an aptamer specific to the protein.

[0024] The present invention also provides a kit for predicting the possibility of gastric cancer development, which comprises the composition.

[0025] As one embodiment of the present invention, the kit may be, but is not limited to, one or more selected from the group consisting of a reverse transcription polymerase chain reaction (RT-PCR) kit, a real-time polymerase chain reaction (qRT-PCR) kit, a DNA chip kit, an enzyme-linked immunosorbent assay (ELISA) kit, and a protein chip kit.

[0026] The present invention also provides a composition for predicting the possibility of metachronous gastric cancer development, which comprises an agent for measuring the level of mRNA or the protein expressed therefrom of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2.

[0027] The present invention also provides a composition for predicting the prognosis of early gastric cancer patients treated by endoscopic submucosal dissection, which comprises an agent for measuring the level of mRNA or the protein expressed therefrom of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2.

[0028] The present invention also provides a method for providing information for predicting the possibility of gastric cancer development, which comprises: (a) measuring the level of mRNA or the protein expressed therefrom of any one or more genes selected from the group consisting of CREB5 and AKT2 in a biological sample isolated from a subject; and

[0029] (b) comparing the measured mRNA or protein level with the level measured in a biological sample isolated from a control group.

[0030] As an embodiment of the present invention, the gastric cancer may include metachronous gastric cancer, and the method may further include, but is not limited to, measuring the mRNA level of any one or more genes selected from the group consisting of CDK1 and KDF1 or the level of the protein expressed therefrom in a biological sample separated from a subject, and comparing the measured level with the level measured in a sample separated from a control group.

[0031] As another embodiment of the present invention, the mRNA level of the gene may be measured by one or more methods selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (competitive RT-PCR), real-time reverse transcription polymerase chain reaction (real time quantitative RT-PCR), multiplex reverse transcription polymerase chain reaction (Multi-plex PCR), real-time polymerase chain reaction (qRT-PCR), RNase protection assay (RNase protection method), Northern blotting, DNA chip technology assay, methylated DNA binding domain sequencing (MBD-seq), and reduced representation bisulfite sequencing (RRBS), but is not limited thereto.

[0032] As yet another embodiment of the present invention, the protein level can be measured by, but is not limited to, one or more methods selected from the group consisting of western blot, ELISA (enzyme linked immunosorbent assay), RIA (Radioimmunoassay), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, Immunohistochemistry (IHC), Immunoprecipitation Assay, Complement Fixation Assay, FACS (Fluorescence Activated Cell Sorter), and protein chip.

[0033] As yet another embodiment of the present invention, the mRNA level or protein level of the gene can be measured in one biological sample, but is not limited thereto.

[0034] As yet another embodiment of the present invention, the biological sample can be one or more selected from the group consisting of tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, urine, and feces separated from a subject, but is not limited thereto.

[0035] As yet another embodiment of the present invention, the method may further include, but is not limited to, a step of determining that the risk of gastric cancer development is high when the level of the mRNA or protein measured in a biological sample separated from a subject is higher than the level measured in a biological sample separated from a control group.

[0036] Furthermore, the present invention includes: (a) measuring the level of mRNA or protein expressed therefrom of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2 in a biological sample separated from a subject; and

[0037] (b) Comparing the measured mRNA or protein level with the level measured in a biological sample isolated from a control group; A method for providing information for predicting the likelihood of developing metachronous gastric cancer is provided.

[0038] Furthermore, the present invention includes (a) measuring the level of mRNA or protein expressed therefrom of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2 in a biological sample isolated from a patient with early gastric cancer; and

[0039] (b) Comparing the measured mRNA or protein level with the level measured in a biological sample isolated from a control group; A method for providing information for predicting the prognosis after endoscopic submucosal dissection treatment in a patient with early gastric cancer is provided.

[0040] Furthermore, the present invention includes (a) measuring the level of mRNA or protein expressed therefrom of any one or more genes selected from the group consisting of CREB5 and AKT2 in a biological sample isolated from a subject; and

[0041] (b) Comparing the measured mRNA or protein level with the level measured in a biological sample isolated from a control group; A method for predicting the likelihood of developing gastric cancer is provided.

[0042] Furthermore, the present invention includes (a) measuring the level of mRNA or protein expressed therefrom of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2 in a biological sample isolated from a subject; and

[0043] (b) Comparing the measured mRNA or protein level with the level measured in a biological sample isolated from a control group; A method for predicting the likelihood of developing metachronous gastric cancer is provided.

[0044] In addition, the present invention provides a method for predicting prognosis after endoscopic submucosal dissection in patients with early gastric cancer, comprising: (a) measuring the level of mRNA or the protein expressed therefrom of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2 in a biological sample isolated from a patient with early gastric cancer; and

[0045] (b) comparing the measured mRNA or protein level with the level measured in a biological sample isolated from a control group.

[0046] The present invention also provides a use of a composition comprising an agent for measuring the level of mRNA or the protein expressed therefrom of any one or more genes selected from the group consisting of CREB5 and AKT2 for predicting the possibility of gastric cancer development.

[0047] The present invention also provides a use of an agent for measuring the level of mRNA or the protein expressed therefrom of any one or more genes selected from the group consisting of CREB5 and AKT2 for manufacturing a preparation for predicting the possibility of gastric cancer development.

[0048] The present invention also provides a use of a composition comprising an agent for measuring the level of mRNA or the protein expressed therefrom of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2 for predicting the possibility of metachronous gastric cancer development.

[0049] The present invention also provides a use of an agent for measuring the level of mRNA or the protein expressed therefrom of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2 for manufacturing a preparation for predicting the possibility of metachronous gastric cancer development.

[0050] Furthermore, the present invention provides a use for predicting the prognosis of patients with early gastric cancer treated by endoscopic submucosal dissection, in a composition comprising an agent for measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2, or a protein expressed therefrom.

[0051] In addition, the present invention provides a use of an agent for measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2, or a protein expressed therefrom, for manufacturing a preparation for predicting the prognosis of patients with early gastric cancer treated by endoscopic submucosal dissection.

[0052] (Blank) [[Effect of the Invention]]

[0053] The composition for predicting the possibility of gastric cancer development according to the present invention not only has high accuracy in predicting the development of gastric cancer, but also enables the prediction of gastric cancer by a single measurement method by using multiplex reverse transcription polymerase chain reaction (multi-plex PCR) or the like in a single tissue specimen, so it has a high utilization rate. In addition, it includes a method for quantifying basal gastritis by a quantitative method that can predict not only metachronous gastric cancer but also the development of gastric cancer, so it provides a method for predicting the risk of gastric cancer development in normal people with a family history or risk factors of gastric cancer. In addition, there is an effect that the prognosis of patients can be predicted by predicting the development of metachronous gastric cancer in patients with early gastric cancer treated by endoscopic submucosal dissection.

[0054] (Blank) [[Brief Description of the Drawings]]

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Mode for Carrying Out the Invention

[0066] In one embodiment of the present invention, among patients with early gastric cancer treated by endoscopic submucosal dissection, patients who obtained and stored normal gastric mucosal tissue at the time of diagnosis were targeted, and differentially expressed genes (DEGs) were identified between patients who developed metachronous gastric cancer and patients who did not develop metachronous gastric cancer during follow-up observation. By performing correlation analysis based on gene expression levels, the number of metachronous gastric cancers, the number of adenomas, the degree of atrophic gastritis (OLGA stage), and the degree of intestinal metaplasia (OLGIM stage), self-organizing map (SOM) analysis, and isoform analysis, etc., CDK1, KDF1, ERCC6L, KLK8, MCM2, POLR2L, PTN, CREB5, and AKT2 isoform were selected as candidate genes (see Example 1).

[0067] In another embodiment of the present invention, for the candidate genes, gene expression differences in various gastric cancer cell lines and normal gastric mucosal cell lines were confirmed through qRT-PCR, and CDK1, KDF1, CREB5, and AKT2 isoform were selected as the final target genes by evaluating the suppression of viability in siRNA-treated gastric cancer cell lines (see Example 2-1). Through Western blot and immunohistochemistry, it was confirmed that the protein expression of CDK1 and KDF1 was higher in patient tissues with metachronous gastric cancer than in patient tissues without metachronous gastric cancer (see Examples 2-2 and 2-3).

[0068] In yet another embodiment of the present invention, as a result of evaluating the effectiveness of the CDK1, KDF1, CREB5, and AKT2 isoform genes, it was confirmed that the accuracy of each gene marker for predicting metachronous gastric cancer was high, and in particular, the accuracy increased most when the CDK1, KDF1, and AKT2 isoform markers were combined. It was also confirmed that CREB5 and AKT2 isoform showed expression differences depending on the OLGIM stage, and it was confirmed that it was possible to predict gastric cancer not only in metachronous gastric cancer but also in normal people with a family history or risk factors of gastric cancer (see Example 3).

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[0070] Hereinafter, the present invention will be described in detail.

[0071] The present invention provides a composition for predicting the likelihood of gastric cancer development, which comprises a preparation for measuring the level of mRNA of any one or more genes selected from the group consisting of CREB5 and AKT2, or the protein expressed therefrom.

[0072] In the present invention, the composition may further comprise, but is not limited to, a preparation for measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1 and KDF1, or the protein expressed therefrom.

[0073] Furthermore, the present invention provides a composition for predicting the likelihood of metachronous gastric cancer development, which comprises a preparation for measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2, or the protein expressed therefrom.

[0074] The composition can include one of the four genes, or all combinations of two, three, or four genes.

[0075] For example, it may be CDK1; KDF1; CREB5; AKT2; CDK1 and KDF1; CDK1 and CREB5; CDK1 and AKT2; KDF1 and CREB5; KDF1 and AKT2; CREB5 and AKT2; CDK1, KDF1, and CREB5; CDK1, KDF1, and AKT2; CDK1, CREB5, and AKT2; KDF1, CREB5, and AKT2; or CDK1, KDF1, CREB5, and AKT2. According to an embodiment of the present invention, CREB5 is a gene highly associated with the OLGIM stage. When compared with other genes, it does not show a large association with mGC itself. However, the AKT2 isoform is a gene highly associated with the OLGIM stage and also shows a high association with mGC. Therefore, in the case of the combination of CDK1, KDF1, and the AKT2 isoform, the accuracy may be the highest in predicting the possibility of metachronous gastric cancer development, but it is not limited to the combination of the above genes.

[0076] When the expression of the above genes increases, the composition of the present invention can measure this and obtain useful information for predicting the possibility of gastric cancer development. This has not been known so far and has been first revealed by the present invention. For patients who have undergone endoscopic submucosal dissection for early gastric cancer, not only can they approach the observation of future metachronous gastric cancer occurrence, but also predict the prognosis, and it is a quantitative method that can predict the possibility of gastric cancer development in normal people. It is very significant in that it can predict gastric cancer by a single measurement method using multiplex reverse transcription polymerase chain reaction (multi-plex PCR) in one tissue specimen.

[0077] In the present invention, "CDK1 (Cyclin Dependent Kinase 1)" is cyclin-dependent kinase 1, also known as cell division cycle protein 2 homolog, which is a highly conserved protein functioning as a serine / threonine protein kinase and is known to play a central role in the regulation of the cell cycle. The CDK1 may include the amino acid sequence of NCBI Reference Sequence: NP_001777.1 (SEQ ID NO: 1) and may be encoded by the nucleotide sequence of NCBI Reference Sequence: NM_001786.5 (SEQ ID NO: 2), but is not limited thereto.

[0078] In one embodiment of the present invention, as a result of analyzing the ROC curve for determining the accuracy of gastric cancer prediction in a group of gastric cancer patients, it was confirmed that the accuracy (AUC 0.829) was high, and it was confirmed that it is a biomarker that can be usefully used for predicting the possibility of gastric cancer development.

[0079] In the present invention, "KDF1 (Keratinocyte Differentiation Factor 1)" is a protein that plays a role in regulating epidermal formation during early development and is known to act as an inhibitor of basal cell proliferation and a promoter of the differentiation of basal progenitor cell progeny. The KDF1 may include the amino acid sequence of NCBI Reference Sequence: NP_689578.2 (SEQ ID NO: 3) and may be encoded by the nucleotide sequence of NCBI Reference Sequence: NM_152365.3 (SEQ ID NO: 4), but is not limited thereto.

[0080] In one embodiment of the present invention, as a result of analyzing the ROC curve for determining the accuracy of gastric cancer prediction in a group of gastric cancer patients, it was confirmed that the accuracy (AUC 0.841) was very high, and it was confirmed that it is a biomarker that can be usefully used for predicting the possibility of gastric cancer development.

[0081] In the present invention, "CREB5 (cAMP responsive element binding protein 5)" is a gene belonging to the CRE-binding protein family and is known to function as a transcriptional activator of eukaryotic cells capable of regulating gene expression. The CREB5 may include the amino acid sequence of NCBI Reference Sequence: NP_878901.2 (SEQ ID NO: 5) and may be encoded by the nucleotide sequence of NCBI Reference Sequence: NM_182898.4 (SEQ ID NO: 6), but is not limited thereto.

[0082] In one embodiment of the present invention, it was confirmed that the expression of CREB5 increased as the stage increased in the OLGIM (Operative Link on Gastritis Assessment based on Intestinal Metaplasia) system based on the evaluation of intestinal metaplasia. In particular, it was confirmed that the expression level of CREB5 significantly increased at stages 3 and 4, which are high-risk groups for gastric cancer development, and it was confirmed that it is possible to predict the possibility of gastric cancer onset.

[0083] In the present invention, "AKT2 (AKT Serine / Threonine Kinase 2, RAC-beta serine / threonine-protein kinase)" functions as a very important mediator in the signal transduction pathway downstream of activated tyrosine kinase and PI3K, and each cellular action regulated by AKT is known to affect the roles of cellular actions such as cell proliferation, cell survival, cell size regulation, responsiveness to available nutrients, intermediate metabolic processes, angiogenesis, and tissue invasion. Specifically, AKT2 in the present invention may mean an AKT2 isoform, but is not limited thereto.

[0084] In the present invention, the AKT2 isoform can include the amino acid sequence of SEQ ID NO: 7 and can be encoded by the nucleotide sequence of SEQ ID NO: 8, but is not limited thereto.

[0085] In one embodiment of the present invention, as a result of measuring the expression of the AKT2 isoform protein, it was confirmed that the expression of the AKT2 isoform protein was significantly increased in the patient group with a higher OLGIM stage compared to the patient group with a lower OLGIM stage, and it was confirmed that it can be used for predicting the possibility of gastric cancer development.

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[0087] In the present invention, the "agent for measuring the level of mRNA" means an agent used in a method for measuring the level of mRNA transcribed from the gene in order to confirm the presence or absence of the expression of the gene of the present invention contained in a sample.

[0088] In the present invention, the agent for measuring the mRNA level of the gene can be a primer or a probe that specifically binds to the gene or mRNA, but is not limited thereto.

[0089] In the present invention, a "primer" is a short single-strand oligonucleotide that acts as a starting point for DNA synthesis. The primer specifically binds to a polynucleotide as a template under appropriate buffer and temperature conditions, and DNA is synthesized by adding nucleoside triphosphates having bases complementary to the template DNA to the primer and ligating them by a DNA polymerase. A primer generally consists of 15 to 30 base sequences, and the melting temperature (Tm) at which it binds to the template strand varies depending on the base composition and length. The sequence of the primer does not necessarily have to have a sequence completely complementary to a part of the base sequence of the template, and it is sufficient if it has a length and complementarity suitable for the purpose of amplifying a specific section of mRNA or cDNA and measuring the amount of mRNA through DNA synthesis. Therefore, in the present invention, a primer pair can be easily designed by referring to the base sequence of the cDNA or genomic DNA of the gene or its mRNA. The primer for the amplification reaction is composed of a set (pair) that binds complementarily to the opposite sides (antisense) of the template (or sense) at both ends of a specific section of the mRNA to be amplified, respectively.

[0090] In the present invention, a "probe" means a fragment of polynucleotide such as RNA or DNA having a length of several to several hundred base pairs that can specifically bind to mRNA, cDNA (complementary DNA), DNA, etc. of a specific gene. Since it is labeled, the presence or absence and expression level of the target mRNA or cDNA to which it binds can be confirmed. The selection of the probe and hybridization conditions can be appropriately selected by techniques known in the art. The probe can be used in a diagnostic method for detecting alleles (or allelic genes, alleles), etc. The diagnostic method includes various detection methods based on nucleic acid hybridization such as Southern blot, and may be provided in a form pre-bound to the substrate of a DNA chip in a method using a DNA chip.

[0091] In the present invention, a primer or a probe can be chemically synthesized using a phosphoramidite solid support synthesis method or other widely known methods. Also, a primer or a probe can be variously modified by methods known in the art as long as it does not interfere with hybridization with the target polynucleotide to be detected. Examples of such modifications include methylation, capping, substitution of one or more natural nucleotides with homologs, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.), and the binding of a labeling material using fluorescence or an enzyme.

[0092] In the present invention, the primer or probe is not limited to a specific sequence as long as it can detect the gene or its mRNA.

[0093] In the present invention, the agent for measuring the protein level can be, but is not limited to, an antibody or an aptamer specific to the protein.

[0094] In the present invention, the "aptamer" refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable three-dimensional structure by itself and can bind to a target molecule with high affinity and specificity. Aptamers for various desired target substances (proteins, sugars, dyes, DNA, metal ions, cells, etc.) can be developed by a method called SELEX (Systematic Evolution of Ligands of Exponential enrichment).

[0095] In the present invention, the "antibody" refers to a protein molecule that is directed against an antigenic site and specifically binds thereto. Antibodies can be produced by various methods commonly practiced in the art, such as fusion methods, recombinant DNA methods, or phage antibody library methods. In some embodiments, the antibody or antibody fragment can be derived from different organisms, including humans, mice, rats, hamsters, rabbits, or camels, etc., and can be, for example, monoclonal or polyclonal antibodies, immunologically active fragments, antibody heavy chains, humanized antibodies, antibody light chains, genetically engineered single-chain Fv molecules, or chimeric antibodies, etc. In the present invention, the antibody is not limited to a specific type of antibody as long as it can detect the protein of the present invention.

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[0097] In the present invention, "gastric cancer" is a general term for cancers that occur in the stomach. Gastric adenocarcinoma, which accounts for most of gastric cancers, originates from glandular cells of the gastric mucosa and can be further divided into many types according to the shape observed under a microscope. In addition, all of lymphoma that occurs in lymphoid tissue, epileptogenic tumors that occur in the nerves and muscle tissues of the stomach, sarcomas (malignant tumors derived from non-epithelial tissues), and neuroendocrine cancers that secrete hormones, etc. can be included in gastric cancer.

[0098] In the present invention, the gastric cancer can include all primary or metachronous gastric cancers. As a result, not only can the occurrence of gastric cancer in normal people with a family history or risk factors be diagnosed and predicted, but also metachronous gastric cancer can be predicted.

[0099] In the present invention, "metachronous gastric cancer (MGC)" means gastric cancer discovered 12 months after the initial diagnosis of gastric cancer. More specifically, it can mean gastric cancer newly occurring at a site other than the original treatment site during follow-up observation after treating early gastric cancer with endoscopic submucosal dissection, but is not limited thereto, and is a concept that includes all gastric cancers newly occurring 12 months after the first diagnosis of gastric cancer.

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[0101] The present invention also provides a composition for predicting the prognosis of a patient with early gastric cancer treated by endoscopic submucosal dissection, which includes a preparation for measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2, or the protein expressed therefrom.

[0102] In the present invention, "endoscopic submucosal dissection" means a procedure for removing gastric polyps, adenomas, and early gastric cancer without lymph node metastasis using an endoscopic device.

[0103] In one embodiment of the present invention, for patients with early gastric cancer treated by endoscopic submucosal dissection, the possibility of developing metachronous gastric cancer was predicted by confirming the expression of CDK1, KDF1, CREB5, or AKT2, and through this, it was confirmed that the prognosis of patients with early gastric cancer can be predicted after treatment by endoscopic submucosal dissection.

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[0105] The present invention also provides a use for predicting the possibility of gastric cancer development of a composition containing an agent for measuring the level of mRNA of any one or more genes selected from the group consisting of CREB5 and AKT2, or a protein expressed therefrom.

[0106] The present invention also provides a use of an agent for measuring the level of mRNA of any one or more genes selected from the group consisting of CREB5 and AKT2, or a protein expressed therefrom, for manufacturing a preparation for predicting the possibility of gastric cancer development.

[0107] The present invention also provides a use for predicting the possibility of metachronous gastric cancer development of a composition containing an agent for measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2, or a protein expressed therefrom.

[0108] The present invention also provides a use of an agent for measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2, or a protein expressed therefrom, for manufacturing a preparation for predicting the possibility of metachronous gastric cancer development.

[0109] The present invention also provides a use for predicting the prognosis of early gastric cancer patients treated with endoscopic submucosal dissection of a composition containing an agent for measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2, or a protein expressed therefrom.

[0110] The present invention also provides a use of an agent for measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2, or a protein expressed therefrom, for manufacturing a preparation for predicting the prognosis of early gastric cancer patients treated with endoscopic submucosal dissection.

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[0112] The present invention also provides a kit for predicting the onset of gastric cancer or metachronous gastric cancer, which contains the above composition.

[0113] In the present invention, the "kit" means a tool that can predict the onset possibility of gastric cancer or metachronous gastric cancer by including a preparation for measuring the mRNA or protein of any one or more genes selected from the group consisting of the above CDK1, KDF1, CREB5, and AKT2. In the present invention, the kit may be one or more selected from the group consisting of a reverse transcription polymerase chain reaction (RT-PCR) kit, a real-time polymerase chain reaction (qRT-PCR) kit, a DNA chip kit, an ELISA (Enzyme-linked immunosorbent assay) kit, and a protein chip kit, but is not limited thereto.

[0114] In addition to the preparation for measuring the above mRNA or protein, the kit of the present invention may include other constituent components, compositions, solutions, devices, etc. that are usually necessary for these detection methods. At this time, the substance for detecting the expression level of the above mRNA or protein can act one or more times without limitation on the number of times, and there is no limitation on the order of application of each substance, and they can be applied simultaneously or at different times.

[0115] Specifically, the kit for measuring the mRNA expression level of the above gene of the present invention can be a kit containing essential elements necessary for performing RT-PCR. In addition to each specific primer pair for the above gene, the RT-PCR kit can include a test tube or other appropriate container, reaction buffers (with various pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitor, DEPC water (DEPC-water), sterile water, etc. In addition, it can include a primer pair specific for the gene used as a quantitative control group.

[0116] In addition, the kit of the present invention can include essential elements necessary for performing DNA chip analysis. The DNA chip analysis kit can include a substrate to which cDNA corresponding to a gene or a fragment thereof is attached as a probe, and reagents, preparations, enzymes, etc. for producing a fluorescently labeled probe. Further, the substrate can include cDNA corresponding to a gene for a quantitative control group or a fragment thereof.

[0117] Together, the kit of the present invention can be a protein chip analysis kit for measuring the level of a protein encoded by the gene. The kit can include, but is not particularly limited thereto, a substrate for immunological detection of an antibody, an appropriate buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, etc. The substrate can be, but is not particularly limited thereto, a nitrocellulose membrane, a 96-well plate synthesized from a polyvinyl resin, a 96-well plate synthesized from a polystyrene resin, a slide glass made of glass, etc. The chromogenic enzyme can be, but is not particularly limited thereto, peroxidase or Alkaline Phosphatase. The fluorescent substance can be, but is not particularly limited thereto, FITC, RITC, etc. The chromogenic substrate solution can be, but is not particularly limited thereto, ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) or OPD (o-phenylenediamine), TMB (tetramethylbenzidine).

[0118] In the present invention, the kit can include a container; an instruction manual; and a detection preparation for the mRNA or protein. The container can serve to package the detection preparation and can also serve to store and fix it. The material of the container can take forms such as, for example, bottles, tubs, sachets, envelopes, tubes, ampoules, etc., and these can be formed, in part or in whole, from plastic, glass, paper, foil, wax, etc. The container can be equipped with a stopper that can be completely or partially separable and that was initially part of the container or can be attached to the container by mechanical, adhesive or other means, and a stopper that allows access to the contents by an injection needle can also be attached. The kit can include an outer package, and the outer package can include an instruction manual regarding the use of each component.

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[0120] In addition, the present invention provides a method for providing information for predicting the likelihood of gastric cancer development or a method for predicting the likelihood of gastric cancer development, including: (a) measuring the level of mRNA or protein expressed therefrom of any one or more genes selected from the group consisting of CREB5 and AKT2 in a biological sample separated from a subject; and

[0121] (b) comparing the measured mRNA or protein level with the level measured in a biological sample separated from a control group.

[0122] In the present invention, the gastric cancer can include metachronous gastric cancer, and the method can further include, but is not limited to, measuring the level of mRNA or protein expressed therefrom of any one or more genes selected from the group consisting of CDK1 and KDF1 in a biological sample separated from a subject and comparing this with the level measured in a sample separated from a control group.

[0123] In addition, the present invention provides a method for providing information for predicting the possibility of metachronous gastric cancer development, or a method for predicting the possibility of metachronous gastric cancer development, comprising: (a) measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2, or the protein expressed therefrom, in a biological sample isolated from a subject; and

[0124] (b) comparing the measured mRNA or protein level with the level measured in a biological sample isolated from a control group.

[0125] In addition, the present invention provides a method for providing information for predicting the prognosis after endoscopic submucosal dissection treatment in patients with early gastric cancer, or a method for predicting the prognosis after endoscopic submucosal dissection treatment in patients with early gastric cancer, comprising: (a) measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2, or the protein expressed therefrom, in a biological sample isolated from a patient with early gastric cancer; and

[0126] (b) comparing the measured mRNA or protein level with the level measured in a biological sample isolated from a control group.

[0127] In the present invention, the mRNA level of the gene can be measured by, but not limited to, one or more methods selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (competitive RT-PCR), real-time reverse transcription polymerase chain reaction (real time quantitative RT-PCR), multiplex reverse transcription polymerase chain reaction (Multi-plex PCR), real-time polymerase chain reaction (qRT-PCR), RNase protection assay, Northern blotting, DNA chip technology assay, methylated DNA binding domain sequencing (MBD-seq) analysis method, and reduced representation bisulfite sequencing (RRBS) analysis method.

[0128] In the present invention, the composition can analyze the expression levels of four genes at once through multiplex reverse transcriptase polymerase reaction (Multi-plex PCR), but is not limited thereto.

[0129] In the present invention, the protein level can be measured by, but not limited to, one or more methods selected from the group consisting of Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry (IHC), immunoprecipitation assay, complement fixation assay, FACS (Fluorescence Activated Cell Sorter), and protein chip.

[0130] In the present invention, the mRNA level or protein level of the gene can be measured in one biological sample, but is not limited thereto.

[0131] In the present invention, the biological sample means any sample capable of confirming the presence or absence or expression level of the mRNA or protein of CDK1, KDF1, CREB5, or AKT2 in the body, and can be, for example, one or more selected from the group consisting of tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, urine, and feces separated from a subject, but is not limited thereto. Specifically, the tissue can be gastric mucosa (GM) tissue or intestinal metaplasia (IM) tissue, but is not limited thereto.

[0132] In the present invention, the method for providing information for predicting the onset possibility of the gastric cancer or metachronous gastric cancer may further include, but is not limited thereto, step (c) of determining that the risk of onset of gastric cancer or metachronous gastric cancer is high when the level of the mRNA or protein measured in the biological sample separated from the subject is higher than the level measured in the biological sample separated from the control group.

[0133] In the present invention, the method for providing information for predicting the prognosis after endoscopic submucosal dissection treatment in the early gastric cancer patient may further include, but is not limited thereto, step (c) of predicting that the prognosis after endoscopic submucosal dissection treatment is poor when the level of the mRNA or protein measured in the biological sample separated from the early gastric cancer patient is higher than the level measured in the biological sample separated from the control group. At this time, the biological sample can be the gastric mucosa tissue around the tumor in the early gastric cancer patient, and can be separated regardless of before, after, or the elapsed time of the endoscopic submucosal dissection treatment, but is not limited thereto.

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[0135] In addition, the present invention includes the step of manufacturing a preparation for measuring the level of mRNA of any one or more genes selected from the group consisting of CREB5 and AKT2, or the protein expressed therefrom;

[0136] (b) the step of measuring the level of mRNA of any one or more genes selected from the group consisting of CREB5 and AKT2, or the protein expressed therefrom, in a biological sample isolated from a subject using the preparation; and

[0137] (c) the step of comparing the measured mRNA or protein level with the level measured in a biological sample isolated from a control group; and provides a method for predicting the likelihood of developing gastric cancer.

[0138] In addition, the present invention includes the step of manufacturing a preparation for measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2, or the protein expressed therefrom;

[0139] (b) the step of measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2, or the protein expressed therefrom, in a biological sample isolated from a subject using the preparation; and

[0140] (c) the step of comparing the measured mRNA or protein level with the level measured in a biological sample isolated from a control group; and provides a method for predicting the likelihood of developing metachronous gastric cancer.

[0141] In addition, the present invention includes the step of manufacturing a preparation for measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2, or the protein expressed therefrom;

[0142] (b) Measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2 or the protein expressed therefrom in a biological sample isolated from a patient with early gastric cancer using the preparation; and

[0143] (c) Comparing the measured mRNA or protein level with the level measured in a biological sample isolated from a control group; A method for predicting the prognosis after endoscopic submucosal dissection treatment in patients with early gastric cancer is provided.

[0144] In the present invention, the preparation for measuring the level of mRNA of the gene or the protein expressed therefrom may be a primer or probe that specifically binds to the gene or mRNA; or an antibody or aptamer specific to the protein, but is not limited thereto.

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[0146] In the present invention, "prediction" may mean, for the purpose of the present invention, confirming the possibility or risk of developing gastric cancer or metachronous gastric cancer, or confirming the prognosis after endoscopic submucosal dissection treatment in patients with early gastric cancer.

[0147] In the present invention, "subject" means a subject who has developed gastric cancer or recurrence of gastric cancer, has been diagnosed with early gastric cancer, and intends to predict the possibility of developing metachronous gastric cancer or prognosis before or after endoscopic submucosal dissection treatment. In the present invention, subjects or individuals include, without limitation, animals that can develop gastric cancer, such as humans, dogs, horses, cows, mice, goats, rabbits, chickens, ducks, geese, etc.

[0148] In the present invention, the "control group" means all individuals not diagnosed with gastric cancer and individuals diagnosed with early gastric cancer who have not developed metachronous gastric cancer after endoscopic submucosal dissection. According to the present invention, by comparing a sample separated from an individual not diagnosed with gastric cancer with a sample separated from a subject for which the possibility of developing gastric cancer is to be predicted, the possibility of developing gastric cancer in the subject can be predicted. Also, by comparing a sample separated from an individual who has not developed metachronous gastric cancer after endoscopic submucosal dissection with a sample separated from a subject for which the possibility of developing metachronous gastric cancer is to be predicted, the possibility of developing metachronous gastric cancer in the subject can be predicted.

[0149] The method for providing information for predicting the possibility of developing gastric cancer according to the present invention can measure the gene expression levels with a single sample by collecting a single sample, instead of the existing method of collecting a plurality of samples from an individual for measuring the expression levels of the four genes described above, measures the gene expression levels using multiplex reverse transcriptase polymerase reaction, and enables the diagnosis and prediction of gastric cancer with only one measurement.

[0150] In addition, the method can be usefully used not only for predicting metachronous gastric cancer, but also for predicting the risk of developing gastric cancer in normal individuals.

[0151] In the present invention, when the term "comprising" is used, this means that other components can be further included, rather than excluding other components, unless otherwise stated to the contrary. The terms "step of ~(doing)" or "step of ~" used throughout the present invention do not mean "step for ~".

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[0153] Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited only to these examples.

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[0155] [Example]

Example

[0156] Example 1. Selection of candidate genes for predicting gastric cancer diagnosis and occurrence

[0157] In patients with early gastric cancer treated by endoscopic submucosal dissection, in order to search for biomarkers for predicting the occurrence of gastric cancer through genomic analysis of gastric mucosal tissue, normal gastric mucosal tissue at the time of diagnosis in patients with early gastric cancer treated by endoscopic submucosal dissection was obtained, and 23 subjects in whom metachronous gastric cancer occurred during follow-up observation and 23 subjects in whom no new gastric cancer or gastric adenoma occurred during a follow-up observation period of 3 years or more were selected from the patients stored in an ultra-low temperature freezer.

[0158] For the selected subjects, RNA sequencing was performed on the normal gastric mucosal tissue stored in an ultra-low temperature freezer at the time of diagnosis, and differentially expressed genes (DEGs) were identified.

[0159] In addition, correlation analysis was performed based on gene expression levels, the number of metachronous gastric cancers, the number of adenomas, atrophic gastritis, and the degree of intestinal metaplasia, and candidate genes were selected by performing self-organizing map (SOM) analysis and homology analysis.

[0160] On the other hand, whole exome sequencing (WES) was performed on tumor tissues using formalin-fixed paraffin-embedded endoscopic submucosal dissection tissues of the subjects. Real-time polymerase chain reaction (qRT-PCR) was performed on various gastric cancer cell lines and normal gastric mucosal cell lines for the candidate genes, and genes showing significant differences in gastric cancer cell lines were selected when compared with normal gastric mucosal cell lines. The difference in cell viability was evaluated by treating the selected genes with siRNA in gastric cancer cell lines.

[0161] For effectiveness evaluation, independently of the 46 individuals, an effectiveness evaluation set consisting of 50 subjects with metachronous gastric cancer and 100 subjects without metachronous gastric cancer after endoscopic submucosal dissection was constructed, and real-time qPCR, Western blot, and immunohistochemistry were performed on the candidate genes for comparison.

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[0163] For the selected subjects, a tissue examination was performed at the time of diagnosis, RNA sequencing was performed on normal gastric mucosal tissue stored in an ultra-low temperature freezer, and differentially expressed genes (DEGs) were identified using DESeq2. A batch effect caused by Helicobacter pylori was confirmed during the analysis process, and batch correction was performed.

[0164] As a result, a total of 24 DEGs were identified, 9 in the mGC (subjects with metachronous gastric cancer after endoscopic submucosal dissection) group and 15 in the control (subjects without metachronous gastric cancer after endoscopic submucosal dissection) group, with significantly increased expression (Figure 1a).

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[0166] In addition, after examining the clinical information of the target patient group, correlation analysis was performed between the number of metachronous gastric cancers, the number of adenomas, neoplasms (gastric cancer + adenoma), the degree of atrophic gastritis (OLGA stage), the degree of intestinal metaplasia (OLGMI stage), and the expression levels of each gene identified using DESeq2. Self-organizing map (SOM) and alternative splicing analysis were performed.

[0167] As a result, the SOM analysis did not show a significant difference in mGC, but a common significant association was confirmed in unit V7 containing CREB5 when analyzed by the OLGIM stage and MLH-1 IHC (Figure 1c).

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[0169] In the analysis of isoform proteins confirmed by alternative splicing, there was no significant difference in isoforms between the mGC group and the control group. However, when analyzing the OLGA stage and the OLGIM stage, significant differences were shown in the isoforms of AKT2 (Figure 1d) and KDM5C. At this time, there was a significant difference in expression depending on the OLGIM stage, and the cDNA sequence (SEQ ID NO: 7) and amino acid sequence (SEQ ID NO: 8) of the AKT2 isoform set as the target are shown in Figure 1e.

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[0171] Based on the results of bulk RNA sequencing analysis performed on normal gastric mucosal tissues around the tumor, the results of correlation analysis, self-organizing maps, isoform analysis, etc. additionally performed based on the analysis results, and the functions and reporting frequencies of each gene confirmed in previous studies, several candidate genes were selected as CDK1, KDF1, ERCC6L, KLK8, MCM2, POLR2L, PTN, CREB5, and the AKT2 isoform (Figure 2).

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[0173] Example 2. Selection of target genes and confirmation of protein expression by confirming the expression levels of candidate genes in gastric cancer cell lines

[0174] 2-1. Confirmation of gene expression by qRT-PCR

[0175] Regarding the candidate genes selected through RNA sequencing performed on normal gastric mucosal tissues around the tumor and the candidate genes selected by WES of the tumor, the gene expression levels in various gastric cancer cell lines (intestinal type: SNU216, MKN1, AGS, diffuse type: SNU1, SNU5, SNU16, SNU488, SNU601, SNU620, SNU638, MKN45) and normal gastric mucosal cell line (HFE145) were compared by real-time polymerase chain reaction (qRT-PCR).

[0176] For the selected genes, siRNA was used to treat gastric cancer cell lines (0 nM, 25 nM, 50 nM) to confirm the suppression of gene expression. The inhibitory effect on cell viability was evaluated and compared in the siRNA-treated gastric cancer cell lines. Using correlation analysis, three target genes (CDK1, KDF1, POLR2L) with high expression in the mGC group, including CDK1, whose expression was confirmed to be higher in the mGC group than in the control group (Figure 1b), were selected (Figure 1a, Figure 1b, and Figure 3). Subsequently, among the total of five genes (CREB5, AKT2 isoform, CDK1, KDF1, POLR2L) in Figures 1a to 1e and Figure 3, CDK1, KDF1, CREB5, and AKT2 isoform, excluding POLR2L with a small expression difference between the control and mGC, were selected as the four final target genes (Figure 4a).

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[0178] 2-2. Confirmation of CDK1 and KDF1 Protein Expression by Western Blot

[0179] Proteins were separated from the tissues of some patients in the evaluation set used in Figure 4a, and the protein expression levels of CDK1 and KDF1, which showed high expression in the mGC group, were confirmed by Western blot.

[0180] After endoscopic submucosal dissection, five subjects with metachronous gastric cancer and five subjects without metachronous gastric cancer were randomly selected, and the protein expression of CDK1 was confirmed. As a result, it was confirmed that the expression of CDK1 was higher in the tissues of subjects with metachronous gastric cancer than in the tissues of subjects without metachronous gastric cancer (upper part of Figure 4b).

[0181] In addition, eight patients were randomly selected from each group, and the protein expression of KDF1 was confirmed. As a result, similar to CDK1, it was confirmed that higher KDF1 expression appeared in the tissues of subjects with metachronous gastric cancer than in the tissues of subjects without metachronous gastric cancer (lower end of Fig. 4b).

[0182] The above results were the same as those obtained by confirming the mRNA expression level through previous qPCR, indicating that both CDK1 and KDF1 were more highly expressed in subjects with metachronous gastric cancer.

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[0184] 2-3. Confirmation of CDK1 and KDF1 protein expression through immunohistochemistry (IHC)

[0185] The expression of the candidate genes CDK1 and KDF1 previously selected in the pathological slides of some of the patients in the evaluation set used in Fig. 4a was confirmed using immunohistochemistry.

[0186] After the treatment of endoscopic submucosal dissection, two subjects with metachronous gastric cancer and two subjects without metachronous gastric cancer were randomly selected, and the protein expression of CDK1 (Fig. 4c) and KDF1 (Fig. 4d) was confirmed. As a result, it was confirmed that both of the two candidate genes were more strongly stained in the tissues of subjects with metachronous gastric cancer than in the tissues of subjects without metachronous gastric cancer (Fig. 4c and Fig. 4d). This means that the proteins of CDK1 and KDF1 are more abundantly present in the tissues of subjects with metachronous gastric cancer, which is the same result as the qPCR in Example 2-1 and the Western blot results in Example 2-2.

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[0188] Example 3. Evaluation of target gene effectiveness

[0189] To evaluate the effectiveness of the target genes selected in Examples 1 and 2, an effectiveness evaluation set independent of the 46 subjects in the target patient group set for candidate gene selection was constructed. This set consisted of 50 subjects with metachronous gastric cancer and 100 subjects without metachronous gastric cancer after endoscopic submucosal dissection, and of course, they were selected with matching of age, gender, and H. pylori infection status.

[0190] For the four genes (CDK1, KDF1, CREB5, and AKT2 isoform) selected in Example 2, ROC curves were drawn to determine the accuracy of gastric cancer prediction in the patient group (Figures 5a to 5d), and as a result, the gene combinations with the highest accuracy are shown in Table 1 below and Figure 5c.

[0191]

Table 1

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[0193] As shown in the results of Table 1 above, for the analysis results of the CDK1 ROC curve, the accuracy was AUC 0.829, for the analysis results of the KDF1 ROC curve, the accuracy was AUC 0.841, and the AUC of the AKT2 isoform ROC curve was 0.771, so it was confirmed that the accuracy of each gene for gastric cancer prediction was high.

[0194] Also, while measuring the expression levels in combination with the above CDK1, KDF1, and AKT2 isoform and analyzing the ROC curve, it was confirmed that the accuracy increased significantly to AUC 0.890, and it was confirmed that the gastric cancer prediction accuracy increased when using the combination of the four genes compared to when using each of the four genes alone.

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[0196] Next, to confirm whether CREB5 and AKT2, which show differential expression according to the OLGIM (Operative Link on Gastritis Assessment based on Intestinal Metaplasia) stage, also show the same results in a 150-person evaluation set, RNA was extracted and qPCR was performed. In the case of the OLGIM stage, the degree and range of atrophy are evaluated and shown divided into a total of 5 stages (stage 0-IV), and through many studies, it has been reported that, together with the OLGA stage, it is often used as an indicator for predicting the possibility of gastric cancer development.

[0197] First, in the case of CREB5, it was confirmed that as the OLGIM stage increases, the expression of CREB5 becomes stronger (Figure 6). That is, in the OLGIM system based on the evaluation of intestinal metaplasia, it was recognized that the expression level of CREB5 significantly increases at stages 3 and 4, which are high-risk groups for gastric cancer development, and it was confirmed that gastric cancer can be predicted.

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[0199] Also, as a result of analyzing alternative splicing, the amount of RNA of AKT2 that is expressed as a whole did not show a large correlation with the OLGIM stage, but since it was confirmed that the expression of a specific isoform shows a large difference according to the OLGIM stage (Figure 1d), thereafter, the experiment was conducted by dividing the patients in the evaluation set samples they had into a group with a high OLGIM stage and a group with a low OLGIM stage.

[0200] As a result of confirming the expression of a specific isoform protein of AKT2 previously confirmed in each group through qPCR, it was confirmed that the expression of a specific isoform of AKT2 is considerably higher in the group of patients with a high OLGIM stage than in the group of patients with a low OLGIM stage (Figure 7).

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[0202] From the above results, the usability of CDK1, KDF1, CREB5, and AKT2 isoform as predictors of gastric cancer onset can be confirmed. The increased expression of each of these genes can be used not only as a predictive marker for the occurrence of metachronous gastric cancer or primary gastric cancer, but also the prediction of gastric cancer onset according to the present invention can be confirmed with a single tissue specimen, and it is possible to predict the onset possibility of gastric cancer by a single measurement method using multiplex reverse transcription polymerase chain reaction (multi-plex PCR) or the like. Therefore, it has a technical advantage in terms of high usability. In addition, since it includes a method capable of quantifying basal gastritis by a quantitative method that can predict not only metachronous gastric cancer but also gastric cancer onset in normal individuals, it simultaneously provides a method capable of predicting the risk of gastric cancer occurrence in normal individuals with a family history or risk factors of gastric cancer. Additionally, by predicting the occurrence of metachronous gastric cancer in early gastric cancer patients treated by endoscopic submucosal dissection, the prognosis after treatment of endoscopic submucosal dissection in patients can be predicted.

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Industrial Applicability

[0204] Any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2 according to the present invention are expected to be usefully utilized for predicting the onset possibility of gastric cancer or metachronous gastric cancer, or predicting the prognosis in early gastric cancer patients treated by endoscopic submucosal dissection, and thus have industrial applicability.

Claims

1. A composition for predicting the possibility of gastric cancer development, comprising a preparation for measuring the level of mRNA of any one or more genes selected from the group consisting of CREB5 and AKT2, or the protein expressed therefrom.

2. The composition for predicting the possibility of gastric cancer development according to claim 1, wherein the gastric cancer includes metachronous gastric cancer.

3. The composition for predicting the possibility of gastric cancer development according to claim 2, wherein the composition further comprises a preparation for measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1 and KDF1, or the protein expressed therefrom.

4. The composition for predicting the possibility of gastric cancer development according to claim 1, wherein the preparation for measuring the mRNA level of the gene is a primer or probe that specifically binds to the gene or mRNA.

5. The composition for predicting the possibility of gastric cancer development according to claim 1, wherein the preparation for measuring the protein level is an antibody or aptamer specific to the protein.

6. A kit for predicting the possibility of gastric cancer development, comprising the composition according to any one of claims 1 to 5.

7. The kit for predicting the possibility of gastric cancer development according to claim 6, wherein the kit is one or more selected from the group consisting of a reverse transcription polymerase chain reaction (RT-PCR) kit, a real-time polymerase chain reaction (qRT-PCR) kit, a DNA chip kit, an ELISA (Enzyme-linked immunosorbent assay) kit, and a protein chip kit.

8. A composition for predicting the possibility of metachronous gastric cancer development, comprising a preparation for measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2, or the protein expressed therefrom.

9. A composition for predicting the prognosis of a patient with early gastric cancer treated by endoscopic submucosal dissection, comprising a preparation for measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2, or the protein expressed therefrom.

10. (a) Measuring the level of mRNA of any one or more genes selected from the group consisting of CREB5 and AKT2, or the protein expressed therefrom, in a biological sample isolated from a subject; and (b) Comparing the measured mRNA or protein level with the level measured in a biological sample isolated from a control group; A method for providing information for predicting the possibility of developing gastric cancer, comprising the steps of:

11. The method for providing information for predicting the possibility of developing gastric cancer according to claim 10, wherein the gastric cancer includes metachronous gastric cancer.

12. The method further comprises measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1 and KDF1, or the protein expressed therefrom, in a biological sample isolated from a subject, and comparing this with the level measured in a sample isolated from a control group. The method for providing information for predicting the possibility of developing gastric cancer according to claim 11.

13. The mRNA level of the gene is measured by one or more methods selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (competitive RT-PCR), real-time reverse transcription polymerase chain reaction (real time quantitative RT-PCR), multiplex reverse transcription polymerase chain reaction (Multiplex PCR), real-time polymerase chain reaction (qRT-PCR), RNase protection assay, Northern blotting, DNA chip technology assay, methylated DNA binding domain sequencing (MBD-seq: Methylated DNA binding domain sequencing) analysis method, and reduced representation bisulfite sequencing (RRBS: Reduced representation bisulfite sequencing) analysis method, The method for providing information for predicting the onset possibility of gastric cancer according to claim 10, characterized in that it is measured by the above method.

14. The protein level is measured by one or more methods selected from the group consisting of Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA: Radioimmunoassay), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry (Immunohistochemistry, IHC), immunoprecipitation assay, complement fixation assay, FACS (Fluorescence Activated Cell Sorter), and protein chip. The method for providing information for predicting the onset possibility of gastric cancer according to claim 10, characterized in that it is measured by the above method.

15. The mRNA level or protein level of the gene is measured in one biological sample. The method for providing information for predicting the onset possibility of gastric cancer according to claim 10.

16. The biological sample is one or more selected from the group consisting of tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, urine, and feces separated from a subject, and is characterized in that, the method for providing information for predicting the onset possibility of gastric cancer according to claim 10.

17. (c) Further including the step of determining that the risk of gastric cancer onset is high when the level of the mRNA or protein measured in the biological sample separated from the subject is higher than the level measured in the biological sample separated from the control group, and is characterized in that, the method for providing information for predicting the onset possibility of gastric cancer according to any one of claims 10 to 16.

18. (a) Measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2 or the protein expressed therefrom in a biological sample separated from a subject; and (b) Comparing the measured mRNA or protein level with the level measured in a biological sample separated from a control group; and including the method for providing information for predicting the onset possibility of metachronous gastric cancer.

19. (a) Measuring the level of mRNA of any one or more genes selected from the group consisting of CDK1, KDF1, CREB5, and AKT2 or the protein expressed therefrom in a biological sample separated from a patient with early gastric cancer; and (b) Comparing the measured mRNA or protein level with the level measured in a biological sample separated from a control group; and including the method for providing information for predicting the prognosis after endoscopic submucosal dissection treatment in patients with early gastric cancer.

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