Method for diagnosing gastric cancer or predicting onset thereof

Abstract

The present invention relates to a method for diagnosing gastric cancer or predicting the onset thereof. The present invention also relates to a novel gene variant; a method for preparing a gastric cancer model comprising a genetic mutation; a gastric cancer model comprising the genetic mutation; a composition for diagnosing gastric cancer or predicting the onset thereof; a kit for diagnosing gastric cancer or predicting the onset thereof; and a method for screening a drug for treating or preventing gastric cancer. The method for diagnosing gastric cancer and predicting the onset thereof enables early diagnosis of gastric cancer or prediction and prevention of the risk of gastric cancer occurrence prior to disease onset, thereby significantly reducing the incidence and mortality of gastric cancer. In addition, by observing the presence or absence of genetic mutations or changes in frequency of genetic mutation in response to drug treatment, the method can be utilized for molecular mechanism studies for gastric cancer treatment or for the development of drug screening platforms.

Description

Methods for diagnosing or predicting the onset of stomach cancer

[0001] This application claims the benefit of priority based on Korean Patent Application No. 10-2024-0123434 filed on September 10, 2024, and all contents disclosed in the document of said Korean patent application are incorporated herein as part of this specification.

[0002] The present invention relates to a method for diagnosing or predicting the onset of gastric cancer. Furthermore, the present invention relates to a novel gene variant, a method for preparing a gastric cancer model comprising the gene variant, a gastric cancer model comprising the gene variant, a composition for diagnosing or predicting the onset of gastric cancer, a kit for diagnosing or predicting the onset of gastric cancer, and a method for screening drugs for treating or preventing gastric cancer.

[0003] Gastric cancer is one of the most common malignant cancers in Korea. According to the incidence status by cancer type from the National Cancer Information Center of Korea, the number of new gastric cancer cases in 2021 was approximately 30,000; notably, the number of male gastric cancer cases was about 20,000, ranking second among all malignant cancers. Furthermore, according to the cancer incidence and mortality status from Korea's e-National Indicators, the number of deaths from gastric cancer in 2021 was confirmed to reach approximately 7,000. Although the number of cases and deaths from gastric cancer is high, it is known that the cure rate is high when detected early. According to the announcement by the Central Cancer Registry of Korea, the 5-year relative survival rate for gastric cancer based on the staging classification of the U.S. National Cancer Institute was 97.4% for the localized stage (where the cancer has not spread beyond the stomach), 61.4% for the regional stage (where the cancer has invaded surrounding organs, tissues, or lymph nodes), and 6.6% for the distant stage (where the cancer has metastasized to other parts of the body), confirming the importance of early detection of gastric cancer. However, gastric cancer often presents no distinct symptoms in its early stages, and symptoms that appear as the disease progresses—such as upper abdominal discomfort, pain, and indigestion—are similar to those of gastric ulcers or gastritis. Furthermore, diagnosing gastric cancer remains challenging because the shape or location of the tumor—such as polyps smaller than 3mm—cannot be identified by endoscopy or ultrasound. Consequently, recent research has focused on precision medicine utilizing patients' genetic information, while there remains an unmet need for the discovery of new genetic mutations related to gastric cancer.

[0004] Accordingly, the inventors have made diligent research efforts to develop a method for diagnosing gastric cancer or efficiently and easily predicting its onset. As a result, the present invention was completed by identifying numerous mutations found in gastric adenomas or gastric cancers that have a high probability of progressing to gastric cancer.

[0005] Each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below.

[0006] Furthermore, terms not specifically defined in this specification should be understood to have the meanings commonly used in the technical field to which the present invention pertains. Additionally, unless specifically defined in the context, the singular includes the plural, and the plural includes the singular.

[0007]

[0008] The present invention provides a method for diagnosing or predicting the onset of gastric cancer, comprising the step of identifying one or more variants selected from the group consisting of the NOTCH2 gene, TSC2 gene, FGFR3 gene, ARID2 gene, CREBBP gene, MSH2 gene, and TET2 gene within a biological sample isolated from a subject.

[0009] The above method may further include the step of obtaining nucleic acid from a biological sample separated from a subject; or the step of determining that gastric cancer has developed or is highly likely to develop if the mutation is confirmed.

[0010] As used herein, the term “subject” includes a human or any non-human animal, and said non-human animal may be a vertebrate, such as a primate, dog, cattle, horse, pig, or rodent, such as a mouse, rat, guinea pig, etc. said subject may be a healthy subject without any disease, a subject at risk of developing gastric cancer such as a patient with gastric adenoma, or a subject that has developed gastric cancer. In this specification, said “subject” is used interchangeably with “individual.”

[0011] As used herein, the term "gastric adenoma" refers to a type of polyp that occurs in the stomach and can be classified into dysplastic, moderate dysplastic, and high-grade dysplastic depending on the degree of tumorimorphic changes in the cellular shape. Although gastric adenomas are asymptomatic, they are treated as a precancerous stage of gastric cancer due to their high potential to progress to gastric cancer. In this specification, the term "gastric adenoma" may be used interchangeably with the term "gastric adenoma."

[0012] As used herein, the term "gastric cancer" refers to a malignant tumor occurring in the stomach. The gastric cancer may be early gastric cancer, advanced or progressive gastric cancer, recurrent gastric cancer, or metastatic gastric cancer.

[0013] The term “early gastric cancer” as used in this specification refers to an early stage of gastric cancer in which the cancer is confined to the mucosa or submucosa and has not invaded deeply.

[0014] As used in this specification, the term "advanced, progressive gastric cancer" refers to gastric cancer in which the cancer has passed the submucosal layer of the stomach and penetrated beyond the muscle layer, and the cancer is not confined to the stomach.

[0015] As used in this specification, the term "recurring gastric cancer" refers to gastric cancer that has recurred after disappearing due to treatment or the like.

[0016] As used in this specification, the term "metastatic gastric cancer" refers to gastric cancer that has progressed and metastasized to other organs.

[0017] As used herein, the term "invasion" refers to the process in which cancer cells grow or expand and penetrate from a primary lesion into surrounding tissues. In this specification, the term "invasion" may be used interchangeably with the terms "infiltration" and "invasion."

[0018] As used herein, the term "biological sample" means a sample in which the gene of the present invention or a variant thereof may be detected. Anything that can be collected from a subject may be included without limitation and may be collected by methods commonly used in the technical field of the present invention. The biological sample may be, but is not limited to, various types of cells or organ secretions such as whole blood, serum, plasma, gastric fluid, ascites, pleural fluid, pericardial fluid, cerebrospinal fluid, lymph fluid, pancreatic fluid, semen, synovial fluid, urine, feces, saliva, sputum, cyst fluid, amniotic fluid, tears, mucus, tissue, circulating tumor cells (CTC); or nucleic acids obtained therefrom. The nucleic acids obtained from the above whole blood, serum, plasma, gastric fluid, ascites, etc. may be, for example, cell-free nucleic acids, bacterial nucleic acids, viral nucleic acids, fetal nucleic acids within the mother, hematopoietic nucleic acids, non-hematopoietic nucleic acids, tumor nucleic acids, non-tumor nucleic acids, etc. The above biological samples may be pretreated by methods commonly used in the art of the present invention. For example, filtration, centrifugation, extraction, distillation, dissociation, concentration, digestive fluid treatment, homogenization, removal or inactivation of interfering factors may be, but are not limited thereto.

[0019] As used herein, the term “nucleic acid” includes a series of naturally occurring nucleosides, nucleotide monomers or nucleotide polymers, or functional analogs thereof, and includes all that are ordinarily known in the art of the present invention. The nucleic acids may be, for example, DNA, RNA, oligonucleotides such as LNA (Locked nucleic acid) or BNA (Bridged nucleic acid), PNA (Peptide nucleic acid), etc., or fragments thereof.

[0020] As used in this specification, the term "variation" means a change in genetic traits resulting from an alteration of a base, nucleotide, polynucleotide, or nucleic acid in an existing base sequence. Such variation may include substitution, insertion, deletion, duplication, inversion, or translocation of one or more bases, nucleotides, polynucleotides, or nucleic acids. In this specification, the term "variation" may be used interchangeably with the term "mutation."

[0021] The term “Insertion” as used in this specification means that a base, nucleotide, polynucleotide, or nucleic acid is added to an existing base sequence to change the number of nucleic acids.

[0022] The term “Deletion” as used in this specification means that a base, nucleotide, polynucleotide, or nucleic acid of an existing base sequence is removed, thereby changing the number of nucleic acids.

[0023] The term "Duplication" as used in this specification means that a nucleotide sequence of a specific length appears repeated two or more times during the genetic recombination process.

[0024] The term "inversion" as used in this specification means that a part of a chromosome is combined by rotating 180º during the gene recombination process.

[0025] As used herein, the term "translocation" means the movement of a part of a specific chromosome to another chromosome.

[0026] The gene variant according to the present invention may be a single nucleotide variant (SNV), a deletion or insertion of one or more nucleotides, a copy number variant (CNV), or a tandem repeat polymorphism (TRP), but is not limited thereto.

[0027] As used herein, the term "single nucleotide variant (SNV)" means that one base, nucleotide, polynucleotide, or nucleic acid is changed to another base, nucleotide, polynucleotide, or nucleic acid. The single nucleotide variant includes point mutations such as nonsense mutations (chain termination mutations) and missense mutations, or single nucleotide polymorphisms (SNPs). The nonsense mutation refers to a mutation in which a stop codon is formed at a corresponding position due to a base change, and the missense mutation refers to a mutation in which the amino acid encoded by the base change is altered due to a base change. In this specification, the term "single nucleotide variant" may be used interchangeably with the term "single nucleotide substitution."

[0028] As used herein, the term "copy number variant (CNV)" refers to a variant region in which a gene sequence of length greater than 1 kb (kilobase) shows a difference in the number of repetitive sequences due to deletion, insertion, duplication, translocation, or translocation when comparing a reference sequence and sequencing reads. In this specification, the term "copy number variant" may be used interchangeably with the term "copy number variation."

[0029] The term “Tandem repeat polymorphism (TRP)” as used in this specification means that a nucleotide sequence is repeated multiple times. The tandem repeat includes short tandem repeats (STRs) in which a short nucleotide sequence of length 2 to 6 bp (Basepair) is repeated 3 to 100 times, and variation number of tandem repeats (VNTRs) in which a long nucleotide sequence of length 15 to 70 bp is repeated 5 to 100 times.

[0030] One or more variants selected from the group consisting of the above NOTCH2, TSC2, FGFR3, ARID2, CREBBP, MSH2, and TET2 genes are each gene variants that appear specifically at a statistically significant level compared to other gene variants in gastric adenoma and / or gastric cancer.

[0031] The above variation may have one or more of the following characteristics:

[0032] Substitution of guanine, the 710th base of the NOTCH2 gene, with adenine, cytosine, or thymine;

[0033] Substitution of cytosine, the 2816th base of the NOTCH2 gene, with thymine, adenine, or guanine;

[0034] Substitution of adenine, the 5065th base of the NOTCH2 gene, with thymine, guanine, or cytosine;

[0035] Substitution of adenine, the 856th base of the TSC2 gene, with guanine, cytosine, or thymine;

[0036] Substitution of guanine, the 2032nd base of the TSC2 gene, with adenine, cytosine, or thymine;

[0037] Substitution of cytosine, the 1349th base of the FGFR3 gene, with thymine, adenine, or guanine;

[0038] Substitution of adenine, the 1759th base of the ARID2 gene, with guanine, cytosine, or thymine;

[0039] Substitution of cytosine, the 1651st base of the CREBBP gene, with adenine, guanine, or thymine;

[0040] Substitution of cytosine, the 23rd base of the MSH2 gene, with thymine, adenine, or guanine;

[0041] Substitution of cytosine, the 1168th base of the MSH2 gene, with thymine, adenine, or guanine; and

[0042] The 3116th base of the TET2 gene, cytosine, is replaced with thymine, adenine, or guanine.

[0043] Preferably, the above variation may have one or more of the following features:

[0044] Substitution of guanine, the 710th base of the NOTCH2 gene, with adenine;

[0045] The 2816th base of the NOTCH2 gene, cytosine, is replaced with thymine;

[0046] Substitution of adenine, the 5065th base of the NOTCH2 gene, with thymine;

[0047] Substitution of adenine, the 856th base of the TSC2 gene, with guanine;

[0048] Substitution of guanine, the 2032nd base of the TSC2 gene, with adenine;

[0049] The 1349th base of the FGFR3 gene, cytosine, is replaced with thymine;

[0050] Substitution of adenine, the 1759th base of the ARID2 gene, with guanine;

[0051] The 1651st base of the CREBBP gene, cytosine, is replaced with adenine;

[0052] The 23rd base of the MSH2 gene, cytosine, is replaced with thymine;

[0053] Substitution of cytosine, the 1168th base of the MSH2 gene, with thymine; and

[0054] The 3116th base of the TET2 gene, cytosine, is replaced with thymine.

[0055] The above NOTCH2 gene may be represented by SEQ ID NO. 1, the above TSC2 gene by SEQ ID NO. 2, the above FGFR3 gene by SEQ ID NO. 3, the above ARID2 gene by SEQ ID NO. 4, the above CREBBP gene by SEQ ID NO. 5, the above MSH2 gene by SEQ ID NO. 6, and the above TET2 gene by SEQ ID NO. 7.

[0056] The above sequence number 1 refers to the 120457929 to 120612020th nucleotide sequence corresponding to the CDS region of the NOTCH2 gene on chromosome 1 of the Human reference genome GRCh37 / hg19.

[0057] The above sequence number 2 refers to the nucleotide sequences from 2098617 to 2138611 corresponding to the CDS region of the TSC2 gene on chromosome 16 of the Human reference genome GRCh37 / hg19.

[0058] The above sequence number 3 refers to the nucleotide sequences from 1795662 to 1809015 corresponding to the CDS region of the FGFR3 gene on chromosome 4 of the Human reference genome GRCh37 / hg19.

[0059] The above sequence number 4 refers to the nucleotide sequences from 46123620 to 46298861, corresponding to the CDS region of the ARID2 gene on chromosome 12 of the Human reference genome GRCh37 / hg19.

[0060] The above sequence number 5 refers to the nucleotide sequences from 3777719 to 3929917, corresponding to the CDS region of the CREBBP gene on chromosome 16 of the Human reference genome GRCh37 / hg19.

[0061] The above sequence number 6 refers to the nucleotide sequences from 47630331 to 47710088, corresponding to the CDS region of the MSH2 gene on chromosome 2 of the Human reference genome GRCh37 / hg19.

[0062] The above sequence number 7 refers to the nucleotide sequences from 106155100 to 106158597, corresponding to the CDS region of the TET2 gene on chromosome 4 of the Human reference genome GRCh37 / hg19.

[0063] The above sequence numbers 1 to 7 should each be interpreted as containing a sequence that is substantially identical. The sequence that is substantially identical may be a sequence corresponding to the CDS region of each of the above genes in the Human reference genome GRCh37 / hg19, and may be a sequence corresponding to the CDS region of each of the above genes in the Human reference genome GRCh38 / hg38. For example, sequence number 1 may be interpreted as containing nucleotide sequences from 119911553 to 120069662 corresponding to the CDS region of the NOTCH2 gene on chromosome 1 in the Human reference genome GRCh38.p14.

[0064] The gene mutation of the present invention may serve as a biomarker for gastric adenoma or gastric cancer. Therefore, by identifying the gene mutation of the present invention, it is possible to determine whether gastric adenoma or gastric cancer has developed, and based on this, gastric cancer can be diagnosed or its onset predicted. The NOTCH2 gene, TSC2 gene, or FGFR3 gene may serve as a biomarker for gastric adenoma, and the ARID2 gene, CREBBP gene, MSH2 gene, or TET2 gene may serve as a biomarker for gastric cancer. Since gastric adenoma is a disease with a high probability of progressing to gastric cancer, gastric cancer diagnosis or onset prediction can be performed by identifying the gene mutation of the present invention.

[0065] As used herein, the term "biomarker" refers to a substance that can be detected, its content measured, its expression level measured, or amplified in biological samples to distinguish the presence of gastric cancer for diagnosis or prediction of onset. For example, it includes all organic biomolecules capable of identifying biological changes, such as polypeptides, proteins, nucleic acids like DNA and RNA, lipids, glycolipids, glycoproteins, etc.

[0066] The above mutation can be identified using analytical methods such as polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), nuclease protection assay (NPA), Northern blot, Southern blot, next-generation sequencing (NGS), in situ hybridization, or microarray.

[0067] As used in this specification, the term "polymerase chain reaction (PCR)" refers to a technique for amplifying a specific region in large quantities using the DNA base sequence of a specific region as a template. The PCR proceeds in the stages of DNA denaturation, primer binding, and DNA synthesis. The PCR includes all types of PCR analysis methods, such as competitive polymerase chain reaction (competitive PCR), real-time polymerase chain reaction (quantitative PCR, qPCR), multiplex polymerase chain reaction (multiplex PCR), inverse polymerase chain reaction (inverse PCR), and digital polymerase chain reaction (dPCR).

[0068] As used herein, the term "competitive polymerase chain reaction (competitive PCR)" refers to a PCR analysis method that simultaneously and competitively amplifies a target DNA whose base sequence is to be determined and an artificial DNA from which the middle portion of the target DNA has been removed, and then quantitatively compares the PCR products.

[0069] The term “real-time polymerase chain reaction (real time PCR, quantitative PCR, qPCR)" as used in this specification refers to a PCR analysis method that measures the amount of PCR product in real time.

[0070] As used herein, the term "multiplex PCR" refers to a PCR analysis method capable of amplifying multiple different DNA base sequences in a single PCR reaction by using multiple primer pairs simultaneously.

[0071] The term “inverse polymerase chain reaction (inverse PCR)” as used in this specification refers to a method of analyzing an unknown sequence linked to the 5’ or 3’ of DNA whose sequence is already known.

[0072] As used in this specification, the term "Digital Polymerase Chain Reaction (Digital PCR)" refers to a PCR analysis method in which the amount of target DNA is measured by diluting the PCR reaction solution until there is approximately one target DNA, amplifying it, and then verifying the number of amplifications.

[0073] The term "reverse transcription polymerase chain reaction (RT-PCR)" as used in this specification refers to a technique of synthesizing cDNA (complementary DNA) using an RNA base sequence of a specific region as a template and then performing PCR. The RT-PCR proceeds through the steps of synthesizing cDNA from RNA using reverse transcriptase, denaturing cDNA, binding primers, and synthesizing cDNA. The RT-PCR includes all types of RT-PCR analysis methods, such as competitive reverse transcription polymerase chain reaction (competitive RT-PCR), real-time reverse transcription polymerase chain reaction (real-time RT-PCR, quantitative RT-PCR, qRT-PCR), multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR), inverse reverse transcription polymerase chain reaction (inverse RT-PCR), and digital reverse transcription polymerase chain reaction (dRT-PCR).

[0074] As used herein, the term "nuclease protection assay (NPA)" refers to an analytical method that detects and quantifies a target RNA among many RNAs through hybridization of a probe or similar substance with the target RNA. Unhybridized RNA or probes or similar substances are removed by a nuclease, and hybridized RNA-probes or similar substances can be separated by precipitation. The above-mentioned nuclease protection assay includes ribonuclease protection assays and S1 nuclease protection assays.

[0075] As used herein, the term "Northern blot" refers to an analytical method for detecting target RNA or quantifying target RNA by the steps of electrophoresing RNA, moving the electrophoresed RNA to a membrane filter, and hybridizing the RNA on the membrane filter with a probe. In this specification, the term "Northern blot" may be used interchangeably with the term "RNA blot."

[0076] As used herein, the term "Southern blot" refers to an analytical method for detecting target DNA or quantifying target RNA by means of the steps of electrophoresing DNA, transferring the electrophoresed DNA to a membrane filter, and hybridizing the DNA on the membrane filter with a probe. In this specification, the term "Southern blot" may be used interchangeably with the term "DNA blot."

[0077] As used herein, the term "Next generation sequencing (NGS)" refers to a technology for high-throughput sequencing of genomes through the massive parallel sequencing of clonally amplified molecules and single nucleic acid molecules. In other words, it is a technology capable of analyzing even small sample volumes and processing hundreds of thousands of reactions simultaneously. The NGS proceeds through the stages of clonal amplification, massively parallel sequencing, and cyclic sequencing.

[0078] The term "in situ hybridization" as used in this specification refers to an analytical method for detecting target DNA or RNA by hybridizing target DNA or RNA with a nucleic acid strand or probe that is complementary thereto, and can be applied to stored samples such as frozen tissues. The above-mentioned in situ hybridization includes fluorescence in situ hybridization (fluorescence in situ hybridization, fluorescence in situ hybridization, FISH), etc. In this specification, the term "in situ hybridization" may be used interchangeably with the terms "in situ hybridization," "in situ matching," "in situ hybridization method," "in situ hybridization," "in situ hybridization," or "in situ matching."

[0079] The term "microarray" as used in this specification refers to an analytical method for confirming DNA expression, DNA methylation, DNA mutations, etc. through hybridization of probes arranged on a chip with single-strand DNA. In this specification, the term "microarray" may be used interchangeably with the terms "DNA chip," "biochip," and "gene array."

[0080] As used herein, the term "diagnosis" means determining the presence or characteristics of gastric cancer or the susceptibility of a subject to gastric cancer, and includes judgment, monitoring, or any type of analysis for the occurrence, metastasis, or recurrence of gastric cancer, the severity of gastric cancer symptoms, the subject's drug responsiveness, drug resistance, or survival rate, and differences in therapeutic or preventive effects against gastric cancer. The above diagnosis does not mean determining the presence or absence of gastric cancer with 100% accuracy. The gastric cancer diagnosis of the present invention may be performed, for example, by identifying mutations in the ARID2 gene, CREBBP gene, MSH2 gene, and / or TET2 gene.

[0081] As used in this specification, the term "treatment" refers to any act in which the symptoms of gastric cancer improve or are completely cured through the administration of drugs, etc.

[0082] As used in this specification, the term "prevention" refers to any act of suppressing or delaying the symptoms of gastric cancer through the administration of drugs, etc.

[0083] As used herein, the term “administration” means the physical introduction of a drug into a subject using any of the various methods and delivery systems known to a person skilled in the art. The route of administration of the drug includes, for example, an oral administration route, or an intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as injection, infusion, or topical administration, but is not limited thereto. The number of administrations of the drug may be, for example, once, multiple times, and / or over one or more extended periods. The dosage of the drug may vary depending on the subject’s age, sex, body weight, route of administration, severity of symptoms, etc. Specifically, depending on the subject’s symptoms, the drug may be administered at a rate of 0.1 to 100 mg / kg once or several times daily, or at intervals of several days to several months.

[0084] As used herein, the term "prediction of onset" means predicting progression to gastric cancer or the likelihood (risk) of developing gastric cancer. This includes judging that the subject's course indicates a higher likelihood (risk) of developing gastric cancer than in cases where it does not, monitoring the subject's course, or any type of analysis for said judgment or monitoring. In the present invention, the occurrence of gastric cancer is predicted, for example, when a gastric adenoma is diagnosed by the gastric cancer diagnosis or onset prediction method according to the present invention. The prediction of gastric cancer onset according to the present invention may be performed, for example, by identifying mutations in the NOTCH2 gene, TSC2 gene and / or FGFR3 gene, or mutations in the NOTCH2 gene, TSC2 gene, FGFR3 gene, ARID2 gene, CREBBP gene, MSH2 gene and / or TET2 gene.

[0085]

[0086] Another aspect of the present invention provides a gene variant comprising one or more variants selected from the group consisting of the NOTCH2 gene, TSC2 gene, FGFR3 gene, ARID2 gene, CREBBP gene, MSH2 gene and TET2 gene.

[0087] In the gene variant for diagnosing or predicting the onset of gastric cancer according to the present invention, each term has the same meaning as the foregoing unless specifically stated otherwise.

[0088] As used herein, the term "gene variant" refers to a gene whose genetic information or genetic trait has changed due to a variation in its base sequence. In this specification, the term "gene variant" may be used interchangeably with the term "variant."

[0089]

[0090] Another aspect of the present invention provides a method for preparing a gastric cancer model, comprising the step of inducing one or more mutations selected from the group consisting of the NOTCH2 gene, TSC2 gene, FGFR3 gene, ARID2 gene, CREBBP gene, MSH2 gene, and TET2 gene in a subject other than a human or a host cell.

[0091] In the method for manufacturing a gastric cancer model according to the present invention, each term has the same meaning as described above unless specifically stated otherwise.

[0092] In this specification, inducing a gene mutation means the introduction or administration of a substance that induces a gene mutation according to the present invention, such as a gene variant, or artificial manipulation, alteration, modification, or engineering of a gene, such as knock-down, knock-out, or knock-in.

[0093] The term "introduction" above refers to introducing a gene variant of the present invention, or a vector containing the same, into a subject other than a human or a host cell through any means, and may be used interchangeably with "infusion" or "injection."

[0094] The above method for manufacturing a gastric cancer model may further include the steps of: performing a pretreatment to induce a gene mutation of the present invention; obtaining a biological sample from a subject other than a human containing the gene mutation; obtaining nucleic acid from the biological sample or a host cell; performing sequencing on the nucleic acid; or selecting a subject other than a human or a host cell into which the gene mutation has been introduced.

[0095] As used herein, the term "vector" refers to a plasmid, virus, nanoparticle, or other medium known in the art that can insert or introduce a gene variant of the present invention into a subject other than a human or a host cell as a means for introducing said gene variant. The vector may be constructed as a vector for expression or a vector for cloning. For example, it may be a plasmid vector, a cosmid vector, a bacteriophage vector, or a viral vector such as an adenovirus vector, a retrovirus vector, an adeno-associated virus (AAV) vector, and a lentivirus vector. Additionally, the vector may be a non-viral vector such as a liposome or a nanoparticle, but is not limited thereto. The above vector may be constructed through various methods known in the art and may include antibiotic resistance genes commonly used in the art as selection markers, e.g., genes for resistance to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline. In the above vector, the gene variant of the present invention may be operatively linked with a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites). In this specification, the term "vector" may be used interchangeably with the term "carrier."

[0096] As used herein, the term "operably connected" refers to a functional coupling between a nucleic acid expression regulatory sequence and another nucleic acid sequence, thereby allowing the regulatory sequence to regulate the transcription and / or translation of the other nucleic acid sequence.

[0097] As used herein, the term "host cell" refers to a cell comprising the vector and capable of stably and continuously expressing or cloning the gene variant of the present invention. For example, prokaryotic host cells such as strains of the genus Bacillus (e.g. Escherichia coli, Bacillus subtilis, and Bacillus thuringiensis), Streptomyces, Pseudomonas, Proteus mirabilis, or Staphylococcus; The host cells may be fungi such as Aspergillus, eukaryotic host cells such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, or Neurospora crassa; lower eukaryotic cells; higher eukaryotic cells such as insect-derived cells; plant cells; or cells derived from mammals such as COS7 cells (Monkey kidney cells), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, or 293 cells, but are not limited thereto, and any host cells commonly used in the art may be used without limitation.

[0098] The above gastric cancer model is not limited to any specific type, such as ex vivo models, in vitro models, or in vivo models, and may be, for example, cell lines, animal models, or biological organ-mimicking models such as spheroids or organoids. The above gastric cancer model may serve as a research subject capable of identifying the etiology and pathogenesis of gastric cancer, or testing the efficacy and toxicity of candidate drugs for the treatment or prevention of gastric cancer.

[0099] As used in this specification, the term "cell line" refers to an individual of each cell line when isolated cells are pure-cultured and subcultured. The cell line is capable of indefinite proliferation, and cells of the same cell line possess identical traits. In the present invention, the cell line may be prepared using a host cell into which a vector containing the gene variant of the present invention has been introduced. In the present invention, the cell line may be prepared using a host cell into which a vector containing the gene variant of the present invention has been introduced.

[0100] As used herein, the term "animal model" refers to a non-human subject having a disease similar in form to a human disease. In the present invention, the animal model may be one into which a vector or cell line containing the gene variant of the present invention is injected or transplanted.

[0101] As used in this specification, the term "spheroid" refers to a cluster of single cells that are assembled to form a 3D spherical shape. The spheroid may be produced from somatic cells. In the present invention, the spheroid may comprise a vector or cell line containing a genetic variant of the present invention.

[0102] As used in this specification, the term "organoid" refers to a mass of cells having a three-dimensional structure. It is defined as a scaled-down and simplified version of an organ produced through an artificial culture process without being collected, acquired, or harvested from animals or the like. The organoid may be produced from stem cells such as embryonic stem cells, induced pluripotent stem cells, or adult stem cells, and the origin of the constituent cells is not limited. Since the cells of the organoid can grow in all directions outside the body, the organoid can mimic an organ interacting within the body. In the present invention, the organoid may comprise a vector or cell line containing a gene variant of the present invention.

[0103]

[0104] Another aspect of the present invention provides a gastric cancer model comprising one or more variants selected from the group consisting of the NOTCH2 gene, TSC2 gene, FGFR3 gene, ARID2 gene, CREBBP gene, MSH2 gene and TET2 gene.

[0105] In the gastric cancer model according to the present invention, each term has the same meaning as the foregoing unless specifically noted otherwise.

[0106] The above-mentioned gene mutation may be induced by the introduction or administration of a substance that induces gene mutations according to the present invention, such as a gene variant according to the present invention, into a gastric cancer model, or by artificial manipulation, alteration, modification, or engineering of genes, such as knockdown, knockout, or knock-in.

[0107]

[0108] Another aspect of the present invention provides a composition for preparing a gastric cancer model comprising a gene variant of the present invention.

[0109] In the composition for preparing a gastric cancer model according to the present invention, each term has the same meaning as described above unless specifically stated otherwise.

[0110] The composition for manufacturing a gastric cancer model according to the present invention may include a substance used to manufacture a gastric cancer model (e.g., a culture medium, an extracellular matrix, etc.), a substance that induces a gene mutation according to the present invention (e.g., a vector, etc.), and a substance used to artificially manipulate, alter, modify, or engineer genes, such as knockdown, knockout, or knock-in, to induce a gene mutation according to the present invention.

[0111]

[0112] Another aspect of the present invention provides a biomarker composition for diagnosing or predicting the onset of gastric cancer, comprising a gene variant of the present invention.

[0113] In the biomarker composition for diagnosing or predicting the onset of gastric cancer according to the present invention, each term has the same meaning as the foregoing unless specifically stated otherwise.

[0114]

[0115] Another aspect of the present invention provides a composition for diagnosing or predicting the onset of gastric cancer, comprising a preparation used to perform detection, content measurement, expression level measurement, or amplification of a gene variant of the present invention.

[0116] In the composition for diagnosing or predicting the onset of gastric cancer according to the present invention, each term has the same meaning as the foregoing unless specifically stated otherwise.

[0117] The above preparation is a substance capable of binding to the gene variant of the present invention for the detection, content measurement, expression level measurement, or amplification of the gene variant of the present invention, and may be a primer, probe, or antisense nucleotide, but is not limited thereto.

[0118] As used herein, the term "primer" refers to a nucleic acid sequence having a free 3' hydroxyl group and capable of binding complementarily to a template strand. The primer can function as a starting point for template strand replication. The primer can synthesize DNA in the presence of a buffer solution, temperature, time, polymerase, and nucleoside triphosphate (NTP). The primer may be one known in the art or a modified version based thereon. The type or form of the primer of the present invention is not particularly limited as long as it can perform detection, content measurement, expression level measurement, or amplification of the gene variant. In this specification, the term "primer" may be used interchangeably with the term "initiator."

[0119] As used herein, the term "probe" refers to a nucleic acid fragment of several to several hundred bases capable of binding complementarily to a target base sequence, and may be labeled with fluorescence, enzymes, biotin, etc., to facilitate the detection, content measurement, or expression level measurement of said gene variant. The probe may be one known in the art or a modified version based thereon. The type or form of the probe of the present invention is not particularly limited as long as it is capable of performing the detection, content measurement, or expression level measurement of said gene variant. In this specification, the term "probe" may be used interchangeably with the term "probe."

[0120] As used herein, the term "antisense nucleotide" refers to a nucleotide capable of binding complementarily to a target base sequence. The antisense nucleotide includes antisense oligonucleotides (ASOs). The antisense nucleotide may be one known in the art or one modified based thereon. The type or form of the antisense nucleotide of the present invention is not particularly limited as long as it can perform detection, content measurement, or expression level measurement of the gene variant.

[0121] The above composition may be used in analysis methods such as polymerase chain reaction, reverse transcription polymerase chain reaction, nuclease protection assay, Northern blot, Southern blot, next-generation sequencing, in situ hybridization, or microarray.

[0122] The composition of the present invention may further include substances or components necessary for the analysis method. The substances or components may be, for example, DNA polymerase, reverse transcriptase, NTP, reaction buffer, distilled water, etc., necessary for performing PCR, and may be restriction enzyme, agarose gel, membrane filter, filter paper, etc., necessary for performing Southern blot.

[0123]

[0124] Another aspect of the present invention provides a kit for diagnosing or predicting the onset of gastric cancer, comprising the above-mentioned composition for diagnosing or predicting the onset of gastric cancer.

[0125] In the kit for diagnosing or predicting the onset of gastric cancer according to the present invention, each term has the same meaning as the foregoing unless specifically noted otherwise.

[0126] The above kit may be a polymerase chain reaction kit, a reverse transcription polymerase chain reaction kit, a nuclease protection analysis kit, a Northern blot kit, a Southern blot kit, a next-generation sequencing library preparation kit, an in situ hybridization kit, or a microarray kit, but is not limited thereto.

[0127]

[0128] Another aspect of the present invention provides a method for screening drugs for the treatment or prevention of gastric cancer, comprising the step of identifying one or more mutations selected from the group consisting of the NOTCH2 gene, TSC2 gene, FGFR3 gene, ARID2 gene, CREBBP gene, MSH2 gene, and TET2 gene in a gastric cancer model of the present invention.

[0129] In the method for screening drugs for the treatment or prevention of gastric cancer according to the present invention, each term has the same meaning as the foregoing unless specifically noted otherwise.

[0130] The above method may further include the steps of: contacting, injecting, or administering a candidate drug to a gastric cancer model of the present invention; isolating a biological sample from the gastric cancer model; obtaining nucleic acids from the gastric cancer model or the biological sample isolated therefrom; or determining the candidate drug as a drug for the treatment or prevention of gastric cancer if the mutation is not identified or if the gene mutation is reduced when compared with a control group. The control group may be a subject having the gene mutation of the present invention.

[0131]

[0132] Another aspect of the present invention provides a method for providing information for the diagnosis or prediction of gastric cancer development, comprising the step of identifying one or more variants selected from the group consisting of the NOTCH2 gene, TSC2 gene, FGFR3 gene, ARID2 gene, CREBBP gene, MSH2 gene, and TET2 gene within a biological sample isolated from a subject.

[0133] In the method for providing information for diagnosing or predicting the onset of gastric cancer according to the present invention, each term has the same meaning as the foregoing unless specifically noted otherwise.

[0134] The above information provision method may further include the step of obtaining nucleic acid from a biological sample separated from a subject; or the step of determining that gastric cancer has developed or is highly likely to develop when the mutation is confirmed.

[0135]

[0136] Another aspect of the present invention provides the use of a composition or kit comprising a gene variant, a gastric cancer model, a composition for preparing a gastric cancer model, a biomarker composition, and a preparation used to perform detection, content measurement, expression level measurement, or amplification of a gene variant, for diagnosing or predicting the onset of gastric cancer in vitro or ex vivo within a biological sample separated from a subject.

[0137] In the use of a gene variant, a gastric cancer model, a composition for manufacturing a gastric cancer model, a biomarker composition, a composition comprising a preparation used to perform detection, content measurement, expression level measurement, or amplification of a gene variant, or a kit according to the present invention, each term has the same meaning as the foregoing unless specifically stated otherwise.

[0138] The gastric cancer diagnosis and onset prediction method of the present invention can significantly reduce the number of gastric cancer cases and deaths by diagnosing gastric cancer early or by predicting the risk of gastric cancer development and preventing it before it occurs. In addition, by observing the presence or changes in frequency of gene mutations following drug treatment, this can be utilized for molecular mechanism research for gastric cancer treatment or for the development of drug screening platforms.

[0139] Figure 1 shows the results of analyzing the correlation between the genome derived from gastric tissue and the genome derived from gastric juice of an endoscopic subject. Type A represents a gene mutation observed in both normal tissue of the gastric antrum and gastric juice, Type B represents a gene mutation observed in both normal tissue of the gastric body and gastric juice, Type C represents a gene mutation observed in both the gastric adenoma tissue of a gastric adenoma subject or the gastric cancer tissue of a gastric cancer subject and gastric juice, Non represents a gene mutation observed only in gastric juice and not in normal gastric tissue, gastric adenoma tissue, or gastric cancer tissue, SGC represents a sample, and the number following SGC represents the sample number.

[0140] Figure 2 shows the results of gene mutations that were specifically high in gastric adenoma and gastric cancer subjects. P represents the significance probability (P-value), Adenoma represents gastric adenoma, Gastric cancer (GC) represents gastric cancer, and NOTCH2, TSC2, FGFR3, MSH2, TET2, ARID2, and CREBBP represent genes.

[0141] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited by these examples.

[0142]

[0143] Example 1. Sample Preparation

[0144] From May 2021 to May 2022, 30 healthy individuals (including patients with gastritis), 40 patients with gastric adenoma, and 44 patients with gastric cancer were recruited to undergo endoscopy for the development of biomarkers for the diagnosis or prediction of gastric cancer. Gastric fluid and tissue samples were collected with the patients' consent and used as samples. It is known that gastric adenomas have the potential to progress to gastric cancer, and depending on the type of gastric adenoma, the likelihood of progression to gastric cancer is high.

[0145]

[0146] Example 2. Confirmation of Gene Variation

[0147] After extracting genomic DNA from the endoscopic subject samples of Example 1, a total of 84 samples containing genomic DNA capable of next-generation sequencing (NGS) were selected. Using NGS on the selected genomic DNA, mutations specific to gastric adenoma and gastric cancer that commonly appear in the gastric fluid samples and tissue samples of the subjects were selected.

[0148] Specifically, the above genomic DNA extracted using the QIAamp DNA Mini Kit (Qiagen) was automatically prepared into a library on the Ion Chef System using the Oncomine Comprehensive Assay Plus V.3 panel, and then sequencing was performed using the Thermosisher Ion S5 System. Subsequently, using Oncomine Comprehensive Plus 2.4 of Ion Reporter Software, the genomes derived from gastric tissue and gastric fluid were compared for the genes of the subject-derived samples according to Example 1, as shown in Figure 1. Gene mutations observed in both normal gastric antral tissue and gastric fluid were designated as Type A; gene mutations observed in both normal gastric body tissue and gastric fluid were designated as Type B; gene mutations observed in both gastric adenoma tissue of a gastric adenoma subject or gastric cancer tissue of a gastric cancer subject and gastric fluid were designated as Type C; and gene mutations observed only in gastric fluid and not in normal gastric tissue, gastric adenoma tissue, or gastric cancer tissue were designated as Non.

[0149] Subsequently, among the genomes and gene mutations of gastric adenoma subjects and gastric cancer subjects, the reference genome was compared to select gene mutations that were specifically high for gastric adenoma and gastric cancer, respectively, and are shown in Figure 2.

[0150] As can be seen in Figure 1, an average similarity of 93.4% was observed between the gene mutations in the genomes derived from normal gastric tissue, gastric adenoma tissue, or gastric cancer tissue of 84 samples and the gene mutations in the genomes derived from gastric juice.

[0151] The above results suggest that not only gastric tissue but also gastric fluid can be used as biological samples to diagnose gastric cancer or predict its onset.

[0152]

[0153] In addition, as shown in Figure 2, NOTCH2, TSC2, and FGFR3 gene mutations were selected, which were statistically significantly higher than other gene mutations at a level of P=0.009476 in gastric adenoma subjects. Furthermore, ARID2, CREBBP, MSH2, and TET2 gene mutations were selected, which were statistically significantly higher than other gene mutations at a level of P=0.0023 in gastric cancer subjects. The above mutations are, respectively, the substitution of guanine at the 710th base of the NOTCH2 gene with adenine, the substitution of cytosine at the 2816th base with thymine, or the substitution of adenine at the 5065th base with thymine; and the substitution of adenine at the 856th base of the TSC2 gene with guanine or the substitution of guanine at the 2032nd base with adenine; Cytosine at the 1349th base of the FGFR3 gene is replaced with thymine; adenine at the 1759th base of the ARID2 gene is replaced with guanine; cytosine at the 1651st base of the CREBBP gene is replaced with adenine; cytosine at the 23rd base of the MSH2 gene is replaced with thymine or cytosine at the 1168th base is replaced with thymine; and cytosine at the 3116th base of the TET2 gene is replaced with thymine.

[0154] Considering the high likelihood that gastric adenomas will progress to gastric cancer, the above results suggest that gastric cancer can be diagnosed or its onset predicted using each gene mutation or combination thereof.

[0155]

[0156] Example 3. Diagnosis of gastric cancer

[0157] In order to confirm whether gastric cancer can actually be diagnosed through the gastric cancer diagnosis and onset prediction method of the present invention, the degree of differentiation and submucosa invasion of gastric cancer cells of gastric cancer subjects having one or more of ARID2 gene mutations, CREBBP gene mutations, MSH2 gene mutations, and TET2 gene mutations, and gastric cancer subjects without the four gene mutations were analyzed, and the T stage of gastric cancer was analyzed to confirm the degree of invasion of cancer cells in more detail.

[0158] Specifically, a paraffin block was prepared by fixing cancer cells in a tissue sample collected from a gastric cancer subject of Example 2 using formalin. Subsequently, the paraffin block was sliced ​​thinly to create slides. These slides were observed under a microscope using immunohistochemical staining appropriate for the characteristics of each cancer cell, and a pathologist identified the characteristics of the gastric cancer cells. Subsequently, the degree of differentiation of the cancer cells is shown in Table 1, the state of invasion into the gastric submucosal layer is shown in Table 2, and the T classification of the gastric cancer is shown in Table 3.

[0159]

[0160] Example 3-1. Degree of differentiation of cancer cells

[0161] The degree of differentiation of cancer cells is a pathological criterion that evaluates how similar the structure and function of cancer cells are to original normal cells, and it is closely related to the prognosis of cancer. It is known that tumors can be classified into four grades based on the degree of differentiation; Grade 1 tumors appear close to normal because the cancer cells are well differentiated. The higher the grade number, the more abnormal the cells appear, and Grade 4 tumors appear the most abnormal (Source: NIH National Cancer Institute and Tatjana Bogdanova et al. (2014), Tumor volume assessment in gastric cancer, Acta chirurgica latviensis, volume 114: issue 1).

[0162] Specifically, well-differentiated cancer cells are similar to normal cells, so their growth rate is slow and the risk of metastasis is low, resulting in a favorable prognosis after treatment and making gastric cancer treatment relatively easier.

[0163] Moderately differentiated cancer cells exhibit a form somewhere between well-differentiated and undifferentiated cancer cells.

[0164] Unlike normal cells, undifferentiated or poorly differentiated cancer cells are immature, grow abnormally and rapidly, and have a very high potential for metastasis; therefore, the prognosis after gastric cancer treatment is poor and the risk of recurrence is also high (Source: NIH National Cancer Institute and AJCC 1st Ed Cancer Staging Manual).

[0165] The results of the analysis of the degree of differentiation of cancer cells for samples taken from the gastric cancer subjects of Example 2 are shown in Table 1.

[0166] Tumor Grade Cancer Cell Differentiation Number of Subjects with Gene Mutations Number of Subjects Without Gene Mutations Grade 1 (Low grade) Well-differentiated 21.0% 52.0% Grade 2 (Intermediate grade) Moderately-differentiated 31.6% 24.0% Grade 3 (High grade) Poorly-differentiated 42.1% 24.0% Grade 4 (High grade) Undifferentiated 5.3% 0%

[0167]

[0168] As can be seen in Table 1, nearly half of the gastric cancer subjects with one or more mutations selected from the group consisting of the ARID2, CREBBP, MSH2, and TET2 genes showed high tumor grades (grade 3 or grade 4). On the other hand, the majority of gastric cancer subjects without gene mutations showed low tumor grades (grade 1 or grade 2).

[0169] That is, gastric cancer subjects with one or more mutations among the four genes mentioned above were found to have a higher cancer growth rate, potential for metastasis, and risk of recurrence, and a poor prognosis after treatment compared to gastric cancer subjects without gene mutations.

[0170] The above results show that each gene mutation or a combination of one or more of these promotes the growth and metastasis of cancer, and that gastric cancer subjects with said mutations have a higher probability of cancer progression compared to gastric cancer subjects without gene mutations.

[0171] Furthermore, it suggests that each gene mutation or a combination of one or more of them can be utilized for the diagnosis of gastric cancer, the prediction of gastric cancer onset, and the prediction of prognosis and recurrence after gastric cancer treatment.

[0172]

[0173] Example 3-2. Gastric submucosal invasion

[0174] The state of gastric submucosal invasion refers to the condition in which gastric cancer has penetrated the gastric mucosa and invaded the submucosal layer; it was judged as negative if there was no invasion into the submucosal layer and positive if there was invasion.

[0175] The stomach is divided from the inside out into the mucosa, submucosa, muscularis propria, subserosa, and serosa, and gastric cancer progresses by invading from the mucosa to the serosa.

[0176] Early gastric cancer is defined as mucosal cancer confined to the mucosal layer and submucosal cancer confined to the submucosal layer. The frequency of lymph node metastasis in mucosal cancer is reported to be around 5%, while the frequency in submucosal cancer is around 20%, which is because lymphatic vessels are more developed in the submucosal layer compared to the mucosa. Lymphatic invasion is reported as one of the important risk factors for lymph node metastasis, and the presence of lymph node metastasis is the most important prognostic factor for patients with early gastric cancer (Sources: Young Gil Son et al. (2009), Predictive Factors for Lymph Node Metastasis in Submucosal Gastric Cancer, Journal of the Korean Surgical Society, 76(6):355-359 and Takuji Gotoda et al. (2000), Incidence of lymph node metastasis from early gastric cancer: estimation with a large number of cases at two large centers, Gastric cancer, 3:219-225).

[0177] The results of the analysis of the cancer cell invasion status into the gastric submucosal layer of the sample taken from the gastric cancer subject of Example 2 are shown in Table 2.

[0178] Gastric submucosal invasion status Number of subjects with gene mutation Number of subjects without gene mutation Negative 57.9% 80% Positive 42.1% 20%

[0179]

[0180] As shown in Table 2, gastric submucosal invasion in gastric cancer subjects with one or more selected mutations from the group consisting of the ARID2, CREBBP, MSH2, and TET2 genes was observed more than twice as often as in subjects without gene mutations. On the other hand, gastric submucosal invasion was not observed in more than half of gastric cancer subjects without gene mutations.

[0181] The above results show that each gene mutation or a combination of one or more of these promotes the invasion of gastric cancer cells into the gastric submucosal layer, and that gastric cancer subjects with said mutations have a higher probability of cancer progression compared to gastric cancer subjects without the gene mutations.

[0182] Furthermore, it suggests that each gene mutation or a combination of one or more of them can be utilized for the diagnosis of gastric cancer, the prediction of gastric cancer onset, and the prediction of prognosis and recurrence after gastric cancer treatment.

[0183]

[0184] Example 3-3. T-classification of gastric cancer

[0185] The T classification of gastric cancer indicates the degree of invasion of cancer cells into the stomach wall and determines the stage of the cancer. The stage of cancer refers to the extent of progression and is determined by combining the degree of invasion into the stomach wall (T classification), the extent of metastasis to surrounding lymph nodes (N classification), and the presence of metastasis to other organs (M classification).

[0186] According to the AJCC 8th edition of the international staging system for gastric cancer, the T classification of gastric cancer is classified into T1a, T1b, T2, T3, T4a, and T4b.

[0187] T1a refers to cases where the tumor has invaded the mucosal layer of the stomach wall, and T1b refers to cases where the tumor has invaded the submucosal layer of the stomach.

[0188] T2 refers to cases where the tumor has invaded the muscular layer of the stomach, and T3 refers to cases where the tumor has invaded the subserosal layer.

[0189] T4a refers to cases where the tumor has invaded the serosa, and T4b refers to a condition where the tumor has penetrated the serosa and invaded surrounding organs such as the spleen, transverse colon, liver, diaphragm, pancreas, abdominal wall, adrenal gland, adrenal gland, small intestine, and retroperitoneum (Source: 8th Edition of the AJCC Gastric Cancer Staging System).

[0190] T1a and T1b are classified as early gastric cancer, and T2 or higher is classified as advanced gastric cancer (Source: Tae-Han Kim et al.(2023), Korean practice guidelines for gastric cancer 2022: an evidence-based, multidisciplinary approach, Journal of gastric cancer, 23(1):3-106), and the negative submucosal invasion status of Example 3-2 corresponds to T1a, and the positive submucosal invasion status corresponds to T1b to T4.

[0191] The results of the T-classification analysis for tissue samples taken from the gastric cancer subjects of Example 2 are shown in Table 3.

[0192] T Classification Number of subjects with gene mutation Number of subjects without gene mutation T1a 5 7.9% 76.0% T1b 2 1.0% 16.0% T20% 4.0% T3 5.3% 4.0% T4 15.8% 0%

[0193]

[0194] As can be seen in Table 3, among gastric cancer subjects with one or more mutations selected from the group consisting of the ARID2 gene, CREBBP gene, MSH2 gene and TET2 gene, the number of subjects corresponding to T1b to T4 was the same as the results of the submucosal invasion status of Example 3-2.

[0195] In addition, T4, in which the tumor invaded the serosa of the stomach or penetrated the serosa to invade surrounding organs, was not observed at all in the group of subjects without gene mutations. On the other hand, it was observed in 15.8% of subjects with one or more mutations selected from the group consisting of the ARID2 gene, CREBBP gene, MSH2 gene, and TET2 gene.

[0196] The above results show that each gene mutation or a combination of one or more of these promotes the invasion of gastric cancer cells into the gastric submucosal layer, and that gastric cancer subjects with said mutations have a higher probability of cancer progression compared to gastric cancer subjects without the gene mutations.

[0197] Furthermore, it suggests that each gene mutation or a combination of one or more of them can be utilized for the diagnosis of gastric cancer, the prediction of gastric cancer onset, and the prediction of prognosis and recurrence after gastric cancer treatment.

Claims

1. A method for diagnosing or predicting the onset of gastric cancer, comprising the step of identifying one or more variants selected from the group consisting of the NOTCH2 gene, TSC2 gene, FGFR3 gene, ARID2 gene, CREBBP gene, MSH2 gene, and TET2 gene within a biological sample isolated from a subject.

2. In Paragraph 1, A method for diagnosing or predicting the onset of gastric cancer, wherein the NOTCH2 gene is represented by SEQ ID NO. 1, the TSC2 gene by SEQ ID NO. 2, the FGFR3 gene by SEQ ID NO. 3, the ARID2 gene by SEQ ID NO. 4, the CREBBP gene by SEQ ID NO. 5, the MSH2 gene by SEQ ID NO. 6, and the TET2 gene by SEQ ID NO.

7.

3. In Paragraph 1, A method for diagnosing or predicting the onset of gastric cancer, wherein the biological sample is whole blood, serum, plasma, gastric fluid, ascites, pleural fluid, pericardial fluid, cerebrospinal fluid, lymph fluid, pancreatic fluid, semen, synovial fluid, urine, feces, saliva, sputum, cystic fluid, amniotic fluid, tears, mucus, tissue, cell or organ secretion; or nucleic acid obtained therefrom.

4. In Paragraph 1, A method for diagnosing or predicting the onset of gastric cancer, wherein the above mutation is identified using polymerase chain reaction, reverse transcription polymerase chain reaction, nuclease protection assay, Northern blot, Southern blot, next-generation sequencing, in situ hybridization, or microarray.

5. In Paragraph 1, A method for diagnosing or predicting the onset of gastric cancer, wherein the above variant is a single nucleotide variant, a deletion or insertion of one or more nucleotides, a copy number variant, or a tandem repeat polymorphism within the gene base sequence.

6. In Paragraph 1, A method for diagnosing or predicting the onset of gastric cancer, wherein the above mutation has one or more of the following characteristics: Substitution of guanine, the 710th base of the NOTCH2 gene, with adenine, cytosine, or thymine; Substitution of cytosine, the 2816th base of the NOTCH2 gene, with thymine, adenine, or guanine; Substitution of adenine, the 5065th base of the NOTCH2 gene, with thymine, guanine, or cytosine; Substitution of adenine, the 856th base of the TSC2 gene, with guanine, cytosine, or thymine; Substitution of guanine, the 2032nd base of the TSC2 gene, with adenine, cytosine, or thymine; Substitution of cytosine, the 1349th base of the FGFR3 gene, with thymine, adenine, or guanine; Substitution of adenine, the 1759th base of the ARID2 gene, with guanine, cytosine, or thymine; Substitution of cytosine, the 1651st base of the CREBBP gene, with adenine, guanine, or thymine; Substitution of cytosine, the 23rd base of the MSH2 gene, with thymine, adenine, or guanine; Substitution of cytosine, the 1168th base of the MSH2 gene, with thymine, adenine, or guanine; and The 3116th base of the TET2 gene, cytosine, is replaced with thymine, adenine, or guanine.

7. In Paragraph 1, A method for diagnosing or predicting the onset of gastric cancer, wherein the above gastric cancer is early gastric cancer, advanced gastric cancer, recurrent gastric cancer, or metastatic gastric cancer.

8. A gene variant comprising one or more variants selected from the group consisting of the NOTCH2 gene, TSC2 gene, FGFR3 gene, ARID2 gene, CREBBP gene, MSH2 gene and TET2 gene.

9. A method for preparing a gastric cancer model comprising the step of inducing one or more mutations selected from the group consisting of the NOTCH2 gene, TSC2 gene, FGFR3 gene, ARID2 gene, CREBBP gene, MSH2 gene and TET2 gene in a subject or host cell other than a human.

10. A gastric cancer model comprising one or more variants selected from the group consisting of NOTCH2 gene, TSC2 gene, FGFR3 gene, ARID2 gene, CREBBP gene, MSH2 gene and TET2 gene.

11. A composition for the diagnosis or prediction of gastric cancer development, comprising a preparation used to perform detection, content measurement, expression level measurement, or amplification of one or more variants selected from the group consisting of NOTCH2 gene, TSC2 gene, FGFR3 gene, ARID2 gene, CREBBP gene, MSH2 gene and TET2 gene.

12. In Paragraph 11, A composition for diagnosing or predicting the onset of gastric cancer, wherein the above preparation is a primer, probe, or antisense nucleotide.

13. In Paragraph 11, The above composition is a composition for the diagnosis or prediction of gastric cancer, which is used in polymerase chain reaction, reverse transcription polymerase chain reaction, nuclease protection assay, Northern blot, Southern blot, next-generation sequencing, in situ hybridization, or microarray.

14. A kit for diagnosing or predicting the onset of gastric cancer, comprising the composition of claim 11.

15. A method for screening drugs for the treatment or prevention of gastric cancer, comprising the step of identifying one or more mutations selected from the group consisting of the NOTCH2 gene, TSC2 gene, FGFR3 gene, ARID2 gene, CREBBP gene, MSH2 gene and TET2 gene in the gastric cancer model of claim 10.

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