Prognostic diagnosis marker and therapeutic target for diffuse gastric cancer

Abstract

The present invention provides a composition and a kit for diagnosis or prognosis prediction of diffusive type gastric cancer, the composition comprising a preparation for measuring the expression level of SYT11 gene. In addition, the present invention provides a method for providing information necessary for diagnosis of diffuse gastric cancer or information necessary for determination of a treatment strategy for diffuse gastric cancer.

Description

Technical Field

[0001] The present invention relates to a composition for diagnosis or prediction of prognosis capable of diagnosing diffuse type gastric cancer or predicting the prognosis of diffuse type gastric cancer and a kit comprising the composition, and also relates to a method for providing information required for diagnosing diffuse type gastric cancer or for determining a treatment strategy for diffuse type gastric cancer. Background Art

[0002] Gastric cancer is the most common cancer in Koreans in the world, and is one of the diseases that requires the most prevention, treatment, and prognosis prediction. According to the Lauren classification based on pathology and histology, gastric cancer can be divided into intestinal type and diffuse type. Diffuse type gastric cancer is characterized by the proliferation of cancer cells in the submucosal layer of the stomach in a diffuse shape, and has the characteristics of infiltrating the stomach wall. However, compared with intestinal type gastric cancer, diffuse type gastric cancer has a poor survival rate, and the incidence rate is high among young people in their twenties and thirties who are relatively less alert to cancer or have relatively few opportunities for physical examinations, so it is difficult to detect it in the early stages of the disease. Therefore, for diffuse type gastric cancer, it is more necessary to detect the onset of gastric cancer at an early stage, and the need for research on the method of diagnosing diffuse type gastric cancer among gastric cancers is increasing in order to formulate treatment strategies.

[0003] Until now, most of the examination methods for diagnosing gastric cancer have been performed by physical means such as X-ray photography or gastroendoscopy, but this has the disadvantages of causing pain to the patient undergoing the examination and requiring a cumbersome process. Accordingly, research is being conducted on genetic markers that are specifically expressed or have increased expression levels for gastric cancer patients, and research is being conducted on measuring the expression levels of gastric cancer markers for diagnosis. However, in research on gastric cancer, the fact is that research on genetic markers related to the diagnosis of diffuse gastric cancer classified by histology or research related to the prognosis prediction of diffuse gastric cancer patients is not complete.

[0004] In addition, the research team of the present invention proposed a study on using the SYT11 gene as a diagnostic marker for "stem-like gastric cancer" through Korean Patent Publication No. 2020-0044695, which involves a composition for treating gastric cancer containing a synaptotagmin 11 (SYT11) inhibitor as an active ingredient and a diagnostic method for stem-like gastric cancer containing the measurement of SYT11 expression, but the correlation between diffuse gastric cancer, which has different characteristics, and the expression level of the SYT11 gene has not been reported. In addition, there has been no research on marker genes that can be used for companion diagnostics (companion diagnostics) for formulating treatment strategies suitable for specific patients by predicting the effects of SYT11-targeting therapeutic agents such as the SYT11 inhibitor in advance when administering them. Therefore, it is necessary to develop a method that provides new markers that can be used when diagnosing diffuse gastric cancer and predicting its prognosis, and establishing treatment strategies such as the use of SYT11-targeted therapeutic agents, and provides information required for diagnosing diffuse gastric cancer and determining treatment strategies for diffuse gastric cancer. Summary of the invention

[0005] Technical issues An object of the present invention is to provide a composition and a kit capable of diagnosing diffuse type gastric cancer among gastric cancers or predicting the prognosis of a patient with diffuse type gastric cancer.

[0006] Furthermore, an object of the present invention is to provide information required for diagnosing diffuse gastric cancer or information required for determining a treatment strategy for a diffuse gastric cancer patient.

[0007] Technical Solution In order to achieve the above object, one aspect of the present invention provides a composition for diagnosis or prognosis prediction of diffuse gastric cancer, comprising an agent for measuring the expression level of the SYT11 gene.

[0008] In addition, another aspect of the present invention provides a kit for diagnosing or predicting the prognosis of diffuse gastric cancer, comprising the composition.

[0009] In addition, another aspect of the present invention provides a method for providing information required for diagnosing diffuse gastric cancer or determining a treatment strategy for diffuse gastric cancer, comprising the step of measuring the expression level of the SYT11 gene from an isolated biological gastric tissue sample, wherein the information required for determining the treatment strategy for the diffuse gastric cancer is selected from at least one of the group consisting of: prognosis prediction of diffuse gastric cancer; prognosis evaluation of diffuse gastric cancer; and effectiveness evaluation of SYT11 targeted therapeutic agents in gastric cancer patients.

[0010] Effects of the Invention According to the present invention, it was confirmed that the expression level of the SYT11 gene showed a high correlation in patients with diffuse gastric cancer between the intestinal type and the diffuse type, and also had a high correlation with the prognosis of patients with diffuse gastric cancer, thereby confirming that the SYT11 gene can be effectively used as a marker for diagnosing diffuse gastric cancer or predicting prognosis. In addition, in addition to the SYT11 gene, the expression levels of 8 genes in patients with diffuse gastric cancer appeared in a pattern similar to the expression level of the SYT11 gene, thereby confirming that they also had a close correlation with the prognosis of patients with diffuse gastric cancer, and the 8 genes can be effectively used together with the SYT11 gene to establish a diagnosis or treatment strategy for diffuse gastric cancer.

[0011] However, the effects of the present invention are not limited to the above-mentioned effects, and those skilled in the art can clearly understand other effects not mentioned from the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The table includes photographs of normal human gastric tissue, intestinal gastric cancer tissue, and diffuse gastric cancer tissue confirmed using a microscope, and the tissues are classified into a group with a low SYT11 gene expression level (SYT11-low) or a group with a high SYT11 gene expression level (SYT11-high) based on the SYT11 gene expression level data measured for the above tissues.

[0013] Figure 2 The results are based on clinical data of 424 gastric cancer patients who were classified as intestinal type or diffuse type gastric cancer, and then reclassified according to the expression level of the SYT11 gene and whether it is stem-like gastric cancer.

[0014] Figure 3 Based on clinical data from 424 gastric cancer patients, the SYT11 gene ( Figure 3 A, B) or SYT4 gene ( Figure 3 C, D) Survival likelihood of expression levels were analyzed and compared in graphs.

[0015] Figure 4 These are tables and graphs comparing and analyzing the expression levels of the SYT11 gene and the expression levels of eight genes (ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and VIMENTIN genes) in patients with diffuse gastric cancer.

[0016] Figure 5This graph analyzes and compares the survival probability based on the type of gastric cancer (intestinal type or diffuse type) and the expression levels of the SYT11 gene and eight genes (ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and VIMENTIN genes) based on clinical data of gastric cancer patients.

[0017] Figure 6 The results are from Western blotting of SYT11 protein and ANGPTL2, THBS4, VIMENTIN, and JAM3 proteins after transfection of gastric cancer cell line SNU484 cells with siSYT11, which functions as a SYT11-targeting therapeutic agent, and siScramble (siSC), a control group.

[0018] Figure 7 The results are shown in Figure 1. The gastric cancer cell line SNU484 cells were transfected with SYT11-ASO and control NC-ASO, which function as SYT11-targeted therapeutic agents, and Western blotting was performed on SYT11 protein, ANGPTL2, THBS4, and JAM3 proteins. DETAILED DESCRIPTION

[0019] First, the terms used in the present invention are defined.

[0020] In the present invention, "prognosis" refers to the expected future medical outcome of a patient's disease (e.g., overall survival rate, recurrence-free survival rate, etc.), improvement of the disease (e.g., recovery of the disease, etc.), degree of improvement of the disease (e.g., tumor regression, etc.), or a positive prognosis including disease stability, a negative prognosis including disease recurrence, degree of disease progression or lethality (e.g., tumor growth, metastasis, drug resistance, etc.).

[0021] As used herein, the term "prediction" means to estimate in advance the future medical outcome of a patient's disease, and includes prediction of the progression of a patient's disease (e.g., improvement, stabilization, relapse, progression, lethality, deterioration, etc.).

[0022] In the present invention, the "biological sample" refers to a tissue obtained from a diffuse gastric cancer patient or a normal human individual, and specifically, may be a gastric tissue.

[0023] Hereinafter, the present invention will be described in detail.

[0024] 1. Composition and kit for diagnosis or prognosis prediction of diffuse gastric cancer The present invention provides a composition for diagnosing diffuse gastric cancer or for predicting the prognosis of diffuse gastric cancer.

[0025] The composition for diagnosis or prognosis prediction of diffuse gastric cancer includes an agent for measuring the expression level of the SYT11 gene.

[0026] When gastric cancer is classified according to Lauren classification based on pathological and histological characteristics, the diffuse type gastric cancer, which is distinguished from the intentional type gastric cancer, is a gastric cancer that exhibits the characteristic of individual cancer cells infiltrating the stomach wall without forming a mass with low adhesion of cancer cells. The intentional type gastric cancer is a gastric cancer that exhibits the characteristic of cancer cells gathering in one location and growing in a mass form.

[0027] The SYT11 gene (Synaptotagmin 11, ILMN_1717934) is a member of the synaptotagmin gene family, encoding a protein similar to other family member proteins known as calcium sensors. The protein expressed by the SYT11 gene can play a role in regulating the calcium-dependent regulation of membrane transport during synaptic transmission. The base sequence of the SYT11 gene and the amino acid sequence of the protein expressed from the SYT11 gene can be obtained from a publicly available database or literature such as the GenBank of the National Center for Biotechnology Information (NCBI) of the United States.

[0028] The composition for diagnosing or predicting the prognosis of diffuse gastric cancer may further include at least one agent for measuring the expression level of a gene selected from the group consisting of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and VIMENTIN.

[0029] The angiopoietin-like protein 2 (ANGPTL2) gene (Angiopoietin-like protein 2, ILMN_1772612), calcium voltage-gated channel auxiliary subunit β1 (CACNB1) gene (Calcium Voltage-Gated ChannelAuxiliary Subunit Beta 1, ILMN_2271149), CC motif chemokine ligand 19 (CCL19) gene (C-CMotif Chemokine Ligand 19, ILMN_1769129), CD34 gene (ILMN_1732799), junctional adhesion molecule C (JAM3) gene (Junctional adhesion molecule C, ILMN_1769575), Slit homolog 2 protein (SLIT2) gene (Slit homolog 2 The base sequences of the thrombospondin-4 (THBS4) gene (ILMN_1736078) and the vimentin (VIMENTIN) gene (ILMN_1782538) and the amino acid sequences of the proteins expressed from the genes can be obtained from publicly available databases or literature such as GenBank of NCBI.

[0030] The preparation for measuring the expression level of the gene may be a preparation for measuring the amount of mRNA present in the gene or the amount of protein expressed by the gene.

[0031] The preparation for measuring the amount of mRNA in the gene refers to a preparation that can specifically bind to the mRNA in the gene to identify or amplify its amount, including a primer pair or probe that specifically binds to the base sequence of the mRNA or cDNA obtained by reverse transcription of the mRNA.

[0032] The primer is a nucleic acid sequence that forms a base pair with a complementary template strand using a short nucleic acid sequence with a free 3' terminal hydroxyl group, thereby providing a starting point when the nucleic acid polymerase replicates the template strand for amplification. In addition, the probe refers to a nucleic acid fragment with a length of several bases to several hundred bases, which is composed of a sequence that can form a specific bond with mRNA or cDNA.

[0033] The amount of mRNA in the gene can be measured by using sense primers and antisense primers of the gene sequence by performing polymerase chain reaction (PCR), 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 RT-PCR) and the like, and can be measured by using a probe having a sequence that can form a specific bond with the mRNA of the gene or the cDNA obtained by reverse transcription by performing Northern blot hybridization (Northern blot), microarray and the like. Furthermore, it can be measured by RNase protection assay, base sequence analysis and the like, but is not limited thereto, and any preparation required for any method known to a person of ordinary skill in the art can be used.

[0034] The agent for measuring the amount of the protein expressed from the gene refers to an agent capable of specifically binding to and recognizing the protein, including an antibody or a nucleic acid aptamer that specifically binds to the protein.

[0035] The antibody refers to an immunoglobulin molecule that is reactive by specifically binding to the epitope of the protein in immunology, and may include monoclonal antibodies, polyclonal antibodies, antibodies with full-length chain structures, antibodies with functional fragments that have at least antigen binding function, and recombinant antibodies. The nucleic acid aptamer refers to a single-stranded nucleic acid molecule that has the characteristic of being able to form specific binding with the protein as a target and form a stable three-dimensional structure. The nucleic acid aptamer that is specific to the protein can be synthesized using the exponential enrichment ligand systematic evolution (SELEX: Systematic Evolution of Ligands by Exponential enrichment) technology and the like.

[0036] The amount of protein expressed from the gene can be measured using the antibody or nucleic acid aptamer by Western blot, protein microarray (protein chip), enzyme-linked immunosorbent assay (ELISA), 2-dimentional electrophoresis, immunochemical staining (IHC), immunofluorescence, co-immunoprecipitation, fluorescence activated cell sorter (FACS), radioimmunoassay (RIA), radioimmunodiffusion, matrix-assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF), etc., but is not limited thereto, and any preparation required by any method known to a person of ordinary skill in the art can be used.

[0037] The composition containing the preparation for measuring the expression level of the SYT11 gene can be used for providing information required for diagnosing diffuse gastric cancer. In a specific embodiment of the present invention, it was confirmed that the expression level of the SYT11 gene is higher in patients with diffuse gastric cancer, unlike intestinal gastric cancer. Based on this, the composition of the present invention can be used for the following purpose: by measuring the expression level of the SYT11 gene in patients whose gastric cancer is unknown or whose histological classification is unknown, when the expression level of the SYT11 gene is high, providing information for diagnosing diffuse gastric cancer ( Figure 1 and Figure 2 ).

[0038] Furthermore, a composition comprising at least one of a preparation for measuring the expression level of the SYT11 gene or a preparation for measuring the expression level of a gene selected from the group consisting of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and VIMENTIN can be used for predicting the prognosis of patients with diffuse gastric cancer. When the expression level of the SYT11 gene and the expression level of at least one gene selected from the group consisting of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and VIMENTIN are measured, if the result is that the expression level of the gene is high, it can be predicted that the prognosis of patients with diffuse gastric cancer is poor, and strategies and measures for treatment can be formulated based on this. In this regard, in a specific example of the present invention, it was confirmed that the expression level of the SYT11 gene was higher in patients with diffuse gastric cancer, and the survival probability of patients with diffuse gastric cancer with high expression levels of the SYT11 gene was higher than that of patients with diffuse gastric cancer with low expression levels of the SYT11 gene ( Figure 3 ). In addition, in patients with diffuse gastric cancer with high expression levels of the SYT11 gene, the expression levels of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and VIMENTIN genes were also measured to be high ( Figure 4 ), and confirmed that the expression level of the gene is also correlated with the survival probability of patients with diffuse gastric cancer ( Figure 5 ).

[0039] The invention provides a kit for diagnosing diffuse gastric cancer or predicting the prognosis of diffuse gastric cancer.

[0040] The kit includes the composition for diagnosis or prognosis prediction of diffuse gastric cancer.

[0041] The kit may include a composition comprising an agent for measuring the expression level of the SYT11 gene, or a composition comprising at least one of an agent for measuring the expression level of the SYT11 gene and an agent for measuring the expression level of a gene selected from the group consisting of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and VIMENTIN.

[0042] The kit may also include other components suitable for measuring the amount of mRNA in the gene. For example, the kit may also include the form of an RT-PCR kit or a DNA chip kit, or the composition of the RT-PCR kit or the DNA chip kit, as long as it is a kit in a form known to those of ordinary skill in the art for measuring the amount of mRNA, it can be applied to the kit of the present invention.

[0043] Specifically, the kit may be a kit including the necessary components required for performing RT-PCR. For example, in addition to the primer pair that can specifically bind to the mRNA of the gene, it may include a test tube or other suitable container, enzymes such as Taq-polymerase and reverse transcriptase, a reaction buffer solution (pH and magnesium concentration are various), deoxynucleotides (dNTP), deoxyribonuclease (DNase) and ribonuclease (RNase) inhibitors, DEPC-water (DEPC-water), sterile water, etc. In addition, it may include a primer pair that can specifically bind to a gene used as a control group during quantification.

[0044] In addition, the kit may include the necessary components for performing DNA chip, and may include reagents, enzymes, preparations, etc. for preparing a glass substrate attached with oligonucleotides capable of specifically binding to the mRNA or reverse transcribed cDNA of the gene and fluorescent labeled probes.

[0045] The kit may include a composition, solution or device of one or more other components suitable for measuring the amount of protein expressed by the gene. For example, the kit may also include the form of an ELISA kit or a protein chip kit, or the composition of the ELISA kit or the protein chip kit, as long as it is a kit in a form known to those of ordinary skill in the art for measuring the amount of protein, it can be applied to the kit of the present invention.

[0046] Specifically, the kit may be a kit including the necessary components required for performing ELISA. For example, it may include a substrate for immunological detection of antibodies, a suitable reaction buffer, a reagent that can detect the formation of an antigen-antibody complex using an antibody that can specifically bind to the protein expressed by the gene, wherein an enzyme that mediates a color development reaction, a secondary antibody labeled with a fluorescent substance, and a color development substrate may be included. Examples of enzymes that mediate the color development reaction may include alkaline phosphatase, acid phosphatase, and peroxidase, and examples of the fluorescent substance may include colloidal gold, fluorescein carboxylic acid (FCA), fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RITC), fluorescein thiourea (FTH), and the like. thiourea), 7-acetoxycoumarin-3-yl, fluorescein-5-yl, fluorescein-6-yl, 2',7'-dichlorofluorescein-5-yl, 2',7'-dichlorofluorescein-6-yl, dihydrotetramethylrosamine-4-yl, tetramethylrhodamine-5-yl, tetramethylrhodamine-6-yl, 4,4-difluoro-5,7-dimethyl-4-borane-3a,4a-diaza-indacen-3-ethyl or 4,4-difluoro-5,7-diphenyl-4-borane-3a,4a-diaza-s-indacen-3-ethyl, etc. In addition, an antibody that specifically binds to a protein expressed by a gene used as a control group in quantification may be included. The ELISA may be direct ELISA, indirect ELISA or competitive ELISA.

[0047] In addition, the kit may be a kit including the necessary components required for performing protein chip. For example, one or more antibodies that can specifically bind to the protein expressed by the gene may be fixedly arranged at a high density at a predetermined position on the substrate. In addition, an antibody that specifically binds to the protein expressed by the gene used as a control group during quantification may be included.

[0048] 2. Method for providing information required for diagnosing diffuse gastric cancer or determining a treatment strategy for diffuse gastric cancer The present invention provides a method for obtaining information required for diagnosing diffuse gastric cancer or determining a treatment strategy for diffuse gastric cancer.

[0049] The method for providing information required for diagnosing diffuse gastric cancer or determining a treatment strategy for diffuse gastric cancer comprises the following steps: measuring the expression level of the SYT11 gene from an isolated biological gastric tissue sample. The information required for determining a treatment strategy for diffuse gastric cancer can be selected from at least one of the group consisting of a prognosis prediction for diffuse gastric cancer; a prognosis evaluation for diffuse gastric cancer; and an effectiveness evaluation of a SYT11 targeted therapeutic agent in gastric cancer patients.

[0050] Furthermore, the method may further include the step of measuring the expression level of at least one gene selected from the group consisting of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4 and VIMENTIN from the isolated biological gastric tissue sample.

[0051] The step of measuring the expression level of the gene may be measuring the amount of mRNA present of the gene or the amount of protein expressed by the gene.

[0052] The amount of mRNA present in the gene can be measured by using a preparation that can specifically bind to the mRNA in the gene and can identify or amplify its amount. As a specific example, the measurement can be performed using a primer pair or a probe that specifically binds to the base sequence of the mRNA or cDNA obtained by reverse transcription of the mRNA, and can be measured using PCR, RT-PCR, competitive RT-PCR, real-time RT-PCR, Northern blot, microarray, RNase protection assay, base sequence analysis, etc., but is not limited thereto, and can be measured using any method known to a person of ordinary skill in the art.

[0053] Measuring the amount of a protein expressed by the gene can be performed by using an agent that can specifically bind to and recognize the protein. As a specific example, the measurement can be performed using an antibody or aptamer that specifically binds to the protein, and can be performed using methods such as Western blot, protein microarray (protein chip), enzyme-linked immunosorbent assay (ELISA), 2-dimentional electrophoresis, immunochemical staining (IHC), immunofluorescence, co-immunoprecipitation assay, fluorescence activated cell sorter (FACS), radioimmunoassay (RIA), radioimmunodiffusion, matrix-assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF), but is not limited thereto, and can be measured using any method known to a person of ordinary skill in the art.

[0054] According to a specific embodiment of the present invention, the SYT11 gene is expressed at a high level in patients with diffuse gastric cancer, thereby showing a statistically significant relationship, while the above-mentioned results are not shown in patients with intestinal gastric cancer ( Figure 2 ). When the expression level of the SYT11 gene in the biological gastric tissue sample to be analyzed is higher than the expression level of the SYT11 gene in the normal control group, the analysis object can be determined to be diffuse gastric cancer. Therefore, the information required for diagnosing diffuse gastric cancer can be provided by the above method.

[0055] Furthermore, according to a specific embodiment of the present invention, diffuse gastric cancer patients with a higher expression level of the SYT11 gene had a higher probability of survival than diffuse gastric cancer patients classified as a group with a low expression level, thereby confirming that the expression level was statistically significantly associated with the prognosis of diffuse gastric cancer, and that such a relationship was not significant in intestinal gastric cancer patients ( Figure 3). In addition, it was confirmed that the expression level of the SYT11 gene and any one of the above-mentioned ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4 and VIMENTIN in patients with diffuse gastric cancer with high expression levels were also correlated with the survival probability ( Figure 5 ). Therefore, when the expression level of the gene is measured in a sample isolated from a patient with diffuse gastric cancer, it can be predicted that the prognosis will not be poor when the expression level is high.

[0056] The method of the present invention may further include the step of comparing the expression level of the gene measured from the biological gastric tissue sample with a reference value measured from a control group sample.

[0057] The control group sample can be a sample isolated from a group that has not suffered from diffuse gastric cancer, a sample isolated from a group of patients with diffuse gastric cancer, or a group of patients with diffuse gastric cancer who have been administered a SYT11 targeted therapeutic agent, but is not limited thereto. For the purpose of diagnosing diffuse gastric cancer or determining a treatment strategy, any sample isolated from a group that can generally be compared in the technical field of the present invention can be used without restriction.

[0058] The reference value measured from the control group sample refers to the benchmark of the value / data obtained by measuring the expression level of the gene with the control group sample as the object, and can be a value used as a benchmark to distinguish whether the expression level of the gene expressed in the sample to be measured is overexpressed or underexpressed. The reference value can be an absolute value; a relative value; a value with an upper limit and / or a lower limit; a range of values; an average value; an intermediate value; a median or a value compared with a specific control group or a reference value. The reference value can be set according to the method commonly used in the technical field of the present invention, and various statistical methods and software for processing statistics can be used, but are not limited to this.

[0059] The diagnosis of diffuse gastric cancer can be a diagnosis of whether the individual being diagnosed is a normal individual who does not suffer from gastric cancer or an individual suffering from diffuse gastric cancer, or a diagnosis of whether the gastric cancer suffered by the individual being diagnosed is diffuse gastric cancer. More specifically, it can be a diagnosis of whether the gastric cancer suffered by the individual being diagnosed is diffuse gastric cancer or intestinal gastric cancer.

[0060] The prognosis prediction of diffuse gastric cancer may be a prediction of whether the prognosis is good or bad, which may be a prediction of the possibility of cure, possibility of improvement, possibility of recurrence, possibility of death, etc. The prognosis prediction may be a prediction of the survival possibility of diffuse gastric cancer patients, for example, based on the average or median survival time of a group of diffuse gastric cancer patients, it may be predicted whether the patient will survive for a relatively long period of time or a relatively short period of time.

[0061] The prognostic evaluation of diffuse gastric cancer is an evaluation of how the prognosis described in relation to the prognostic prediction appears in diffuse gastric cancer patients. For example, it can be an assessment of whether the prognosis improves or worsens over time for patients who are treated with a specific treatment method or medication or for patients who are not treated with it.

[0062] The method of the present invention may further include the step of determining diffuse gastric cancer when the expression level of the gene measured from the biological gastric tissue sample is higher than the average or median value of the expression level of the gene measured from a group that has never suffered from diffuse gastric cancer.

[0063] In addition, the method may also include the following steps: when the expression level of the gene measured from the biological gastric tissue sample is higher than the average or median value of the expression level of the gene measured from a group that has never suffered from diffuse gastric cancer; or higher than the average or median value of the expression level of the gene measured from patients with diffuse gastric cancer, it is determined to be a poor prognosis.

[0064] The evaluation of the effectiveness of the SYT11 targeted therapeutic agent in the gastric cancer patient may include at least one selected from the group consisting of the necessity of administering the SYT11 targeted therapeutic agent; the possibility of expressing drug resistance to the SYT11 targeted therapeutic agent; the sensitivity to the SYT11 targeted therapeutic agent; and the prognosis prediction based on the treatment of the SYT11 targeted therapeutic agent.

[0065] The effectiveness evaluation of the SYT11 targeted therapeutic agent may be one of the diagnostic tests for confirming the possibility of various results of applying the SYT11 targeted therapeutic agent to a specific diffuse gastric cancer patient, and may be called a companion diagnostic.

[0066] The SYT11 targeted therapeutic agent may be, for example, a SYT11 inhibitor, which is used to collectively refer to all preparations that reduce the expression or activity of SYT11, and specifically may include all agents that reduce the expression of SYT11, directly act on SYT11, or indirectly act on its ligand, thereby reducing the expression amount, mRNA level or translation level of transcription SYT11, or hinder the activity of SYT11, thereby reducing the activity of SYT11.

[0067] The SYT1 inhibitor may be a compound, nucleic acid, peptide, virus or vector including the nucleic acid that can inhibit the expression of SYT11 or the activity of SYT11, and the form thereof is not limited. The SYT11 inhibitor is not limited thereto and may include siRNA or shRNA that degrades the mRNA of the SYT11 gene, and antisense oligonucleotides that reduce the expression of the SYT11 protein. In addition, the SYT11 inhibitor that binds to the SYT11 protein to inhibit the function may be a nucleic acid aptamer or a low molecular weight compound.

[0068] In a specific embodiment of the present invention, it was confirmed that the expression level of the SYT11 gene in diffuse gastric cancer patients and the expression level of the gene selected from the group consisting of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4 and VIMENTIN showed the same pattern, thus having a statistically significant correlation. In addition, it was confirmed that the expression level of the SYT11 gene and the expression level of the gene selected from the group consisting of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4 and VIMENTIN were also closely correlated with the prognosis of diffuse gastric cancer patients and showed the same pattern. Therefore, when a therapeutic agent for diffuse gastric cancer targeting SYT11 is administered, the expression level of a gene selected from the group consisting of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4 and VIMENTIN is measured, so that the measurement results of the expression levels can be used in evaluating the possibility of expression of drug resistance to the SYT11 targeted therapeutic agent; sensitivity to the SYT11 targeted therapeutic agent; and prognosis prediction based on treatment with the SYT11 targeted therapeutic agent.

[0069] The necessity of administration is to determine whether the therapeutic agent is administered to an individual who can obtain the therapeutic effect for the therapeutic agent or an individual who is predicted to be likely to obtain the therapeutic effect.

[0070] The method may further include the following step: when the expression level of a gene selected from the group consisting of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4 and VIMENTIN measured in a biological gastric tissue sample isolated from a diffuse type patient is higher than a reference value, determining that the individual providing the sample may correspond to at least one of the following situations: requiring administration of a SYT11 targeted therapeutic agent; having a low possibility of expressing drug resistance to the SYT11 targeted therapeutic agent; having a high sensitivity to the SYT11 targeted therapeutic agent; or having a good prognosis based on treatment with the SYT11 targeted therapeutic agent.

[0071] In a specific example of the present invention, a biological gastric sample obtained from a diffuse gastric cancer patient is used as an object, and the SYT11 gene expression is confirmed. After measuring the expression level of at least one gene selected from the group consisting of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4 and VIMENTIN, it can be determined whether to prescribe a SYT11 targeted therapeutic agent. Specifically, when the expression level of the gene of the patient is measured to be above the reference value, it can be predicted that the patient has high sensitivity to the SYT11 targeted therapeutic agent, or low resistance to the SYT11 targeted therapeutic agent, thereby providing information required for determining whether to prescribe a SYT11 targeted therapeutic agent.

[0072] In addition, according to a specific embodiment of the present invention, when a gastric cancer cell line is treated with a SYT11 targeted inhibitor (e.g., siSYT11 or SYT11-ASO) that targets SYT11 and inhibits its expression or activity, not only the amount of SYT11 protein in the cell is reduced, but also the amounts of ANGPL2, THBS4, VIMENTIN, and JAM3 proteins are reduced ( Figure 6 and Figure 7 ). Therefore, it can be seen that the marker is regulated so that the expression or activity is suppressed simultaneously with the inhibition of SYT11. When the SYT11 targeted therapeutic agent is administered, the sensitivity, resistance, etc. to the SYT11 targeted therapeutic agent can be confirmed by monitoring the marker, and the marker can be used to evaluate the effectiveness of the SYT11 targeted therapeutic agent.

[0073] Hereinafter, the details will be described based on embodiments and experimental examples.

[0074] However, the following Examples and Experimental Examples are examples for specifically illustrating the present invention, and the contents of the present invention are not limited to the following Examples and Experimental Examples.

[0075] [Example 1] Confirmation of the correlation between SYT11 gene expression level and diffuse gastric cancer The expression levels of the SYT11 gene expressed in gastric cancer tissue and normal human gastric tissue were measured and compared, thereby confirming the correlation between the expression level of the SYT11 gene and gastric cancer.

[0076] Cancer and normal tissues were purchased from Biomax US (USA). Immunohistochemistry was performed on 70 gastric cancer tissues and 10 normal tissues to confirm the expression level of SYT11.

[0077] Specifically, in order to remove the paraffin of each tissue, it was immersed in a xylene solution for 10 minutes, and then in order to remove the residual xylene, it was immersed in 100%, 95%, 80%, and 70% ethanol solutions for 5 minutes, and then washed with water. The tissue slide was immersed in a 3% hydrogen peroxide (H2O2) solution for 10 minutes, and then rinsed in water for 15 minutes. After boiling a 0.01M sodium citrate solution to 95°C, the tissue slide was placed, left for 10 minutes, and then cooled at room temperature for 30 minutes. After washing with water for 10 minutes, the sample prepared by immersing in a buffer solution (phosphate buffered saline) for 10 minutes was reacted with anti-SYT11 antibody, biotin-conjugated secondary antibody (biotin-conjugated secondary antibody, vector lab, USA), and avidin + biotinylated horseradish peroxidase (Abidin + Biotinylated horseradish peroxidase, vector lab, USA), and the expression level of SYT11 protein was confirmed using DAB peroxidase substrate (Vectorlab, USA). In addition, in order to distinguish the nuclear part of the tissue, the tissue slide was reacted in a hematoxylin solution for 5 minutes and then washed with water. Then, the tissue slide was immersed in 0.1% hydrogen chloride (HCl) and 0.1% ammonia (NH4OH) aqueous solution in sequence and washed with water. The tissue slide was immersed in 70%, 85%, 95%, and 100% ethanol for 5 minutes in sequence, and then dried after reacting in a xylene solution for 5 minutes.

[0078] According to the degree of coloration of the SYT11 gene measured by immunohistochemistry, the SYT11 gene expression level was classified into a group with a low expression level (SYT11-low) and a group with a high expression level (SYT11-high) based on the median or average value. As a result, the expression level of the SYT11 gene was high in only 30% of normal gastric tissues, but the expression level of the SYT11 gene was high in 64.3% of gastric cancer patients' gastric tissues. Therefore, it can be confirmed that the expression level of the SYT11 gene is correlated with the occurrence of gastric cancer ( Figure 1 ).

[0079] In addition, the 70 gastric cancer tissues were histologically classified into 30 intestinal type gastric cancer tissues and 40 diffuse type gastric cancer tissues, and the expression level of the SYT11 gene was measured as a color level, and similarly classified into a group with a low expression level of the SYT11 gene (SYT11-low) and a group with a high expression level (SYT11-high). As a result, in the intestinal type gastric cancer tissue, the expression level of the SYT11 gene was high in 46.7% of the patients, but in the gastric tissue of the diffuse type gastric cancer patient, the expression level of the SYT11 gene was high in 77.5% of the patients. Therefore, it can be confirmed that the expression level of the SYT11 gene is associated with diffuse type gastric cancer ( Figure 1 ).

[0080] [Example 2] Comparison of SYT11 gene expression levels in diffuse gastric cancer and intestinal gastric cancer The microarray results of 239 patients with intestinal gastric cancer and 185 patients with diffuse gastric cancer obtained from Yonsei University Severance Hospital were analyzed. The receiver operating characteristic curve based on the SYT11 expression level in the microarray results was analyzed, and the gastric cancer patient group was classified into a high or low SYT11 expression level group based on the median or average value. In addition, whether the gastric cancer was stem-like gastric cancer or non-stem-like gastric cancer was also confirmed.

[0081] The results showed that SYT11 gene expression levels were high in 77 out of 239 patients with intestinal type gastric cancer, and SYT11 gene expression levels were high in 27 out of 31 patients with stem cell-like cancer. In contrast, SYT11 gene expression levels were high in 100 out of 185 patients with diffuse type gastric cancer, and SYT11 gene expression levels were high in 67 out of 80 patients with stem cell-like cancer ( Figure 2 ).

[0082] The above results not only reconfirmed that the expression of the SYT11 gene is associated with stem-like gastric cancer as in previous studies, but also newly confirmed that the expression of the SYT11 gene is lower in intestinal type gastric cancer and higher in diffuse type gastric cancer. Therefore, it can be confirmed that the expression level of the SYT11 gene is associated with diffuse type gastric cancer and can be used as a diagnostic marker for diffuse type gastric cancer.

[0083] [Example 3] Confirmation of the correlation between the expression level of SYT11 gene and the prognosis of patients with diffuse gastric cancer Based on the clinical data of 424 gastric cancer patients in Example 2, the correlation between the survival probability of gastric cancer patients and the expression level of the SYT11 gene was analyzed.

[0084] Specifically, 239 patients with intestinal type gastric cancer and 185 patients with diffuse type gastric cancer were classified, and based on the expression levels of the SYT11 gene and SYT4 gene, the microarray results of these patients were used to divide them into a group with high expression levels and a group with low expression levels. Furthermore, the survival probability data of each group were compared to confirm the correlation between the expression level of the gene and the prognosis of gastric cancer patients.

[0085] The results, such as Figure 3 As shown, among 239 patients with intestinal type gastric cancer, there was no statistically significant difference in survival probability between the group with high SYT11 gene expression level and the group with low SYT11 gene expression level (p=0.3363), but among 185 patients with diffuse type gastric cancer, the group with high SYT11 gene expression level showed a significantly lower survival probability (p=0.0067), confirming that the expression level of the SYT11 gene is closely related to the prognosis of diffuse type patients. In addition, from the results of the control group in which the relationship between the expression level of the SYT4 gene belonging to the same family as the SYT11 gene and the survival probability was confirmed, it was confirmed that the prognosis of diffuse type patients was not significantly correlated with the expression level of the SYT4 gene (p=0.1618), confirming that diffuse type gastric cancer is only related to the expression level of the SYT11 gene.

[0086] [Example 4] Discovery and confirmation of 8 companion diagnostic markers and their association with diffuse gastric cancer Through the above-described examples, it was confirmed that the expression level of the SYT11 gene was high in patients with diffuse gastric cancer, and that diffuse patients with high expression of the SYT11 gene had a poor prognosis. Thus, by confirming genes whose expression levels were regulated in the same tendency as the SYT11 gene, eight new markers ( Figure 4 ).

[0087] Specifically, based on the microarray results of gastric cancer patients, the receiver operating characteristic curves of the expression levels of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and VIMENTIN genes were analyzed, and then the patients were divided into a high expression level group and a low expression level group based on the expression level of each gene and the median and average values. In addition, the survival rate of patients based on the expression levels of SYT11 and the above genes was presented in the form of a Kaplan-Meier graph.

[0088] As in Figure 4 It was confirmed that in patients with high expression of the SYT11 gene, the expression levels of eight genes, including ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and VIMENTIN, also increased in proportion to the expression level of the SYT11 gene, showing a statistically significant relationship.

[0089] Furthermore, the survival probability of patients with intestinal type gastric cancer and diffuse type gastric cancer was confirmed when the expression levels of the SYT11 gene and the ANGPL2 gene were both high (SYT11(H)-ANGPL2(H)) or both were low (SYT11(L)-ANGPL2(L)), and the correlation between the other 7 gene markers except the ANGPL2 gene and the survival probability was compared in the same way. The results showed that the survival probability of diffuse type patients with high expression levels of the SYT11 gene and each of the above 8 genes was low and the prognosis was poor, and the results were also statistically significant. On the contrary, in intestinal type gastric cancer, it was confirmed that there was no significant correlation between the survival probability and the expression level in all the 8 markers, and it was found that the 8 gene markers were correlated with the expression level of the SYT11 gene only in diffuse type gastric cancer ( Figure 5 ).

[0090] [Example 5] Confirmation of expression levels of companion diagnostic markers based on treatment with SYT11-targeted therapeutic agents Gastric cancer cell line SNU484 cells were used as the target, and siSYT11 (5'-GCAGAAAGCGCAUUGCCAA(dTdT)-3', d represents deoxyribonucleic acid) as a type of siRNA was transfected (transfection) to introduce a SYT11 targeted therapeutic agent that can bind to the mRNA of the SYT11 gene and inhibit its expression. As a control group, siScramble (5'-CCUACGCCACCAAUUUCGU(dTdT)-3', d represents deoxyribonucleic acid) was transfected. 48 hours after the introduction, proteins were extracted using radioimmunoprecipitation assay (RIPA buffer) buffer, separated by electrophoresis (SDS-PAGE) on polyacrylamide gel, and Western blotting was performed using each antibody of the companion diagnostic marker gene discovered in Example 4.

[0091] As a result, it was confirmed that as the amount of SYT11 protein in cells was reduced using siSYT11, a SYT11-targeted therapeutic agent, the amounts of ANGPL2, THBS4, VIMENTIN2, and JAM3 were also reduced ( Figure 6 ).

[0092] Furthermore, the gastric cancer cell line SNU484 was used as the target, and SYT11-ASO (5'-mU mxD G G C A A T G C G C T T T C T mxD mC-3', m means methylated, As a control group, NC-ASO (5'-mC mxD T A C G C C A C C A A T T T C mxD mU-3', m means methylated, (denotes phosphorothioate). 48 hours after the introduction, proteins were extracted using RIPA buffer and separated by SDS-PAGE electrophoresis on polyacrylamide gel, and then Western blotting was performed using antibodies against the companion diagnostic marker genes discovered in Example 4.

[0093] As a result, similar to the case of using siSYT11, it was confirmed that as the amount of SYT11 protein in cells was reduced using SYT11-ASO, the amounts of ANGPL2, THBS4, VIMENTIN2, and JAM3 were also reduced ( Figure 7 ).

[0094] From the above results, it can be expected that ANGPL2, THBS4, VIMENTIN2 and JAM3 are hypostatic genes of SYT11 regulated by SYT11, and for the genes of Example 4 that have a significant correlation with the expression level of the SYT11 gene, when regulating the expression of SYT11, it can be expected that their expression levels will be regulated together. Therefore, when using a targeted therapeutic agent that reduces SYT11 expression or inhibits SYT11 activity, by measuring the expression levels of ANGPL2, THBS4, VIMENTIN and JAM3 to confirm whether they are reduced, it is possible to monitor the therapeutic effect or resistance of the SYT11 targeted therapeutic agent, and it is expected that the genes can be used as companion diagnostic markers for SYT11 targeted therapeutic agents.

[0095] [Example 6] in conclusion Based on the results of the above-mentioned embodiments, the expression level of the SYT11 gene in patients with diffuse gastric cancer is high, and it can be confirmed that the SYT11 gene can be used as a marker for diagnosing diffuse gastric cancer. In addition, it is confirmed that the expression level of the SYT11 gene is also associated with the prognosis of diffuse patients. When the expression level of the SYT11 gene is high, it can be predicted that the prognosis of diffuse patients is poor. In addition, it can be seen that the expression level of the SYT11 gene in patients with diffuse gastric cancer has a similar tendency to the expression levels of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4 and VIMENTIN 8 genes, and is associated, thereby confirming that it is also associated with the prognosis of patients with diffuse gastric cancer. Therefore, it is confirmed that the 8 genes can also be used as markers for predicting the prognosis of diffuse gastric cancer.

[0096] Furthermore, existing studies have shown that SYT11 can be used as a target for treating gastric cancer, and it can be confirmed experimentally that when a gastric cancer cell line is treated with siSYT11 or SYT11-ASO that can reduce the amount of SYT11, the amount of the marker gene is also reduced. Therefore, for the treatment of diffuse gastric cancer using SYT11 as a target, it can be confirmed that the eight markers can be used as companion diagnostic markers for obtaining information such as whether SYT11 targeted therapy is needed, sensitivity prediction to administration of SYT11 targeted therapeutic agents, and prognosis prediction after administration of therapeutic agents.

[0097] The present invention has only been described in detail with respect to the described embodiments. However, it is obvious to those skilled in the art that various deformations and modifications can be made within the technical concept of the present invention, and such deformations and modifications obviously fall within the scope of the claims.

Claims

1. A composition for diagnosis or prognosis prediction of diffuse gastric cancer, wherein: A preparation for measuring the expression level of the SYT11 gene is included.

2. The composition for diagnosis or prognosis prediction of diffuse gastric cancer according to claim 1, further comprising at least one agent for measuring the expression level of a gene selected from the group consisting of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4 and VIMENTIN.

3. The composition for diagnosis or prognosis prediction of diffuse gastric cancer according to claim 1 or 2, wherein: The preparation for measuring the expression level of the gene is at least one of a preparation for measuring the amount of mRNA of the gene and a preparation for measuring the amount of protein expressed by the gene.

4. The composition for diagnosis or prognosis prediction of diffuse gastric cancer according to claim 3, wherein: The preparation for measuring the amount of the mRNA of the gene includes a primer pair or a probe that specifically binds to the mRNA or cDNA of the gene.

5. The composition for diagnosis or prognosis prediction of diffuse gastric cancer according to claim 3, wherein: The agent for measuring the amount of the protein expressed by the gene includes an antibody or a nucleic acid aptamer specific to the protein expressed by the gene.

6. A kit for diagnosing or predicting the prognosis of diffuse gastric cancer, wherein: The kit comprises the composition according to any one of claims 1 to 5.

7. A method for providing information required for diagnosing diffuse gastric cancer or determining a treatment strategy for diffuse gastric cancer, the method comprising the step of measuring the expression level of the SYT11 gene from an isolated biological gastric tissue sample, wherein: The information required to determine the treatment strategy for the diffuse gastric cancer is at least one selected from the group consisting of: Prognosis prediction for diffuse gastric cancer; Prognostic evaluation of diffuse gastric cancer; and Evaluation of the effectiveness of SYT11-targeted therapeutics in patients with gastric cancer.

8. The method for providing information required for diagnosing diffuse gastric cancer or determining a treatment strategy for diffuse gastric cancer according to claim 7, further comprising the following steps: The expression level of at least one gene selected from the group consisting of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4 and VIMENTIN is measured from the isolated biological gastric tissue sample.

9. The method for providing information required for diagnosing diffuse gastric cancer or determining a treatment strategy for diffuse gastric cancer according to any one of claims 7 or 8, further comprising the following steps: The expression level of the gene measured from the biological gastric tissue sample is compared with a reference value measured from a control group sample.

10. The method for providing information required for diagnosing diffuse gastric cancer or determining a treatment strategy for diffuse gastric cancer according to claim 9, further comprising the following steps: When the expression level of the gene measured from the biological gastric tissue sample is higher than the average or median value of the expression levels of the gene measured from a group of people who do not suffer from diffuse gastric cancer, it is determined that the person suffers from diffuse gastric cancer.

11. The method for providing information required for diagnosing diffuse gastric cancer or determining a treatment strategy for diffuse gastric cancer according to claim 9, further comprising the following steps: When the expression level of the gene measured from the biological gastric tissue sample is higher than the average or median value of the expression levels of the gene measured from a group of patients who do not suffer from diffuse gastric cancer; or higher than the average or median value of the expression levels of the gene measured from a group of patients with diffuse gastric cancer, it is judged to be a poor prognosis.

12. The method for providing information required for diagnosing diffuse gastric cancer or determining a treatment strategy for diffuse gastric cancer according to claim 7, wherein: The evaluation of the effectiveness of the SYT11 targeted therapeutic agent for the gastric cancer patient is to evaluate at least one selected from the group consisting of: the necessity of administering SYT11-targeted therapeutics; the potential for expression of drug resistance to SYT11-targeted therapeutics; sensitivity to SYT11-targeted therapeutics; and Prognostic prediction based on treatment with SYT11-targeted therapeutic agents.

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