Prognostic diagnostic marker and treatment target for diffuse-type gastric cancer

Abstract

The present invention provides a composition and a kit for diagnosing or predicting the prognosis of diffuse type gastric cancer, which contain a preparation for measuring the expression level of the SYT11 gene. Further, in the present invention, a method for providing information necessary for diagnosing diffuse type gastric cancer and information necessary for determining a treatment strategy for diffuse type gastric cancer is provided.

Description

Technical Field

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

Background Art

[0002] Gastric cancer is a cancer with the highest incidence rate among Koreans in the world, and is one of the diseases with a great need for prevention, treatment, and prognosis prediction. Gastric cancer is classified into intestinal type and diffuse type according to the Lauren classification based on pathology and histology. Diffuse type gastric cancer grows in a shape that spreads under the gastric mucosa and has the characteristic that cancer cells infiltrate the gastric wall. However, in the case of diffuse type gastric cancer, it has a characteristic of poor survival rate compared to intestinal type gastric cancer. In particular, the incidence rate is high among young people in their 20s and 30s where the awareness of cancer and the opportunity for health check-ups are relatively insufficient, and it is difficult to be detected in the early stage of onset. Therefore, there is an even higher need to detect the onset of diffuse type gastric cancer at an early stage, and there is an increasing need for research on methods for diagnosing diffuse type gastric cancer among gastric cancers in order to establish a treatment strategy.

[0003] To date, as a test method for diagnosing gastric cancer, physical means such as X-ray imaging and gastroscopy have been mostly used, but this has the disadvantage that it is accompanied by pain for the patient undergoing the test and requires a cumbersome process. As a result, research has been conducted on gene markers that are specifically expressed or have an increased expression level in gastric cancer patients, and research has continued to use this for diagnosis by measuring the expression level of gastric cancer markers. However, research on gene markers related to the diagnosis of diffuse type gastric cancer by histological classification and research related to the prognosis prediction of diffuse type gastric cancer patients are insufficient.

[0004] On the one hand, the research team of the present invention presented a study on using the SYT11 (Synaptotagmin 11) gene as a diagnostic marker for "diffuse type gastric cancer" through Korean Patent Publication No. 2020-0044695, which relates to a composition for treating gastric cancer containing an SYT11 inhibitor as an active ingredient and a method for diagnosing diffuse type gastric cancer including measurement of SYT11 expression. However, the relationship between SYT11 gene expression levels and diffuse type gastric cancer, which has different characteristics, has not been reported. In addition, regarding the administration of a therapeutic agent targeting SYT11 such as the SYT11 inhibitor, research on marker genes that can be used for companion diagnostics to predict the effect of the therapeutic agent in advance and establish a treatment strategy suitable for specific patients has not been conducted. Therefore, there is a need to develop a new marker that can be used for diagnosing diffuse type gastric cancer, predicting its prognosis, and establishing treatment strategies such as the application of SYT11-targeted therapeutic agents, and a method for providing information necessary for determining the diagnosis and treatment strategy of diffuse type gastric cancer through this marker.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a composition and a kit that can diagnose diffuse type gastric cancer among gastric cancers or predict the prognosis of diffuse type gastric cancer patients.

[0007] Another object of the present invention is to provide information necessary for diagnosing diffuse type gastric cancer and information necessary for determining a treatment strategy for diffuse type gastric cancer patients.

Means for Solving the Problems

[0008] To achieve the above object, one aspect of the present invention provides a composition for diagnosing or predicting the prognosis of diffuse gastric cancer, which comprises a preparation for measuring the expression level of the SYT11 gene.

[0009] Another aspect of the present invention provides a kit for diagnosing or predicting the prognosis of diffuse gastric cancer, which comprises the above composition.

[0010] Another aspect of the present invention provides a method for providing information necessary for diagnosing diffuse gastric cancer or determining a treatment strategy for diffuse gastric cancer, which comprises the step of measuring the expression level of the SYT11 gene from a separated biological gastric tissue sample, wherein the information necessary for determining the treatment strategy for diffuse gastric cancer is at least one selected from the group consisting of predicting the prognosis of diffuse gastric cancer, evaluating the prognosis of diffuse gastric cancer, and evaluating the efficacy of an SYT11-targeted therapeutic agent in gastric cancer patients.

Advantages of the Invention

[0011] According to the present invention, it is confirmed that the expression level of the SYT11 gene shows a high correlation in patients with diffuse gastric cancer among intestinal type and diffuse type, and is highly related to the prognosis of patients with diffuse gastric cancer. Thus, it is confirmed that the SYT11 gene can be usefully utilized as a marker for diagnosing diffuse gastric cancer or predicting the prognosis. In addition, it is confirmed that the expression levels of 8 genes other than the SYT11 gene are shown in a pattern similar to the expression level of the SYT11 gene in patients with diffuse gastric cancer and are closely related to the prognosis of patients with diffuse gastric cancer. Therefore, the above 8 genes can be usefully utilized together with the SYT11 gene for diagnosing diffuse gastric cancer and formulating a treatment strategy.

[0012] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

Brief Description of the Drawings

[0013]

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Mode for Carrying Out the Invention

[0014] First, the terms used in the present invention are defined. In the present invention, "prognosis" means predicting the future medical outcome of a patient's disease (e.g., overall survival rate, survival rate without recurrence, etc.), including favorable prognosis including remission of the disease (e.g., recovery of the disease, etc.), degree of improvement of the disease (e.g., regression of the tumor, etc.), or stabilization of the disease, and poor prognosis including recurrence of the disease, degree of progression of the disease or lethality (e.g., growth of the tumor, metastasis, drug resistance, etc.).

[0015] In the present invention, "prediction" means predicting in advance the future medical outcome of a patient's disease, including prediction of the course of the patient's disease (e.g., remission of the disease, degree of improvement, stabilization, recurrence, degree of progression, lethality, deterioration, etc.).

[0016] In the present invention, "biological sample" means tissue obtained from a diffuse-type gastric cancer patient or a normal individual, and specifically may be gastric tissue.

[0017] Hereinafter, the present invention will be described in detail. 1. Composition and kit for diagnosing or predicting the prognosis of diffuse-type gastric cancer The present invention provides a composition for diagnosing diffuse-type gastric cancer or predicting the prognosis of diffuse-type gastric cancer.

[0018] The composition for diagnosing or predicting the prognosis of diffuse-type gastric cancer includes an agent for measuring the expression level of the SYT11 gene.

[0019] The diffuse type gastric cancer is classified as intestinal type gastric cancer when classifying gastric cancer based on pathological and histological characteristics of the Lauren classification, and is a gastric cancer that shows the characteristic that individual cancer cells infiltrate the gastric wall without a clear form of tumor mass formation due to low adhesion of cancer cells. The intestinal type gastric cancer is a gastric cancer that shows the characteristic that cancer cells gather at one position and grow in a mass form.

[0020] The SYT11 gene (Synaptotagmin 11, ILMN_1717934) is one of the Synaptotagmin gene family and encodes a protein similar to other family member proteins known as calcium sensors. The protein expressed from the SYT11 gene can play a role in regulating the calcium-dependent regulation of membrane transport during synaptic transmission. The nucleotide sequence of the SYT11 gene and the amino acid sequence of the protein expressed from the SYT11 gene can be obtained from publicly available databases such as NCBI's GenBank and literature.

[0021] The composition for diagnosing or predicting the prognosis of the diffuse type gastric cancer can 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.

[0022] The nucleotide sequences of the ANGPTL2 gene (Angiopoietin-like protein 2, ILMN_1772612), CACNB1 gene (Calcium Voltage-Gated Channel Auxiliary Subunit Beta 1, ILMN_2271149), CCL19 gene (C-C Motif Chemokine Ligand 19, ILMN_1769129), CD34 gene (ILMN_1732799), JAM3 gene (Junctional adhesion molecule C, ILMN_1769575), SLIT2 gene (Slit homolog 2 protein, ILMN_1676449), THBS4 gene (Thrombospondin-4, ILMN_1736078) and VIMENTIN gene (ILMN_1782538), and the amino acid sequences of the proteins expressed from the said genes can be obtained from publicly available databases such as GenBank of NCBI or literature.

[0023] The preparation for measuring the expression level of the said gene may be a preparation for measuring the amount of the mRNA of the said gene or the amount of the protein expressed from the said gene.

[0024] The preparation for measuring the amount of the mRNA of the said gene means a preparation that can specifically bind to and recognize the mRNA of the said gene or amplify the amount, and includes a pair of primers or a probe that specifically binds to the nucleotide sequence of the said mRNA or cDNA made by reverse transcribing the mRNA.

[0025] The primer is a short-length nucleic acid sequence having a free 3’ hydroxyl group, and serves as a starting point when a nucleic acid polymerase replicates and amplifies a template strand by forming base pairs with the complementary template strand. The probe means a nucleic acid fragment having a length of several bases to several hundred bases, which consists of a sequence capable of specifically binding to mRNA or cDNA.

[0026] The amount of the mRNA of the gene present can be measured by performing methods such as PCR, RT-PCR, Competitive RT-PCR, Real-time RT-PCR, etc. using the sense and antisense primers of the gene sequence, and can be measured by performing methods such as Northern blot, Microarray, etc. using a probe having a sequence capable of specifically binding to the mRNA of the gene or the cDNA produced by reverse transcription. Furthermore, it can be measured by methods such as RNase protection assay, sequencing, etc., but is not limited thereto, and any formulation necessary for using any method known to an ordinary technician is also possible.

[0027] The formulation for measuring the amount of the protein expressed from the gene means a formulation capable of specifically binding to and recognizing the protein, and includes an antibody or an aptamer that specifically binds to the protein.

[0028] The antibody means an immunoglobulin molecule that immunologically binds specifically to the epitope of the protein and has reactivity, and can include all monoclonal antibodies, polyclonal antibodies, antibodies with a full-length chain structure, antibodies of functional fragments having at least an antigen-binding function, and recombinant antibodies. The aptamer means a single-stranded nucleic acid molecule having a stable tertiary structure and having the characteristic of being able to form a specific binding targeting the protein, and an aptamer specific to the protein can be synthesized using techniques such as SELEX (Systematic Evolution of Ligands by Exponential enrichment).

[0029] The amount of the protein expressed from the gene can be measured by methods such as Western blot, protein microarray (protein chip), ELISA (Enzyme Linked Immunosorbent Assay), two-dimensional electrophoresis, immunohistochemistry (IHC), immunofluorescence, co-immunoprecipitation assay, FACS (Fluorescence activated cell sorter), radioimmunoassay (RIA), radioimmunodiffusion, MALDI-TOF analysis (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) using the antibody or aptamer, but is not limited thereto, and any formulation necessary for using any method known to an ordinary technician is also possible.

[0030] A composition containing a preparation for measuring the expression level of the SYT11 gene can be used for the purpose of providing information necessary for the diagnosis of diffuse gastric cancer. In a specific embodiment of the present invention, unlike intestinal gastric cancer, it was confirmed that the expression level of the SYT11 gene was high in patients with diffuse gastric cancer. By measuring the expression level of the SYT11 gene in patients whose onset of gastric cancer is unknown or in gastric cancer patients whose histological classification is unknown, when the expression level of the SYT11 gene is shown to be high, the composition of the present invention can be used to provide information for diagnosing diffuse gastric cancer (FIG. 1 and FIG. 2).

[0031] In addition, a composition containing a preparation for measuring the expression level of the SYT11 gene or at least one further 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. As a result of measuring 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, when the expression level of the gene is high, it enables the prediction that the prognosis of patients with diffuse gastric cancer is poor, and strategies and countermeasures for treatment can be established. In this regard, in a specific embodiment of the present invention, the SYT11 gene expression level was shown to be even higher in diffuse type patients among gastric cancer patients, and it was confirmed that the survival possibility of diffuse gastric cancer patients with a high SYT11 gene expression level was even higher than that of diffuse gastric cancer patients with a low SYT11 gene expression level (FIG. 3). Also, in diffuse gastric cancer patients with a high SYT11 gene expression level, the expression levels of the ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and Vimentin genes were also measured to be high together (FIG. 4), and it was confirmed that the expression level of the gene is also related to the survival possibility of diffuse gastric cancer patients (FIG. 5).

[0032] The present invention provides a kit for diagnosing diffuse gastric cancer or predicting the prognosis of diffuse gastric cancer.

[0033] The kit includes a composition for diagnosing or predicting the prognosis of the diffuse gastric cancer.

[0034] The kit can include a composition containing a preparation for measuring the expression level of the SYT11 gene, a preparation for measuring the expression level of the SYT11 gene, and at least one composition containing 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.

[0035] The kit can further include other components suitable for measuring the amount of the mRNA of the gene present. For example, the kit can be in the form of an RT-PCR kit or a DNA chip kit, or can further include the components of the RT-PCR kit or DNA chip kit, and can be applied to the kit of the present invention as long as it is in a form used for measuring the amount of mRNA known to ordinary technicians.

[0036] Specifically, the kit may be a kit including the essential components necessary for performing RT-PCR. For example, in addition to the primer pair that can specifically bind to the mRNA of the gene, it can include test tubes or other appropriate containers, enzymes such as Taq-polymerase and reverse transcriptase, reaction buffer solutions (with various pH and magnesium concentrations), deoxynucleotides (dNTP), DNase and RNase inhibitors, DEPC-water, sterile water, etc. It can also include a primer pair that can specifically bind to a gene used as a control group during quantification.

[0037] Alternatively, the kit may be a kit containing essential components necessary for performing a DNA chip. It may include a glass substrate to which oligonucleotides capable of specifically binding to the mRNA of the gene or reverse-transcribed cDNA are attached, and reagents, enzymes, formulations, etc. for producing fluorescently labeled probes.

[0038] The kit may include a composition, solution or device containing one or more other components suitable for measuring the amount of the protein expressed from the gene. For example, the kit may be in the form of an ELISA kit, a protein chip kit, or may further include the components of the ELISA kit, the protein chip kit, and can be applied to the kit of the present invention as long as it is in a form used for measuring the amount of a protein known to ordinary technicians.

[0039] Specifically, the kit may be a kit containing the essential components necessary for performing ELISA. For example, it can include a substrate for immunological detection of an antibody, an appropriate reaction buffer solution, and a reagent capable of detecting the formation of an antigen-antibody complex using an antibody that can specifically bind to the protein expressed from the gene. Here, it can include an enzyme that mediates a color reaction, a secondary antibody labeled with a fluorescent substance, a chromogenic substrate, and the like. Examples of the enzyme that mediates the color reaction can include alkaline phosphatase, acid phosphatase, peroxidase. Examples of the fluorescent substance can include Coloid gold, Fluorescein carboxylic acid (FCA), Fluorescein isothiocyanate (FITC), RITC (Rhodamine-B-isothiocyanate), Fluorescein thiourea (FTH), 7-acetoxycoumarin-3-yl, Fluorescein-5-yl, Fluorescein-6-yl, 2’,7’-dichlorofluorescein-5-yl, 2’,7’-dichlorofluorescein-6-yl, Dihydrotetramethylrhodamine-4-yl, Tetramethylrhodamine-5-yl, Tetramethylrhodamine-6-yl, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-ethyl, or 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-ethyl, and the like. Also, it can include an antibody that specifically binds to the protein expressed from the gene used as a control group during quantification. The ELISA may be direct ELISA, indirect ELISA, or competitive ELISA.

[0040] Alternatively, the kit may be a kit containing essential components necessary for performing a protein chip. For example, it may be one in which one or more antibodies capable of specifically binding to a protein expressed from the gene are immobilized and arrayed at high density at defined positions on a substrate. Further, it can contain an antibody that specifically binds to a protein expressed from a gene used as a control group during quantification.

[0041] 2. Method for providing information necessary for diagnosing diffuse-type gastric cancer or determining a treatment strategy for diffuse-type gastric cancer The present invention provides a method for providing information necessary for the diagnosis of diffuse gastric cancer or information necessary for determining a treatment strategy for diffuse gastric cancer.

[0042] The method for providing information necessary for the diagnosis of diffuse gastric cancer or for determining a treatment strategy for diffuse gastric cancer includes the step of measuring the expression level of the SYT11 gene from a separated biological gastric tissue sample. The information necessary for determining a treatment strategy for diffuse gastric cancer is at least one selected from the group consisting of prognosis prediction of diffuse gastric cancer, prognosis evaluation of diffuse gastric cancer, and evaluation of the effectiveness of an SYT11-targeted therapeutic agent in gastric cancer patients.

[0043] Further, 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 a separated biological gastric tissue sample.

[0044] The step of measuring the expression level of the gene may be to measure the amount of mRNA of the gene present or to measure the amount of protein expressed from the gene.

[0045] Measuring the amount of the mRNA of the gene may be performed by using a preparation that can specifically bind to and recognize the mRNA of the gene or can amplify the amount. As a specific example, it may also be performed by using a primer pair or a probe that specifically binds to the base sequence of the mRNA or cDNA created by reverse-transcribing the mRNA. It can be measured by methods such as PCR, RT-PCR, competitive RT-PCR, real-time RT-PCR, Northern blot, microarray, RNase protection assay, and sequencing, but is not limited thereto, and it is possible to measure using any method known to ordinary technicians.

[0046] Measuring the amount of the protein expressed from the gene may be performed by measuring using a preparation that can specifically bind to and recognize the protein. As a specific example, it may be measured using an antibody or aptamer that specifically binds to the protein, and may be measured by methods such as Western blot, protein microarray (protein chip), ELISA (Enzyme Linked Immunosorbent Assay), two-dimensional electrophoresis, immunohistochemistry (IHC), immunofluorescence, co-immunoprecipitation assay, FACS (Fluorescence activated cell sorter), radioimmunoassay (RIA), radioimmunodiffusion, MALDI-TOF analysis (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry), etc., but is not limited thereto, and it is possible to measure using any method known to an ordinary technician.

[0047] According to a specific embodiment of the present invention, the SYT11 gene is shown to be expressed at a high level and have a statistically significant relationship in patients with diffuse-type gastric cancer, and in intestinal-type gastric cancer, such results are not shown (Figure 2). When the expression level of the SYT11 gene is shown to be high in the biological gastric tissue sample to be analyzed compared to the expression level of the SYT11 gene in the normal control group, the analysis target can be determined to be diffuse-type gastric cancer. Therefore, information necessary for the diagnosis of diffuse-type gastric cancer can be provided through the above-described method.

[0048] Moreover, according to a specific embodiment of the present invention, in the case of diffuse-type gastric cancer patients with a high SYT11 gene expression level, the survival probability is even higher compared to diffuse-type gastric cancer patients in the group classified as having a low expression level. It was confirmed that there is a statistically significant relationship between the expression level and the prognosis of diffuse-type gastric cancer, and such a relationship was not significantly shown in intestinal-type gastric cancer patients (Figure 3). Also, it was confirmed that there is a relationship with survival probability in diffuse-type gastric cancer patients in whom the expression levels of both the SYT11 gene and any one of the ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and VIMENTIN are all high (Figure 5). Therefore, when the expression level is high when measuring the expression level of the gene in a sample isolated from a diffuse-type gastric cancer patient, it can be predicted that the prognosis is not poor.

[0049] The method of the present invention may further include a step of comparing the gene expression level measured from the biological gastric tissue sample with a reference value measured from a control group sample.

[0050] The control group sample may be a sample isolated from a population without diffuse-type gastric cancer, a sample isolated from a diffuse-type gastric cancer patient population, or a diffuse-type gastric cancer patient population administered with an SYT11-targeted therapeutic agent, but is not limited thereto. As long as it is a sample isolated from a population that can be normally compared in the technical field of the present invention for the diagnosis and treatment strategy determination of diffuse-type gastric cancer, it can be used without limitation.

[0051] The reference value measured from the control group sample means a reference for the value / data obtained by measuring the expression level of the gene for the control group sample, and may be a value serving as a criterion for determining whether the expression level of the gene expressed in the sample to be measured is overexpressed or underexpressed. The reference value may be an absolute value; a relative value; a value having an upper limit and / or a lower limit; a range of values; an average value; a median value; a central value; or a value compared with a specific control group or reference value. The reference value can be set by a method commonly used in the technical field of the present invention, and various statistical methods and software for processing statistics can be used, but it is not limited thereto.

[0052] The diagnosis of the diffuse gastric cancer is to diagnose whether the individual to be diagnosed is a normal individual without gastric cancer or an individual with diffuse gastric cancer, or to diagnose whether the gastric cancer possessed by the individual to be diagnosed is diffuse gastric cancer. More specifically, it may be to diagnose whether the gastric cancer possessed by the individual to be diagnosed is diffuse gastric cancer or intestinal-type gastric cancer.

[0053] The prognosis prediction of the diffuse gastric cancer may be to predict whether the prognosis is good or bad, which may be to predict the possibility of complete recovery, improvement, recurrence, death, etc. The prognosis prediction may be to predict the survival possibility of diffuse gastric cancer patients. For example, based on the average value or median value of the survival period of the diffuse gastric cancer patient population, it may be to predict whether the patient will survive relatively long or relatively short.

[0054] The prognosis evaluation of the diffuse gastric cancer is to evaluate how the prognosis described in relation to the prognosis prediction appears in diffuse gastric cancer patients. For example, it may be to evaluate whether the prognosis of patients to whom a specific treatment method or administration of a pharmaceutical has been applied, or patients to whom it has not been applied, has improved or worsened over time.

[0055] The method of the present invention may further include a step of determining that it is diffuse gastric cancer when the gene expression level measured from the biological gastric tissue sample is higher than the average value or the median value of the gene expression levels measured from a population without diffuse gastric cancer.

[0056] In addition, the method may further include a step of determining a poor prognosis when the gene expression level measured from the biological gastric tissue sample is higher than the average value or the median value of the gene expression levels measured from a population without diffuse gastric cancer; or the average value or the median value of the gene expression levels measured from a population of diffuse gastric cancer patients.

[0057] The evaluation of the efficacy of the SYT11-targeted therapeutic agent in the gastric cancer patient may be to evaluate at least one selected from the group consisting of the necessity of administering the SYT11-targeted therapeutic agent; the possibility of developing drug resistance to the SYT11-targeted therapeutic agent; the sensitivity to the SYT11-targeted therapeutic agent; and the prognosis prediction of the treatment with the SYT11-targeted therapeutic agent.

[0058] The evaluation of the efficacy of the SYT11-targeted therapeutic agent may be one of the diagnostic tests for confirming the possibility of various results by applying the SYT11-targeted therapeutic agent to specific diffuse gastric cancer patients, and may be referred to as a companion diagnostic.

[0059] The SYT11-targeted therapeutic agent may be, for example, an SYT11 inhibitor, and is used in the general sense of collectively referring to all preparations that reduce the expression or activity of SYT11. Specifically, it includes all preparations that reduce the expression level of SYT11 at the transcription, mRNA level, or translation level, or reduce the activity of SYT11 by interfering with the SYT11 activity, in such a way as affecting the reduction of SYT11 expression, acting directly on SYT11, or acting indirectly on its ligand.

[0060] The inhibitor of SYT1 can be used in any form without limitation, such as a compound, nucleic acid, peptide, virus, or vector containing the nucleic acid, which can suppress the expression of SYT11 or target SYT11 to inhibit its activity. The SYT11 inhibitor includes, but is not limited to, siRNA or shRNA that degrades the mRNA of the SYT11 gene, and antisense oligonucleotides that reduce the expression of the SYT11 protein. Further, as an SYT11 inhibitor that binds to the SYT11 protein to inhibit its function, it may be an aptamer or a small molecule compound.

[0061] In a specific embodiment of the present invention, it was confirmed that in patients with diffuse gastric cancer, the expression level of the SYT11 gene and the expression level of a gene selected from the group consisting of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and Vimentin were shown in the same pattern and there was a statistically significant correlation. Further, the expression level of the SYT11 gene and the expression level of a gene selected from the group consisting of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and Vimentin were also closely related to the prognosis of patients with diffuse gastric cancer and were confirmed to show the same pattern. Therefore, when administering a therapeutic agent for diffuse gastric cancer targeting SYT11, by measuring the expression level of a gene selected from the group consisting of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and Vimentin, the possibility of the expression of drug resistance to the SYT11-targeted therapeutic agent; the sensitivity to the SYT11-targeted therapeutic agent; and the prognosis prediction of the treatment with the SYT11-targeted therapeutic agent; can be evaluated by using the measurement results of the expression level.

[0062] The necessity of the administration is for determining whether to administer the therapeutic agent to an individual who can obtain or is expected to obtain a therapeutic effect from the therapeutic agent.

[0063] When the expression level of a gene selected from the group consisting of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and Vimentin, measured from a biological gastric tissue sample isolated from a diffuse-type patient, is higher than a reference value, the method may further include a step of determining that the individual who provided the sample may correspond to at least one of the following: administration of an SYT11-targeted therapeutic agent is necessary; the possibility of developing drug resistance to the SYT11-targeted therapeutic agent is low; the sensitivity to the SYT11-targeted therapeutic agent is high; or the prognosis of treatment with the SYT11-targeted therapeutic agent is good.

[0064] In one specific example of the present invention, it is possible to confirm that the SYT11 gene is expressed in a biological gastric sample obtained from a diffuse-type gastric cancer patient, measure the expression level of at least one gene selected from the group consisting of ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and Vimentin, and then determine the prescription of an SYT11-targeted therapeutic agent. Specifically, when the expression level of the gene of the patient is measured to be equal to or higher than a reference value, the patient can be predicted to be highly sensitive to the SYT11-targeted therapeutic agent or have low resistance to the SYT11-targeted therapeutic agent, and provide information necessary for determining the prescription of the SYT11-targeted therapeutic agent.

[0065] In addition, according to a specific embodiment of the present invention, a SYT11-targeted therapeutic agent that suppresses the expression of SYT11 or suppresses its activity against gastric cancer cell lines, for example, when treated with siSYT11 or SYT11-ASO, not only the amount of intracellular SYT11 protein, but also the amounts of ANGPTL2, THBS4, Vimentin, and JAM3 proteins were both confirmed to decrease (Figures 6 and 7). Therefore, it was found that the markers are co-expressed or regulated by suppression of SYT11, and when administering a SYT11-targeted therapeutic agent, by monitoring the markers, sensitivity, resistance, etc. to the SYT11-targeted therapeutic agent can be confirmed, and the markers can be used for evaluating the effectiveness of the SYT11-targeted therapeutic agent.

[0066] Hereinafter, the present invention will be described in detail with reference to examples and experimental examples. However, the following examples and experimental examples are specific illustrations of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

[0067] [Example 1] Confirmation of the relationship between the expression level of the SYT11 gene and diffuse-type gastric cancer By measuring and comparing the expression levels of the SYT11 gene expressed from gastric cancer tissues and normal human gastric tissues, the relationship between the SYT11 gene expression level and gastric cancer was confirmed.

[0068] Cancer and normal tissues were purchased from Biomax (Biomax US, USA), and immunohistochemistry was performed on 70 gastric cancer tissues and 10 normal tissues to confirm the SYT11 expression level.

[0069] Specifically, for the removal of paraffin from each tissue, the tissue was immersed in a xylene solution for 10 minutes, and then successively immersed in 100%, 95%, 80%, and 70% ethanol solutions for 5 minutes each for the removal of the remaining xylene, followed by rinsing with water. The tissue slides were immersed in a 3% hydrogen peroxide (H2O2) solution for 10 minutes, and then immersed in water for 15 minutes for rinsing. After boiling a 0.01M sodium citrate solution at 95°C, the tissue slides were placed in it and left for 10 minutes, and then cooled to room temperature for 30 minutes. After further rinsing with water for 10 minutes, the prepared samples were placed in a phosphate buffered saline for 10 minutes, and then reacted successively with an anti-SYT11 antibody, a biotin-conjugated secondary antibody (Vector lab, USA), and an avidin-biotin conjugated enzyme (Abidin + Biotinylated horseradish peroxidase, Vector lab, USA). Subsequently, the expression level of SYT11 protein was confirmed using a DAB peroxidase substrate (Vector lab, USA). Also, to distinguish the nuclear part of the tissue, the tissue was reacted with a hematoxylin solution for 5 minutes, rinsed with water, and then the tissue slides were successively immersed in 0.1% hydrochloric acid (HCl) and 0.1% aqueous ammonia (NH4OH), followed by rinsing with water. After successively immersing in 70%, 85%, 95%, and 100% ethanol for 5 minutes each, the tissue slides were reacted with a xylene solution for 5 minutes and then dried.

[0070] According to the degree of color development of the SYT11 gene measured by immunohistochemistry, the groups were classified into a group with a low expression level of the SYT11 gene (SYT11-low) and a group with a high expression level of the SYT11 gene (SYT11-high) based on the median or average value. As a result, in the case of normal gastric tissue, only 30% showed a high expression level of the SYT11 gene, while in the case of gastric tissue from gastric cancer patients, 64.3% of the patients showed a high expression level of the SYT11 gene. Therefore, it was confirmed that there is a correlation between the expression level of the SYT11 gene and the occurrence of gastric cancer (Figure 1).

[0071] Also, the 70 gastric cancer tissues were histologically classified into 30 intestinal type gastric cancer (diffuse type) tissues and 40 diffuse type gastric cancer (diffuse type) tissues, and the expression levels of the SYT11 gene were measured by the degree of coloring in each case. Similarly, they were classified into a group with a low SYT11 gene expression level (SYT11-low) and a group with a high expression level (SYT11-high). As a result, in the case of intestinal type gastric cancer tissues, it was shown that the expression level of the SYT11 gene was high in 46.7% of cases, while in the case of the gastric tissues of diffuse type gastric cancer patients, it was shown that the expression level of the SYT11 gene was high in 77.5% of the patients. Therefore, it was confirmed that the expression level of the SYT11 gene is related to diffuse type gastric cancer (Figure 1).

[0072] [Example 2] Comparison of the difference in SYT11 gene expression levels between diffuse-type gastric cancer and intestinal-type gastric cancer The microarray results of 239 intestinal type gastric cancer patients and 185 diffuse type gastric cancer patients provided by Yonsei University Severance Hospital were analyzed. After receiver operating characteristic analysis based on the SYT11 expression level in the microarray results, the gastric cancer patient group was classified into a group with a high or low SYT11 expression level based on the median or average value. Also, whether the gastric cancer was stem-like gastric cancer or non-stem-like gastric cancer was confirmed together.

[0073] As a result, in the case of intestinal type gastric cancer, 77 out of 239 patients were shown to have a high SYT11 gene expression level, and among them, 27 out of 31 stem-like patients were analyzed to have a high SYT11 gene expression level. In contrast, in the case of diffuse type gastric cancer, 100 out of 185 patients were shown to have a high SYT11 gene expression level, and among them, 67 out of 80 stem-like patients were analyzed to have a high SYT11 gene expression level (Figure 2).

[0074] Through the above results, in addition to further confirming that the expression of the SYT11 gene is associated with gastric cancer of the stem type as in previous studies, by newly confirming that the expression of the SYT11 gene is low in intestinal gastric cancer and high in diffuse gastric cancer, it was confirmed that the expression level of the SYT11 gene is associated with diffuse gastric cancer and can be used as a diagnostic marker for diffuse gastric cancer.

[0075] [Example 3] Confirmation of the relationship between the SYT11 gene expression level and the prognosis of patients with diffuse-type gastric cancer Based on the clinical data of 424 gastric cancer patients according to Example 2 above, the relationship between the survival probability of gastric cancer patients and the SYT11 gene expression level was analyzed.

[0076] Specifically, 239 intestinal gastric cancer patients and 185 diffuse gastric cancer patients were classified, and based on the expression levels of the SYT11 gene and the SYT4 gene using the microarray results of these patients, they were divided into a high-expression group and a low-expression group. Also, the survival probability data of each group were compared to confirm the relationship between the gene expression level and the prognosis of gastric cancer patients.

[0077] As a result, as can be seen from Figure 3, in the case of 239 intestinal gastric cancer patients, there was no statistically significant difference in survival probability between the high-expression group and the low-expression group of the SYT11 gene expression level (p = 0.3363), while in the case of 185 diffuse gastric cancer patients, the survival probability was significantly lower in the high-expression group of the SYT11 gene expression level (p = 0.0067), and it was confirmed that there was a close relationship between the SYT11 gene expression level and the prognosis of diffuse gastric cancer patients. Also, from the results of the control group that confirmed the relationship between the SYT4 gene expression level belonging to the same family as the SYT11 gene and survival probability, it was confirmed that there was no significant relationship between the prognosis of diffuse gastric cancer patients and the SYT4 gene expression level (p = 0.1618), and it was confirmed that diffuse gastric cancer is only related to the expression level of the SYT11 gene.

[0078] [Example 4] Discovery of eight companion diagnostic markers and confirmation of their relationship with diffuse-type gastric cancer Through the above examples, it was confirmed that the expression level of the SYT11 gene was high in diffuse-type gastric cancer patients, and that diffuse-type patients with a high expression level of the SYT11 gene had a poor prognosis. Therefore, genes whose expression levels are regulated in a trend like the SYT11 gene were identified, and eight new markers that could potentially be used as companion diagnostic markers were discovered (Figure 4).

[0079] Specifically, based on the microarray results of gastric cancer patients, after performing Receiver operating characteristic analysis according to the expression levels of the ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and VIMENTIN genes, the patients were classified into a high-expression group and a low-expression group based on the median and average values of the expression levels of each gene. In addition, the survival rates of the patients according to the expression levels of SYT11 and each of the above genes were shown using a Kaplan Meier graph.

[0080] As can be confirmed from Figure 4, in patients with a high expression level of the SYT11 gene, the expression levels of the eight genes ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and VIMENTIN all increased in proportion to the expression level of the SYT11 gene, and a statistically significant relationship was shown.

[0081] In addition, the survival probabilities of patients with intestinal-type gastric cancer and patients with diffuse-type gastric cancer were examined for cases where the expression levels of both the SYT11 gene and the ANGPTL2 gene were high (SYT11(H)-ANGPL2(H)) or low (SYT11(L)-ANGPL2(L)). The correlations with survival were also compared in the same manner for seven other gene markers in addition to the ANGPTL2 gene. As a result, it was shown that diffuse-type patients with high expression levels of both the SYT11 gene and each of the eight genes had low survival probability and poor prognosis, and the results were statistically significant. In contrast, in the case of intestinal-type gastric cancer, it was confirmed that there was no significant correlation between survival probability and expression level for all eight markers. It was found that the eight gene markers were correlated with the expression level of the SYT11 gene only in diffuse-type gastric cancer (Figure 5).

[0082] [Example 5] Confirmation of the expression levels of companion diagnostic markers by treatment with SYT11-targeted therapeutic agents For gastric cancer cell line SNU484 cells, siSYT11 (5′-GCAGAAAGCGCAUUGCCAA(dTdT)-3′, where d means deoxyribonucleic acid), which is a type of siRNA and can bind to the mRNA of the SYT11 gene to suppress its expression, was transfected and introduced as a SYT11-targeted therapeutic agent. As a control group, siScramble (5′-CCUACGCCACCAAUUUCGU(dTdT)-3′, where d means deoxyribonucleic acid) was transfected. 48 hours after introduction, proteins were extracted using RIPA buffer, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then Western blotting was performed using each antibody of the companion diagnostic marker genes discovered through Example 4 above.

[0083] As a result, it was confirmed that the amounts of ANGPTL2, THBS4, VIMENTIN2, and JAM3 were decreased by reducing the amount of SYT11 protein in the cells using siSYT11, which is a SYT11-targeted therapeutic agent (Figure 6).

[0084] In addition, for gastric cancer cell line SNU484 cells, as another type of SYT11-targeted therapeutic agent, SYT11-ASO (5′-mU * mU * G * G * C * A * A * T * G * C * G * C * T * T * T * C * T * mG * mC-3′, m means methylated, * means phosphorothioated) was introduced by transfection. As a control group, NC-ASO (5′-mC * mC * T * A * C * G * C * C * A * C * C * A * A * T * T * T * C * mG * mU-3′, m means methylated, * means phosphorothioated) was transfected. 48 hours after introduction, proteins were extracted using RIPA buffer, separated by SDS-PAGE on a polyacrylamide gel, and then Western blotting was performed using each antibody of the companion diagnostic marker gene discovered through Example 4.

[0085] As a result, it was confirmed that, similar to when siSYT11 was used, the amounts of ANGPL2, THBS4, VIMENTIN2, and JAM3 were decreased by reducing the amount of intracellular SYT11 protein using SYT11-ASO (Figure 7).

[0086] From the above results, it can be predicted that the ANGPL2, THBS4, VIMENTIN2, and JAM3 genes are downstream genes of SYT11 regulated by SYT11, and it could be predicted that the genes of Example 4, which have a significant correlation with the expression level of the SYT11 gene, would have their expression levels co-regulated when the expression of SYT11 was regulated. Therefore, when using a target therapeutic agent that reduces SYT11 expression or suppresses SYT11 activity, the therapeutic effect and resistance of the SYT11 target therapeutic agent can be monitored by measuring the expression levels of ANGPL2, THBS4, VIMENTIN, and JAM3 and confirming whether their expression is decreased. Thus, it is expected that the said genes can be utilized as companion diagnostic markers for SYT11 target therapeutic agents.

[0087] [Example 6] Conclusion Taking the above-mentioned results of the examples into consideration, it was confirmed that the expression level of the SYT11 gene is high in patients with diffuse-type gastric cancer, and that the SYT11 gene can be used as a marker for diagnosing diffuse-type gastric cancer. In addition, it was confirmed that the expression level of the SYT11 gene is also related to the prognosis of diffuse-type patients, and that if the expression level of the SYT11 gene is high, the prognosis of diffuse-type patients can be predicted to be poor. In addition, it was confirmed that the expression level of the SYT11 gene and the expression levels of eight genes, ANGPTL2, CACNB1, CCL19, CD34, JAM3, SLIT2, THBS4, and VIMENTIN, showed a similar tendency in patients with diffuse-type gastric cancer, and were related to the prognosis of patients with diffuse-type gastric cancer. Therefore, it was confirmed that the eight genes can also be used as markers for predicting the prognosis of diffuse-type gastric cancer.

[0088] Furthermore, previous research has revealed that SYT11 can be a target for treating gastric cancer, and experiments have confirmed that the amount of the marker gene is also reduced when gastric cancer cell lines are treated with siSYT11 or SYT11-ASO, which can reduce the amount of SYT11. Therefore, in relation to the treatment of diffuse gastric cancer targeting SYT11, it has been confirmed that the above eight markers can be used as accompanying diagnostic markers to obtain information such as whether SYT11 targeted therapy is necessary, prediction of sensitivity to administration of SYT11 targeted therapeutic agents, and prediction of prognosis after administration of therapeutic agents.

[0089] Although the present invention has been described in detail above only with reference to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the technical scope of the present invention, and it is natural that such modifications and variations fall within the scope of the appended claims.

Claims

1. A composition for diagnosing or predicting the prognosis of diffuse gastric cancer, comprising a preparation for measuring the expression level of the SYT11 gene.

2. The composition for diagnosing or predicting the prognosis of diffuse gastric cancer according to claim 1, further comprising at least one preparation 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 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 from the gene, and the composition for diagnosing or predicting the prognosis of diffuse gastric cancer according to claim 1 or claim 2.

4. The preparation for measuring the amount of mRNA of the gene contains a primer pair or a probe that specifically binds to the mRNA or cDNA of the gene, and the composition for diagnosing or predicting the prognosis of diffuse gastric cancer according to claim 3.

5. The preparation for measuring the amount of protein expressed from the gene contains an antibody or an aptamer specific to the protein expressed from the gene, and the composition for diagnosing or predicting the prognosis of diffuse gastric cancer according to claim 3.

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

7. A method for providing information necessary 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 a separated biological gastric tissue sample; wherein the information necessary for determining the treatment strategy for diffuse gastric cancer is at least one selected from the group consisting of predicting the prognosis of diffuse gastric cancer, evaluating the prognosis of diffuse gastric cancer, and evaluating the effectiveness of an SYT11-targeted therapeutic agent in gastric cancer patients.

8. The method for providing information necessary for diagnosing diffuse gastric cancer or determining a treatment strategy for diffuse gastric cancer according to claim 7, further comprising 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 separated biological gastric tissue sample.

9. The method for providing information necessary for the diagnosis of diffuse gastric cancer or determination of a treatment strategy for diffuse gastric cancer according to any one of claims 7 or 8, further comprising the step of comparing the gene expression level measured from the biological gastric tissue sample with a reference value measured from a control group sample.

10. The method for providing information necessary for the diagnosis of diffuse gastric cancer or determination of a treatment strategy for diffuse gastric cancer according to claim 9, further comprising the step of determining diffuse gastric cancer when the gene expression level measured from the biological gastric tissue sample is higher than the average value or median value of the gene expression levels measured from a group without diffuse gastric cancer.

11. The method for providing information necessary for the diagnosis of diffuse gastric cancer or determination of a treatment strategy for diffuse gastric cancer according to claim 9, further comprising the step of determining a poor prognosis when the gene expression level measured from the biological gastric tissue sample is higher than the average value or median value of the gene expression levels measured from a group without diffuse gastric cancer or the average value or median value of the gene expression levels measured from a group of diffuse gastric cancer patients.

12. The evaluation of the efficacy of the SYT11-targeted therapeutic agent in the gastric cancer patient is to evaluate at least one selected from the group consisting of the necessity of administration of the SYT11-targeted therapeutic agent, the possibility of expression of drug resistance to the SYT11-targeted therapeutic agent, the sensitivity to the SYT11-targeted therapeutic agent, and the prognosis prediction of treatment with the SYT11-targeted therapeutic agent. The method for providing information necessary for the diagnosis of diffuse gastric cancer or determination of a treatment strategy for diffuse gastric cancer according to claim 7.

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