KR20150072207A - A Method for diagnosis of gastric cancer using metabolomics - Google Patents
A Method for diagnosis of gastric cancer using metabolomics Download PDFInfo
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Abstract
Description
The present invention relates to a method for diagnosing gastric cancer using metabolism, and more particularly, to a method for diagnosing stomach cancer by analyzing a macro environment-derived metabolism body and a local environment-derived metabolism body alone or simultaneously.
Despite a gradual decline in cancer incidence and mortality, gastric cancer is the fourth most common cancer worldwide and the second leading cause of cancer deaths. The incidence of gastric cancer is particularly high in East Asia, Eastern Europe and Latin America.
Symptoms of gastric cancer range from no symptoms at all to severe pain. In addition, the symptoms of gastric cancer do not have any characteristics but general digestive symptoms. In general, most of the stomach cancer in the early stages of the disease is mostly mild, even if a little digestive failure or upper abdominal discomfort is felt to the degree that most people are overlooked it can cause the death rate of stomach cancer is also increased.
Until now, most of the examination methods of stomach cancer were physical. The first is gastrointestinal X-ray, double contrast, compression, and mucosal imaging. Second, the upper gastrointestinal endoscope allows us to look directly at the periosteum, revealing a very small lesion that does not appear in X-ray examination In addition to being able to perform biopsy directly at the suspicious site of stomach cancer, the diagnosis rate is increasing. However, this method has the disadvantage of suffering hygiene problems and suffering from the patient during the examination. Recently, studies have been conducted to diagnose gastric cancer by measuring the expression level of a gene marker specifically expressed in gastric cancer. However, studies on gene markers for predicting the prognosis of gastric cancer patients are relatively less studied. In addition, the best treatment for the ongoing gastric cancer to date is to excise the lesion by surgery, and therefore, there is no surgical resection method for the cure. There are various methods of surgical resection, but in the surgery aiming at cure, it is necessary to exclude as wide a range as possible, but the scope of ablation can be determined considering the aftereffects due to extensive resection after surgery. In this case, not only the stomach, but also the surrounding lymph nodes, including many organs, because it is a fairly large surgery, it may be difficult to guarantee the prognosis of the patient. In addition, when gastric cancer is metastasized to other organs, it is impossible to perform radical surgery. Therefore, other methods such as administration of anticancer drug are used. In this case, the anticancer drugs that have been on the market until now have been effective in suppressing recurrence after the temporary symptom relief or resection, Although it has a temporary effect, side effects and economic burden of administration of anticancer drugs may cause double pain in patients. The development of a diagnostic agent for stomach cancer, which enables accurate identification of the onset and progression of gastric cancer, and the selection of biomarkers for the development of therapeutic agents that complement the disadvantages of surgical resection or anticancer drugs, .
Accordingly, the present inventors have made efforts to develop a method for rapidly and accurately diagnosing stomach cancer by using metabolomics. As a result, by analyzing the macro-environment-derived metabolism and the metabolism from the local environment of the gastric cancer patient alone or simultaneously, Screening, monitoring, development of therapeutic agent, diagnosis kit and prognosis of stomach cancer using biomarkers according to the present invention.
It is an object of the present invention to provide a method for diagnosing, screening and monitoring gastric cancer by analyzing a macro environment-derived metabolite and a local environment-derived metabolism alone or simultaneously.
It is another object of the present invention to provide a biochip or kit capable of diagnosing, screening and monitoring gastric cancer by analyzing a macro environment-derived metabolism substance and a local environment-derived metabolism substance alone or simultaneously.
In order to achieve the above object,
The present invention relates to a pharmaceutical composition for preventing or treating a disease or condition in a sample isolated from a subject by using 4-hydroxyphenylacetate, alanine, phenylacetylglycine, acetate, lactate, phenylalanine, It is known that mannitol, formate, 3-indoxylsulfate, glycolate, arginine, methionine, tyrosine, O-acetyl carnitine, measuring the concentration of at least one selected from the group consisting of acetylcarnitine, glycine, acetone and hypoxanthine;
2) the relationship of comparing the concentration of metabolites measured in step 1) with a normal control; And
3) In step 2), 4-hydroxyphenylacetate, alanine, phenylacetylglycine, acetate, lactate, phenylalanine, mannitol, ), Formate, 3-indoxylsulfate, glycolate, arginine, methionine, tyrosine, O-acetylcarnitine, When the concentration of any one or more selected from the group consisting of glycine and acetone is increased as compared with the normal control group or the concentration of hypoxanthine is decreased as compared with the normal control group, Or determining that the risk of gastric cancer is high. The present invention also provides a method for analyzing a metabolite for providing information on gastric cancer diagnosis.
In addition,
1) In a sample isolated from a test sample, isoleucine, glutamate, leucine, valine, alanine, lysine, phenylalanine, lactate, measuring the concentration of at least one selected from the group consisting of lactate and taurine;
2) comparing the concentration of the metabolite measured in step 1) with a normal control; And
3) determining that the subject is at risk for gastric cancer or having a high risk of dignity if the concentration of the metabolite measured in the step 2) is increased as compared with the normal control group; And provides a method for analyzing metabolites.
In addition,
1) A method for detecting glycolated, glycine, or glycine in a sample selected from the group consisting of urine, blood, serum, plasma and saliva separated from a subject, and cells or tissues separated from the subject, , Methionine, lactate, alanine, phenylalanine, tyrosine, agrinine, valine, glutamate, lysine, Measuring the concentration of at least one selected from the group consisting of leucine, phenylacetylglycine, acetylcarnitine, and acetone;
2) the relationship between the concentration of the metabolites measured in each sample in step 1) and the concentration of the metabolites measured in the same type of sample from the normal control, respectively; And
3) In step 2), each sample is mixed with a solution containing glycolate, glycine, methionine, lactate, alanine, phenylalanine, tyrosine, Which is selected from the group consisting of agrinine, valine, glutamate, lysine, leucine, phenylacetylglycine, acetylcarnitine and acteone. And determining that the subject is at risk for stomach cancer if the one or more concentrations are increased compared to the normal control group.
The present invention also relates to a pharmaceutical composition comprising 4-hydroxyphenylacetate, alanine, phenylacetylglycine, acetate, lactate, phenylalanine, mannitol, Formate, 3-indoxylsulfate, glycolate, arginine, methionine, tyrosine, O-acetylcarnitine, glycine, glycine, acetone, and hypoxanthine. The present invention also provides a diagnostic kit for a gastric cancer using a macro-environment metabolite, which comprises a metering device for at least one metabolite selected from the group consisting of glycine, acetone and hypoxanthine.
The present invention also relates to a pharmaceutical composition comprising isoleucine, glutamate, leucine, valine, alanine, lysine, phenylalanine, lactate, And a metering device for any one or more metabolites selected from the group consisting of taurine, and a gastric cancer diagnostic kit using a local environmental metabolism.
The present invention also relates to a pharmaceutical composition comprising at least one compound selected from the group consisting of glycollate, glycine, methionine, lactate, alanine, phenylalanine, tyrosine, agrinine, the macro environment metabolism and the local environment metabolism, including quantitative devices for any one or more metabolites selected from the group consisting of valine, glutamate, lysine, leucine, A gastric cancer diagnostic kit used.
In addition,
1) confirming a pattern of a metabolite through NMR analysis in any one sample selected from the group consisting of urine, blood, serum, plasma and saliva separated from the subject;
2) confirming the pattern of the metabolite through HR-MAS analysis in a sample of cells or tissues separated from the subject; And
3) screening metabolic markers having matched patterns in macroscopic and local environments, comprising the step of selecting metabolites that match the pattern of the metabolites in steps 1) and 2) above.
In addition,
1) confirming a pattern of a metabolite through NMR analysis in any one sample selected from the group consisting of urine, blood, serum, plasma and saliva separated from the subject;
2) confirming the pattern of the metabolite through HR-MAS analysis in a sample of cells or tissues separated from the subject;
3) selecting the metabolites in which the pattern of the metabolism coincides in the step 1) and the step 2); And
4) A method for diagnosing, monitoring or screening gastric cancer is provided by analyzing patterns of increase and decrease of metabolites selected in step 3).
The present invention relates to a method for diagnosing gastric cancer using metabolism. More specifically, the present invention relates to a method for diagnosing a gastric cancer using a sample of urine, blood, serum, plasma and saliva, Thus, the novel biomarker metabolite of the present invention can be usefully used for the diagnosis, screening, monitoring, diagnostic kits and therapeutic agent development of gastric cancer. In addition, the biomarker metabolism of the present invention can more accurately determine the diagnosis and prognosis of gastric cancer by comparing the macro environment-derived metabolism of the gastric cancer patient with the local environment-derived metabolism.
FIG. 1A shows that the metabolites of the stomach cancer patient group and the control group are clearly separated in the macro environment.
Fig. 1B is a diagram showing a metabolite clearly distinguished from the gastric cancer patient group and the control group through VIP analysis.
2A shows that the gastric cancer tissue and the normal tissue metabolite are clearly separated in the local environment.
FIG. 2B is a diagram showing a metabolite clearly distinguished from gastric cancer tissue and normal tissue through VIP analysis. FIG.
FIG. 3 is a schematic diagram illustrating a comparison of the change in metabolism of macro-environment metabolism and the change in local environmental metabolism in cancer.
FIG. 4 is a graph showing the OPLS-DA model validation using urine metabolites in the gastric cancer patient group and the control group.
Hereinafter, the present invention will be described in detail.
The present invention relates to a method for the preparation of 4-hydroxyphenylacetate (4-hydroxyphenylacetate), alanine, phenylacetylglycine, acetate, lactate, phenylalanine phenylalanine, mannitol, formate, 3-indoxylsulfate, glycolate, arginine, methionine, tyrosine, O-acetylcarnitine Measuring the concentration of at least one selected from the group consisting of O-acetylcarnitine, glycine, acetone and hypoxanthine;
2) the relationship of comparing the concentration of metabolites measured in step 1) with a normal control; And
3) In step 2), 4-hydroxyphenylacetate, alanine, phenylacetylglycine, acetate, lactate, phenylalanine, mannitol, ), Formate, 3-indoxylsulfate, glycolate, arginine, methionine, tyrosine, O-acetylcarnitine, When glycine and acetone concentrations are increased compared to the normal control group or when the concentration of hypoxanthine is lower than that of the normal control group, the subject is judged to have a stomach cancer or a high risk of gastric cancer A method for analyzing a metabolite for providing information on gastric cancer diagnosis.
The sample of step 1) is preferably one selected from the group consisting of urine, blood, serum, plasma and saliva, but is not limited thereto.
The concentration of the step 1) is preferably measured by any one selected from the group consisting of nuclear magnetic resonance (NMR), chromatography and mass spectrometry, but is not limited thereto.
In a specific example of the present invention, the present inventors recruited 50 patients with progressive dialysis gastric cancer (stomach cancer patients) and 14 patients with early gastric cancer (control group) from Korea University Hospital in order to analyze cancer macro-environment metabolites, Urine samples were collected from patients. In order to identify representative gastric cancer NMR spectra in the collected urine samples, 1 H-NMR-MS was used and a total of 44 NMR spectra were obtained from the urine samples (see Table 1). (PCA) and orthogonal partial least squares discriminant analysis (PCA) were used to compare the metabolic patterns of the gastric cancer patients and the control group using a total of 44 cancer macrophysiological metabolites identified in urine metabolites ) Method, it was confirmed that metabolites of gastric cancer patient group and control group were clearly separated in macroscopic environment in both PCA analysis and OPLS-DA analysis (see FIG. 1A and Table 1). In addition, VIP (variable importance of projection) method was used to identify metabolites different from gastric cancer patient group and control group by using the above-identified 44 cancer macroenvironmental metabolites. As a result, Phenylacetylglycine, acetate, lactate, phenylalanine, mannitol, formate, 3- (4-hydroxyphenylacetate), alanine, phenylacetylglycine, (3-indoxylsulfate), glycolate, arginine, methionine, tyrosine, O-acetylcarnitine, glycine and acetone metabolites And hypoxanthine metabolites were found to be lower in gastric cancer patients (see FIG. 1B).
Accordingly, the present invention confirms 44 metabolites derived from the gastric cancer macro environment using urine collected from gastric cancer patients, and can be used as a diagnostic method of gastric cancer using the metabolic pathology according to the present invention.
In addition,
1) In a sample isolated from a test sample, isoleucine, glutamate, leucine, valine, alanine, lysine, phenylalanine, lactate, measuring the concentration of at least one selected from the group consisting of glucose, lactate, and taurine;
2) comparing the concentration of the metabolite measured in step 1) with a normal control; And
3) determining that the subject is at risk for gastric cancer or having a high risk of dignity if the concentration of the metabolite measured in the step 2) is increased as compared with the normal control group; And provides a method for analyzing metabolites.
The sample of step 1) is preferably a cell or a tissue, but is not limited thereto
The concentration of the step 1) is preferably measured by any one selected from the group consisting of nuclear magnetic resonance (NMR), chromatography and mass spectrometry, but is not limited thereto.
In a specific example of the present invention, the present inventors analyzed cancer metastasis from a total of 30 out of 50 advanced gastric cancer patients (stomach cancer patients) and 14 early gastric cancer patients (control group) Normal tissues at least 50-100 mm away from the tissue boundary were collected. The collected tissues were subjected to metabolite analysis using a VNMRS 500 NMR spectrometer (Aglient Technologies Inc., Santa Clara, Calif.). As a result, 19 representative NMR spectra were obtained from gastric cancer tissues and normal tissues (see Table 2) . PCA (Principal Components Analysis) and OPLS-DA (orthogonal partial least squares discriminant analysis) methods were used to compare the metabolic patterns of gastric cancer tissues and normal tissues using the above-identified 19 cancer local metabolites. As a result, it was confirmed that both the PCA analysis and the OPLS-DA analysis clearly differentiated the gastric cancer tissue and the normal tissue metabolite from the local environment (see FIG. 2A). As a result of the variable importance of projection (VIP) method, the lipid metabolites were found to be present in a large number of normal tissues in order to identify metabolites clearly distinguished from gastric cancer tissues and normal tissues using 19 NMR spectra Gastric cancer tissues of patients with gastric cancer were found to contain isoleucine, glutamate, leucine, valine, alanine, lysine, phenylalanine, Lactate, and taurine metabolites were all high (see Fig. 2B and Table 2).
Accordingly, the present invention has identified a total of 19 gastric cancer-derived metabolites from gastric cancer tissues and normal tissues collected from gastric cancer patients, and can be used as a diagnostic method of gastric cancer using the metabolomics according to the present invention.
In addition,
1) A method for detecting glycolated, glycine, or glycine in a sample from a group consisting of urine, blood, serum, plasma and saliva separated from a subject, and cells or tissues separated from the subject, But are not limited to, methionine, lactate, alanine, phenylalanine, tyrosine, agrinine, valine, glutamate, lysine, Measuring the concentration of one or more selected from the group of metabolites consisting of leucine, phenylacetylglycine, acetylcarnitine and acetone;
2) the relationship between the concentrations of the metabolites measured in each sample in step 1) above with the concentrations of the metabolites measured in the same type of sample from a normal control; And
3) In step 2), each sample is mixed with a solution containing glycolate, glycine, methionine, lactate, alanine, phenylalanine, tyrosine, Concentrations of agrinine, valine, glutamate, lysine, leucine, phenylacetylglycine, acetylcarnitine and acteone were determined in normal controls The method comprising the step of judging that the subject has a stomach cancer or that the risk of stomach cancer is high, the method comprising the steps of:
The concentration of the step 1) is preferably measured by any one selected from the group consisting of nuclear magnetic resonance (NMR), chromatography and mass spectrometry, but is not limited thereto.
In a specific embodiment of the present invention, it was not possible to distinguish whether the gastrointestinal macroscopic environmental metabolite was altered by being affected by other conditions, such as a drug or diet. This is a common problem that occurs in various macroscopic metabolites. As a result of comparing the change of the metabolism of the metabolism of the local environment with the metabolism of the macro environment, Amino acid and lipid were identified in macro environment and local environment metabolism. Glycine, glycine, methionine, lactate, alanine, phenylalanine, tyrosine, agrinine, valine, glue, Amino acids such as glutamate, lysine and leucine increased in both macroscopic and local environments in response to the rapid growth rate of the cancer and lipid was degraded The amount of lipid in the cancer tissue was decreased while phenylacetylglycine, acetylcarnitine and acteone contained in the lipid metabolism by-products were found to be high in the macro environment (see Table 3) . These results confirmed that the metabolites identified in the macro environment were changed by reflecting the cancerous local environment. Then, in order to select gastric cancer-related metabolites from urine of gastric cancer patients and to evaluate the gastric cancer predicting ability through the metabolized metabolism by comparing cancer macro-environment metabolism with cancer local metabolism, Were used to generate OPLS-DA models. Urine samples were collected from 31 patients in the control group and 23 patients in the gastric cancer patients, and the metabolites were analyzed. As a result, 22 out of 23 gastric cancer patients were diagnosed as gastric cancer and 29 of 31 control patients were normal. Also, it was confirmed that 96.65% sensitivity and 93.35% specificity were shown (see FIG. 4).
Accordingly, it has been confirmed through the results of the present invention that cancer diagnosis and judgment can be performed using the above-described metabolite.
The present invention also relates to a pharmaceutical composition comprising 4-hydroxyphenylacetate, alanine, phenylacetylglycine, acetate, lactate, phenylalanine, mannitol, Formate, 3-indoxylsulfate, glycolate, arginine, methionine, tyrosine, O-acetylcarnitine, glycine, glycine, acetone, and hypoxanthine. The present invention also provides a diagnostic kit for a gastric cancer using a macro-environment metabolite, comprising a quantification device for a metabolite selected from the group consisting of glycine, acetone and hypoxanthine.
The present invention also relates to a pharmaceutical composition comprising isoleucine, glutamate, leucine, valine, alanine, lysine, phenylalanine, lactate, And a metering device for a metabolite selected from the group consisting of taurine, and a gastric cancer diagnostic kit using a local environmental metabolism.
The present invention also relates to a pharmaceutical composition comprising at least one compound selected from the group consisting of glycollate, glycine, methionine, lactate, alanine, phenylalanine, tyrosine, agrinine, Diagnosis of gastric cancer using macro environment metabolism and local environmental metabolism simultaneously, including quantification device for metabolites selected from the group consisting of valine, glutamate, lysine, leucine Kits.
Preferably, the quantification device is measured by any one selected from the group consisting of nuclear magnetic resonance (NMR), chromatography and mass spectrometry, but is not limited thereto.
In addition,
1) confirming a pattern of a metabolite through NMR analysis in any one sample selected from the group consisting of urine, blood, serum, plasma and saliva separated from the subject;
2) confirming the pattern of the metabolite through HR-MAS analysis in a sample of cells or tissues separated from the subject; And
3) screening metabolic markers having matched patterns in macroscopic and local environments, comprising the step of selecting metabolites that match the pattern of the metabolites in steps 1) and 2) above.
In addition,
1) confirming a pattern of a metabolite through NMR analysis in any one sample selected from the group consisting of urine, blood, serum, plasma and saliva separated from the subject;
2) confirming the pattern of the metabolite through HR-MAS analysis in a sample of cells or tissues separated from the subject;
3) selecting the metabolites in which the pattern of the metabolism coincides in the step 1) and the step 2); And
4) A method for diagnosing, monitoring or screening gastric cancer is provided by analyzing patterns of increase and decrease of metabolites selected in step 3).
The selected metabolites may be selected from the group consisting of Glycolate, Glycine, Methionine, Lactate, Alanine, Phenylalanine, Tyrosine, Agrinine, But is not limited to, any one selected from the group consisting of valine, glutamate, lysine, and leucine.
Hereinafter, the present invention will be described in detail with reference to examples.
However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.
< Example 1> Cancer macro environment Metabolism analysis
<1-1> Urine sample collection
In order to analyze cancer macro - environment metabolites, 50 patients with advanced gastric cancer (stomach cancer) and 14 patients with early gastric cancer (control group) were recruited from Korea University Hospital. A urine sample was collected one week after the cancer removal surgery sample and cancer operation from the above patients. All collected urine samples were stored in a freezer maintained at -80 ° C until analysis. Each urine sample was then centrifuged at 12,000 g × force at 4 ° C for 10 minutes to obtain a 400 uL supernatant. The obtained supernatant was mixed with 230 uL sodium phosphate buffer (0.2 mol / L, pH 7.0, 0.018% NAN3) and 70 uL of 5 mmol / L DSS-dissolved D20. The pH was then adjusted to 7.0 ± 0.1 and then 600 μL samples were transferred to an NMR tube.
<1-2> Urine In metabolism Typical gastric cancer NMR Spectrum identification
In order to confirm a typical gastric cancer NMR spectrum in urine samples collected by the method described in the above Example <1-1>, the following method was performed.
Specifically, the urine metabolism collected by the method described in Example <1-1> was analyzed with a Varian VnmrS-600 MHz (Aglient) instrument equipped with a triple-resonance 5-mm HCN salt tolerant cold probe Technologies Inc., Santa Clara, Calif.). A NOESYPRESAT pulse sequence was used to obtain the 1 H-NMR spectrum of the pretreated urine sample. In order to minimize the water peak, which is relatively sensitive to the sample peak, the saturation power and saturation frequency parameters related to the saturation were measured while varying the parameters. Also, at 298K, the spectrum width was 6720.4 Hz, the data point was 32K, the tracking time was 4 seconds, the relaxation delay was 2 seconds, and the number of transients was 64. All spectra were subjected to Fourier Transform (FT) with line broadening at 0.5 Hz.
As a result, as shown in Table 1, a total of 44 NMR spectra were obtained from the urine metabolites collected from the gastric cancer patient group and the control group according to the method described in Example <1-1> (Table 1).
<1-3> Identified in urine of patients with gastric cancer Metabolism Pattern analysis
PCA (Principal Components Analysis, PCA) was used to compare the metabolic patterns of the gastric cancer patient group and the control group using a total of 44 cancer macroenvironmental metabolites identified in the urine metabolism by the method described in Example <1-2> And orthogonal partial least-squares discriminant analysis (OPLS-DA).
Specifically, a total of 44 metabolites obtained by the method described in the above Example <1-2> were analyzed by 2D NMR analysis, spiking experiment, already reported documents, and Chenomx NMR compound library, , The concentration was measured using Chenomx NMR Suite 6.0 software (Chenomx Inc. Edmonton, Canada). Multivariate analysis was performed using SIMCA-P + version 12.0 (Umetrics AB, Umea, Sweden). First, the SIMCA data were analyzed by principal component analysis (PCA), which is a non - glycemic method, to confirm the pattern difference between the gastric cancer patient metabolism and the control metabolism. Then, using the orthogonal partial least-squares discriminant analysis (OPLS-DA) method, we identified a substance that distinguishes between gastric cancer patients and controls more reliably.
As a result, as shown in Fig. 1A and Table 1, it was confirmed that metabolites of gastric cancer patient group and control group were clearly separated in macroscopic environment in both PCA analysis and OPLS-DA analysis (Fig. 1A and Table 1).
<1-4> Gastric Cancer Patients Metabolism Confirm
In order to identify metabolites that differ between the gastric cancer patient group and the control group using a macro-environment metabolite of 44 cancers confirmed by the method described in Example <1-2>, a variable importance of projection (VIP) method Respectively.
Specifically, VIP (variable importance of projection) was performed using a macro-environment metabolite in a total of 44 cancers confirmed in the urine metabolism by the method described in Example <1-2> above. Only metabolites with a VIP value of greater than or equal to 1 and a t-test P-value of less than 0.05 were selected.
As a result, as shown in FIG. 1B, in the gastric cancer patient group, 4-hydroxyphenylacetate, alanine, phenylacetylglycine, acetate, lactate, phenylalanine phenylalanine, mannitol, formate, 3-indoxylsulfate, glycolate, arginine, methionine, tyrosine, O-acetylcarnitine (O-acetylcarnitine), glycine and acetone metabolites were all high, and hypoxanthine metabolites were found to be low in gastric cancer patients (FIG. 1B).
< Example 2> Cancer local environment Metabolism analysis
<2-1> Gastric cancer sample collection
To analyze the cancerous local environment metabolism, normal tissues at least 50 to 100 mm or more on the cancer tissue and cancer tissue boundary were collected from a total of 30 patients who collected urine samples according to the method described in Example <1-1> Respectively. Stomach cancer stage of gastric cancer tissue was confirmed by H & E staining method.
Specifically, stomach cancer tissue was fixed with heparin flame solution containing 3.7% formaldehyde and then cut. A continuous tissue fragment (4 μm thick) was obtained and stained with hematoxylin and eosin (HE). The patients were free of chemotherapy and radiotherapy, and all samples were stored in a freezer maintained at -80 ° C before analysis.
<2-2> Typical gastric cancer in gastric cancer NMR Spectrum identification
In order to analyze the metabolites of cancer tissues collected by the method described in the above Example <2-1>, the following methods were performed.
Specifically, the cancer tissue collected by the method described in Example <2-1> was weighed to 7.5 mg, deuterium was added thereto, and the HR-MAS nano-probe (Agilent, Walnut Creek, And the volume was adjusted to 40 [mu] l. The pretreated samples were analyzed for metabolites using a VNMRS 500 NMR spectrometer (Aglient Technologies Inc., Santa Clara, Calif.). Spin of the magic angle spining probe was set to 2000 Hz, N2 was used to obtain spectra at 283K to minimize the deformation of the spectrum. The CPMG pulse sequence was used to obtain the 1 H-NMR spectrum. The spectrum width was 6720.4 Hz, the data point was 32 K, the tracking time was 4 seconds, the relaxation delay was 2 seconds, the T2 relaxation time was 0.1 second, 128. All spectra were subjected to Fourier Transform (FT) with line broadening at 0.5 Hz. Spectral binning was performed using Chenomx NMR Suite 6.0 software (Chenomx Inc. Edmonton, Canada) except for the water and urea sites (4.7-6.8 ppm) before normalizing and conditioning all spectra. The spectral binning is performed by scrambling the spectrum obtained in the embodiment <2-2> to a certain range with a narrow section, and then comparing the integrated values for each section. Bin size was set to 0.003 ppm. All spectra were adjusted in MATLAB (R2008a, The Mathworks, Inc. Matick, Mass.) By applying a Correlation Optimized Warping (COW) method based on the binning result.
As a result, a total of 19 representative NMR spectra were obtained from stomach cancer tissues and normal tissues as shown in Table 2 (Table 2).
<2-3> Identified in gastric cancer patient tissue Metabolism Pattern analysis
In order to compare the metabolism patterns of gastric cancer tissues and normal tissues using a total of 19 cancer local metabolites obtained from stomach cancer tissues and normal tissues by the method described in the above Example <2-2>, PCA (Principal Components Analysis , PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA).
Specifically, the total of 19 metabolites obtained by the method described in the above Example <2-2> were confirmed using 2D NMR analysis, already reported literature references and spiking experiments. The relative quantitative value of the metabolite was used as the integration value of the NMR spectral binning site. Multivariate analysis was also performed using SIMCA-P + version 12.0 (Umetrics AB, Umea, Sweden). The relative quantitative data of 19 metabolites, called SIMCA, were first analyzed by principal component analysis (PCA), which is a non - biochemical method, to confirm pattern differences between gastric cancer patients and normal tissue metabolites. Then, OPLS-DA (orthogonal partial least-squares discriminant analysis) analysis was performed to identify metabolites that differentiate between gastric cancer tissues and normal tissues.
As a result, as shown in Fig. 2A, it was confirmed that both the PCA analysis and the OPLS-DA analysis clearly differentiated the gastric cancer tissue and normal tissue metabolites in the local environment (Fig. 2A).
≪ 2-4 > Cancer-related Metabolism Confirm
In order to identify the metabolites clearly differentiated from gastric cancer tissues and normal tissues using representative metabolites in the NMR spectra obtained from gastric cancer tissues and normal tissues by the method described in the above Example <2-2>, VIP (variable importance of projection method.
Specifically, VIP (variable importance of projection) was performed using a total of 19 local environmental metabolites identified in the gastric cancer tissue metabolism by the method described in the above Example <2-2>. Only the metabolites with a VIP value of 1 or more and a P-value of 0.05 or less in the t-test were selected.
As a result, as shown in FIG. 2B and Table 2, it was confirmed that lipid metabolites were abundant in normal tissues. In gastric cancer patients, isoleucine, glutamate, leucine ), Valine, alanine, lysine, phenylalanine, lactate, and taurine metabolites were found to be high (FIG. 2B and Table 2 ).
< Example 3> Cancer macro environment Metabolism Cancer local environment Metabolism compare
In order to compare the macro-environment metabolites identified in gastric cancer patient urine by the method described in the above Example <1-4> and the local environmental metabolites identified in the gastric cancer tissues by the method described in Example <2-4> The same method was used.
Specifically, the macroscopic environment obtained in Tables 1 and 2 and the metabolism list obtained in the local environment were compared first in terms of substances having the same tendency to increase or decrease while they were common in the first place. The second compares the substances that are present in the same metabolic pathway and increase or decrease in the same metabolic direction. We could not distinguish whether the gastrointestinal macroscopic environmental metabolism reflects cancer status, or whether it has been influenced by other factors such as medication or dietary habits. This is a common problem that arises in various macroscopic environmental metabolites, compared with the tendency of local environmental metabolism change and macroscopic environmental metabolism (Fig. 3).
As a result, as shown in Table 3, amino acids and lipids were commonly found in macroscopic and local environmental metabolites. Glycine, glycine, methionine, lactate, alanine, phenylalanine, tyrosine, agrinine, valine, glue, Amino acids such as glutamate, lysine and leucine increased in both macroscopic and local environments in response to the rapid growth rate of the cancer and lipid was degraded The amount of lipid in cancer tissues decreased and phenylacetylglycine, acetylcarnitine and acteone contained in lipid metabolism by-products were found to be high in macro environment (Table 3).
Therefore, it was confirmed that the metabolites, which were waxed in the macro environment, were changed by reflecting the local environment of the cancer.
< Example 4> Determined cancer macro environment Metabolism Evaluation of Stomach Cancer Prediction
The following method was performed to evaluate the stomach cancer prediction ability through the cancer macro environment metabolite selected in Example <1-4> and discriminated in <Example 3>.
Specifically, in order to verify the OPLS-DA model produced using the metabolites selected in Example <1-4> and confirmed in Example 3, urine samples of 31 control subjects and 23 gastric cancer patients were used. Metabolite analysis was performed separately.
As a result, as shown in FIG. 4, 22 out of 23 gastric cancer patients were diagnosed as gastric cancer, and 29 of the 31 control patients were normal. Also, it was confirmed that the sensitivity was 96.65% and the specificity was 93.35% (FIG. 4).
Therefore, it was confirmed through the result of <Example 4> that cancer diagnosis and judgment can be performed using the verified metabolite.
The present invention provides a method for evaluating the metabolism of a gastric cancer patient using a metabolite of the present invention or a metabolite derived from a local environment using a sample such as urine, blood, serum, plasma and saliva, Environment, and local environment at the same time to develop diagnostic, screening, monitoring, diagnostic kits and therapeutic agents for gastric cancer.
Claims (15)
2) the relationship between the concentration of the metabolites measured in each sample in step 1) and the concentration of the metabolites measured in the same type of sample from the normal control, respectively; And
3) In step 2), each sample is mixed with a solution containing glycolate, glycine, methionine, lactate, alanine, phenylalanine, tyrosine, Which is selected from the group consisting of agrinine, valine, glutamate, lysine, leucine, phenylacetylglycine, acetylcarnitine and acteone. And determining that the subject is at risk for stomach cancer if the one or more concentrations are increased relative to the normal control group.
2) the relationship of comparing the concentration of metabolites measured in step 1) with a normal control; And
3) In step 2), 4-hydroxyphenylacetate, alanine, phenylacetylglycine, acetate, lactate, phenylalanine, mannitol, ), Formate, 3-indoxylsulfate, glycolate, arginine, methionine, tyrosine, O-acetylcarnitine, When the concentration of any one or more selected from the group consisting of glycine and acetone is increased as compared with the normal control group or the concentration of hypoxanthine is decreased as compared with the normal control group, Or determining that the risk of gastric cancer is high.
2) comparing the concentration of the metabolite measured in step 1) with a normal control; And
3) determining that the subject is at risk for gastric cancer or having a high risk of dignity if the concentration of the metabolite measured in the step 2) is increased as compared with the normal control group; Method of analysis of metabolites.
2) confirming the pattern of the metabolite through HR-MAS analysis in a sample of cells or tissues separated from the subject; And
3) screening metabolic markers having a matching pattern in a macroscopic and local environment, comprising the step of selecting metabolites with a pattern of the metabolites in the above-mentioned steps 1) and 2)
2) confirming the pattern of the metabolite through HR-MAS analysis in a sample of cells or tissues separated from the subject;
3) selecting the metabolites in which the pattern of the metabolism coincides in the step 1) and the step 2); And
4) A method for diagnosing, monitoring or screening gastric cancer by analyzing the pattern of increase / decrease of metabolites selected in step 3).
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