Helicobacter pylori infection-related metabolic marker composition for predicting gastric cancer onset risk and application of helicobacter pylori infection-related metabolic marker composition

Abstract

The invention discloses a helicobacter pylori infection related metabolic marker composition for predicting the onset risk of gastric cancer and application thereof. The invention discloses a helicobacter pylori infection related metabolic marker combination for predicting the onset risk of gastric cancer. The helicobacter pylori infection related metabolic marker combination consists of 26 substances such as kynurenine, glutaric acid and citrulline. According to the invention, through cross section research of 1800 cases of research objects, metabolic characteristics related to H.pylori infection are analyzed, and in nested case-control research, it is found that a metabolic marker with the metabolic characteristics of H.pylori infection is significantly related to gastric cancer risk and plays a mediation role in gastric cancer occurrence. A blood sample of a participating object can be collected in a gastric cancer screening item, plasma is separated, 26 plasma metabolites in the scheme are detected, a metabolism score is constructed according to an LASSO regression coefficient, and the gastric cancer risk is evaluated.

Description

Technical Field

[0001] The present invention belongs to the field of medical diagnosis and relates to a Helicobacter pylori infection-related metabolic marker combination for predicting the risk of gastric cancer and an application thereof. Background Art

[0002] Gastric cancer is the fifth most common cancer in the world and the third leading cause of cancer-related death. Known environmental risk factors for the development of gastric cancer include Helicobacter pylori (H.pylori) infection, smoking and drinking. Unhealthy lifestyles, such as smoking, drinking, large intake of pickled foods, and insufficient intake of fresh fruits and vegetables are all important causes of the high incidence of gastric cancer. In addition, the most important environmental risk factor for gastric cancer is H.pylori infection, which is a Class I carcinogen confirmed by the International Agency for Research on Cancer. Improving detection and eradication of H.pylori infection is one of the focuses of primary prevention of gastric cancer. Gastric cancer patients are often discovered in the late stage, and gastric cancer still brings a serious disease burden. Therefore, understanding the molecular characteristics of gastric cancer is crucial for identifying high-risk populations and implementing prevention and screening measures. However, there are limited biomarkers for predicting the incidence of gastric cancer.

[0003] Emerging plasma metabolomics studies provide insights into predicting disease risk and its clinical applications. Various plasma metabolic signatures have been found to be potential biomarkers for precancerous lesions and gastric cancer. Changes in short-chain fatty acids and steroids in plasma can be used to assess the progression of gastric cancer. These metabolic signatures are involved in the processes leading to gastric cancer through complex cellular functions and mechanisms. However, most studies are based on hospital-derived subjects and are conducted using targeted metabolomics approaches through case-control studies. The European Prospective Investigation into Cancer and Nutrition cohort (EPIC-EURGAST) found through a nested case-control study that elevated concentrations of certain plasma phospholipid fatty acids were associated with an increased risk of gastric cancer. Therefore, it is necessary to systematically identify biomarkers associated with gastric cancer risk using non-targeted metabolomics through prospective nested case-control studies.

[0004] Plasma metabolic characteristics can reflect internal disturbances caused by environmental factors, and these metabolic characteristics can also be regarded as markers of cancer risk. Many studies have shown that H. pylori infection can induce the occurrence and development of gastric cancer through a variety of known mechanisms, such as chronic inflammation, virulence factors, and DNA damage. Inducing metabolic changes is also one of the important mechanisms by which Helicobacter pylori causes gastric cancer. H. pylori infection can directly or indirectly affect internal metabolic characteristics and play an important role in the development of cancer. However, there are relatively few studies on metabolic characteristics related to environmental factors that closely affect the risk of gastric cancer (such as H. pylori infection, smoking, and drinking). Summary of the invention

[0005] The purpose of the present invention is to provide a combination of Helicobacter pylori infection-related metabolic markers for predicting the risk of gastric cancer in view of the above-mentioned deficiencies in the prior art.

[0006] Another object of the present invention is to provide a use of the combination.

[0007] Another object of the present invention is to provide the use of a reagent for detecting the metabolic marker combination.

[0008] The fourth object of the present invention is to provide a plasma test kit for Helicobacter pylori infection for predicting the risk of gastric cancer.

[0009] The purpose of the present invention can be achieved through the following technical solutions:

[0010] A combination of Helicobacter pylori infection-related metabolic markers for predicting the risk of gastric cancer, comprising kynurenine, glutaric acid, citrulline, lysine, valine, betaine, tyrosine, adenine, sarcosine, butyric acid, creatine, glycerol, isobutyrylcarnitine, N-acetyltyrosine, propionylcarnitine, N-acetylarginine, erythritol, glycol lithocholate sulfate, α-hydroxycaproic acid, 4-hydroxyglutamic acid, glycine deoxycholic acid, methionine sulfone, ethanol cholesterol-glucuronide, lactosyl-N-ceramide (d18:1 / 24:1), bilirubin degradation products, C 16 H 18 It is composed of N2O5 and tetrahydrocortisone glucuronide.

[0011] The invention relates to the use of the combination of Helicobacter pylori infection-related metabolic markers in the preparation of a kit for assisting in predicting the risk of gastric cancer.

[0012] As a preferred embodiment of the present invention, the combination of Helicobacter pylori infection-related metabolic markers is used in the preparation of a plasma test kit for assisting in predicting the risk of gastric cancer.

[0013] The use of a reagent for detecting the combination of metabolic markers related to Helicobacter pylori infection in the preparation of a kit for assisting in predicting the risk of gastric cancer.

[0014] As a preferred embodiment of the present invention, the combination of Helicobacter pylori infection-related metabolic markers is used in the preparation of a plasma test kit for assisting in predicting the risk of gastric cancer.

[0015] As a preferred embodiment of the present invention, the reagent for detecting the combination of metabolic markers associated with Helicobacter pylori infection is a reagent for detecting the combination of metabolic markers associated with Helicobacter pylori infection in plasma using an ultra-high performance liquid chromatography combined with quadrupole-orbitrap high-resolution mass spectrometry technology platform.

[0016] A Helicobacter pylori-infected plasma kit for predicting the risk of gastric cancer comprises the Helicobacter pylori infection-related metabolic marker combination as a standard combination.

[0017] As a preferred embodiment of the present invention, the plasma test kit further comprises a reagent for detecting the combination of Helicobacter pylori infection-related metabolic markers as described in claim 1 by combining ultra-high performance liquid chromatography with quadrupole-orbitrap high-resolution mass spectrometry.

[0018] As a preferred embodiment of the present invention, the mobile phase used in the ultra-high performance liquid chromatography detection comprises mobile phase A and mobile phase B, mobile phase A is 10mM ammonium acetate / methanol / formic acid with a volume ratio of 95:5:0.1, and mobile phase B is methanol / formic acid with a volume ratio of 9.9:0.1.

[0019] As a preferred embodiment of the present invention, the mobile phase for the quadrupole-orbitrap high-resolution mass spectrometry comprises mobile phase A and mobile phase B, mobile phase A is an aqueous solution of 10 mM ammonium formate and 0.1% formic acid, and mobile phase B is acetonitrile containing 0.1% formic acid.

[0020] Beneficial effects:

[0021] The present invention analyzes the metabolic characteristics associated with H.pylori infection through a cross-sectional study involving 1,800 research subjects, and finds in a nested case-control study (326 gastric cancer cases and 326 healthy controls) that the metabolic characteristics of H.pylori infection are significantly correlated with the risk of gastric cancer, and play a mediating role in the occurrence of gastric cancer. To this end, we provide a class of H.pylori-related plasma metabolite combinations for gastric cancer risk prediction and their applications. Blood samples from participating subjects can be collected in gastric cancer screening projects, plasma can be separated and 26 plasma metabolites in the scheme can be detected, and a metabolic score can be constructed based on the LASSO regression coefficient to assess gastric cancer risk. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 ROC Curve DETAILED DESCRIPTION

[0023] Example 1 Collection of samples and organization of sample data

[0024] 1. Selection of research samples:

[0025] A total of 1,800 subjects from two prospective cohorts in Nantong and Changzhou, Jiangsu Province, were included in the cross-sectional study. Among them, 326 new gastric cancer cases were matched with healthy controls 1:1 by age ± 2 years, gender and region, and a nested case-control study was designed. A total of 1,800 samples that met the criteria were included in this study, including 824 cases in Nantong and 976 cases in Changzhou.

[0026] 2. Extraction of plasma samples:

[0027] From each research subject, 5 ml of fasting venous blood was collected in the morning using a vacuum anticoagulation (EDTA) blood collection tube. The plasma was immediately separated according to the standard method and frozen at -80°C for later use. 100 μL of plasma sample was transferred to an EP tube, 300 μL of extraction solution (methanol, containing an isotope-labeled internal standard mixture) was added, and the mixture was vortexed for 30 seconds; ultrasonicated for 10 minutes (ice-water bath); allowed to stand at -40°C for 1 hour; the sample was centrifuged at 4°C, 12000 rpm for 15 minutes; the supernatant was taken into a sampling bottle and tested on the machine.

[0028] Example 2 Plasma Metabolome Detection

[0029] Metabolomics testing:

[0030] Sample detection: Ultra-high performance liquid chromatography (Waters ACQUITY) and quadrupole-orbitrap high-resolution mass spectrometry (Thermo Scientific Q Exactive) were used for detection.

[0031] (1) A Waters C18 column (UPLC BEH C18-2.1x100 mm, 1.7 μm) was used, and eluted with 80% mobile phase A (95:5:0.1 vol / vol / vol 10 mM ammonium acetate / methanol / formic acid) for 1 min, 80% mobile phase B (99.9:0.1 vol / vol methanol / formic acid) for 2 min, and 100% mobile phase B for 7 min; mass spectrometry analysis was performed in electrospray ionization negative ion mode, full scan analysis was performed in the range of 200-1000 m / z, the resolution was 70000, and the data acquisition rate was 3 Hz; other parameters: sheath gas flow rate 50, in-source CID 5ev, sweep gas 5, spray voltage 3kV, capillary temperature 300°C, s-lens RF voltage 50V, heater temperature 300°C.

[0032] (2) A Waters HILIC column (UPLC BEH Amide 2.1x150 mm, 1.7 μm) was used, and eluted with 5% mobile phase A (aqueous solution of 10 mM ammonium formate and 0.1% formic acid) for 0.5 min and 40% mobile phase B (acetonitrile containing 0.1% formic acid) for 10 min; mass spectrometry analysis was performed in electrospray ionization positive ion mode, with full scan analysis in the range of 70-800 m / z, a resolution of 70,000, and a data acquisition rate of 3 Hz; other parameters: sheath gas flow rate 40, sweep gas 2, spray voltage 3.5 kV, capillary temperature 350°C, s-lens RF voltage 40, and heater temperature 300°C.

[0033] Data processing:

[0034] Peak identification, peak extraction, peak alignment and integration were performed, and substance annotation was performed to determine 615 named metabolites in baseline plasma; outliers were filtered based on relative standard deviation; missing data were filled with half of the minimum value; and normalization was performed using an internal standard.

[0035] Example 3

[0036] Statistical analysis:

[0037] Linear regression was performed in the cross-sectional study, and after multiple correction (FDR) to avoid false positive results, 46 metabolites were found to be significantly different between H. pylori positive and negative subjects, satisfying PFDR < 0.2.

[0038] Lasso regression (LASSO regression) was used to remove collinearity, and 26 metabolic markers with independent predictive value were further discovered (Table 1). The metabolic score associated with H. pylori infection was constructed by multiplying the coefficient of LASSO regression with the expression level of the metabolite. The formula is as follows:

[0039] Metabolic score = ∑(S0*expressionM)

[0040] S0 represents the LASSO regression coefficient, and expressionM represents the expression level of plasma metabolites.

[0041] Conditional logistic regression was performed in a nested case-control study, and it was found that the metabolic score associated with H.pylori infection was associated with the risk of gastric cancer (OR=1.66, 95%CI: 1.32-2.09, P=1.62×10-5), and the effect was stable in the two regions (Nantong: OR=1.52, 95%CI: 1.09-2.12, P=0.014; Changzhou: OR=1.54, 95%CI: 1.20-1.97, P=0.001). The association between the metabolic score associated with H.pylori infection and the risk of gastric cancer was not affected by other risk factors.

[0042] Based on the data of all 1800 samples in the above cohort, a prediction model was established using metabolic scores. Receiver operating characteristic (ROC) analysis showed that the area under the ROC curve (AUC) of the model in the cohort was 0.87 (95% CI: 0.85-0.88), and the sensitivity and specificity were 75.2% and 84.3%, respectively. Figure 1). The 26 significantly different metabolites between H. pylori positive and negative subjects are shown in Table 1.

[0043] Table 1

[0044]

[0045]

Claims

1. A combination of Helicobacter pylori infection-related metabolic markers for predicting the risk of gastric cancer, characterized in that: The combination of metabolic markers related to Helicobacter pylori infection consists of kynurenine, glutaric acid, citrulline, lysine, valine, betaine, tyrosine, adenine, sarcosine, butyric acid, creatine, glycerol, isobutyrylcarnitine, N-acetyltyrosine, propionylcarnitine, N-acetylarginine, erythritol, glycol lithocholate sulfate, α-hydroxycaproic acid, 4-hydroxyglutamic acid, glycine deoxycholic acid, methionine sulfone, ethanol cholesterol-glucuronide, lactosyl-N-ceramide (d18:1 / 24:1), bilirubin degradation products, C16H18N2O5, and tetrahydrocortisone glucuronide.

2. Use of the combination of Helicobacter pylori infection-related metabolic markers according to claim 1 in the preparation of a kit for assisting in predicting the risk of gastric cancer.

3. The use according to claim 2, characterized in that Use of the combination of Helicobacter pylori infection-related metabolic markers as described in claim 1 in the preparation of a plasma kit for assisting in predicting the risk of gastric cancer.

4. Use of a reagent for detecting the combination of metabolic markers associated with Helicobacter pylori infection according to claim 1 in the preparation of a kit for assisting in predicting the risk of gastric cancer.

5. The use according to claim 4, characterized in that Use of the combination of Helicobacter pylori infection-related metabolic markers as described in claim 1 in the preparation of a plasma kit for assisting in predicting the risk of gastric cancer.

6. The use according to claim 5, characterized in that: The reagent for detecting the combination of metabolic markers associated with Helicobacter pylori infection according to claim 1 is a reagent for detecting the combination of metabolic markers associated with Helicobacter pylori infection according to claim 1 derived from plasma using an ultra-high performance liquid chromatography combined with a quadrupole-orbitrap high-resolution mass spectrometry technology platform.

7. A plasma kit for predicting the risk of gastric cancer by Helicobacter pylori infection, characterized in that: Contains the Helicobacter pylori infection-related metabolic marker combination as described in claim 1 as a standard combination.

8. The plasma kit according to claim 7, characterized in that The invention also contains a reagent for detecting the combination of Helicobacter pylori infection-related metabolic markers as described in claim 1 by combining ultra-high performance liquid chromatography with quadrupole-orbital trap high-resolution mass spectrometry.

9. The plasma kit according to claim 8, characterized in that The mobile phase used in the ultra-high performance liquid chromatography detection comprises mobile phase A and mobile phase B, wherein mobile phase A is 10 mM ammonium acetate / methanol / formic acid in a volume ratio of 95:5:0.1, and mobile phase B is methanol / formic acid in a volume ratio of 9.9:0.

1.

10. The plasma kit according to claim 8, characterized in that The mobile phase for the quadrupole-orbital trap high-resolution mass spectrometry comprises mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of 10 mM ammonium formate and 0.1% formic acid, and mobile phase B is acetonitrile containing 0.1% formic acid.

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