Serum marker combination for gastric cancer diagnosis and application thereof

Abstract

This invention provides a combination of serum biomarkers for the diagnosis of gastric cancer and their applications. This invention is the first to discover that CHI3L1, FCGBP, TFF2, and VSIG2 are significantly elevated in the serum of gastric cancer patients. ROC curve analysis verified that the combination of these four proteins as serum biomarkers can effectively distinguish between gastric cancer patients and healthy controls, and that combined use can better differentiate between early and late-stage gastric cancer patients, demonstrating superior diagnostic value for gastric cancer. Compared with traditional methods for gastric cancer diagnosis, the serum biomarker combination established in this invention is non-invasive, improving the accuracy of gastric cancer diagnosis. It can be used as a health screening test and provides an efficient and non-invasive detection method for gastric cancer patients who cannot undergo pathological examination.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to gastric cancer biomarkers, specifically to a combination of serum biomarkers for the diagnosis of gastric cancer and its application. Background Technology

[0002] Gastric cancer is currently the fourth most common malignant tumor worldwide and the second leading cause of cancer-related deaths. Currently, the diagnosis and screening of gastric cancer still rely on gastroscopy, but its application is greatly limited due to its invasiveness, high cost, and the need for specialized endoscopists. Furthermore, due to the lack of typical symptoms in the early stages, the specificity and sensitivity of clinical disease biomarkers are poor, and more than 60% of patients have local or distant metastases at the time of diagnosis. The 5-year survival rate for patients with localized early-stage gastric cancer exceeds 90%, while the 5-year survival rate for patients with intermediate or advanced-stage cancer is less than 30%. Therefore, there is an urgent need to develop novel and reliable predictive biomarkers for gastric cancer, especially non-invasive serum biomarkers, which can provide valuable clinical information for the diagnosis, treatment, monitoring, and prognosis of gastric cancer.

[0003] CHI3L1 belongs to the glycoside hydrolase family 18 and is synthesized and secreted by various cell types, including immune-related cells, fibroblast-like cells, smooth muscle cells, and tumor cells. Serum CHI3L1 protein expression is significantly elevated in liver cancer patients, and higher CHI3L1 protein expression is associated with lower overall survival rates. TFF2 (trefoil factor 2), a member of the trefoil factor family, has potential importance in the diagnosis and prognostic prediction of gastric cancer due to its methylated form. FCGBP, or IgG Fc-binding protein, was initially discovered in the intestinal epithelium and thus plays a crucial role in innate mucosal epithelial defense, metastasis, and tumor immunity. Previous studies have found that FCGBP is poorly expressed in colorectal cancer patients and acts as an independent risk factor for predicting prognosis. VSIG2, also known as V-Set immunoglobulin domain-containing protein 2, belongs to the immunoglobulin superfamily (IgSF) and can act as a co-inhibitory molecule mediating tumor immune escape.

[0004] Studies have shown that biomarkers play a crucial role in the diagnosis and prognostic monitoring of gastric cancer. However, commonly used serum biomarkers in clinical practice are mostly broad-spectrum markers with poor organ specificity. They cannot simultaneously possess both high sensitivity and specificity, and can only serve as one of the detection methods for disease progression, recurrence, and prognostic assessment. Therefore, their diagnostic value for gastric cancer is very limited. Given the high mortality rate and low diagnostic rate of gastric cancer, and based on a thorough understanding of its pathogenesis, there is an urgent need to develop low-cost, highly specific, and sensitive non-invasive biomarkers to meet the needs of precision medicine in clinical practice. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a combination of serum biomarkers for the diagnosis of gastric cancer and its application.

[0006] In a first aspect of the invention, a combination of serum protein markers for the diagnosis of gastric cancer is provided, characterized in that the combination of serum protein markers includes CHI3L1, FCGBP, TFF2, and VSIG2.

[0007] Furthermore, the gastric cancer mentioned is early-stage gastric cancer.

[0008] Furthermore, the amino acid sequence of the CHI3L1 protein is shown in SEQ ID NO.1.

[0009] Furthermore, the amino acid sequence of the FCGBP protein is shown in SEQ ID NO.2.

[0010] Furthermore, the amino acid sequence of the TFF2 protein is shown in SEQ ID NO.3.

[0011] Furthermore, the amino acid sequence of the VSIG2 protein is shown in SEQ ID NO.4.

[0012] In a second aspect of the invention, a kit for the diagnosis of gastric cancer is provided, characterized in that the kit contains reagents for detecting the expression of CHI3L1, FCGBP, TFF2, and VSIG2 proteins.

[0013] Furthermore, the gastric cancer mentioned is early-stage gastric cancer.

[0014] Furthermore, the amino acid sequence of the CHI3L1 protein is shown in SEQ ID NO.1.

[0015] Furthermore, the amino acid sequence of the FCGBP protein is shown in SEQ ID NO.2.

[0016] Furthermore, the amino acid sequence of the TFF2 protein is shown in SEQ ID NO.3.

[0017] Furthermore, the amino acid sequence of the VSIG2 protein is shown in SEQ ID NO.4.

[0018] In a third aspect of the invention, the use of the serum protein marker combination described in the first aspect of the invention in the preparation of products for the diagnosis of gastric cancer is provided.

[0019] Furthermore, the gastric cancer mentioned is early-stage gastric cancer.

[0020] Furthermore, the diagnosis is a non-invasive diagnostic testing method.

[0021] In a fourth aspect of the invention, the use of the serum protein biomarker combination described in the first aspect of the invention is provided in the preparation of a product for distinguishing between gastric cancer and healthy individuals.

[0022] In a fifth aspect of the invention, the use of the serum protein biomarker combination described in the first aspect of the invention is provided in the preparation of a product for distinguishing between patients with early and late gastric cancer.

[0023] In a sixth aspect of the invention, a kit for diagnosing gastric cancer is provided, characterized in that the kit comprises reagents for detecting VSIG2 protein expression.

[0024] Furthermore, the amino acid sequence of the VSIG2 protein is shown in SEQ ID NO.4.

[0025] In a seventh aspect of the invention, the use of the VSIG2 protein in the preparation of products for the diagnosis of gastric cancer is provided.

[0026] Furthermore, the amino acid sequence of the VSIG2 protein is shown in SEQ ID NO.4.

[0027] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0028] Compared with the prior art, the present invention has the following advantages and advancements: This invention is the first to discover that CHI3L1, FCGBP, TFF2, and VSIG2 are significantly elevated in the serum of gastric cancer patients. ROC curve analysis verified that the combination of CHI3L1-FCGBP-TFF2-VSIG2 serum markers can effectively distinguish between gastric cancer patients and healthy controls. Furthermore, the combined use can better differentiate between early and late-stage gastric cancer patients, demonstrating superior diagnostic value for gastric cancer. Compared with traditional methods for diagnosing gastric cancer, the serum biomarker combination established in this invention is a non-invasive procedure, which can improve the accuracy of gastric cancer diagnosis. It can be used as a health screening test and provide an efficient and non-invasive detection method for some gastric cancer patients who cannot undergo pathological examination. Compared with the serum biomarkers currently used in clinical practice, the serum biomarker combination established in this invention has higher sensitivity and specificity for diagnosing gastric cancer, comparable to the combined use of 11 tumor biomarkers in clinical practice, including AFP, CEA, CA199, CA724, CA125, CYFRA211, CA153, CA242, NSE, SCC, and CA50. In addition, the detection method established in this patent uses ELISA technology, which has low requirements for researchers and equipment, can be operated in a routine laboratory, and the results are easy to interpret and analyze. The detection cost is low, the testing process is short, and it can basically meet the various clinical needs for testing. Attached Figure Description

[0029] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0030] Figure 1 The multi-parameter Cox model was shown to include 18 genes, including CHI3L1, FCGBP, TFF2, and VSIG2.

[0031] Figure 2 The ROC curve analysis of the combination of CHI3L1, FCGBP, TFF2 and VSIG2 in gastric cancer tissues and healthy individuals is shown in the TCGA database.

[0032] Figure 3 The expression levels of CHI3L1, FCGBP, TFF2, and VSIG2 in peripheral blood serum from gastric cancer patients and healthy individuals were shown.

[0033] Figure 4 ROC curve analysis of CHI3L1, FCGBP, TFF2 and VSIG2 used individually and in combination is shown.

[0034] Figure 5The ROC curve analysis shows the combination of CHI3L1, FCGBP, TFF2 and VSIG2 with 11 tumor markers in gastric cancer patients and healthy individuals.

[0035] Figure 6 Growth curve analysis of the gastric cancer cell line HGC-27 after interference with the expression of CHI3L1, FCGBP, TFF2 and VSIG2 is shown. Detailed Implementation

[0036] This invention provides a combination of serum biomarkers for the diagnosis of gastric cancer and its application. The invention is described in detail below with reference to embodiments to facilitate further understanding by those skilled in the art. However, the embodiments described below are only a part of the embodiments of this invention and should not be considered as any limitation on this invention. It should be noted that adjustments and improvements made by those skilled in the art based on the concept of this invention should be considered within the scope of protection of this invention. Specific technical operation steps and operators not specified in the embodiments are all performed in accordance with the general technical conditions described in the literature or relevant product instructions.

[0037] Example 1: Establishing a multiparameter Cox regression model for gastric cancer patients based on the TCGA database The Cancer Genome Atlas (TCGA) database is a project jointly launched in 2006 by the National Cancer Institute (NCI) and the National Human Genome Research Institute (NHGRI). Its aim is to improve our understanding of cancer biology and advance cancer diagnosis, treatment, and prevention through comprehensive genomic analysis. The TCGA database includes clinical data, genomic variants, mRNA expression, miRNA expression, and methylation data for various cancer types.

[0038] By downloading mRNA expression data from 375 gastric cancer patients and 32 normal tissue samples from the TCGA database, along with clinical information from 368 gastric cancer patients, and using R language (version 3.6.0) and its limma package, differentially expressed gene sets were obtained from gastric cancer patients compared to the normal population, with a corrected p-value (FDR) less than 0.05 and a log(Foldchange, FC) greater than 0.5 as screening criteria. Based on the expression of these differentially expressed gene sets, multiparameter Cox regression analysis was used to screen gene sets related to the prognosis of gastric cancer patients, and nomograms were constructed to establish the hazard ratio (HR) between genes and patient prognosis. An HR value greater than 1 indicates that the covariate is positively correlated with survival time, meaning that an increase in the covariate increases the risk of death; an HR value less than 1 indicates that the covariate is negatively correlated with survival time, meaning that an increase in the covariate decreases the risk of death; and an HR value equal to 1 indicates that the covariate is not related to survival time. The p-value represents the significance test result for each covariate, indicating whether the covariate has a significant impact on survival time. The smaller the p-value, the more significant the effect of the covariate on survival time. Generally, a p-value < 0.05 is considered significant. The concordance index (C-index) is a measure of model prediction accuracy, representing the consistency between the model's predicted survival time and the actual survival time. The C-index value ranges between 0.5 and 1; the closer to 1, the higher the model's prediction accuracy. This model ultimately screened 18 genes (…). Figure 1 (CHI3L1 and TFF2 have P-values ​​< 0.05 in this model; although FCGBP and VSIG2 have P-values ​​> 0.05, they still have a significant impact on the accuracy of this risk assessment model.)

[0039] Example 2: Screening the diagnostic combination with the largest AUC using stepwise regression ROC curves Based on the mRNA gene expression profiles of gastric cancer patients and normal individuals screened in the TCGA database using the method in Example 1, the 18 genes obtained in Example 1 were arranged and combined, and the AUC values ​​of different combinations were analyzed using ROC curves. The combination with the highest AUC was finally obtained, namely the combination of CHI3L1, FCGBP, TFF2, and VSIG2. The results are as follows: Figure 2 As shown, its AUC value is 0.749, indicating that this combination has certain diagnostic value.

[0040] The amino acid sequence of CHI3L1 protein is shown in SEQ ID NO.1, the amino acid sequence of FCGBP protein is shown in SEQ ID NO.2, the amino acid sequence of TFF2 protein is shown in SEQ ID NO.3, and the amino acid sequence of VSIG2 protein is shown in SEQ ID NO.4.

[0041] Example 3: Expression of CHI3L1, FCGBP, TFF2 and VSIG2 in the serum of gastric cancer patients This patent collected peripheral blood serum from 20 healthy controls and 60 gastric cancer patients, and used ELISA to detect the expression of CHI3L1, FCGBP, TFF2, and VSIG2 proteins in the serum samples; the results are as follows. Figure 3 As shown, compared with healthy controls, CHI3L1, FCGBP, TFF2, and VSIG2 proteins were all significantly upregulated in the serum of gastric cancer patients, and the differences were statistically significant.

[0042] The specific procedures for the ELISA method are as follows (the biological materials or reagents used in the following procedures were obtained from commercially available kits: Chondroitin 39 (CHI3L1) ELISA kit, Wuhan Feiyue Biotechnology Co., Ltd., FY-EH5750; IgG-Fc fragment binding protein (FCGBP) ELISA kit, Wuhan Feiyue Biotechnology Co., Ltd., FY-EH9322; Trefoil factor 2 (TFF2) ELISA kit, Wuhan Feiyue Biotechnology Co., Ltd., FY-EH5486; V-Set immunoglobulin domain protein 2 (VSIG2) ELISA kit, Wuhan Feiyue Biotechnology Co., Ltd., FY-EH9492): (1) Numbering: Except for the fixed blank and positive standard areas, the remaining wells are test sample wells and should be numbered as needed; (2) Sample addition: Except for the blank and positive standard wells, add 200 uL of serum sample to the corresponding wells in the order of their numbers. After sealing the plate with sealing film, place the microplate at 37 degrees Celsius for 1 hour. (3) Washing: Wash the plate 5 times with cleaning solution on the plate washing machine. Take out the washed plate and pat it on a paper towel if necessary to remove residual liquid in the hole. (4) Sealing: Add 100 μL of pre-prepared sealing solution to each well, including blank and positive standard wells, seal the plate with sealing film, and place the microwells at 37 degrees Celsius for 40 minutes; (5) Washing: Same as (3); (6) Add binding antibody: Add 50uL of CHI3L1, FCGBP, TFF2 or VSIG2 binding antibody to each well, seal the plate with sealing film and place the microwells at 37 degrees Celsius for 40 minutes; (7) Washing: Same as (3); (8) Add enzyme-labeled antibody: Add 100uL of enzyme-labeled antibody to each well, seal the plate with sealing film, and place the microwells at 37 degrees Celsius for 20 minutes; (9) Washing: Same as (3); (10) Color development: Add 50 μL of the corresponding color development solution to each well and rotate at room temperature in the dark for 20 minutes; (11) Termination: Add 50 μL of termination solution to each well; (12) Measurement: Place the microplate in an enzyme immunoassay analyzer and measure the OD value of each well using dual wavelengths of 450nm / 630nm and record the results.

[0043] Example 4: Clinical value of serum CHI3L1, FCGBP, TFF2 and VSIG2 in the diagnosis of gastric cancer The expression levels of CHI3L1, FCGBP, TFF2, and VSIG2 proteins in the serum of gastric cancer patients and healthy individuals, as determined in Example 3, were analyzed using ROC curves to assess their independent diagnostic value for gastric cancer. The results are as follows: Figure 4 As shown in the left figure, serum expression of CHI3L1, FCGBP, TFF2, and VSIG2 all demonstrate good diagnostic efficacy for gastric cancer. Specifically, CHI3L1 protein expression is significantly elevated in the serum of gastric cancer patients and exhibits excellent diagnostic value, with the highest area under the ROC curve (AUC). Simultaneously, FCGBP, TFF2, and VSIG2 expression are all significantly elevated in the serum of gastric cancer patients, and their expression levels can effectively differentiate between gastric cancer patients and healthy individuals. Furthermore, as... Figure 4 As shown in the right figure, the combined use of serum CHI3L1, FCGBP, TFF2, and VSIG2 showed the best diagnostic effect in differentiating between early and late-stage gastric cancer patients. This indicates that while serum CHI3L1, FCGBP, TFF2, and VSIG2 may have some diagnostic value when used individually, their combined use has greater diagnostic value.

[0044] The specific process of analyzing the results obtained using ELISA technology using ROC curves is as follows: (1) Calculation of sensitivity and specificity: Calculate the sensitivity (number of true positives / (number of true positives + number of false negatives)) and specificity (number of true negatives / (number of true negatives + number of false positives)) according to different thresholds. (2) Plot the ROC curve: plot the ROC curve with 1-specificity as the x-axis and sensitivity as the y-axis; (3) Calculation of AUC value: Calculate the area under the ROC curve, i.e., AUC; the larger the AUC value, the better the model performance; (4) Select the optimal threshold: On the ROC curve, the optimal threshold can be selected, which is the point with the highest sensitivity and the lowest specificity. The sensitivity and specificity corresponding to this optimal threshold are the optimal performance points. The steeper the rising part of the ROC curve, the better the performance of the model. The closer the AUC value is to 1, the better the performance of the serum marker or model in distinguishing between gastric cancer and healthy control populations.

[0045] Example 5: Diagnostic value of the combination of serum CHI3L1, FCGBP, TFF2 and VSIG2 with 11 tumor markers for gastric cancer The expression levels of CHI3L1, FCGBP, TFF2, and VSIG2 proteins in the serum of 60 gastric cancer patients were determined using Example 3. The expression levels of 11 other tumor markers, including AFP, CEA, CA199, CA724, CA125, CYFRA211, CA153, CA242, NSE, SCC, and CA50, were also analyzed. ROC curve analysis was used to assess the clinical value of the combination of CHI3L1, FCGBP, TFF2, and VSIG2, and their combination with the 11 tumor markers, in the diagnosis of gastric cancer. Results are as follows: Figure 5 As shown, the combination of serum CHI3L1, FCGBP, TFF2, and VSIG2 has good diagnostic efficacy for gastric cancer, and its use in combination with 11 tumor markers does not increase its clinical diagnostic efficacy for gastric cancer. Therefore, the combination of serum CHI3L1, FCGBP, TFF2, and VSIG2 can be used independently for the diagnosis of gastric cancer.

[0046] Example 6: CHI3L1, FCGBP, TFF2, and VSIG2 affect the growth of gastric cancer cells. Human gastric cancer cell line HGC-27 was cultured in high-glucose DMEM medium containing 10% fetal bovine serum (containing 100 U / ml penicillin and 0.1 mg / ml streptomycin) at 37°C and 5% CO2 Incubate in an incubator.

[0047] HGC-27 cells were transfected with small interfering RNAs (siRNAs) of CHI3L1, FCGBP, TFF2, and VSIG2. The optical density (OD) values ​​of the cells were measured at 24, 48, and 72 hours using a CCK8 assay. The mean optical density (mOD) values ​​at each time point were then displayed and statistically analyzed using GraphPad. Here, ns represents no statistically significant difference. Figure 6As shown, interference with CHI3L1 expression in HGC-27 cells significantly inhibited cell growth, with a statistically significant difference. However, interference with FCGBP, TFF2, and VSIG2 had no effect on the growth of gastric cancer cells, but simultaneous interference with CHI3L1, FCGBP, TFF2, and VSIG2 significantly inhibited the growth of gastric cancer cells. This indicates that the combined effect of CHI3L1, FCGBP, TFF2, and VSIG2 is more significant.

[0048] The above description is merely a preferred embodiment of the present invention and does not limit the invention in any way. Modifications and alterations made to the above embodiments by those skilled in the art based on the technical essence of the present invention should all fall within the protection scope of the present invention.

Claims

1. A combination of serum protein biomarkers for the diagnosis of gastric cancer, characterized in that, The serum protein biomarker combination includes CHI3L1, FCGBP, TFF2, and VSIG2.

2. The serum protein marker combination according to claim 1, characterized in that, The amino acid sequence of the CHI3L1 protein is shown in SEQ ID NO.

1.

3. The serum protein marker combination according to claim 1, characterized in that, The amino acid sequence of the FCGBP protein is shown in SEQ ID NO.

2.

4. The serum protein marker combination according to claim 1, characterized in that, The amino acid sequence of the TFF2 protein is shown in SEQ ID NO.

3.

5. The serum protein marker combination according to claim 1, characterized in that, The amino acid sequence of the VSIG2 protein is shown in SEQ ID NO.

4.

6. A reagent kit for diagnosing gastric cancer, characterized in that, The kit contains reagents for detecting the expression of CHI3L1, FCGBP, TFF2, and VSIG2 proteins.

7. Use of the serum protein marker combination according to any one of claims 1-4 in the preparation of a product for the diagnosis of gastric cancer.

8. Use of the serum protein marker combination according to any one of claims 1-4 in the preparation of a product for distinguishing between patients with early and late gastric cancer.

9. A reagent kit for diagnosing gastric cancer, characterized in that, The kit contains reagents for detecting VSIG2 protein expression.

10. Use of VSIG2 protein in the preparation of products for gastric cancer diagnosis.

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