Application of D-sorbitol in screening of esophageal squamous cell carcinoma

Abstract

The present invention belongs to the field of biomedicine technology, and discloses a marker for esophageal squamous cell carcinoma screening and its application. The marker for esophageal squamous cell carcinoma screening provided by the present invention is D-sorbitol or N-o-toluic acid glycine or a combination of D-sorbitol and N-o-toluic acid glycine, and the detection reagent of the marker can be used to prepare an esophageal squamous cell carcinoma screening product. The present invention also provides a kit for esophageal squamous cell carcinoma screening, and the kit contains a detection reagent for detecting the above-mentioned marker. The present invention can effectively detect esophageal squamous cell carcinoma, especially early esophageal squamous cell carcinoma, by detecting the expression level of D-sorbitol or N-o-toluic acid glycine or a combination of D-sorbitol and N-o-toluic acid glycine in human serum; when D-sorbitol is combined with N-o-toluic acid glycine, its detection sensitivity is as high as 90.0%, and the specificity reaches 85.7%, which can be used for large-scale screening of asymptomatic people in high-incidence areas of esophageal squamous cell carcinoma, which is conducive to screening and early detection of asymptomatic high-risk people.

Description

[0001] This application is a divisional application of the invention patent with application number CN 202110738053.3, application date 2021-06-30, and name “A marker and detection kit for early screening of esophageal squamous cell carcinoma”. Technical Field

[0002] The invention belongs to the field of medical biotechnology, and specifically discloses an early diagnosis marker for esophageal squamous cell carcinoma based on metabolomics and a kit thereof. Background Art

[0003] Esophageal squamous cell carcinoma is one of the six most common malignant tumors in the world, with significant regional distribution differences and familial clustering. my country is the country with the highest incidence and mortality of esophageal squamous cell carcinoma in the world. There are about 500,000 new cases of esophageal squamous cell carcinoma in the world each year, more than half of which occur in China. In China, half of the patients occur in the Taihang Mountain area at the junction of Henan, Hebei and Shanxi provinces. Among them, Lin County, Henan Province is the area with the highest incidence and mortality of esophageal squamous cell carcinoma in the world, which is 100 times higher than that in Western countries. In China, the pathological type of esophageal squamous cell carcinoma is mainly squamous cell carcinoma, accounting for 97%, which is very different from the main pathological type in the West (80% is adenocarcinoma). In addition, there are huge differences in epidemic characteristics, risk factors for disease occurrence, etc. with the population in Western countries.

[0004] The prognosis of esophageal squamous cell carcinoma is poor. The 5-year survival rate of patients in the middle and late stages after surgery is only about 10%; while the 5-year survival rate of early esophageal squamous cell carcinoma (lesions are limited to the mucosal layer and submucosal layer, without lymph node metastasis) can be as high as 95%. It can be seen that early diagnosis and early treatment of esophageal squamous cell carcinoma are important factors affecting the prognosis of esophageal squamous cell carcinoma. However, since early patients have no specific symptoms, most patients are already in the middle and late stages when they seek medical treatment. The detection rate of early esophageal squamous cell carcinoma is less than 2%, resulting in no significant improvement in the mortality rate of the disease in the high-incidence areas of esophageal squamous cell carcinoma in China. Endoscopic screening is an important method for detecting early esophageal cancer. In areas with a high incidence of esophageal squamous cell carcinoma, the country generally defines "asymptomatic people over 40 years old, male, smokers, drinkers, and those with a positive family history" as "high-risk or high-risk groups", and these people are screened early through chromoendoscopy and mucosal biopsy. However, endoscopic examination is invasive and expensive, and the early cancer detection rate is only 2%. More than 90% of asymptomatic people are not accompanied for examination. These factors have seriously hindered the progress of early screening for esophageal squamous cell carcinoma. At the same time, finding less invasive and less expensive examination methods to improve the efficiency of early screening for esophageal squamous cell carcinoma is an important trend in the prevention and treatment of esophageal squamous cell carcinoma.

[0005] With the rapid development of metabolomics technology, a large number of large-scale association studies on esophageal squamous cell carcinoma have emerged, and a number of highly expressed metabolites of esophageal squamous cell carcinoma have been discovered, laying a solid foundation for early warning and accurate screening of asymptomatic high-risk populations and early diagnosis of esophageal squamous cell carcinoma. However, there are currently no effective preparations for early diagnosis and screening of esophageal squamous cell carcinoma. If metabolic markers that can be used for screening of esophageal squamous cell carcinoma can be screened, it will surely be a powerful boost to the current status of early diagnosis of esophageal squamous cell carcinoma in my country, and it will also open up new avenues for drug screening and accurate treatment. Summary of the invention

[0006] In view of the problems and shortcomings of the prior art, one of the objects of the present invention is to provide a marker for screening of esophageal squamous cell carcinoma. A second object of the present invention is to provide a detection reagent for the marker for screening of esophageal squamous cell carcinoma and its application in the preparation of esophageal squamous cell carcinoma screening products. A third object of the present invention is to provide a kit for screening of esophageal squamous cell carcinoma.

[0007] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0008] A first aspect of the present invention provides a marker for screening of esophageal squamous cell carcinoma, wherein the marker is at least one of N-o-toluylglycine, D-sorbitol, and N-acetyl-D-mannosamine.

[0009] According to the above-mentioned marker, preferably, the marker is a serum marker.

[0010] The second aspect of the present invention provides a use of a detection reagent for the marker described in the first aspect in the preparation of a screening product for esophageal squamous cell carcinoma.

[0011] According to the above application, preferably, the test sample of the product is serum.

[0012] According to the above application, preferably, the detection reagent is a reagent for detecting the marker in a sample by chromatography, mass spectrometry or chromatography-mass spectrometry.

[0013] According to the above application, preferably, the chromatography is gas chromatography, liquid chromatography or high performance liquid chromatography.

[0014] According to the above application, preferably, the chromatography-mass spectrometry is gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, high performance liquid chromatography-mass spectrometry, gas chromatography-tandem mass spectrometry, liquid chromatography-tandem mass spectrometry or high performance liquid chromatography-tandem mass spectrometry.

[0015] The third aspect of the present invention provides a kit for screening esophageal squamous cell carcinoma, the kit containing a detection reagent for detecting the marker described in the first aspect; the marker is at least one of N-o-toluic acid glycine, D-sorbitol, and N-acetyl-D-mannosamine.

[0016] According to the above-mentioned kit, preferably, the detection reagent is a reagent for detecting the marker in a sample by chromatography-mass spectrometry or chromatography-mass spectrometry.

[0017] According to the above-mentioned kit, preferably, the chromatography is gas chromatography, liquid chromatography or high performance liquid chromatography; the chromatography-mass spectrometry is gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, high performance liquid chromatography-mass spectrometry, gas chromatography-tandem mass spectrometry, liquid chromatography-tandem mass spectrometry or high performance liquid chromatography-tandem mass spectrometry.

[0018] According to the above-mentioned kit, preferably, the kit further contains a standard product of the marker described in the first aspect above.

[0019] According to the above-mentioned kit, preferably, the detection sample of the kit is serum.

[0020] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0021] (1) The present invention first discovered through a metabolomics method that N-o-toluic acid glycine, D-sorbitol, and N-acetyl-D-mannosamine can be used for early screening and detection of esophageal squamous cell carcinoma. By detecting the expression levels of N-o-toluic acid glycine, D-sorbitol, and N-acetyl-D-mannosamine in human serum, esophageal squamous cell carcinoma, especially early esophageal squamous cell carcinoma, can be effectively detected; it has been verified that when the present invention uses any one of N-o-toluic acid glycine, D-sorbitol, and N-acetyl-D-mannosamine alone for esophageal squamous cell carcinoma screening, the AUC value of the ROC curve is above 0.8; when multiple markers are used in combination, the AUC value of the ROC curve is closer to 1 than a single indicator, and the differentiation effect is good, and the diagnostic effect is good. Therefore, the marker for esophageal squamous cell carcinoma screening of the present invention can be used for early screening of esophageal squamous cell carcinoma.

[0022] (2) When the present invention uses the three markers of N-o-toluylglycine, D-sorbitol, and N-acetyl-D-mannosamine as a combination for early esophageal squamous cell carcinoma screening, the detection sensitivity is as high as 95.0% (i.e., the rate of patients with early esophageal squamous cell carcinoma who are correctly diagnosed as early esophageal squamous cell carcinoma when these three markers are used for diagnosis is 95.%), and the specificity is 90.0% (i.e., the rate of patients without esophageal squamous cell carcinoma who are correctly diagnosed as not suffering from esophageal squamous cell carcinoma when these three markers are used for diagnosis is 90.0%). Therefore, the markers of the present invention are The biopharmaceutical has high sensitivity and specificity, which greatly improves the detection rate of early esophageal squamous cell carcinoma. Moreover, the detection rate of esophageal squamous cell carcinoma is much higher than the detection rate of existing clinical endoscopic screening for esophageal squamous cell carcinoma (2%-3%). It can be used for large-scale screening of asymptomatic high-risk people in areas with a high incidence of esophageal squamous cell carcinoma. It also provides an important detection method for long-term follow-up of asymptomatic high-risk people in areas with a high incidence of esophageal squamous cell carcinoma, which is conducive to the early detection of asymptomatic high-risk people of esophageal squamous cell carcinoma, thereby greatly reducing the mortality rate of patients with esophageal squamous cell carcinoma and bringing great benefits to patients with esophageal squamous cell carcinoma and their families.

[0023] (3) The marker used for esophageal squamous cell carcinoma screening in the present invention is a serum detection marker, which can avoid invasive diagnosis. The risk of esophageal squamous cell carcinoma can be obtained at an early stage by taking serum for testing in a minimally invasive manner, thereby providing a basis for clinicians to conduct further in-depth examinations, and providing support for quickly and accurately grasping the patient's disease status and severity of the disease, and timely adopting more personalized prevention and treatment plans, thereby delaying and preventing disease progression.

[0024] (4) The test sample of the kit for screening esophageal squamous cell carcinoma of the present invention is serum, which requires a small amount of blood, causes little pain to the public, and has a high degree of acceptance; moreover, the operation is simple, the detection result takes a short time, and it has broad market prospects and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The ROC curves of using N-o-toluic acid glycine, D-sorbitol, and N-acetyl-D-mannosamine to diagnose and distinguish between esophageal squamous cell carcinoma patients and normal control groups, respectively; A is the ROC curve of N-o-toluic acid glycine to diagnose and distinguish between esophageal squamous cell carcinoma patients and normal control groups, B is the ROC curve of D-sorbitol to diagnose and distinguish between esophageal squamous cell carcinoma patients and normal people, and C is the ROC curve of N-acetyl-D-mannosamine to diagnose and distinguish between esophageal squamous cell carcinoma patients and normal control groups;

[0026] Figure 2 ROC curve for the combination of N-o-toluic acid glycine and D-sorbitol in differentiating esophageal squamous cell carcinoma patients from normal controls;

[0027] Figure 3ROC curve for the combination of N-o-toluylglycine and N-acetyl-D-mannosamine in differentiating esophageal squamous cell carcinoma patients from the normal control group;

[0028] Figure 4 ROC curve for the combined diagnosis of D-sorbitol and N-acetyl-D-mannosamine to differentiate between esophageal squamous cell carcinoma patients and normal controls;

[0029] Figure 5 This is the ROC curve of the combined diagnosis of N-o-toluic acid glycine, D-sorbitol, and N-acetyl-D-mannosamine to distinguish esophageal squamous cell carcinoma group from the normal control group. DETAILED DESCRIPTION

[0030] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. It will be understood by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0031] The experimental methods described in the following examples, unless otherwise specified, are all conventional techniques in the art, or are based on the conditions recommended by the manufacturers; the reagents, materials and instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0032] Example 1: Screening of serum differential metabolic markers for esophageal squamous cell carcinoma

[0033] 1. Collection of serum samples

[0034] A total of 79 serum samples were collected from the Key Open Laboratory of Esophageal Squamous Cell Carcinoma of Henan Province, the First Affiliated Hospital of Zhengzhou University, including 39 normal human sera (normal control group) and 40 esophageal squamous cell carcinoma patient sera (esophageal squamous cell carcinoma group). The 39 normal human sera were from the normal physical examination population of the physical examination center of the laboratory's cooperative hospital, without any tumor-related evidence. The 40 esophageal squamous cell carcinoma patient sera were from early-stage (stage 0+stage I) esophageal squamous cell carcinoma patients confirmed by histopathology, who did not receive radiotherapy or chemotherapy.

[0035] 2. Experimental methods

[0036] (1) Serum sample pretreatment:

[0037] Serum samples were stored in a -80 °C refrigerator. Before metabolomics analysis, all samples were thawed at room temperature, 50 μL of serum samples were taken, and then 150 μL of methanol, 3 times the amount, was added, and vortexed for 30 seconds to mix. After mixing, it was placed in a high-speed centrifuge for centrifugation at 13000 rpm for 10 minutes. 75 μL of the supernatant after centrifugation was taken and placed in two 1.5 ml centrifuge tubes, and the supernatant was blown dry with a nitrogen blower. After drying, it was re-dissolved with 100 μL of methanol containing an internal standard solution (L-2-chlorophenylalanine) or 100 μL of methanol containing an internal standard solution (ketoprofen). The final concentrations of L-2-chlorophenylalanine and ketoprofen were 100 ng / mL and 1 μg / mL, respectively, and used as positive and negative ion mode detection samples, respectively. After re-dissolution, vortex for 30 seconds to mix, then place in a high-speed centrifuge for centrifugation at 13,000 rpm for 10 minutes, aspirate the supernatant after centrifugation, and then place the supernatant in a liquid chromatography-mass spectrometry vial for UPLC-Q / TOF-MS detection.

[0038] (2) UPLC-Q / TOF-MS detection:

[0039] 1) Chromatographic conditions:

[0040] Instrument: Rapid high-resolution liquid chromatography (UPLC, Agilent 1290, USA);

[0041] The chromatographic column was an Inertsil ODS-3C18 column with a size of 250 mm × 4.6 mm (Shimadzu, Japan); mobile phase: phase A was water, phase B was acetonitrile (each containing 0.1% formic acid); flow rate: 1 mL / min; column temperature: 25°C; injection volume: 10 μL. The linear gradient elution conditions were as follows:

[0042]

[0043] 2) Mass spectrometry conditions:

[0044] Instrument: quadrupole / time-of-flight mass spectrometer detector (Agilent 6530Q-TOF / MS, USA);

[0045] ESI ion source, Agilent MasshunterQualitative 4.0 software was used for online data acquisition, and Agilent Masshunter Qualitative 6.0 software was used for offline data processing. The detailed mass spectrometry conditions are as follows: drying gas: nitrogen, temperature 325°C, flow rate 12L / min, nebulizer pressure 35psi; capillary voltage: +4000V; fragmentation voltage: 100V; separator voltage: 60V; mass collection range: 50-1000Da.

[0046] (3) Data processing and analysis:

[0047] 1) Data pre-processing

[0048] Based on the data obtained by UPLC-Q / TOF-MS, XCMS program code was used under the R software platform for peak extraction, alignment and deconvolution analysis, and peak screening was performed according to the 80% principle to obtain a three-dimensional visualization matrix containing variables (retention time Rt, mass-to-charge ratio m / z), observations and peak intensities. This data matrix was imported into SIMCA-P software (version 13.0) for multivariate statistical analysis.

[0049] 2) Multivariate statistical analysis

[0050] In order to investigate the metabolic changes in the esophageal squamous cell carcinoma group compared with the normal control group, unsupervised principal component analysis (PCA) was first performed on all variables to observe the clustering of each group of data and remove outliers. Finally, the orthogonal partial least squares discriminant analysis (PLS-DA) model was used for supervised data analysis to amplify the differences between the groups and obtain the most significant separation between the groups.

[0051] 3) Mining and identification of differential metabolites between esophageal squamous cell carcinoma group and normal control group based on UPLC-Q / TOF-MS detection:

[0052] The VIP values ​​of the esophageal squamous cell carcinoma group and the normal control group under the PLS-DA model were combined with the P values ​​of the univariate statistical analysis. Variables with VIP>1.0 and P<0.05 were considered to have significant differences, and variables with significant differences were considered to be differential metabolic biomarkers.

[0053] The screened differential variables need to be attributed to the biomarkers they represent. Metabolite identification based on UPLC-Q / TOF-MS technology is mainly matched through metabolite spectral libraries: find the mass spectrum of the differential variable on the UPLC-Q / TOF-MS total ion flow chart, compare the precise molecular weight of the differential metabolite with the network database, such as HMDB (http: / / www.hmdb.ca), METLIN (http: / / metlin.scripps.edu) and KEGG (http: / / www.kegg.jp), preliminarily identify the structure of the above differential metabolites, and finally determine the structure of the differential metabolites by purchasing standard products, using the molecular weight, chromatographic retention time and corresponding multi-stage MS fragmentation spectrum of the standard products, and prepare a series of standard solutions with known concentrations, and further determine the content of the differential metabolites through the standard curve.

[0054] 3. Experimental results

[0055] According to the above ideas, three differential metabolites that distinguished the esophageal squamous cell carcinoma group and the normal control group were finally identified, as shown in Table 1. Moreover, compared with the normal control group, the levels of N-o-toluylglycine, D-sorbitol, and N-acetyl-D-mannosamine in the serum of patients with esophageal squamous cell carcinoma were significantly increased.

[0056] Table 1 Statistical data of differential metabolites between esophageal squamous cell carcinoma group and normal control group based on UPLC-Q / TOF-MS detection

[0057] serial number Differential metabolites VIP P-value Multiple 1 N-O-toluoylglycine 1.97 0.02 2.50 2 D-Sorbitol 2.04 0.01 3.08 3 N-Acetyl-D-mannosamine 1.93 0.01 2.38

[0058] Example 2: Evaluation of the ability of differential metabolite diagnosis to distinguish esophageal squamous cell carcinoma patients from healthy subjects

[0059] 1. The ability of a single differential metabolite to diagnose and distinguish between esophageal squamous cell carcinoma patients and normal subjects:

[0060] Based on the content data of N-o-toluylglycine, D-sorbitol, and N-acetyl-D-mannosamine in the serum samples of the esophageal squamous cell carcinoma group (40 esophageal squamous cell carcinoma patients) and the normal control group (39 normal people) detected by UPLC-Q / TOF-MS in Example 1, the receiver operating characteristic curve (ROC curve) was used to evaluate the ability of each differential metabolite to diagnose and distinguish esophageal squamous cell carcinoma patients from normal people alone.

[0061] ROC curves of N-o-toluylglycine, D-sorbitol, and N-acetyl-D-mannosamine for distinguishing esophageal squamous cell carcinoma patients from normal subjects Figure 1 According to the ROC curve, the area under the curve (AUC), sensitivity and specificity of the ROC curve of each differential metabolite were statistically analyzed, and the results are shown in Table 2.

[0062] Table 2 AUC of the three differential metabolites for distinguishing esophageal squamous cell carcinoma patients from normal subjects

[0063] serial number Differential metabolites AUC Sensitivity Specificity 1 N-O-toluoylglycine 0.80 78.3％ 93.7％ 2 D-Sorbitol 0.88 86.9％ 79.8％ 3 N-Acetyl-D-mannosamine 0.84 79.0％ 90.0％

[0064] The area under the ROC curve (AUC) has been widely recognized as an inherent accuracy indicator for evaluating the authenticity of diagnostic tests. When AUC is 0.5, it means that there is no diagnostic significance; when AUC is between 0.5 and 0.7, it means that the diagnostic accuracy is low; when AUC is between 0.7 and 0.9, it means that the diagnostic accuracy is moderate; when AUC>0.9, it means that the diagnosis has a high accuracy. Figure 1As shown in Table 2, the AUC of the ROC curve of N-o-toluic acid glycine, D-sorbitol, and N-acetyl-D-mannosamine, which are used alone to distinguish between patients with esophageal squamous cell carcinoma and normal subjects, can reach above 0.8, indicating that N-o-toluic acid glycine, D-sorbitol, and N-acetyl-D-mannosamine can diagnose and distinguish patients with esophageal squamous cell carcinoma and normal subjects alone with good accuracy.

[0065] The Youden index (Youden index = sensitivity + specificity - 1) was further calculated based on the coordinates of the ROC curve. The relative content of the metabolites corresponding to the maximum Youden index was the optimal cutoff value for diagnosing and distinguishing esophageal squamous cell carcinoma patients from normal people, as shown in Table 3.

[0066] Table 3 Youden index and cutoff value of three differential metabolites for distinguishing esophageal squamous cell carcinoma patients from normal subjects

[0067] serial number Differential metabolites Youden Index Optimal cutoff value 1 N-(o-Toluoyl)glycine 0.589 0.67 2 D-Sorbitol 0.714 0.40 3 N-Acetyl-D-mannosamine 0.693 0.73

[0068] 2. The ability of multiple differential metabolites to diagnose and distinguish between esophageal squamous cell carcinoma patients and normal people:

[0069] (1) The ability of the combination of N-o-toluylglycine and D-sorbitol to distinguish between patients with esophageal squamous cell carcinoma and normal subjects:

[0070] The relative contents of N-o-toluic acid glycine and D-sorbitol in the serum samples of the esophageal squamous cell carcinoma group (40 patients with esophageal squamous cell carcinoma) and the normal control group (39 normal people) detected by UPLC-Q / TOF-MS in Example 1 were used as independent variables (assuming X1 = relative content of N-o-toluic acid glycine, X2 = relative content of D-sorbitol), and the groups (esophageal squamous cell carcinoma group and normal control group) were used as dependent variables to analyze the relative contents of N-o-toluic acid glycine and D-sorbitol in the serum samples of the esophageal squamous cell carcinoma group and the normal control group. The binary logistic regression equation was obtained by performing binary logistic regression on the content of N-o-toluic acid glycine and D-sorbitol in each serum sample: logit[p]=-5.28+0.27X1+0.26X2; the relative content of N-o-toluic acid glycine and D-sorbitol in each serum sample was then substituted into the binary logistic regression equation to obtain the regression value logit[p] of each serum sample. The possible regression value logit[p] was used as the diagnostic point to calculate the sensitivity and specificity. Then, the ROC curve was drawn according to the calculated sensitivity and specificity. The ROC curve is shown in FIG. Figure 2 shown.

[0071] From the ROC curve, it can be seen that the area under the ROC curve AUC of the combination of N-o-toluic acid glycine and D-sorbitol for diagnosing and distinguishing esophageal squamous cell carcinoma patients from normal people is 0.93, which has a high accuracy. The Youden index (Youden index = sensitivity + specificity - 1) is further calculated based on the coordinates of the ROC curve. The logit [p] value corresponding to the maximum Youden index is the optimal cutoff value for diagnosing and distinguishing esophageal squamous cell carcinoma patients from normal people, and the optimal cutoff value is 0.77.

[0072] (2) The ability of the combination of N-o-toluylglycine and N-acetyl-D-mannosamine to distinguish esophageal squamous cell carcinoma patients from normal subjects:

[0073] The relative contents of N-o-toluoylglycine and N-acetyl-D-mannosamine in the serum samples of the esophageal squamous cell carcinoma group (40 patients with esophageal squamous cell carcinoma) and the normal control group (39 normal people) detected by UPLC-Q / TOF-MS in Example 1 were used as independent variables (assuming X1 = relative content of N-o-toluoylglycine, X2 = relative content of N-acetyl-D-mannosamine), and the groups (esophageal squamous cell carcinoma group and normal control group) were used as dependent variables. The relative contents of N-o-toluoylglycine and N-acetyl-D-mannosamine in the serum samples of the esophageal squamous cell carcinoma group and the normal control group were analyzed. The relative contents of N-o-toluoyl glycine and N-acetyl-D-mannosamine in each serum sample were subjected to binary logistic regression to obtain a binary logistic regression equation: logit[p]=-8.23+0.41X1+0.32X2; the relative contents of N-o-toluoyl glycine and N-acetyl-D-mannosamine in each serum sample were then substituted into the binary logistic regression equation to obtain the regression value logit[p] of each serum sample. The possible regression value logit[p] was used as the diagnostic point to calculate the sensitivity and specificity, and then the ROC curve was drawn according to the calculated sensitivity and specificity. The ROC curve is shown in FIG. Figure 3 shown.

[0074] From the ROC curve, it can be seen that the area under the ROC curve AUC of the combination of N-o-toluyl glycine and N-acetyl-D-mannosamine for diagnosing and distinguishing esophageal squamous cell carcinoma patients from normal people is 0.94, which has a high accuracy. The Youden index (Youden index = sensitivity + specificity - 1) is further calculated based on the coordinates of the ROC curve. The logit [p] value corresponding to the maximum Youden index is the optimal cutoff value for diagnosing and distinguishing esophageal squamous cell carcinoma patients from normal people, and the optimal cutoff value is 0.47.

[0075] (3) The ability of the combination of D-sorbitol and N-acetyl-D-mannosamine to distinguish between patients with esophageal squamous cell carcinoma and normal subjects:

[0076] The relative contents of D-sorbitol and N-acetyl-D-mannosamine in the serum samples of the esophageal squamous cell carcinoma group (40 patients with esophageal squamous cell carcinoma) and the normal control group (39 normal people) detected by UPLC-Q / TOF-MS in Example 1 were used as independent variables (assuming X1 = relative content of D-sorbitol, X2 = relative content of N-acetyl-D-mannosamine), and the groups (esophageal squamous cell carcinoma group and normal control group) were used as dependent variables to analyze the relative contents of D-sorbitol and N-acetyl-D-mannosamine in the serum samples of the esophageal squamous cell carcinoma group and the normal control group. The relative contents were subjected to binary logistic regression to obtain the binary logistic regression equation: logit[p]=-7.81+0.32X1+0.61X2; the relative contents of D-sorbitol and N-acetyl-D-mannosamine in each serum sample were then substituted into the binary logistic regression equation to obtain the regression value logit[p] of each serum sample. The possible regression value logit[p] was used as the diagnostic point to calculate the sensitivity and specificity, and then the ROC curve was drawn according to the calculated sensitivity and specificity. The ROC curve is shown in FIG. Figure 4 shown.

[0077] From the ROC curve, it can be seen that the area under the ROC curve AUC of D-sorbitol and N-acetyl-D-mannosamine combined for distinguishing esophageal squamous cell carcinoma patients from normal people is 0.94, which has a high accuracy. The Youden index (Youden index = sensitivity + specificity - 1) is further calculated based on the coordinates of the ROC curve. The logit [p] value corresponding to the maximum Youden index is the optimal cutoff value for distinguishing esophageal squamous cell carcinoma patients from normal people, and the optimal cutoff value is 0.78.

[0078] (4) The ability of the combination of N-o-toluylglycine, D-sorbitol and N-acetyl-D-mannosamine to distinguish between patients with esophageal squamous cell carcinoma and normal subjects:

[0079] The relative contents of N-o-toluoylglycine, D-sorbitol and N-acetyl-D-mannosamine in the serum samples of the esophageal squamous cell carcinoma group (40 patients with esophageal squamous cell carcinoma) and the normal control group (39 normal people) detected by UPLC-Q / TOF-MS in Example 1 were used as independent variables (assuming X1 = relative content of N-o-toluoylglycine, X2 = relative content of D-sorbitol, X3 = relative content of N-acetyl-D-mannosamine), and the groups (esophageal squamous cell carcinoma group and normal control group) were used as dependent variables. The relative contents of N-o-toluoylglycine, D-sorbitol and N-acetyl-D-mannosamine in the esophageal squamous cell carcinoma group and the normal control group were analyzed. Binary logistic regression was performed on the relative contents in the serum samples of the control group to obtain the binary logistic regression equation: logit[p]=-13.82+0.30X1+0.43X2+0.50X3; the relative contents of N-o-toluic acid glycine, D-sorbitol and N-acetyl-D-mannosamine in each serum sample were then substituted into the binary logistic regression equation to obtain the regression value logit[p] of each serum sample. The possible regression value logit[p] was used as the diagnostic point to calculate the sensitivity and specificity, and then the ROC curve was drawn according to the calculated sensitivity and specificity. The ROC curve and scatter plot are shown in FIG. Figure 5 shown.

[0080] From the ROC curve, it can be seen that the area under the ROC curve AUC of the combination of N-o-toluic acid glycine, D-sorbitol and N-acetyl-D-mannosamine for diagnosing and distinguishing esophageal squamous cell carcinoma patients from normal people is 0.96, which has a high accuracy. The Youden index (Youden index = sensitivity + specificity - 1) is further calculated based on the coordinates of the ROC curve. The logit [p] value corresponding to the maximum Youden index is the optimal cutoff value for diagnosing and distinguishing esophageal squamous cell carcinoma patients from normal people, and the optimal cutoff value is 0.72.

[0081] The ROC curve AUC values, sensitivity, specificity, Youden index and optimal cutoff values ​​for the above-mentioned single and multiple differential metabolite combinations in distinguishing esophageal squamous cell carcinoma patients from normal subjects were statistically analyzed, as shown in Table 4.

[0082] Table 4 AUC values ​​of different differential metabolite combinations for diagnosis and differentiation of esophageal squamous cell carcinoma patients and normal subjects

[0083]

[0084] As shown in Table 4, compared with a single differential metabolite, when the three differential metabolites of N-o-toluoyl glycine, D-sorbitol and N-acetyl-D-mannosamine were used to diagnose and distinguish esophageal squamous cell carcinoma patients from normal people, the area under the ROC curve AUC reached a maximum of 0.96, which was significantly higher than the single differential metabolite diagnosis and the combination of any two differential metabolites; moreover, when the three differential metabolites of N-o-toluoyl glycine, D-sorbitol and N-acetyl-D-mannosamine were used to diagnose esophageal squamous cell carcinoma, the sensitivity of diagnosis reached 92.2% and the specificity reached 98.0%, indicating that the combination of the three differential metabolites had the best diagnostic effect on esophageal squamous cell carcinoma and high diagnostic accuracy. In addition, the Youden index in statistics refers to the sum of sensitivity and specificity minus 1, and its value range is 0 to 1. The closer the Youden index is to 1, the greater its diagnostic value, indicating that the application value of this method is higher. When the three differential metabolites of N-o-toluic acid glycine, D-sorbitol and N-acetyl-D-mannosamine were combined, their Youden index was significantly higher than that of a single differential metabolite, indicating that the method of using the combination of N-o-toluic acid glycine, D-sorbitol and N-acetyl-D-mannosamine to diagnose esophageal squamous cell carcinoma has a good diagnostic value.

[0085] Example 3: Application of three differential metabolites in screening for esophageal squamous cell carcinoma

[0086] 1. Collection of serum samples

[0087] In addition, 20 sera from patients with esophageal squamous cell carcinoma and 20 normal human sera were selected from the Key Open Laboratory of Esophageal Squamous Cell Carcinoma of Henan Province, the First Affiliated Hospital of Zhengzhou University; among them, the sera of the 20 patients with esophageal squamous cell carcinoma were from patients with early (stage 0+stage I) esophageal squamous cell carcinoma confirmed by histopathology, none of whom received radiotherapy or chemotherapy, and the sera of the 20 normal humans were from the normal physical examination population of the physical examination center of the laboratory's cooperative hospital, without any tumor-related evidence.

[0088] 2. Analysis methods

[0089] The UPLC-Q / TOF-MS technique described in Example 1 was used to qualitatively and quantitatively determine the three metabolites (N-o-toluylglycine, D-sorbitol, and N-acetyl-D-mannosamine) in the sera of 20 patients with esophageal squamous cell carcinoma and 20 normal subjects.

[0090] Among them, when N-o-toluic acid glycine, D-sorbitol or N-acetyl-D-mannosamine is used alone for the diagnosis of esophageal squamous cell carcinoma, the negative and positive of the sample are judged according to the content of N-o-toluic acid glycine, D-sorbitol or N-acetyl-D-mannosamine in the serum sample and the optimal cutoff value of the corresponding differential metabolite calculated in Example 2. If the content of the differential metabolite in the serum sample is higher than the optimal cutoff value, the patient is judged to be an esophageal squamous cell carcinoma patient, otherwise, the patient is judged to be a normal person.

[0091] When any two or three of the three differential metabolites of N-o-toluic acid glycine, D-sorbitol, and N-acetyl-D-mannosamine are used in combination for the diagnosis of esophageal squamous cell carcinoma, the contents of N-o-toluic acid glycine, D-sorbitol, and N-acetyl-D-mannosamine in the serum sample are substituted into the Logistic regression equation of the corresponding combination in Example 2. The calculated logit [p] value and the optimal cutoff value for distinguishing esophageal squamous cell carcinoma patients from normal people are used to diagnose and distinguish the negative and positive of the sample. If the logit [p] value of the combination is higher than the corresponding optimal cutoff value, it is judged as esophageal squamous cell carcinoma. Otherwise, it is judged as a normal person.

[0092] 3. Test results

[0093] The diagnostic results of the 40 serum samples are shown in Table 5 .

[0094] Table 5 Results of the combination of three metabolic markers in the diagnosis of esophageal squamous cell carcinoma

[0095]

[0096] The diagnostic results of 40 serum samples are shown in Table 5. When N-o-toluoyl glycine, D-sorbitol or N-acetyl-D-mannosamine was used alone for the diagnosis of esophageal squamous cell carcinoma, its positive predictive value could reach more than 70%; when any two of the differential metabolites were used in combination for the diagnosis of esophageal squamous cell carcinoma, its positive predictive value and negative predictive value were significantly higher than those of a single differential metabolite; moreover, when N-o-toluoyl glycine, D-sorbitol and N-acetyl-D-mannosamine were used in combination for the diagnosis of esophageal squamous cell carcinoma, its positive predictive value and negative predictive value reached the highest, 90.4% and 94.7% respectively. This shows that the use of N-o-toluoyl glycine, D-sorbitol and N-acetyl-D-mannosamine in combination for the diagnosis of esophageal squamous cell carcinoma has the best effect.

[0097] Example 4: Preparation of a screening kit for esophageal squamous cell carcinoma based on the metabolic markers of the present invention

[0098] Based on the three metabolic markers related to esophageal squamous cell carcinoma obtained by screening of the present invention, an esophageal squamous cell carcinoma screening kit is designed, and the kit includes the following components:

[0099] Marker standard: at least one of N-o-toluic acid glycine, D-sorbitol, and N-acetyl-D-mannosamine. The kit may contain one, any multiple, or three metabolic marker standard products, which may be combined as required. When more than one metabolic marker standard product is involved, the standard products of each marker may be packaged separately, or the standard products of each marker may be mixed to form a mixture and packaged.

[0100] Kit application process: Collect the serum of the subjects and freeze it in a -80℃ refrigerator. Before the experiment, thaw the serum sample in a 4℃ refrigerator, take 50μL of the serum sample, then add 150μL of 3 times the amount of methanol, vortex for 30s to mix, and then put it into a high-speed centrifuge for centrifugation at 13000rpm for 10min. Take 75μL of the supernatant after centrifugation and put it into two 1.5ml centrifuge tubes, blow dry the supernatant with a nitrogen blower, and then re-dissolve it with 100μL of methanol containing internal standard solution (L-2-chlorophenylalanine or ketoprofen), vortex for 30s to mix, and then put it into a high-speed centrifuge for centrifugation at 13000rpm for 10min. Take the supernatant after centrifugation and put it into a liquid mass spectrometer vial for UPLC-Q / TOF-MS detection.

[0101] The treated serum samples were analyzed according to the UPLC-Q / TOF-MS instrument setting method of Example 1, and the markers were quantitatively and qualitatively analyzed referring to the data processing method of Example 1.

[0102] When using this esophageal squamous cell carcinoma detection kit, it is recommended to detect 3 markers at the same time to further improve the detection efficiency.

[0103] In summary, the present invention effectively overcomes the deficiencies in the prior art and has a high industrial utilization value. The above embodiments serve to illustrate the substantive content of the present invention, but do not limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. Application of a detection reagent of a combination of D-sorbitol and N-o-toluic acid glycine in the preparation of a screening product for esophageal squamous cell carcinoma; the detection sample of the product is serum.

2. The use according to claim 1, characterized in that: The detection reagent is a reagent for detecting the combination of D-sorbitol and N-o-toluic acid glycine in a sample by chromatography-mass spectrometry.

3. The use according to claim 2, characterized in that: The chromatography-mass spectrometry method is liquid chromatography-mass spectrometry.

4. The use according to any one of claims 1 to 3, characterized in that: The product is a test kit.

5. The use according to claim 4, characterized in that: The kit also contains a standard of a combination of D-sorbitol and N-o-toluylglycine.

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