Metabolomics-based diagnostic markers for lymph node metastasis in esophageal squamous cell carcinoma and their application

Abstract

The present invention discloses metabolomics-based diagnostic markers for esophageal squamous cell carcinoma lymph node metastasis and their applications. The present invention provides the application of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide as markers for developing detection, auxiliary detection, screening, or auxiliary screening of esophageal squamous cell carcinoma lymph node metastasis. Experiments of the present invention have demonstrated that the use of three target compound concentrations can quickly diagnose whether esophageal squamous cell carcinoma patients have lymph node metastasis. This method has the advantages of high accuracy, high sensitivity, and strong universality, and can provide a strong scientific basis for clinical diagnosis and treatment.

Description

Technical Field

[0001] The present invention belongs to the field of clinical testing and diagnosis, and specifically relates to a metabolomics-based diagnostic marker for esophageal squamous cell carcinoma lymph node metastasis and its application. Background Art

[0002] Early-stage esophageal cancer presents with subtle clinical symptoms and is difficult to detect. Most patients with esophageal cancer are already in the locally advanced stage or have distant metastases at the time of diagnosis. Recent data indicate that lymph node metastasis is a key factor affecting the clinical prognosis of esophageal cancer patients. The 5-year overall survival rate for patients with non-metastatic esophageal cancer after surgery is 70-92%, while that for patients with metastatic esophageal cancer is only 18-47%. Although significant progress has been made through surgery and adjuvant chemoradiotherapy, the overall prognosis for patients with esophageal cancer remains poor, likely due to the lack of early symptoms and the current lack of sensitive early diagnostic methods. Therefore, it is crucial to diagnose early-stage esophageal cancer patients based on lymph node metastasis using more sensitive detection methods to support clinicians in adjusting treatment plans and improve the prognosis of esophageal cancer patients in my country.

[0003] Metabolomics is the study of all small molecule metabolites (such as amino acids, fatty acids, and lipids) in biological samples (such as plasma, serum, urine, feces, and saliva) or cells, and the identification of their relative relationships with pathophysiological changes. Because information transmission within organisms proceeds step by step through DNA, mRNA, proteins, and metabolites, metabolomics can be considered the end product and phenotype of genomics and proteomics. While genomics and proteomics can reveal intrinsic differences within organisms, these differences do not necessarily lead to phenotypic differences due to compensatory mechanisms. Small molecule metabolites, on the other hand, can reflect both intrinsic differences and the effects of external factors on the organism. The pathogenesis of esophageal cancer is not yet fully understood, but it is associated with factors such as diet, smoking, and alcohol consumption. Therefore, using metabolomics to identify metabolite changes that characterize esophageal cancer lymph node metastasis is consistent with its pathogenesis.

[0004] At present, researchers have used plasma metabolomics technology to study the lymph node metastasis of esophageal cancer, such as Jin H et al. (Jin H, Qiao F, Chen L, Lu C, Xu L, Gao X. Serum metabolomic signatures of lymph node metastasis of esophageal squamous cell carcinoma. J Proteome Res. 2014 Sep 5; 13(9): 4091-103), Zhang H et al. (Zhang H, Wang L, Hou Z, Ma H, Mamtimin B, Hasim A, Sheyhidin I. Metabolomic profiling reveals potential biomarkers in esophageal cancer progression using liquid chromatography-mass spectrometry platform. Biochem Biophys Res Commun. 2017 Sep 9; 491(1): 119-125.) Plasma samples were analyzed using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) techniques, and the obtained data were analyzed using traditional statistical methods such as logistic regression analysis and principal component analysis (PCA) to search for biomarkers related to esophageal cancer metastasis.

[0005] However, most of these studies used only small sample sizes and universal chromatographic methods to screen a series of small-molecule metabolites as biomarkers. The sensitivity and specificity of these metabolites for diagnosing esophageal cancer lymph node metastasis were not reported, making their clinical significance very limited. Therefore, using large-scale clinical samples for plasma metabolomics studies to identify sensitive, specific, safe, and cost-effective plasma metabolite biomarkers for diagnosing esophageal cancer lymph node metastasis has important clinical application value. Summary of the Invention

[0006] The purpose of the present invention is to provide the use of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide and Nicotinamide.

[0007] In one aspect, the present invention provides the use of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide as markers for developing detection or auxiliary detection or screening or auxiliary screening of esophageal squamous cell carcinoma lymph node metastasis.

[0008] The above-mentioned 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide are metabolites derived from plasma.

[0009] In another aspect, the present invention provides the use of a substance for detecting 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, or Nicotinamide in at least one of the following:

[0010] 1) Detection or auxiliary detection of esophageal squamous cell carcinoma lymph node metastasis;

[0011] 2) Screening or auxiliary screening for lymph node metastasis of esophageal squamous cell carcinoma.

[0012] The above-mentioned substances for detecting 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide are for detecting the concentrations of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide in plasma metabolites.

[0013] The above substances include the instruments and / or reagents required for ultra-high performance liquid chromatography-mass spectrometry, and also include a readable carrier recording the conditions of the ultra-high performance liquid chromatography-mass spectrometry;

[0014] In the above, the above substances also include instruments and / or reagents required for plasma metabolite extraction.

[0015] In another aspect, the present invention provides an ultra-high performance liquid chromatography-mass spectrometry instrument and / or reagent for detecting the concentrations of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and nicotinamide in plasma metabolites, and a readable carrier recording the operating parameters of the ultra-high performance liquid chromatography-mass spectrometry instrument, for use in preparing a product having at least one of the following functions:

[0016] 1) Detection or auxiliary detection of esophageal squamous cell carcinoma lymph node metastasis;

[0017] 2) Screening or auxiliary screening for lymph node metastasis of esophageal squamous cell carcinoma.

[0018] The subjects of the above screening are patients with esophageal squamous cell carcinoma.

[0019] In the above, the ultra-high performance liquid chromatography-mass spectrometry instrument parameters recorded in the readable carrier are as follows:

[0020] 1) Mobile Phase Conditions: This project used an ACQUITY UPLC-I / CLASS-TQ-S (Waters) ultra-high performance liquid chromatography tandem mass spectrometer, using a Waters ACQUITY UPLC HSS T3 column (100×2.1 mm, 1.8 μm, Waters). Phase A consisted of a solution containing 10 mM ammonium formate and 0.02% (volume percentage) formic acid (solvent: water, solute: ammonium formate and formic acid), and phase B consisted of methanol. The column oven temperature was 40°C, the sample tray was set to 10°C, and the injection volume was 10 μL.

[0021] 2) Mass spectrometry conditions:

[0022] This project used a Waters Xevo TQ-S triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode for mass spectrometry analysis. Ion source parameters were as follows: Capillary voltages = 2.5 kV, Cone voltages = 30 V, Desolvation Temperature = 550°C, Desolvation gas flow = 1100 (L / Hr), Cone gas flow = 150 (L / Hr), Nebuliser gas flow = 7.0 (Bar).

[0023] In another aspect, the present invention provides a kit for screening or assisting in screening for lymph node metastasis of esophageal squamous cell carcinoma, comprising the following components: the substance described above for detecting the concentration of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide in plasma metabolites, and a carrier having a model recorded thereon or a device loaded with the model;

[0024] The model is established by input information (1) and input information (2) and is used to demonstrate the relationship between the input information (1) and the input information (2); the input information (1) is: information on the concentrations of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide in the plasma metabolites of all subjects in the modeling group; (2) the phenotypes of all subjects in the modeling group, namely, patients with esophageal squamous cell carcinoma with lymph node metastasis or patients with esophageal squamous cell carcinoma without lymph node metastasis; the modeling group is composed of n patients with esophageal squamous cell carcinoma with lymph node metastasis and m patients with esophageal squamous cell carcinoma without lymph node metastasis, where n is a natural number (in the embodiment of the present invention, n is 26), and m is a natural number (in the embodiment of the present invention, m is 53).

[0025] In another aspect, the present invention provides a device for screening or assisting in screening of esophageal squamous cell carcinoma lymph node metastasis, comprising a detection device and a result output device;

[0026] The detection device is used to detect the concentrations of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide in the plasma metabolites of the subject;

[0027] The result output device is used to receive the concentrations of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide in the plasma metabolites of the subject output by the detection device, and input the information into the model, which outputs the result to predict whether the subject has esophageal squamous cell carcinoma lymph node metastasis;

[0028] The model is established by input information (1) and input information (2) and is used to demonstrate the relationship between the input information (1) and the input information (2); the input information (1) is: information on the concentrations of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide in the plasma metabolites of all subjects in the modeling group; (2) the phenotypes of all subjects in the modeling group, namely, patients with esophageal squamous cell carcinoma lymph node metastasis or patients with esophageal squamous cell carcinoma without lymph node metastasis; the modeling group consists of n patients with esophageal squamous cell carcinoma lymph node metastasis and m patients with esophageal squamous cell carcinoma without lymph node metastasis, where n is a natural number and m is a natural number.

[0029] In another aspect, the present invention provides a device for screening or assisting in screening of esophageal squamous cell carcinoma lymph node metastasis, comprising a model loading device, a detection device, and a result output device;

[0030] The model loading device is a device for loading the model; the model is established by input information (1) and input information (2) and is used to demonstrate the relationship between the input information (1) and the input information (2); the input information (1) is: information on the concentrations of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide in the plasma metabolites of all subjects in the modeling group; (2) the phenotypes of all subjects in the modeling group, namely, patients with esophageal squamous cell carcinoma lymph node metastasis or patients with esophageal squamous cell carcinoma without lymph node metastasis; the modeling group is composed of n patients with esophageal squamous cell carcinoma lymph node metastasis and m patients with esophageal squamous cell carcinoma without lymph node metastasis, where n is a natural number and m is a natural number;

[0031] The detection device is used to detect the concentrations of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide in the plasma metabolites of the subject;

[0032] The result output device is used to receive the information on the concentrations of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide in the plasma metabolites of the subject output by the detection device, and input the information into the model loading device to predict whether the subject has esophageal squamous cell carcinoma lymph node metastasis.

[0033] In the above, the relationship between the input information (1) and the input information (2) is established and used to show the input information (1) and the input information (2). In an embodiment of the present invention, it is performed according to the random forest algorithm. The parameters of the random forest algorithm are as follows: the R language software used is version 4.2.0, the randomForest algorithm comes from the R language randomForest package version 4.7-1.1, the random seed is set to 008, and the R language pROC package (version 1.18.0) is used to evaluate the performance of the model.

[0034] To address the current situation where lymph node metastasis in esophageal cancer is common but effective liquid diagnostic methods are lacking, this paper provides a biomarker suitable for diagnosing lymph node metastasis in esophageal cancer. This marker has high sensitivity and specificity for diagnosing lymph node metastasis in esophageal cancer and can be used for esophageal cancer staging and surgical decision-making, with significant implications for improving esophageal cancer prognosis and patient survival.

[0035] The present invention analyzed plasma samples from 130 esophageal cancer patients, including 43 patients with lymph node metastasis and 87 patients without. Using UHPLC-MRM-MS / MS with internal standard quantification, the plasma metabolite concentrations of three target compounds in the samples—1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and nicotinamide—were measured. Using a random forest algorithm, the plasma metabolite concentrations of these three metabolites were used as a feature to develop a model for diagnosing esophageal cancer lymph node metastasis. Using the concentrations of these three target compounds in the test samples, the model can rapidly diagnose whether esophageal cancer patients have lymph node metastasis. This model demonstrates high accuracy, sensitivity, and universal applicability, providing a robust scientific basis for clinical diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Figure 1A shows the single-cell metabolic profile of esophageal squamous cell carcinoma lymph node metastasis and the metabolic pathway analysis results of the non-targeted metabolome of esophageal squamous cell carcinoma tissue (1B).

[0037] Figure 2 Figure 2 is an example of an extracted ion chromatogram for targeted metabolomics; 2A is the extracted ion chromatogram of the standard solution, and 2B is the extracted ion chromatogram of the sample. The chromatographic peaks in the figure are marked in the example in the figure.

[0038] Figure 3 Box plots showing the abundance differences of three metabolites, nicotinamide (3A), 1-methylnicotinamide (3B), and 1-methyl-2-pyridone-5-carboxamide (3C), in the training set.

[0039] Figure 4 The principal component analysis diagram of samples stratified by the abundance of three metabolites.

[0040] Figure 5 Figure 5 is the ROC curve for the diagnosis of esophageal cancer lymph node metastasis using three biomarkers. 5A is the ROC result of the training set, and 5B is the ROC result of the validation set. DETAILED DESCRIPTION

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0042] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0043] The following examples include a total of 130 plasma samples from esophageal cancer patients, including 43 patients with lymph node metastasis and 87 patients without lymph node metastasis. The diagnostic criterion for lymph node metastasis is that the intraoperative lymph node sampling is positive as confirmed by postoperative pathology report.

[0044] The instruments and reagents in the following examples are as follows:

[0045] Table 1 shows the experimental instruments

[0046]

[0047] Table 2 is the experimental reagents

[0048]

[0049]

[0050] Table 3 shows the standard products

[0051]

[0052] Example 1. Acquisition and application of plasma metabolite markers

[0053] 1. Discovery of Plasma Metabolite Markers

[0054] The applicant used single-cell transcriptome technology to perform single-cell transcriptome sequencing on tumor tissues and corresponding metastatic lymph nodes of 2 cases of esophageal squamous cell carcinoma with lymph node metastasis, and tumor tissues and lymph nodes of 3 cases of esophageal squamous cell carcinoma without lymph node metastasis. In the esophageal squamous cell carcinoma lymph node metastasis group, it was found that compared with other cell types, tumor cells underwent significant metabolic reprogramming ( Figure 1 A). Furthermore, the applicant used non-targeted metabolomics technology to sequence and analyze 13 esophageal squamous cell carcinoma tissues with lymph node metastasis and 8 esophageal squamous cell carcinoma tissues without lymph node metastasis. Pathway analysis of differential metabolites revealed significant differences in the niacin and nicotinamide metabolic pathways between the lymph node metastasis group and the non-lymph node metastasis group ( Figure 1 B), and the abundance of 1-methylnicotinamide was significantly increased in the tumor tissues of the lymph node metastasis group.

[0055] The detection difficulty and stability of each metabolite in the niacin and nicotinamide metabolic pathway were evaluated, and 1-methyl-6-oxopyridine-3-carboxamide, nicotinamide and 1-methylnicotinamide were selected as candidate markers for esophageal squamous cell carcinoma with lymph node metastasis.

[0056] 2. Plasma targeted metabolomics detection method using ultra-high performance liquid chromatography-mass spectrometry

[0057] The following uses plasma samples from 130 esophageal cancer patients, including 43 patients with lymph node metastasis (recorded as metastasis group, Group A or Group A) and 87 patients without lymph node metastasis (recorded as non-metastasis group, Group B or Group B).

[0058] Plasma samples were centrifuged and stored at -80°C. During the study, plasma samples were removed and, after sample pretreatment, subjected to targeted metabolomics analysis using ultra-performance liquid chromatography-mass spectrometry. Plasma concentrations of target metabolites were determined for diagnosis of esophageal cancer lymph node metastasis and screening of esophageal cancer lymph node metastasis markers.

[0059] The specific operations are as follows:

[0060] 1. Metabolite extraction

[0061] 1) Thaw the plasma sample on ice and vortex for 30 seconds after it is completely thawed;

[0062] 2) Place 100 μL of the plasma sample to be tested in a 1.5 mL EP tube and add 400 μL of the extract (1:1 volume ratio of acetonitrile to methanol, pre-cooled at -40°C, containing a certain concentration of isotope internal standard);

[0063] 3) Vortex for 30 seconds to mix, and sonicate for 15 minutes in an ice-water bath;

[0064] 4) Let it stand at -40℃ for one hour;

[0065] 5) Centrifuge the sample at 4°C, 12,000 rpm (13,800 × g, radius 8.6 cm) for 15 min, and collect the supernatant.

[0066] 6) Take 440 μL of the supernatant, blow dry with nitrogen, and reconstitute with 110 μL of 10% aqueous methanol. Centrifuge the sample at 12,000 rpm (13,800 × g, radius 8.6 cm) for 15 min at 4°C. Collect 80 μL of the supernatant as the metabolite sample for UHPLC-MS / MS analysis.

[0067] 2. Preparation of standard solution

[0068] Accurately weigh the corresponding amount of the standard substances shown in Table 3 into a 10 mL volumetric flask, and prepare 10 mmol / L standard stock solutions using 10% methanol aqueous solution.

[0069] Take the corresponding amount of standard stock solution in a 10mL volumetric flask and prepare the standard solution with 10% methanol aqueous solution, which is recorded as the calibration solution of different standards.

[0070] The calibration solution was diluted in sequence with 10% methanol aqueous solution to obtain a series of calibration solutions of different standard concentrations.

[0071] 3. On-machine testing

[0072] The internal standard solution (name of internal standard: Nicotinamide-2,4,5,6-d4; manufacturer and product number: ISOREAG IR-22309; concentration on the instrument: 1.6 μM) was added to the test samples or calibration solutions of a series of different standard concentrations obtained in step 1 above from each plasma sample, and then subjected to UHPLC-MS / MS detection:

[0073] 1) Mobile Phase Conditions: This project used an ACQUITY UPLC-I / CLASS-TQ-S (Waters) ultra-high performance liquid chromatography tandem mass spectrometer, using a Waters ACQUITY UPLC HSS T3 column (100×2.1 mm, 1.8 μm, Waters). Phase A consisted of a solution containing 10 mM ammonium formate and 0.02% (volume percentage) formic acid (solvent: water, solute: ammonium formate and formic acid), and phase B consisted of methanol. The column oven temperature was 40°C, the sample tray was set to 10°C, and the injection volume was 10 μL.

[0074] 2) Mass spectrometry conditions:

[0075] This project used a Waters Xevo TQ-S triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode for mass spectrometry analysis. Ion source parameters were as follows: Capillary voltages = 2.5 kV, Cone voltages = 30 V, Desolvation Temperature = 550°C, Desolvation gas flow = 1100 (L / Hr), Cone gas flow = 150 (L / Hr), Nebuliser gas flow = 7.0 (Bar).

[0076] For each target compound, several parent ion-daughter ion pairs (transitions) with the highest signal intensity were selected, their MRM parameters were optimized, and the ion pairs with the best response were selected for quantitative analysis, while the other ion pairs were used for qualitative analysis of the target compound.

[0077] In this project, all mass spectrometry data acquisition and quantitative analysis of target compounds were completed using skyline software.

[0078] 4. Calibration curve

[0079] The method described in step 3 was used to perform UPLC-MRM-MS / MS analysis on a series of calibration solutions (0.98 nmol / L, 1.95 nmol / L, 3.91 nmol / L, 7.81 nmol / L, 15.63 nmol / L, 31.25 nmol / L, 62.50 nmol / L, 125.00 nmol / L, 250.00 nmol / L, 500.00 nmol / L, 1000.00 nmol / L, 2000.00 nmol / L) of different concentrations of the standard obtained in step 2 above to obtain a calibration curve.

[0080] The calibration curve y represents the peak area of the target compound, and x represents the concentration of the target compound (nmol / L).

[0081] In this experiment of the present invention, the standard curve formula of 1-methylnicotinamide is y=6.5298*10 - 7 X 2 +2.1270*10 -3 X+8.1473*10 -4 The standard curve formula for 1-methyl-2-pyridone-5-carboxamide is y = -6.8590*10 -9 X 2 +3.5648*10 -4 X+6.1525*10 -3 ; Nicotinamide standard curve formula is y = -2.8720*10 -9 X 2 +3.2149*10 -4 X+4.0703*10 -4 .

[0082] In the above standard curve formula, X is the concentration of the corresponding substance in nmol / L, and y is the peak area.

[0083] The least squares method was used for regression analysis. When the weight was set to 1 / x, the recovery rate (accuracy) and correlation coefficient (R 2 If the signal-to-noise ratio (S / N) of a calibration concentration is close to or less than 20, or the recovery rate is outside the range of 80-120%, then the calibration point is excluded.

[0084] 5. Method detection limit and quantification limit

[0085] UHPLC-MRM-MS analysis was performed on a series of calibration solutions (the dilution concentration was the same as 4) after serial dilution. The signal-to-noise ratio was used to calculate the limit of quantification (LLOQ). The lower limit of quantification (LLOQ) was defined as the compound concentration that achieved a signal-to-noise ratio of 10 (according to the US FDA guideline for bioanalytical method validation).

[0086] 6. Method precision and accuracy

[0087] The precision of the method was evaluated by the relative standard deviation (RSD) of replicate injections of QC samples. The accuracy was evaluated by the recovery of spiked QC samples, which was calculated as the percentage of the measured concentration to the spiked concentration.

[0088] 2. Results Analysis: Obtaining Diagnostic Markers for Lymph Node Metastasis in Esophageal Squamous Cell Carcinoma

[0089] 1. Identification of diagnostic markers for lymph node metastasis in esophageal squamous cell carcinoma

[0090] Using the method described in step 3 above, the metabolites of the test samples obtained in step 1 above were analyzed by UHPLC-MS / MS to obtain the original metabolic fingerprint of all samples. The concentrations of the targeted metabolites in different groups were then analyzed in the hope of identifying biomarkers that could distinguish esophageal cancer from lymph node metastasis.

[0091] The specific operations are as follows:

[0092] 1) Chromatographic separation

[0093] Extracted ion chromatograms (EICs) of calibrator solution and sample are as follows Figure 2 As shown, Figure 2 A is the ion chromatogram of the calibration solution, Figure 2B is the ion chromatogram of a test sample; the chromatographic peaks are labeled as examples in the figure. It can be seen that the target compound 1-methylnicotinamide (denoted as 1-m…e_4 in the figure) in the calibrator has a response time of 0.90 and a peak area of 687937.75; 1-methyl-2-pyridone-5-carboxamide (denoted as 1-m…e_3 in the figure) has a response time of 3.27 and a peak area of 18743962; and nicotinamide (denoted as Nic…d_1 in the figure) has a response time of 3.28 and a peak area of 852123.0625. There are no significant differences in retention times and chromatographic peak shapes between the test sample and the calibrator solution. This indicates that the target compounds 1-methylnicotinamide, 1-methyl-2-pyridone-5-carboxamide, and nicotinamide were obtained in the test sample.

[0094] After testing, all 130 samples were found to contain the target compounds 1-methylnicotinamide, 1-methyl-2-pyridone-5-carboxamide and Nicotinamide.

[0095] 2) Method quantitative parameters

[0096] The quantitative parameters of the target compounds in the above-mentioned samples are shown in Table 4 below. The lower limits of detection (LLODs) of each target compound in the 130 samples ranged from 0.24–4.88 nmol / L, and the lower limits of quantification (LLOQs) ranged from 0.98–39.06 nmol / L. The correlation coefficients (R2) of all target compounds were greater than 0.9989, indicating a good quantitative relationship between the chromatographic peak area and the compound concentration, which can meet the requirements of targeted metabolomics analysis.

[0097] Table 4 shows the quantitative parameters of the target compounds

[0098]

[0099] The recoveries and relative standard deviations (RSDs) of the QC samples are shown in Table 5 below. The QC samples were injected 20 times. As shown in Table 5, the average recoveries of all target compounds ranged from 92.3% to 103.6%, and the RSDs were all less than 5.8%.

[0100] Table 5 shows the recovery and relative standard deviation (RSD) of the QC samples.

[0101] Target compound Concentration (nmol / L) Recovery rate RSD 1-methyl-2-pyridone-5-carboxamide 1000.00 102.5％ 4.3％ 1-methylnicotinamide 200.00 92.3％ 5.8％ Nicotinamide 200.00 99.3％ 4.0％

[0102] The above data show that this method can accurately and reliably detect the content of target metabolites in samples within the concentration range shown above.

[0103] 3) Detection results of target metabolite concentration in the sample to be tested

[0104] According to the method in step 3 above, the metabolite content of the sample to be tested is detected, and then the concentration C is obtained by inserting it into the calibration curve obtained in step 4 above. C值 .

[0105] The final concentration of the sample was C F (Final Concentration, nmol / L) is the concentration C directly measured by the instrument C (Calculated Concentration, nmol / L) multiplied by the Dilution Factor (Dil), expressed in nmol / L. The Dilution Factor refers to the dilution factor of the sample during testing. For this test, the Dilution Factor is 110.

[0106] The concentration of target metabolite in the sample C M (Metabolite Concentration, also known as abundance) is equal to the final measured concentration of the sample C F Multiply by the final sample volume V F (Final Volume, μL) and the sample experimental concentration factor CF, divided by the sample volume V S (Sample Volume, μL) (Specific formula is as follows); unit is nmol / L.

[0107] Calculation formula

[0108] For the method of the present invention, in This is the experimental concentration factor CF.

[0109] The concentration of each target metabolite in each sample to be tested C M The quantitative results are shown in Table 6.

[0110] Table 6 shows the concentrations of each target metabolite in each sample to be tested. M Quantitative results

[0111]

[0112]

[0113]

[0114]

[0115] The concentrations of the target compounds in the metastatic group and the non-metastatic group of the above 130 samples were averaged, and the results are shown in Table 7. It can be seen that 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide were significantly different in the metastatic group and the non-metastatic group, further proving that these three markers can be used as markers to distinguish whether esophageal cancer patients have lymph node metastasis.

[0116] Table 7 shows the quantitative results of target metabolites in the samples of the metastasis group and the non-metastasis group.

[0117]

[0118] Example 2: Application of three diagnostic markers for esophageal cancer lymph node metastasis

[0119] The 130 enrolled samples were divided into training and test sets at a ratio of 3:2. The training set consisted of 79 cases, including 26 cases in the esophageal cancer lymph node metastasis group (Group A) and 53 cases in the esophageal cancer non-lymph node metastasis group (Group B). The test set consisted of 51 cases, including 17 cases in the esophageal cancer lymph node metastasis group (Group A) and 34 cases in the esophageal cancer non-lymph node metastasis group (Group B).

[0120] 1. Training Set

[0121] The training set consisted of 79 cases, including 26 cases in the esophageal cancer lymph node metastasis group (Group A) and 53 cases in the esophageal cancer non-lymph node metastasis group (Group B).

[0122] 1. Extraction of metabolites

[0123] Extraction was performed according to the method of Example 1 to obtain metabolites of different test samples.

[0124] 2. On-machine testing

[0125] UHPLC-MS / MS detection was performed according to the method of Example 1-3. The concentrations (also known as abundances) of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide in the metabolites of different test samples were calculated based on the calibration curve obtained by the method of Example 1-4.

[0126] The results are as follows Figure 3As shown, the abundance of the three metabolite markers tested was compared at the metabolite abundance level. Compared with the non-metastatic group, the abundance of 1-methyl-2-pyridone-5-carboxamide and 1-methylnicotinamide was higher in the metastatic group, while the abundance of nicotinamide was significantly lower. The difference in the abundance of 1-methyl-2-pyridone-5-carboxamide and nicotinamide between the two groups was statistically significant.

[0127] 3. Cluster analysis

[0128] To further evaluate the predictive ability of the identified metabolites, principal component analysis (PCA) was performed on the 79 samples in the training set.

[0129] The results are as follows Figure 4 As shown, significant clustering differences were found between the metastatic and non-metastatic groups. Based on the differences in the abundance of the three metabolites in the training set, the metastatic and non-metastatic groups could be distinguished. The first and second principal components of the PCA explained 56.7% and 33.5% of the total variance, respectively, indicating that these three metabolites showed significant clustering differences between the metastatic and non-metastatic groups, suggesting that the selected markers have good discrimination between metastatic and non-metastatic cases.

[0130] 4. Random Forest Algorithm

[0131] The training set was trained using a random forest algorithm, with the abundance of the three identified metabolites used as features to construct the model. R software version 4.2.0 was used, and the randomForest algorithm was derived from the R package randomForest, version 4.7-1.1. The random seed was set to 0.08. The performance of the model was evaluated using the R pROC package (version 1.18.0).

[0132] A random forest model was trained to evaluate the lymph node metastasis of esophageal squamous cell carcinoma patients with lymph node metastasis. The final evaluation model accurately predicted the lymph node metastasis of the enrolled cases.

[0133] The results are as follows Figure 5 As shown in A, the area under the ROC curve (AUC) was 0.828, the sensitivity was 53.85%, the specificity was 88.68%, the positive predictive value was 70.00%, and the negative predictive value was 79.66%.

[0134] From the above, it can be seen that the model obtained by using the random forest algorithm, characterized by the abundance of the three identified metabolites, can identify and evaluate whether patients with esophageal squamous cell carcinoma with lymph node metastasis have lymph node metastasis.

[0135] 2. Validation Set

[0136] To further evaluate whether the selected markers can assess lymph node metastasis in other esophageal squamous cell carcinomas, a test set was used for validation.

[0137] The test set consists of 51 cases, 17 cases in the esophageal cancer lymph node metastasis group (Group A), and 34 cases in the esophageal cancer lymph node non-metastasis group (Group B).

[0138] 1. Extraction of metabolites

[0139] Extraction was performed according to the method of Example 1 to obtain metabolites of different test samples.

[0140] 2. On-machine testing

[0141] UHPLC-MS / MS detection was performed according to the method of Example 1-3. The concentrations (also known as abundances) of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide in the metabolites of different test samples were calculated based on the calibration curve obtained by the method of Example 1-4.

[0142] 3. Model Evaluation

[0143] The abundance of the three metabolite markers in the test set samples was input into the random forest model constructed above to evaluate the model obtained by the sub-model identification algorithm and assess whether patients with esophageal squamous cell carcinoma have lymph node metastasis.

[0144] The ROC curve was drawn based on the abundance, and the results were as follows Figure 5 As shown in Figure B, it can be seen that a fairly good prediction rate (AUC=0.774) is obtained, wherein the sensitivity and specificity are 58.82% and 76.47% respectively.

Claims

1. Use of substances for detecting 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide in the preparation of products for detecting or screening lymph node metastasis of esophageal squamous cell carcinoma.

2. The use according to claim 1, characterized in that: The substances for detecting 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide and Nicotinamide are for detecting the concentrations of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide and Nicotinamide in plasma metabolites.

3. The use according to claim 1 or 2, characterized in that: The substances include instruments and / or reagents required for ultra-high performance liquid chromatography-mass spectrometry.

4. The use according to claim 1 or 2, characterized in that: The subjects of the screening are patients with esophageal squamous cell carcinoma.

5. A device for screening or assisting in screening for lymph node metastasis of esophageal squamous cell carcinoma, comprising a detection device and a result output device; The detection device is used to detect the concentrations of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide in the plasma metabolites of the subject; The result output device is used to receive the concentrations of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide in the plasma metabolites of the subject output by the detection device, and input the concentration information into the model, which outputs the result to predict whether the subject has esophageal squamous cell carcinoma lymph node metastasis; The model is established by input information (1) and input information (2) and is used to demonstrate the relationship between the input information (1) and the input information (2); the input information (1) is: information on the concentrations of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide and Nicotinamide in the plasma metabolites of all subjects in the modeling group; the input information (2) is: the phenotypes of all subjects in the modeling group, that is, patients with esophageal squamous cell carcinoma lymph node metastasis or patients with esophageal squamous cell carcinoma without lymph node metastasis; the modeling group is composed of n patients with esophageal squamous cell carcinoma lymph node metastasis and m patients with esophageal squamous cell carcinoma without lymph node metastasis, where n is a natural number and m is a natural number.

6. A device for screening or assisting in screening for esophageal squamous cell carcinoma lymph node metastasis, comprising a model loading device, a detection device, and a result output device; The model loading device is a device for loading the model; the model is established by input information (1) and input information (2) and is used to demonstrate the relationship between the input information (1) and the input information (2); the input information (1) is: information on the concentrations of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide and Nicotinamide in the plasma metabolites of all subjects in the modeling group; the input information (2) is: the phenotypes of all subjects in the modeling group, namely, patients with esophageal squamous cell carcinoma lymph node metastasis or patients with esophageal squamous cell carcinoma without lymph node metastasis; the modeling group is composed of n patients with esophageal squamous cell carcinoma lymph node metastasis and m patients with esophageal squamous cell carcinoma without lymph node metastasis, where n is a natural number and m is a natural number; The detection device is used to detect the concentrations of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide, and Nicotinamide in the plasma metabolites of the subject; The result output device is used to receive the concentration information of 1-methyl-2-pyridone-5-carboxamide, 1-methylnicotinamide and Nicotinamide in the plasma metabolites of the subject output by the detection device, and input the concentration information into the model loading device to predict whether the subject has esophageal squamous cell carcinoma lymph node metastasis.

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