Biomarker combination for detecting liver cirrhosis, liver cancer, hepatitis-combined liver cancer and liver cirrhosis-combined liver cancer diseases and application of biomarker combination

Abstract

The invention belongs to the technical field of liver disease detection markers, and particularly relates to a biomarker combination for detecting liver cirrhosis, liver cancer, hepatitis combined with liver cancer and liver cirrhosis combined with liver cancer and application, the combined marker comprises chitosanase 3-like protein 1, alpha fetoprotein, alpha fetoprotein variant L3 ratio, abnormal prothrombin or Golgi protein 73, alpha fetoprotein, alpha fetoprotein variant L3 ratio, alpha fetoprotein variant L3 ratio and alpha fetoprotein variant L3 ratio. When the level of each component of the detected person is higher than a preset critical value, the corresponding disease is evaluated to be high in risk, and when the level of each component of the detected person is lower than the preset critical value, the corresponding disease is evaluated to be low in risk. The kit has the characteristics of high accuracy, strong specificity and high sensitivity, is also beneficial to detection of liver cirrhosis, liver cancer or hepatitis combined liver cancer which is not easy to detect, and can help patients to diagnose liver cirrhosis, liver cancer, hepatitis combined liver cancer or liver cirrhosis combined liver cancer as early as possible, so that the healing and survival probability is increased.

Description

Technical Field

[0001] This invention belongs to the technical field of liver disease detection biomarkers, specifically relating to a combination of biomarkers for the detection of liver cirrhosis, liver cancer, hepatitis combined with liver cancer, or liver cirrhosis combined with liver cancer, and their applications. Background Technology

[0002] Liver cancer ranks sixth in global cancer incidence and second in cancer-related mortality. Commonly occurring against a background of chronic liver inflammation and fibrosis, hepatitis B virus infection is a major risk factor for liver cancer development, progressing through multiple stages in individual patients. Due to difficulties in early diagnosis, rapid progression, and a lack of targeted therapies, liver cancer has an extremely low survival rate. The diverse risk factors, genetic susceptibility, morphological diversity, and microenvironmental differences significantly limit the progress in early detection, molecular classification, and targeted therapy for liver cancer. Therefore, early diagnosis remains crucial for improving prognosis, and early surveillance of liver cancer is particularly important for high-risk populations with chronic liver disease and cirrhosis.

[0003] Currently, while pathological biopsy remains the gold standard for diagnosing liver cancer, it carries risks of bleeding and tumor spread, and may also result in false negatives due to poor sampling location. Therefore, other auxiliary diagnostic methods are also quite important. Currently, in the "Guidelines for the Diagnosis and Treatment of Primary Liver Cancer (2022 Edition)," the main hematological molecular markers for liver cancer are alpha-fetoprotein (AFP), des-gamma carboxyprothrombin (DCP), and lensculinaris agglutinin-reactive fraction of AFP (AFP-L3). However, these markers have a high false negative rate, low sensitivity, and low diagnostic efficacy in liver cirrhosis, hepatitis complicated with liver cancer, or liver cirrhosis complicated with liver cancer.

[0004] Existing technologies contain numerous studies on liver cancer biomarkers. For example, Chinese patent CN114487426A discloses a combination of biomarkers for predicting liver cancer risk in male hepatitis B virus infected individuals and its applications, including at least one of gamma-glutamyl transferase, platelets, white blood cells, albumin, and cirrhosis indicators, as well as alpha-fetoprotein and abnormal prothrombin. Chinese patent CN115369101A discloses a combination of biomarkers for liver disease detection and its applications; the combination of chitinase 3-like protein 1 (CHI3L1), alpha-fetoprotein, alpha-fetoprotein isoform L3, and abnormal prothrombin can improve the specificity, sensitivity, and accuracy of liver cancer diagnosis. However, biomarkers for applications in cirrhosis, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer need further development. Therefore, new detection biomarkers and detection methods or products with higher specificity and sensitivity require further research. Summary of the Invention

[0005] The purpose of this invention is to provide a combination of biomarkers for the detection of liver cirrhosis, liver cancer, hepatitis combined with liver cancer, or liver cirrhosis combined with liver cancer, and their applications, in order to solve the above-mentioned technical problems.

[0006] To achieve the above-mentioned technical objectives, the technical solution of the present invention is as follows: A combination biomarker for detecting cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer, comprising chitosanase 3-like protein 1, alpha-fetoprotein, alpha-fetoprotein isoform L3 ratio, abnormal prothrombin, and Golgi protein 73.

[0007] As a further improvement, when the levels of each component in the tested individual are above a pre-set threshold, the individual is assessed as having a high risk of the corresponding disease; when the levels of each component are below a pre-set threshold, the individual is assessed as having a low risk of the corresponding disease. The threshold values ​​for each component in the combined biomarker are: Alpha-fetoprotein (AFP): 5.94 ng / mL for cirrhosis, 5.79 ng / mL for hepatocellular carcinoma, 5.83 ng / mL for hepatitis complicated with hepatocellular carcinoma, and 5.83 ng / mL for cirrhosis complicated with hepatocellular carcinoma. Alpha-fetoprotein isoform L3 ratio: cirrhosis 6.87%, hepatocellular carcinoma 6.90%, hepatitis combined with hepatocellular carcinoma 6.90%, cirrhosis combined with hepatocellular carcinoma 6.90%; Chitosan polysaccharide enzyme 3-like protein 1: cirrhosis 57.42 ng / mL, hepatocellular carcinoma 83.74 ng / mL, hepatitis combined with hepatocellular carcinoma 83.74 ng / mL, cirrhosis combined with hepatocellular carcinoma 93.87 ng / mL; Abnormal prothrombin levels: 15.42 ng / mL for cirrhosis, 70.05 ng / mL for liver cancer, 73.80 ng / mL for hepatitis combined with liver cancer, and 80.31 ng / mL for cirrhosis combined with liver cancer; Golgi protein 73: 72.89 ng / mL for cirrhosis, 70.37 ng / mL for liver cancer, 78.09 ng / mL for hepatitis combined with liver cancer, and 90.57 ng / mL for cirrhosis combined with liver cancer.

[0008] A diagnostic model for cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer, named the A3CDG model, is provided. The liver disease and corresponding model scoring formulas are as follows: Cirrhosis A3CDG score = 0.122×AFP + 2.943×AFP-L3 + 0.001×DCP + 0.031×GP73 + 0.018×CHI3L1-18.678; Liver cancer A3CDG score = 0.360×AFP + 0.796×AFP-L3 + 0.013×DCP + 0.018×GP73 + 0.031×CHI3L1-10.319; The A3CDG score for hepatitis complicated with liver cancer is calculated as follows: 0.083×AFP + 1.950×AFP-L3 + 0.014×DCP + 0.001×GP73 + 0.020×CHI3L1-14.046. A3CDG score for cirrhosis complicated with hepatocellular carcinoma = -0.005×AFP + 107.775×AFP-L3 + 0.032×DCP + 0.012×GP73 + 0.020×CHI3L1-545.002; In the above formula: AFP is the content of alpha-fetoprotein in the tested sample (ng / mL). AFP-L3 is the percentage of alpha-fetoprotein isoform L3 in the tested sample (%). DCP is the content of abnormal prothrombin in the test sample (ng / mL). GP73 represents the content (ng / mL) of Golgi protein 73 in the tested sample. CHI3L1 represents the content (ng / mL) of chitosanase 3-like protein 1 in the tested sample.

[0009] As a further improvement, the diagnostic criteria for cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer are as follows: the patient's score is higher than the median score of the liver cirrhosis model in healthy individuals. The median scores for the liver cirrhosis model in healthy individuals are: -1.377, -2.870, -2.890, and -4.316.

[0010] A method for constructing diagnostic models for cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer, based on the aforementioned methods, includes the following steps: Step 1: Extract body fluids from healthy individuals and patients with cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer. Step 2: Measure the content of the aforementioned biomarkers in body fluids; Step 3: Perform statistical analysis on the content of biomarkers; Step 4: Construct the A3CDG model based on the statistical analysis results.

[0011] As a further improvement, the statistical analysis includes regression analysis, which uses a logistic regression model and plots ROC curves for analysis. The coefficients of each marker in the scoring formula are the regression coefficients obtained from the logistic regression model analysis. Specific parameters are determined based on the need for sensitivity or specificity priority, and statistical analysis results are produced.

[0012] As a further improvement, the body fluid is serum, and the markers are chitosanase 3-like protein 1, alpha-fetoprotein, alpha-fetoprotein isoform L3 ratio, abnormal prothrombin, and Golgi protein 73.

[0013] As a further improvement, the sample size of the control population and patients with cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer required to construct the A3CDG model shall not be less than 40 people.

[0014] As a further improvement, the variables in the statistical analysis included the content of chitosanase 3-like protein 1, the content of alpha-fetoprotein, the ratio of alpha-fetoprotein isoforms L3, the content of abnormal prothrombin, and the content of Golgi protein 73.

[0015] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows: A combination of biomarkers for the detection of cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer and its application have the characteristics of high accuracy, strong specificity, and high sensitivity. It is also beneficial for detecting cirrhosis, liver cancer, or hepatitis combined with liver cancer that are not easily detected, and can help patients be diagnosed with cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer as early as possible, thereby increasing the chances of cure and survival. Attached Figure Description

[0016] Figure 1 This is the ROC curve for liver cirrhosis; Figure 2 This is the ROC curve for liver cancer; Figure 3 This is the ROC curve for hepatitis combined with liver cancer; Figure 4 This is the ROC curve for liver cirrhosis combined with liver cancer. Detailed Implementation

[0017] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0018] Example 1: Verify that there are differences in serum levels of five biomarkers—chitosanase 3-like protein 1 (CHI3L1), alpha-fetoprotein (AFP), alpha-fetoprotein isoform (AFP-L3), abnormal prothrombin (DCP), or Golgi protein 73 (GP73)—between patients with cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer and healthy individuals.

[0019] Patients with cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer, as well as healthy individuals, were selected and divided into experimental and healthy groups. Serum levels of CHI3L1, AFP, AFP-L3, DCP, and GP73 were detected using kits.

[0020] The detection method is as follows (taking the detection of CHI3L1 as an example; the detection methods for other markers can refer to the detection method for CHI3L1): The kit includes: 1. Reagent 1 (R1): 2 bottles × 3.5 mL; magnetic microparticles coated with chitosanase 3-like protein 1 antibody, containing Tris buffer and preservative; 2. Reagent 2 (R2): 2 bottles × 4.0 mL; Chitosan polysaccharide enzyme 3-like protein 1 antibody acridine-labeled conjugate, containing PBS buffer and preservative; 3. Reagent 3 (R3): 2 bottles × 10.0 mL; sample diluent containing Tris buffer and preservative; 4. Calibrator 1 (CAL1): 1 vial × 1.0 mL; contains protein stabilizer, Tris buffer, and preservative; 5. Calibrator 2 (CAL2): 1 vial × 1.0 mL; containing chitosan polysaccharide enzyme 3-like protein 1, protein stabilizer, Tris buffer, and preservative; 6. Calibrator 3 (CAL3): 1 vial × 1.0 mL; containing chitosan polysaccharide enzyme 3-like protein 1, protein stabilizer, Tris buffer, and preservative; The concentrations of chitosanase 3-like protein 1 in the calibrators are shown in Table 1: Table 1. Concentration of chitosanase 3-like protein 1 in calibrators

[0021] The specific testing methods are as follows: 1. Equilibrate all components of the kit to room temperature.

[0022] 2. Load the CHI3L1 reagent bottle, place the reagent bottle on the instrument, scan the barcode on the reagent bottle, and read the relevant test parameters.

[0023] 3. Apply for calibration, start the test, confirm the calibration result, and it will show as successful.

[0024] 4. Apply for quality control, start testing, check the quality control data, which shows as under control.

[0025] 5. Perform sample settings, select the application menu, enter the sample number, rack number and cup position, select the test item CHI3L1, select the sample quantity, and select save.

[0026] 6. Load the sample, place the sample rack on the sample holder, and then place the sample rack on the sample tray of the instrument.

[0027] 7. Start the test and check the sample results.

[0028] The test results are shown in Table 2-6.

[0029] Table 2. Detection results of the healthy population

[0030] Table 3 Detection results in the cirrhosis group

[0031] Table 4 Detection results of liver cancer group

[0032] Table 5. Detection results in the hepatitis-complicated liver cancer group

[0033] Table 6. Detection results in the group with cirrhosis and liver cancer

[0034] As shown in Table 2-6, except for DCP (p=0.535), the serum levels of the four biomarkers CHI3L1 (P<0.0001), AFP (P<0.05), AFP-L3 (P<0.001), and GP73 (P<0.0001) in the cirrhosis group were higher than those in the healthy group. Except for DCP (p=0.068), the serum levels of the four biomarkers CHI3L1, AFP, AFP-L3, and GP73 in the liver cancer group were higher than those in the healthy group (P<0.0001). DCP cannot be used alone as a biomarker for detecting cirrhosis and liver cancer, but it can be used in combination with CHI3L1, AFP, AFP-L3, and GP73 as a biomarker for detecting cirrhosis and liver cancer.

[0035] The serum levels of five biomarkers, CHI3L1, AFP, AFP-L3, DCP, and GP73, were higher in the hepatitis-associated-hepatocellular carcinoma group and the cirrhosis-associated-hepatocellular carcinoma group than in the healthy group (P<0.0001).

[0036] The mean values ​​of CHI3L1 in the cirrhosis, hepatocellular carcinoma, hepatitis complicated with hepatocellular carcinoma, and cirrhosis complicated with hepatocellular carcinoma groups were 106.28 ng / mL, 224.61 ng / mL, 269.82 ng / mL, and 330.53 ng / mL, respectively, while the mean value in healthy individuals was 56.26 ng / mL. The mean values ​​of AFP in the cirrhosis, hepatocellular carcinoma, hepatitis complicated with hepatocellular carcinoma, and cirrhosis complicated with hepatocellular carcinoma groups were 19.42 ng / mL, 261.76 ng / mL, 26967.62 ng / mL, and 36601.57 ng / mL, respectively, while the mean value in healthy individuals was 2.86 ng / mL. The mean values ​​of AFP-L3 in the cirrhosis, hepatocellular carcinoma, hepatitis complicated with hepatocellular carcinoma, and cirrhosis complicated with hepatocellular carcinoma groups were 6.34%, 10.10%, 17.54%, and 18.22%, respectively, while the mean value in healthy individuals was 56.26 ng / mL. The mean value of DCP in the hepatitis-associated hepatocellular carcinoma (HCC) and cirrhosis-associated HCC groups was 4037.48 ng / mL and 6999.49 ng / mL, respectively, while the mean value in the healthy population was 14.86 ng / mL. The mean values ​​of GP73 in the cirrhosis, HCC, hepatitis-associated HCC, and cirrhosis-associated HCC groups were 92.50 ng / mL, 126.56 ng / mL, 139.45 ng / mL, and 184.04 ng / mL, respectively, while the mean value in the healthy population was 48.85 ng / mL. There were significant differences between the groups (P<0.05).

[0037] Therefore, CHI3L1, AFP, AFP-L3, and GP73 can be used as markers for detecting cirrhosis or liver cancer, and CHI3L1, AFP, AFP-L3, DCP, and GP73 can be used as markers for detecting hepatitis complicated with liver cancer or cirrhosis complicated with liver cancer.

[0038] Example 2: Verification of the ability of CHI3L1, AFP, AFP-L3, DCP and GP73 to detect cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer.

[0039] Based on the serum levels of the biomarkers in Example 1, receiver operating characteristic (ROC) curves were constructed to further evaluate the ability of biomarkers CHI3L1, AFP, AFP-L3, DCP, and GP73 to differentiate between patients with cirrhosis, liver cancer, hepatitis combined with liver cancer, cirrhosis combined with liver cancer, and healthy individuals.

[0040] The ROC curve reflects the balance between sensitivity and specificity. The area under the ROC curve is an important indicator of test accuracy. The larger the area under the ROC curve, the greater the diagnostic value of the test.

[0041] Sensitivity (true positive rate): The percentage of patients who are actually cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer, but are correctly diagnosed as cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer according to experimental standards. The higher the sensitivity, the better, with an ideal sensitivity of 100%.

[0042] Specificity (true negative rate): The percentage of patients who are actually not cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer, but are correctly judged as not cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer according to experimental criteria. The higher the specificity, the better, with an ideal specificity of 100%.

[0043] ROC analysis results are as follows: Figure 1-4 As shown in Table 7-10.

[0044] Table 7. ROC Analysis Results for Liver Cirrhosis

[0045] Table 8. ROC analysis results for liver cancer

[0046] Table 9. ROC analysis results of hepatitis complicated with liver cancer

[0047] Table 10. ROC analysis results of liver cirrhosis complicated with hepatocellular carcinoma

[0048] ROC analysis showed that the AUCs of CHI3L1 for diagnosing cirrhosis, hepatocellular carcinoma, hepatitis combined with hepatocellular carcinoma, and cirrhosis combined with hepatocellular carcinoma were 0.818, 0.961, 0.973, and 0.985, respectively, with Kappa values ​​of 0.511, 0.851, 0.872, and 0.915, respectively. The optimal cutoff values ​​for the dataset were 57.42 ng / mL, 83.74 ng / mL, 83.74 ng / mL, and 93.87 ng / mL, respectively. Combining CHI3L1 with AFP, AFP-L3, DCP, and GP73 further improved the performance, with AUCs of 0.738, 0.983, 0.990, and 0.994, respectively. This demonstrates that the combination of CHI3L1 with AFP, AFP-L3, DCP, and GP73, i.e., the A3CDG model, can be used to diagnose cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer. Furthermore, the A3CDG model improves the sensitivity and specificity of detection.

[0049] The analysis results of Example 2 show that, based on the biomarker combination of the present invention, the further developed detection reagents and kits also have a more accurate indication effect on cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer with mild clinical symptoms that are not easily detected, and have extremely high medical and practical value.

[0050] Example 3: Verify the ability of CHI3L1 to improve the detection accuracy of cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer.

[0051] Further ROC analysis of CHI3L1 with AFP, AFP-L3, DCP, or GP73 yielded the results shown in Tables 11-14.

[0052] Table 11 ROC Analysis Results for Liver Cirrhosis

[0053] Table 12 Results of ROC analysis for liver cancer

[0054] Table 13 Results of ROC analysis for hepatitis complicated with hepatocellular carcinoma

[0055] Table 14. ROC analysis results of liver cirrhosis complicated with liver cancer

[0056] Comparing the data in Table 11-14 with the single biomarker data in Table 7-10, any combination with CHI3L1 significantly improved the AUC. The AUCs for CHI3L1+AFP in diagnosing cirrhosis, hepatocellular carcinoma, hepatitis complicated with hepatocellular carcinoma, and cirrhosis complicated with hepatocellular carcinoma were 0.824, 0.971, 0.986, and 0.992, respectively; the AUCs for CHI3L+AFP-L3 were 0.840, 0.971, 0.987, and 0.993; the AUCs for CHI3L+DCP were 0.819, 0.972, 0.981, and 0.994; and the AUCs for CHI3L1+GP73 were 0.863, 0.969, 0.975, and 0.986. This indicates that CHI3L1 has the ability to improve detection accuracy, sensitivity, and specificity.

[0057] In addition, CHI3L1 and GP73 were used as biomarkers to detect serum levels of CHI3L and GP73 in patients with triple-negative AFP, AFP-L3, and DCP cirrhosis, hepatocellular carcinoma, hepatitis complicated with hepatocellular carcinoma, or cirrhosis complicated with hepatocellular carcinoma. The cutoff values ​​for CHI3L were 57.42 ng / mL, 83.74 ng / mL, 83.74 ng / mL, and 93.87 ng / mL, respectively, and the cutoff values ​​for GP73 were 72.89 ng / mL, 70.37 ng / mL, 78.09 ng / mL, and 90.57 ng / mL, respectively. The results are shown in Tables 15-17.

[0058] Table 15. Detection results of CHI3L1 and GP73 in patients with triple-negative AFP, AFP-L3, and DCP cirrhosis.

[0059] Table 16. Detection results of CHI3L1 and GP73 in hepatocellular carcinoma cases that are triple-negative for AFP, AFP-L3, and DCP.

[0060] Table 17 Results of CHI3L1 detection in cases of hepatocellular carcinoma complicated with AFP, AFP-L3, and DCP triple-negative hepatitis.

[0061] As shown in Tables 15-17, CHI3L1 and GP73 helped identify 17 cases (36.17%), 10 cases (21.28%), and 5 cases (10.64%) of AFP, AFP-L3, and DCP triple-negative liver cirrhosis, liver cancer, or hepatitis complicated with liver cancer in 47 cases, respectively. This further demonstrates that CHI3L1 and GP73 have the ability to improve the accuracy, sensitivity, and specificity of detection, and can detect patients with mild or no symptoms, or those that are easily missed.

[0062] The analysis results of Example 3 show that, based on the biomarker combination of the present invention, the further developed detection reagents and kits also have a more accurate indication effect on cirrhosis, liver cancer, hepatitis combined with liver cancer, or AFP, AFP-L3 and DCP triple negative cirrhosis, liver cancer or hepatitis combined with liver cancer with mild clinical symptoms that are not easily detected, and have extremely high medical and practical value.

[0063] The conventional operations in the operation steps of this invention are well known to those skilled in the art and will not be described in detail here.

[0064] Example 4: Diagnostic model for cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer.

[0065] I. Model Construction Step 1: Blood was drawn from healthy individuals, patients with cirrhosis, liver cancer, hepatitis combined with liver cancer, and patients with cirrhosis combined with liver cancer, and serum was separated. Step 2: The levels of the patient's serum inner shell polysaccharide enzyme 3-like protein 1, alpha-fetoprotein, alpha-fetoprotein isoform, abnormal prothrombin, and Golgi protein 73 were measured. Step 3: Perform statistical analysis on the biomarker content using a logistic regression model, plot the ROC curve, and calculate the area under the curve; Step 4: Construct the following model based on the statistical analysis results: CHI3L1-AFP model: The CHI3L1-AFP score for cirrhosis is calculated as follows: 0.175 × AFP + 0.025 × CHI3L1 - 2.563. Liver cancer CHI3L1-AFP score = 0.322 × AFP + 0.037 × CHI3L1 - 5.211; The CHI3L1-AFP score for hepatitis complicated with liver cancer is calculated as follows: CHI3L1-AFP score = 0.274 × AFP + 0.030 × CHI3L1 - 4.883. The CHI3L1-AFP score for cirrhosis complicated with hepatocellular carcinoma is calculated as follows: CHI3L1-AFP score = 0.293 × AFP + 0.031 × CHI3L1 - 5.724. CHI3L1-AFP-L3 model: The CHI3L1-AFP-L3 score for cirrhosis is calculated as follows: 4.486 × AFP-L3 + 0.024 × CHI3L1 - 24.449. Liver cancer CHI3L1-AFP-L3 score = 3.955 × AFP-L3 + 0.039 × CHI3L1 - 24.046; The CHI3L1-AFP-L3 score for hepatitis complicated with liver cancer is calculated as follows: 3.837 × AFP-L3 + 0.031 × CHI3L1 - 23.296. The CHI3L1-AFP-L3 score for cirrhosis complicated with hepatocellular carcinoma is calculated as follows: CHI3L1-AFP-L3 = 4.011 × AFP-L3 + 0.032 × CHI3L1 - 24.881. CHI3L1-DCP model: Cirrhosis CHI3L1-DCP score = 0.001 × DCP + 0.028 × CHI3L1 - 2.064; Liver cancer CHI3L1-DCP score = 0.014 × DCP + 0.042 × CHI3L1 - 4.661; The CHI3L1-DCP score for hepatitis complicated with liver cancer is calculated as follows: CHI3L1-DCP score = 0.015 × DCP + 0.037 × CHI3L1 - 4.625. Cirrhosis complicated with hepatocellular carcinoma CHI3L1-DCP score = 0.032 × DCP + 0.030 × CHI3L1 - 5.734; CHI3L1-GP73 model: Cirrhosis CHI3L1-GP73 score = 0.030 × GP73 + 0.022 × CHI3L1 - 3.639; Liver cancer CHI3L1-GP73 score = 0.021 × GP73 + 0.038 × CHI3L1 - 5.246; The CHI3L1-GP73 score for hepatitis complicated with liver cancer is calculated as follows: CHI3L1-GP73 = 0.018 × GP73 + 0.038 × CHI3L1 - 5.164. The CHI3L1-GP73 score for cirrhosis complicated with hepatocellular carcinoma is calculated as follows: CHI3L1-GP73 = 0.034 × GP73 + 0.024 × CHI3L1 - 6.252. A3CDG model: Cirrhosis A3CDG score = 0.122×AFP + 2.943×AFP-L3 + 0.001×DCP + 0.031×GP73 + 0.018×CHI3L1-18.678; Liver cancer A3CDG score = 0.360×AFP + 0.796×AFP-L3 + 0.013×DCP + 0.018×GP73 + 0.031×CHI3L1-10.319; The A3CDG score for hepatitis complicated with liver cancer is calculated as follows: 0.083×AFP + 1.950×AFP-L3 + 0.014×DCP + 0.001×GP73 + 0.020×CHI3L1-14.046. A3CDG score for cirrhosis complicated with hepatocellular carcinoma = -0.005×AFP + 107.775×AFP-L3 + 0.032×DCP + 0.012×GP73 + 0.020×CHI3L1-545.002; In the above formula: AFP is the content of AFP in the tested sample (ng / mL). AFP-L3 is the percentage of AFP-L3 in the tested sample (%). DCP is the concentration of DCP in the tested sample (ng / mL). GP73 represents the content of GP73 in the tested sample (ng / mL). CHI3L1 represents the content (ng / mL) of CHI3L1 in the tested sample.

[0066] The diagnostic criteria for cirrhosis, liver cancer, hepatitis complicated with liver cancer, or cirrhosis complicated with liver cancer are: the subject's score is higher than the median score of the healthy population.

[0067] After extensive sampling and statistical analysis, the median scores for the liver cirrhosis model in healthy individuals were: -1.377, for the liver cancer model: -2.870, for the hepatitis combined with liver cancer model: -2.890, and for the liver cirrhosis combined with liver cancer model: -4.316.

[0068] II. Case Diagnosis Taking a certain patient as an example, this patient was diagnosed with hepatitis before being diagnosed with cirrhosis, was diagnosed with cirrhosis before being diagnosed with liver cancer, and was diagnosed with hepatitis before being diagnosed with hepatitis combined with liver cancer. At enrollment on September 2, 2022, the serum levels of CHI3L1, AFP, AFP-L3, DCP, and GP73 in this patient were measured. The results are shown in Tables 18-20. Table 18 Results of biomarker detection in patients with cirrhosis

[0069] Table 19 Results of biomarker detection in liver cancer patients

[0070] Table 20. Biomarker Detection Results in Patients with Hepatitis Complicated with Liver Cancer

[0071] As shown in Tables 18-20, patients with AFP levels less than 20 ng / mL, AFP-L3 levels less than 10%, and DCP levels less than 40 ng / mL were all below the current diagnostic criteria for cirrhosis, hepatocellular carcinoma (HCC), or hepatitis complicated with HCC. Therefore, a definitive diagnosis based on these criteria would be difficult. However, patients with higher CHI3L1 or GP73 levels indicated a risk of cirrhosis, HCC, or hepatitis complicated with HCC. Therefore, the A3CDG model was used for assessment, and A3CDG scores were calculated for each patient. During the same period, A3CDG scores were assessed in patients with hepatitis and cirrhosis (47 patients each). When assessing patients with cirrhosis or hepatitis complicated with HCC, the median A3CDG scores for hepatitis patients were -1.121 and -2.499, respectively. When assessing patients with HCC, the median A3CDG score for cirrhosis patients was 0.493. The A3CDG scores of patients with cirrhosis, HCC, or hepatitis complicated with HCC were significantly higher than these medians, confirming a diagnosis of cirrhosis, HCC, or hepatitis complicated with HCC.

[0072] In reality, Patient 14 in Table 18 was not diagnosed with cirrhosis according to the A3CDG model on October 25, 2022, but was not diagnosed with cirrhosis until April 26, 2023, according to existing diagnostic criteria for cirrhosis. Similarly, Patient 11 in Table 19 was not diagnosed with liver cancer according to the A3CDG model on September 2, 2022, but was not diagnosed with liver cancer until October 6, 2023, according to existing diagnostic criteria for liver cancer. This demonstrates that A3CDG can diagnose cirrhosis in Patient 14 of Table 18 six months earlier and liver cancer in Patient 11 of Table 19 one year and one month earlier, showing its ability to identify cirrhosis and liver cancer, which are difficult to detect, and thus has value in the early diagnosis of these two conditions.

[0073] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A combined biomarker for detecting cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer, characterized in that, The composite biomarkers include chitosanase 3-like protein 1, alpha-fetoprotein, alpha-fetoprotein isoform L3 ratio, abnormal prothrombin, and Golgi protein 73.

2. The combined biomarker for detecting cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer according to claim 1, characterized in that, When the levels of any component in the tested individual are above a pre-set threshold, the individual is assessed as having a high risk of the corresponding disease; when the levels of any component are below a pre-set threshold, the individual is assessed as having a low risk of the corresponding disease. The threshold values ​​for each component in the composite biomarker are: Alpha-fetoprotein (AFP): 5.94 ng / mL for cirrhosis, 5.79 ng / mL for hepatocellular carcinoma, 5.83 ng / mL for hepatitis complicated with hepatocellular carcinoma, and 5.83 ng / mL for cirrhosis complicated with hepatocellular carcinoma. Alpha-fetoprotein isoform L3 ratio: cirrhosis 6.87%, hepatocellular carcinoma 6.90%, hepatitis combined with hepatocellular carcinoma 6.90%, cirrhosis combined with hepatocellular carcinoma 6.90%; Chitosan polysaccharide enzyme 3-like protein 1: cirrhosis 57.42 ng / mL, hepatocellular carcinoma 83.74 ng / mL, hepatitis combined with hepatocellular carcinoma 83.74 ng / mL, cirrhosis combined with hepatocellular carcinoma 93.87 ng / mL; Abnormal prothrombin levels: 15.42 ng / mL for cirrhosis, 70.05 ng / mL for liver cancer, 73.80 ng / mL for hepatitis combined with liver cancer, and 80.31 ng / mL for cirrhosis combined with liver cancer; Golgi protein 73: 72.89 ng / mL for cirrhosis, 70.37 ng / mL for liver cancer, 78.09 ng / mL for hepatitis combined with liver cancer, and 90.57 ng / mL for cirrhosis combined with liver cancer.

3. A diagnostic model for cirrhosis, liver cancer, hepatitis complicated with liver cancer, or cirrhosis complicated with liver cancer, named the A3CDG model, characterized in that, The liver diseases measured and the corresponding model scoring formulas are as follows: Cirrhosis A3CDG score = 0.122×AFP + 2.943×AFP-L3 + 0.001×DCP + 0.031×GP73 + 0.018×CHI3L1-18.678; Liver cancer A3CDG score = 0.360×AFP + 0.796×AFP-L3 + 0.013×DCP + 0.018×GP73 + 0.031×CHI3L1-10.319; The A3CDG score for hepatitis complicated with liver cancer is calculated as follows: 0.083×AFP + 1.950×AFP-L3 + 0.014×DCP + 0.001×GP73 + 0.020×CHI3L1-14.

046. A3CDG score for cirrhosis complicated with hepatocellular carcinoma = -0.005×AFP + 107.775×AFP-L3 + 0.032×DCP + 0.012×GP73 + 0.020×CHI3L1-545.002; In the above formula: AFP is the content of alpha-fetoprotein in the tested sample (ng / mL). AFP-L3 is the percentage of alpha-fetoprotein isoform L3 in the tested sample (%). DCP is the content of abnormal prothrombin in the test sample (ng / mL). GP73 represents the content (ng / mL) of Golgi protein 73 in the tested sample. CHI3L1 represents the content (ng / mL) of chitosanase 3-like protein 1 in the tested sample.

4. The diagnostic model for cirrhosis, liver cancer, hepatitis complicated with liver cancer, or cirrhosis complicated with liver cancer according to claim 3, characterized in that, The diagnostic criteria for diagnosing cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer are as follows: the patient's score is higher than the median score of the liver cirrhosis model in healthy individuals. The median score of the liver cirrhosis model in healthy individuals is -1.377, the liver cancer model score is -2.870, the hepatitis combined with liver cancer model score is -2.890, and the liver cirrhosis combined with liver cancer model score is -4.

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5. A method for constructing a diagnostic model for cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer, based on claims 3-4, characterized in that, Includes the following steps: Step 1: Extract body fluids from healthy individuals and patients with cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer. Step 2: Measure the content of the aforementioned biomarkers in body fluids; Step 3: Perform statistical analysis on the content of biomarkers; Step 4: Construct the A3CDG model based on the statistical analysis results.

6. The method for constructing a diagnostic model for cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer according to claim 5, characterized in that, The statistical analysis includes regression analysis, which uses a logistic regression model and plots ROC curves for analysis. The coefficients of each marker in the scoring formula are the regression coefficients obtained from the logistic regression model analysis. Specific parameters are determined based on the need for sensitivity or specificity priority, and statistical analysis results are produced.

7. The method for constructing a diagnostic model for cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer according to claim 6, characterized in that, The body fluid is serum, and the markers are chitosanase 3-like protein 1, alpha-fetoprotein, alpha-fetoprotein isoform L3 ratio, abnormal prothrombin, and Golgi protein 73.

8. The method for constructing a diagnostic model for cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer according to claim 6, characterized in that, The number of control subjects and patients with cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer required to construct the A3CDG model shall not be less than 40 each.

9. The method for constructing a diagnostic model for cirrhosis, liver cancer, hepatitis combined with liver cancer, or cirrhosis combined with liver cancer according to claim 7, characterized in that, The variables included in the statistical analysis were chitosanase 3-like protein 1 content, alpha-fetoprotein content, alpha-fetoprotein isoform L3 ratio, abnormal prothrombin content, and Golgi protein 73 content.

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