NOVEL LIVER CIRRHOSIS OR LIVER FIBROSIS MARKER

Abstract

The invention relates to the field of diagnosis and treatment of cirrhosis and liver fibrosis. Specifically, the invention relates to the use of Golgi protein 73 (GP73) and a substance of detecting the level of Golgi protein 73 (GP73) for preparing the products for screening, auxiliarily diagnosing liver diseases or auxiliarily determining the prognosis of liver diseases. The invention proves that Golgi protein 73 (GP73) can be used as a novel marker of cirrhosis, and has diagnostic value especially for cirrhosis resulting from non-hepatitis B virus infection, but has no diagnostic value for primary hepatocellular carcinoma.

Claims

1. Use of a substance of detecting Golgi protein 73 level for preparing the products for screening, auxiliarily diagnosing liver diseases or auxiliarily determining the prognosis of liver diseases.

2. The use of claim 2, wherein the substance of detecting Golgi protein 73 level comprises Golgi protein 73 and/or Golgi protein 73 antibody.

3. The use of claim 3, wherein the substance of detecting Golgi protein 73 level further comprises a reagent and a required instrument for detecting the Golgi protein 73 level.

4. Use of recombinant vector, expression cassette, transgenic cell line or recombinant strain containing Golgi protein 73 encoding gene as a biomaterial of preparing Golgi protein 73 for preparing the products for screening, auxiliarily diagnosing liver diseases or auxiliarily determining the prognosis of liver diseases.

5. Use of recombinant vector, expression cassette, transgenic cell line, recombinant strain containing Golgi protein 73 antibody encoding gene or immunogen or antigen of Golgi protein 73 antibody as a biomaterial of preparing Golgi protein 73 antibody for preparing the products for screening, auxiliarily diagnosing liver diseases or auxiliarily determining the prognosis of liver diseases.

6. The use of any one of claims 1 to 5, wherein the disease is cirrhosis or liver fibrosis, and preferably the cirrhosis or the liver fibrosis is cirrhosis or liver fibrosis resulting from non-hepatitis B virus infection.

7. The use of any one of claims 1 to 5, wherein subject in the screening or diagnosing by the product is a patient with cirrhosis, liver fibrosis or healthy person.

8. The use of any one of claims 1 to 5, wherein the product is tested in serum, plasma or interstitial fluid, and preferably human serum, human plasma or human interstitial fluid.

9. The use of any one of claims 1 to 5, wherein the product is a kit.

10. A kit for screening, auxiliarily diagnosing liver diseases or auxiliarily determining the prognosis of liver diseases, and the kit comprises a substance for detecting Golgi protein 73 level.

11. The kit of claim 10, wherein the substance of detecting the Golgi protein 73 level comprises Golgi protein 73 and/or Golgi protein 73 antibody.

12. The kit of any one of claim 10 or 11, wherein the disease is cirrhosis or liver fibrosis, and preferably the cirrhosis or liver fibrosis is cirrhosis or liver fibrosis resulting from non-hepatitis B virus infection.

13. The kit of any one of claim 10 or 11, wherein detection methods by the kit include enzyme-linked immunosorbent assay, chemiluminescence, electrochemiluminescence, enzymatic chemiluminescence, time-resolved fluorescence, or upconversion luminescence.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 shows the ROC curve of the diagnostic value of GP73 for hepatocellular carcinoma. (A) GP73 concentrations were measured in non-cirrhosis chronic liver disease group, cirrhosis group, hepatocellular carcinoma (HCC) group, non-cirrhosis HCC group and HCC group transformed from cirrhosis. (B) The sensitivity in distinguishing HCC from non-cirrhosis chronic liver disease. (C) The sensitivity in distinguishing HCC from cirrhosis. (D)-(E) Immunohistochemical images of liver in different degrees of disease.

[0027] FIG. 2 shows the ROC curve of the diagnostic value of GP73 for cirrhosis. (A) The diagnostic value of GP73 in recruited patients with cirrhosis. (B) ROC curves of different markers in chronic hepatitis C (CHC). (C) ROC curves of different markers in chronic alcoholic liver disease (ALD). (D) The diagnostic value of GP73 for cirrhosis in all other populations (autoimmune liver disease, nonalcoholic fatty liver disease and primary biliary cirrhosis, etc.).

[0028] FIG. 3 shows a comparison of the diagnostic value of serum GP73 for liver fibrosis. (A) The GP73 concentrations in samples were measured in different degrees of liver fibrosis (F=0, 1, 2, 3 and 4). (B)-(D) ROC curves for different degrees of liver fibrosis. (E) Immunohistochemical images of liver in different degrees of fibrosis.

DETAILED DESCRIPTION

[0029] The following makes further explanations to the present invention by specific implements and experimental data. Although the terminologies hereinafter are used for the purpose of clearness, they are not intended to define or limit the scope of the present disclosure.

[0030] As used in the text, the term sensitivity is also called true-positive rate, which refers to the percentage of patients who are actually ill and are correctly judged as ill according to the testing criteria. The greater the sensitivity is the better. The ideal sensitivity is 100%.

[0031] As used in the text, the term specificity is also called true-negative rate, which refers to the percentage of patients who are actually disease-free and are correctly judged as disease-free according to the testing criteria. The greater the specificity is the better. The ideal sensitivity is 100%.

[0032] Youden index is also called correct diagnostic index. Its value ranges between 01, and the closer it is to 1, the better the diagnostic accuracy. In the method of the invention, the diagnostic criterion is set at the maximum of the correct diagnostic index.

[0033] The receiver (relative) operating characteristic (ROC) reflects the balance between sensitivity and specificity. The area under the ROC curve is an important test accuracy indicator. Calculate the area under the ROC line of each test (AUCROC) for comparison. The larger the area, the greater the diagnostic value of the test.

[0034] Unless otherwise specified, the experimental methods in the following Examples are conventional methods.

[0035] Unless otherwise specified, the materials, reagents, etc. used in the following Examples can be purchased commercially.

EXAMPLES

Example 1. Comparison of Diagnostic Values of Serum GP73, AFP, AFP-L3 and AFP-L3 in Primary Hepatocellular Carcinoma

[0036] 1) Baseline Data of Subjects and Samples

[0037] Blood samples were obtained from: 805 patients with hepatitis; 2015 patients with liver cirrhosis; 1102 patients with liver cancer, a total of 3922 patients. See Table 1 for patient's specific information.

[0038] The samples were all serum of clinically confirmed patients. The three clinical samples were provided by the People's Liberation Army No. 302 Hospital. The patients were treated in the People's Liberation Army No. 302 Hospital from December 2013 to December 2014.

[0039] 2) Statistics

[0040] SPSS 21.0 (SPSS, Chicago, Ill., USA) statistical software was used for statistical analysis. Descriptive methods of continuous variables are selected according to their distribution characteristics. Quantitative data approximates the normal distribution is expressed by xS. The distribution characteristics of quantitative data with skewed distribution are described by median (M) and interquartile range. 2 test was used for comparison among qualitative data sets, Mann-WhitnyU test was used for comparison between two independent samples, and Kruskal Wallis test was used among multiple sets of samples. The receiver operating characteristic curve (ROC curve) was established, and the value of different indicators for the diagnosis of primary hepatocellular carcinoma was compared according to the area under the curve, taking =0.05 as testing standard.

[0041] 3) Results

[0042] The commercial ELISA kit of Beijing Rejing Biotechnology Co. Ltd. was used to detect the level of GP73 in serum samples according to the specific experimental procedures of the kit instruction. The results showed that the level of GP73 in chronic hepatitis, cirrhosis and hepatocellular carcinoma were 45.200.78 ng/mL, 150.321.78 ng/mL and 121.322.47 ng/mL respectively, as shown in Table 1, while the level of alpha-fetoprotein AFP3 in chronic hepatitis, cirrhosis and hepatocellular carcinoma were 3.120.23 ng/mL, 33.514.64 ng/mL and 567.3640.71 ng/mL respectively.

TABLE-US-00001 TABLE 1 Testing results of the subjects Hepatocellular Hepatitis Cirrhosis carcinoma (n = 805) (n = 2015) (n = 1102) Variate mean SEM mean SEM mean SEM P Age 44.68 0.39 50.58 0.25 52.09 0.31 0.000 BMI (kg/m.sup.2) 24.12 0.12 24.14 0.09 24.00 0.10 0.785 GP73 (ng/mL) 45.20 4.78 150.32 1.78 121.32 2.47 0.000 AFP3 (ng/mL) 3.12 0.23 33.51 4.64 567.36 40.71 0.000 ALT (U/L) 46.47 0.14 93.64 5.31 72.23 4.68 0.000 AST (U/L) 35.27 1.61 87.47 3.61 79.36 4.25 0.000 PLT (10{circumflex over ()}9/L) 187.71 12.20 104.14 1.47 144.85 2.75 0.000 APRI 0.56 0.04 2.71 0.14 2.05 0.16 0.000 FIB-4 1.57 0.07 6.92 0.21 4.61 0.17 0.000 Note: ALT, alanine aminotransferase; AST, glutamic oxaloacetic aminotransferase; PLT, platelet; APRI, a scoring method, the ratio index of aspartate aminotransferase (AST) and platelet (PLT); FIB-4, the evaluation index of liver disease.

[0043] 4) Comparison of the Diagnostic Values of Serum GP73, AFP, AFP-L3 and AFP-L3 in Primary Hepatocellular Carcinoma

[0044] Using the pathological diagnosis as the gold standard, the ROC curve was used to calculate the sensitivity, specificity, positive predictive value, negative predictive value, false positive rate and Youden index of serum GP73, AFP, AFP-L3 and AFP-L3, and calculated the best diagnostic value. The results showed that the area under the ROC curves of serum Golgi protein 73 (GP73), alpha-fetoprotein (AFP) and alpha-fetoprotein heterogenous (AFP-L3) were 0.527 (95% CI, 0.503-0.552), 0.826 (95% CI, 0.808-0.843 P<0.001) and 0.824 (95% CI, 0.806-0.841 P<0.001) respectively, as shown in FIG. 1 and Table 2.

TABLE-US-00002 TABLE 2 The diagnostic values of GP73, AFP, AFP-L3 (%) and AFP-L3 in hepatocellular carcinoma Area under Youden the curve Variate (%) Sensitivity Specificity index 95% CI AFP 68.56% 82.45% 0.510 0.826 0.808-0.843 AFP-L3 67.60% 82.65% 0.503 0.824 0.806-0.841 AFP-L3 54.58% 88.02% 0.462 0.677 0.653-0.701 GP73 85.25% 21.89% 0.071 0.527 0.503-0.552 indicates data missing or illegible when filed

Example 2. The Distribution Characteristics of Serum GF73, AFP and AFP-L3(%) in Patients with Hepatitis, Cirrhosis and Hepatocellular Carcinoma

[0045] The subjects and statistical methods were the same as in Example 1. In the experiment, the changes of AFP, AFP-L3(%) and GP73 in patients with hepatitis, cirrhosis and hepatocellular carcinoma were described by interquartile range. The results showed that the trend of GP73 change in hepatitis, cirrhosis and hepatocellular carcinoma was obvious, but it could not distinguish patients with hepatocellular carcinoma from those with hepatitis and cirrhosis. AFP and AFP-L3(%) showed significant change in patients with liver cancer only, and could distinguish patients with hepatocellular carcinoma from those with hepatitis and cirrhosis, as shown in Table 3.

TABLE-US-00003 TABLE 3 Interquartile ranges of GP73, AFP and AFP-L3 (%) in patients with hepatitis, cirrhosis and hepatocellular carcinoma Interquartile range Variate Disease 5 10 25 50 75 90 93 GP73 Hepatitis 15.291 20.568 33.500 57.480 90.573 159.230 219.535 Cirrhosis 26.950 39.260 72.220 121.600 179.200 246.400 277.900 Hepatocellular 29.432 38.750 61.960 103.800 168.350 240.080 291.640 Carcinoma AFP Hepatitis 0.896 1.049 1.490 2.260 3.600 10.697 52.828 cirrhosis 0.822 1.030 1.600 2.660 5.820 20.170 72.520 Hepatocellular 1.740 2.268 5.145 40.850 885.850 1300.000 2245.300 Carcinoma AFP-L3 Hepatitis 0.045 0.053 0.075 0.114 0.181 0.690 5.419 Cirrhosis 0.041 0.052 0.081 0.135 0.299 1.665 7.130 Hepatocellular 0.087 0.111 0.256 3.800 152.600 1018.300 1100.000 carcinoma

Example 3. Comparison of the Diagnostic Values of Serum GP73, LSM, FIB4 and APRI in Cirrhosis

[0046] The subjects and statistical methods were the same as in Example 1.

[0047] The results of this part showed that the sensitivity and specificity of GP73 in the diagnosis of cirrhosis were 83.97% and 93.54% respectively, and the area under curve was 0.927 (95% CI, 0.917-0.937) (FIG. 2). The diagnostic value of GP73 was better than that of liver stiffness measurement (LSM), FIB-4 index and platelet (PLT) ratio index APRI, as shown in Table 4, indicating that GP73 had a stronger ability to distinguish patients with cirrhosis.

TABLE-US-00004 TABLE 4 The diagnostic values of GP73, LSM, APRI and FIB4 in cirrhosis Positive Negative Area cut-off Sensitivity Specificity predictive Predictive Youden under Variate value (%) (%) value % value % index the curve 95% CI P GP73 75.16 83.97 93.54 97.02 69.99 0.775 0.927 0.917-0.937 <0.0001 ng/ml LSM 11.8 74.19 86.75 90.63 66.06 0.609 0.885 0.866-0.901 <0.0001 kPa FIB-4 2.0 83.17 82.75 92.35 66.27 0.659 0.900 0.888-0.910 <0.0001 APRI 0.64 78.92 81.95 91.63 60.84 0.609 0.871 0.858-0.883 <0.0001

Example 4. Comparison of the Diagnostic Values of Serum GP73 in Liver Fibrosis

[0048] Serum GP73 could well distinguish between significant liver fibrosis (F2), severe liver fibrosis (F3) and early cirrhosis (F=4). Serum GP73 level (MeansSE) increased with the degree of liver fibrosis F0 (n=21, 5.74%): 35.182.95; F1 (n=97, 26.50%): 42.732.57; F2 (n=67, 18.31%): 50.593.48; F3 (n=66, 18.03%): 79.935.67; F4 (n=115, 31.42%): 169.267.31 (P<0.001). The sensitivity, specificity and AUROC of serum GP73 in the diagnosis of F2, F3 F=4 were F2: 64.92%, 90.68%, 0.828 (95% CI: 0.785-0.865); F3: 80.66%, 86.49%, 0.895 (95% CI: 0.859-0.924); F=4: 82.61%, 91.24%, 0.933 (95% CI: 0.902-0.956), as shown in FIG. 3.

[0049] The above results indicate that GP73 can be used as a potential marker for screening and discriminating liver fibrosis and cirrhosis (non-hepatitis B virus infection) from healthy people and patients with liver fibrosis and cirrhosis, and the method of screening and diagnosing different degrees of liver fibrosis or cirrhosis (non-hepatitis B virus infection) by detecting the GP73 protein concentration in samples of the screening target is accurate and easy to operate.

[0050] The Examples of the present invention have been described above, but the present invention is not limited thereto, and those skilled in the art can understand that modifications and changes can be made within the scope of the purport of the present invention. The manner of modifications and changes should fall within the scope of protection of the present invention.