CN113484517A

CN113484517A - Biomarker for diagnosing early hepatocellular carcinoma and construction method of diagnosis mode

Info

Publication number: CN113484517A
Application number: CN202110759086.6A
Authority: CN
Inventors: 王强
Original assignee: Affiliated Hospital of North Sichuan Medical College
Current assignee: Affiliated Hospital of North Sichuan Medical College
Priority date: 2021-07-05
Filing date: 2021-07-05
Publication date: 2021-10-08
Anticipated expiration: 2041-07-05

Abstract

The invention discloses a biomarker for diagnosing early hepatocellular carcinoma and a method for constructing a diagnosis mode, wherein the biomarker comprises the following components: abnormal prothrombin, alpha-fetoprotein, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase and total bilirubin. The construction method of the diagnosis mode for diagnosing the early hepatocellular carcinoma comprises the following steps: collecting serum, detecting the concentrations of abnormal prothrombin, alpha-fetoprotein, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase and total bilirubin in the serum, establishing different diagnosis modes according to the concentration difference of biomarkers in the serum of patients with early hepatocellular carcinoma and benign liver diseases, and then carrying out ROC curve analysis through statistical software to establish a diagnosis mode for diagnosing the early hepatocellular carcinoma. The diagnosis mode constructed by the invention can obtain the highest diagnosis value when diagnosing early HBV-related HCC, the diagnosis sensitivity exceeds 80 percent, and the probability of HCC being discovered in the early stage is greatly improved.

Description

Biomarker for diagnosing early hepatocellular carcinoma and construction method of diagnosis mode

Technical Field

The invention relates to the technical field of liver cancer, in particular to a biomarker for diagnosing early hepatocellular carcinoma and a method for constructing a diagnosis mode.

Background

Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor in the world and the third most common cause of cancer-related death, and more than 50 ten thousand new cases occur each year, and 30 to 50 ten thousand people die each year. HBV infection is a very important cause for the development of hepatocellular carcinoma, and more than 50% of the world and 60% to 80% of the Asia and Africa regions have liver cancer development related to HBV infection. The 5-year survival rate of HCC patients is closely related to the diagnosis period, the 5-year survival rate of HCC patients at early stage exceeds 75%, and the 5-year survival rate of HCC patients at middle and advanced stages is far lower than 10%. Since it is very difficult to diagnose HCC in early stages, most patients are diagnosed at a middle-to-late stage, which is a major cause of poor prognosis of HCC.

The liver has only a single tumor with a tumor diameter of ≤ 5.0cm, or 2-3 tumor nodules are present, with a single tumor diameter of <3.0cm, and no vascular invasion and extrahepatic metastasis are defined as early stage HCC. The diagnosis of early HCC relies primarily on imaging detection techniques such as ultrasound, CT and MRI, as well as the detection of tumor markers. The imaging detection technology is a detection technology for diagnosing tumors with wide clinical application, however, the accuracy of detection is often limited by the technical ability and experience of operators. According to different selected diagnostic techniques, the detection performance of the imaging detection technology for detecting early HCC has certain differences, for example, the sensitivity of the ultrasonic detection technology is about 20-68%, the sensitivity of CT detection is about 60%, while the sensitivity of MRI for diagnosing early HCC is about 70%, and the specificity is between 70-85%.

The research on tumor diagnosis markers is carried out, and the sensitivity and specificity of the markers for early diagnosis of hepatocellular carcinoma in the prior art are poor, so that the research on the biomarkers with better sensitivity and specificity for diagnosing the early hepatocellular carcinoma and a new diagnosis mode is imperative.

Disclosure of Invention

In order to solve the above-mentioned deficiencies of the prior art, the present invention aims to provide a biomarker for diagnosing early hepatocellular carcinoma and a method for constructing a diagnosis mode, so as to solve the problems of poor sensitivity and specificity of the existing marker for early diagnosis of hepatocellular carcinoma.

The technical scheme for solving the technical problems is as follows: provided is a biomarker for diagnosing early hepatocellular carcinoma, comprising: abnormal prothrombin, alpha-fetoprotein, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase and total bilirubin.

The invention has the beneficial effects that: the invention combines abnormal prothrombin, alpha fetoprotein, glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase and total bilirubin to be cooperated for diagnosing early hepatocellular carcinoma, and has the advantages of high sensitivity and good specificity.

On the basis of the technical scheme, the invention can be further improved as follows:

further, the biomarker is derived from serum.

The beneficial effect of adopting the further technical scheme is as follows: the level of abnormal prothrombin, alpha-fetoprotein, glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase and total bilirubin in serum is detected, the detection source is easy to obtain, and the sampling is convenient.

The application of the biomarker in preparing a medicament for diagnosing early hepatocellular carcinoma is provided.

A kit for diagnosing early hepatocellular carcinoma comprises the biomarker. The marker can be made into a standard substance, the standard substance is used for carrying out rapid and accurate qualitative and quantitative analysis on the biomarker in the biological sample, and the kit is beneficial to realizing detection standardization and improving the convenience and the importance of detection.

The construction method of the diagnosis mode for diagnosing the early hepatocellular carcinoma comprises the following steps:

collecting serum, detecting the concentrations of abnormal prothrombin, alpha-fetoprotein, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase and total bilirubin in the serum, establishing different diagnosis modes according to the concentration difference of biomarkers in the serum of patients with early hepatocellular carcinoma and benign liver diseases, and then carrying out ROC curve analysis through statistical software to establish a diagnosis mode for diagnosing the early hepatocellular carcinoma.

Further, different diagnostic modes are established by the ratio of the concentration of the biomarkers of increased and decreased concentration.

Further, the area under the curve of different diagnosis modes is analyzed by adopting an ROC curve, and the diagnosis mode with the largest area under the curve is determined as the diagnosis mode for diagnosing the early hepatocellular carcinoma.

Further, the constructed diagnostic pattern was the [1.5 XPIVKA-II/(AST × T-Bil) + AFP/(ALT × T-Bil) ] index.

The invention has the following beneficial effects:

the invention takes abnormal prothrombin, alpha fetoprotein, glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase and total bilirubin as biomarkers, the diagnostic value of early HBV related HCC is diagnosed by detecting the level contents of the abnormal prothrombin, alpha fetoprotein, glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase and total bilirubin in serum and utilizing an ROC curve to evaluate a diagnostic mode established by the combined application of the markers, the highest diagnostic value can be obtained when the [1.5 x PIVKA-II/(AST x T-Bil) + AFP/(ALT x T-Bil) ] indexes are used for diagnosing the early HBV related HCC, the diagnostic sensitivity exceeds 80 percent, and the probability of being discovered in the early HCC is greatly improved.

Drawings

FIG. 1 shows the comparison of serum PIVKA-II (A), AFP (B), AST (C), ALT (D) and T-Bil (E) concentrations in patients with early stage HBV-associated HCC (tumor sizes ≦ 5cm and <3cm) and patients with HBV-associated benign liver disease.

FIG. 2 is a ROC curve for diagnosis of early stage HBV-associated HCC of different tumor sizes for different diagnostic modalities; wherein, the graph A is an ROC curve of early HBV-related HCC with the tumor size less than or equal to 5cm diagnosed by different diagnosis modes; panel B is ROC curve for diagnosis of early stage HBV-associated HCC with tumor size <3cm for different diagnostic modalities.

FIG. 3 is a new diagnostic model comparing the serum PIVKA-II (A), AFP (B), AST (C), ALT (D) and T-Bil (E) concentrations and tumor size (F) for patients diagnosed with early HBV-associated HCC positive and negative (tumor sizes ≦ 5cm and <3cm) and HBV-associated benign liver disease.

FIG. 4 is a graph showing the results of the ability of the new diagnostic modality to differentially diagnose early HBV-associated HCC from different control populations; wherein panel A is a comparison of index levels for new diagnostic patterns in patients with early stage HBV-associated HCC (tumor sizes ≤ 5cm and <3cm), biliary tract disease, gallbladder disease, hepatitis, cirrhosis and other benign liver diseases; FIGS. B-F are ROC curves of HBV-associated HCC with a tumor size of 5cm or less in the new diagnosis mode, using biliary tract disease patients (B), gallbladder disease patients (C), hepatitis patients (D), cirrhosis patients (E) and other benign liver disease patients (F) as control groups; FIG. G-K are ROC curves for HBV-associated HCC with tumor size <3cm in the new diagnosis mode using biliary tract disease patients (G), gallbladder disease patients (H), hepatitis patients (I), cirrhosis patients (J) and other benign liver disease patients (K) as control groups.

FIG. 5 is ROC curve for diagnosing early HBV-associated HCC with serum PIVKA-II ≥ 40mAU/mL in the new diagnostic mode; wherein, the graph A is an ROC curve of early HBV related HCC with the tumor size of less than or equal to 5cm and the serum PIVKA-II of more than or equal to 40mAU/mL diagnosed in a new diagnosis mode; FIG. B is ROC curve of early stage HBV associated HCC with tumor size <3cm for new diagnostic mode diagnosis serum PIVKA-II ≥ 40 mAU/mL.

FIG. 6 is a ROC curve for diagnosing early HBV-associated HCC with serum AFP ≥ 20ng/mL in the new diagnostic mode; wherein, the graph A is an ROC curve of early HBV related HCC with the tumor size of less than or equal to 5cm and AFP of more than or equal to 20ng/mL in a new diagnosis mode; FIG. B is ROC curve for new diagnostic model diagnosis of early stage HBV associated HCC with tumor size <3cm with serum AFP ≥ 20 ng/mL.

FIG. 7 is ROC curve for new diagnostic model diagnosis of early HBV-associated HCC with serum PIVKA-II ≥ 40mAU/mL and/or AFP ≥ 20 ng/mL; wherein, the graph A is an ROC curve of early HBV related HCC with the tumor size of less than or equal to 5cm and the serum PIVKA-II of more than or equal to 40mAU/mL and/or AFP of more than or equal to 20ng/mL diagnosed in a new diagnosis mode; FIG. B is ROC curve for new diagnostic model diagnosis of early stage HBV associated HCC with tumor size <3cm with serum PIVKA-II ≥ 40mAU/mL and/or AFP ≥ 20 ng/mL.

Detailed Description

The following examples are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

In addition, some english abbreviations and representatives have the following meanings:

HCC: hepatocellular carcinoma (hepatocellular carcinoma);

PIVKA-II: abnormal prothrombin (Protein induced by vitamin K absence or antagonist-II);

AFP: alpha-fetoprotein;

AST: glutamic-oxalacetic transaminase;

ALT: glutamic-pyruvic transaminase;

TBIL: total bilirubin.

Example 1:

1. materials and methods

1.1 study object

148 cases (64 cases of patients with tumor size less than 3.0 cm) of early stage HCC patients diagnosed by the affiliated hospitals of the medical college of Chuanbei from 2017 to 2019 in 12 months, and all the patients with early stage HCC in the group have chronic hepatitis B infection. HCC diagnosis is carried out according to the standard of a primary liver cancer diagnosis and treatment guideline (2018.V1) published by the Chinese clinical oncology society. 940 chronic hepatitis B virus infected patients comprise 39 bile duct diseases, 76 gallbladder diseases, 378 hepatitis diseases, 407 liver cirrhosis, 19 hepatic cysts and 21 hepatic hemangiomas. The diagnosis of chronic hepatitis B infection and liver cirrhosis conforms to the guideline for preventing and treating chronic hepatitis B infection revised by the Chinese society for hepatopathy.

1.2 serum PIVKA-II, AFP, AST, ALT and TBIL level detection

Serum PIVKA-ii concentrations were determined by the microparticle chemiluminescence method using the Architect i1000 detection system (Abbott, USA), serum AFP concentrations were determined by the electrochemiluminescence method using the Elecsys e602 detection system (Roche, Germany), and serum AST, ALT and TBIL concentrations were determined by the rate method using the AU5800 detection system (Beckman Coulter, USA).

1.3 data processing for PIVKA-II and AFP Joint detection

The diagnostic cutoff value (cut-off) of PIVKA-II and AFP for HBV-related early HCC was determined by ROC curve analysis using SPSS 19.0 statistical software. The fold of serum PIVKA-II and AFP levels relative to their corresponding diagnostic thresholds was determined using M_cut-offAnd (4) showing.

The invention relates to a tumor marker_cut-offAnd the analysis of the sum to evaluate the diagnostic value of the PIVKA-II and AFP combined detection in HBV-related early HCC.

1.4 determination of New diagnostic mode

According to the expression levels of serum PIVKA-II, AFP, AST, ALT and TBIL of early HBV related HCC patients and CHB patients, the ratio of the serum level with obviously increased indexes and the serum level with obviously decreased indexes in early HCC is used as the principle of establishing different diagnosis modes to carry out the diagnosis mode, thus being beneficial to enlarging the difference between liver cancer of liver cells and benign liver diseases, and achieving the purposes of diagnosis and differential diagnosis. And then analyzing the areas under the curves of different diagnosis modes by adopting an ROC curve, determining a new diagnosis mode for diagnosing the early HBV related HCC by using the diagnosis mode with the largest area under the curves, and evaluating the diagnosis value of the new diagnosis mode in the early HBV related HCC with abnormal PIVKA-II and/or AFP.

1.5 statistical analysis

The method is completed by SPSS 19.0 statistical software, test data are expressed by median (quartile distance), a diagnosis mode is established by combining multiple items, and the data are expressed by indexes obtained by calculating corresponding index detection results. The sex comparison of two groups of people adopts Pearson Chi-square test, the data comparison between the two groups adopts Mann-Whitney U test, and the ROC curve determines the diagnosis critical value, the area under the curve, the diagnosis sensitivity and the specificity. Area under the curve comparison was performed using Medcalc, version 12.3. Statistical significance of all tests was determined by two-tailed test as P < 0.05.

2. Results

2.1 characteristics of the patient

The detection results of the patients with early HCC and the CHB control group are expressed by median (four-quadrant spacing), and data analysis shows that the ages of the two groups of people are concentrated in middle-aged and elderly people, mainly male (128 and 701), and the age of the people with early HCC is 56(48-65) years old and is higher than 50(41-59) years old of the CHB control group (P is less than 0.001). Serum PIVKA-II and AFP levels were higher in HCC ≦ 5.0cm and HCC <3.0cm cases than in CHB control (P <0.001) (FIGS. 1A, B), while serum AST, ALT and T-Bil were lower in CHB control (P <0.001) (FIGS. 1C, D, E), serum PIVKA-II in HCC ≦ 5.0cm cases was higher in HCC <3.0cm cases (P <0.05) (FIG. 1A), while serum AFP, AST, ALT and T-Bil levels were compared between the two and were all statistically insignificant (P >0.05) (FIGS. 1B, C, D, E). The specific clinical characteristics of 1088 study populations are shown in Table 1.

Table 1 clinical characteristics of the study population (n ═ 1088)

Note: data is represented in median (interquartile) or numeric form; p <0.001, compared with chronic hepatitis B group.

2.2 optimal diagnostic modality for HBV-associated HCC in early stage

The diagnostic threshold value is set to 82.72 × 10 by calculating the result of the index or diagnostic mode when the highest diagnostic accuracy is obtained by using the CHB population as a control, and setting the diagnostic threshold value as the index or diagnostic mode^-3In the diagnostic mode [1.5 XT Bil) + AFP/(ALT XT Bil ]]The maximum AUROC (0.925) can be obtained when diagnosing early HBV-associated HCC with tumor size less than or equal to 5.0cm, is obviously higher than PIVKA-II, AFP, PIVKA-II combined with AFP, AFP/(AST × T-Bil) and AUROC (0.925vs 0.826, 0.666, 0.821, 0.853 and 0.859) of AFP/(ALT × T-Bil) (P<0.001). The differences were not statistically significant (0.925vs 0.905 and 0.901) (P) compared to the AUROC of the PIVKA-II/(AST × T-Bil) ratio and the PIVKA-II/(ALT × T-Bil) ratio>0.05) (table 2 and fig. 2A). The diagnostic threshold was set at 194.37 × 10^-3In the diagnostic mode [1.5 XT Bil) + AFP/(ALT XT Bil ]]The sensitivity and specificity of HBV-associated HCC in early stage, diagnosed with a tumor size of less than or equal to 5.0cm, were 67.57% and 95.32%, respectively.

The diagnostic threshold value is set to 60.37 × 10 by calculating the result of the index or diagnostic mode when the highest diagnostic accuracy is obtained by using the CHB population as a control, and setting the diagnostic threshold value as the index or diagnostic mode^-3In this case, the diagnosis is made by [ 1.5X PIVKA-II/(AST X T-Bil) + AFP/(ALT X T-Bil)]When the early HBV-related HCC with the tumor size less than 3.0cm is diagnosed, the maximum AUROC (0.896) which is obviously higher than PIVKA-II, AFP and PIV can be obtainedAUROC (0.896vs 0.741, 0.651 and 0.765) of KA-II in combination with AFP (P)<0.001), no statistical difference (0.896vs 0.854, 0.850, 0.848 and 0.856) (P) compared to the AUROC ratio of PIVKA-II/(AST-Bil), PIVKA-II/(ALT T-Bil), AFP/(AST × T-Bil) and AFP/(ALT × T-Bil) (P896 vs 0.854, 0.850, 0.848 and 0.856)>0.05) (table 2 and fig. 2B). The diagnostic threshold was set at 199.93 × 10^-3In this case, the diagnosis is made by [ 1.5X PIVKA-II/(AST X T-Bil) + AFP/(ALT X T-Bil)]The sensitivity and specificity of HBV-associated HCC in the early stage, diagnosed with tumor size <3.0cm, were 57.81% and 95.43%, respectively.

As can be seen from the above, 1.5 XPIVKA-II/(AST × T-Bil) + AFP/(ALT × T-Bil) can be identified as a novel diagnostic model for the diagnosis of early HBV-related HCC.

TABLE 2 diagnostic value of different diagnostic modalities for early stage HBV-associated HCC

The diagnostic modes in the above table are as follows:

the diagnosis mode is (i) PIVKA-II (mAU/mL)

Diagnostic mode-AFP (ng/mL)

The diagnosis mode is PIVKA-II + AFP

The diagnosis mode is PIVKA-II/(AST × T-Bil) (× 10)^-3)

The diagnostic mode is PIVKA-II/(ALT × T-Bil) (. times.10)^-3)

The diagnosis mode is AFP/(AST × T-Bil) (× 10^-3)

The diagnosis mode is AFP/(ALT multiplied by T-Bil) (. times.10)^-3)

The diagnosis mode (1.5 times PIVKA-II/(AST times T-Bil) + AFP/(ALT times T-Bil) (. times.10)^-3)

2.3 comparison of clinical characteristics of HBV-associated HCC cases in early stages of negative and positive diagnosis

The diagnostic threshold is set at 82.72×10^-3In the new diagnosis mode, 28 cases and 120 cases of early HBV related HCC negative cases and 120 cases of early HBV related HCC positive cases with the tumor size less than or equal to 5.0cm are diagnosed. Negative cases serum PIVKA-II and AFP levels are significantly lower than positive cases (P)<0.001) compared to the CHB control population, none of the differences were statistically significant (P)>0.05) (fig. 3A and B). Negative cases serum AST, ALT and T-Bil levels were all higher than positive cases (P)<0.05) but all were lower than the CHB control population (P)<0.05) (fig. 3C, D and F). In addition, the tumor size in the negative case was 2.70(1.43-3.85) cm, which was significantly lower than that in the positive case [3.50(2.60-4.20) cm ]](P<0.01) (fig. 3E).

2.4 ability of novel diagnostic model to differentially diagnose early HBV-associated HCC from different control populations

The new diagnostic pattern index results for early HBV-associated HCC were significantly higher than those of the different control populations (P <0.001) (fig. 4A). The new diagnosis mode diagnoses HBV related HCC in early stage, the AUROC obtained by using hepatitis population as control population is highest (HCC is less than or equal to 5.0cm: 0.948; HCC is less than 3.0cm:0.927), and the AUROC obtained by using bile duct disease population and other populations (hepatic cyst and hemangioma) as control population is lowest (HCC is less than or equal to 5.0cm:0.867 and 0.869; HCC is less than 3.0cm:0.823 and 0.817) (Table 3 and FIGS. 4B and 4C).

TABLE 3 Performance values of novel diagnostic models for different control groups

2.5 diagnostic value of early HBV-associated HCC with PIVKA-II abnormality in New diagnostic model

196 cases of CHB with serum PIVKA-II more than or equal to 40mAU/mL are taken as control population, and the diagnosis threshold is set to be 82.70 multiplied by 10^-3In the new diagnosis mode, early HBV related HCC with serum PIVKA-II more than or equal to 40 mAUU/mL is diagnosed, AUROC for 115 HCC less than or equal to 5.0cm is 0.934 (figure 5A), sensitivity is 93.04%, and specificity is 80.10%; for 41 HCC cases<AUROC at 3.0cm was 0.920 (FIG. 5B), sensitivity was 92.68%, and specificity was 80.10%. AUROC in the new diagnosis mode is obviously higher than that of PIVKA-II, AFP and PIVKA-II combined AFP (HCC is less than or equal to 5.0cm:0.934vs 0.768,0.650 and 0.746；HCC<3.0cm 0.920vs 0.731,0.619, and 0.695) (P<0.001) (table 4).

TABLE 4 PIVKA-II, diagnostic value of AFP and PIVKA-II in combination with AFP for early HBV-related HCC

2.6 diagnostic value of HBV-associated HCC in early stage for AFP abnormality diagnosis in New diagnostic mode

The new diagnosis mode diagnoses the early HBV-related HCC with serum AFP more than or equal to 20ng/mL by using 408 CHB with serum AFP more than or equal to 20ng/mL as a control population, and the diagnosis threshold is set to be 173.28 multiplied by 10^-3Then, AUROC of 89 cases of HCC is less than or equal to 5.0cm is 0.960 (FIG. 6A), sensitivity is 83.15%, and specificity is 95.34%; the diagnostic threshold was set at 54.87X 10^-3For 40 HCC cases<The AUROC at 3.0cm was 0.933 (FIG. 6B), the sensitivity was 77.50% and the specificity was 90.69%. AUROC in the new diagnosis mode is obviously higher than that of PIVKA-II, AFP and PIVKA-II combined AFP (HCC is less than or equal to 5.0cm:0.960vs 0.837,0.701 and 0.841; HCC<3.0cm:0.933vs0.719,0.644 and 0.752) (P<0.001) (table 5).

TABLE 5 PIVKA-II, diagnostic value of AFP and PIVKA-II in combination with AFP for early HBV-related HCC

2.7 diagnostic value of the novel diagnostic model for the diagnosis of early HBV-associated HCC with abnormalities of PIVKA-II and/or AFP

500 cases of CHB with serum PIVKA-II more than or equal to 40mAU/mL and/or AFP more than or equal to 20ng/mL are taken as control population, the new diagnosis mode diagnoses the early HBV-related HCC with serum PIVKA-II more than or equal to 40mAU/mL and/or AFP more than or equal to 20ng/mL, the diagnosis threshold is set to be 76.88 multiplied by 10^-3In this case, AUROC of 0.942 for 134 cases of HCC ≤ 5.0cm (FIG. 7A), sensitivity of 89.55%, and specificity of 85.00%; for 56 cases of HCC<AUROC at 3.0cm was 0.918 (FIG. 7B), sensitivity was 89.29%, and specificity was 81.40%. AUROC in the optimal diagnosis mode is obviously higher than PIVKA-II, and AU of AFP and PIVKA-II combined with AFPROC (HCC ≦ 5.0cm:0.942vs 0.822,0.547 and 0.805; HCC<3.0cm:0.918vs 0.725,0.543 and 0.730) (P<0.001) (table 6).

TABLE 6 PIVKA-II, diagnostic value of AFP and PIVKA-II in combination with AFP for early HBV-related HCC

Discussion of 3

The invention compares the serum PIVKA-II, AFP, AST, ALT and T-Bil levels of early HBV related HCC and CHB control population, and utilizes the ROC curve to evaluate the diagnostic value of the indexes for diagnosing early HBV related HCC by using different diagnostic modes established alone or in combination. The analysis of test data shows that the clinical common tumor markers PIVKA-II and AFP are combined with AST, ALT and T-Bil related to liver metabolism, and the diagnosis value of the combined tumor markers PIVKA-II and AFP for diagnosing the HCC related to the early HBV is superior to that of the independently used tumor markers. And the index [1.5 multiplied PIVKA-II/(AST multiplied by T-Bil) + AFP/(ALT multiplied by T-Bil) ] can obtain the highest diagnostic value when diagnosing early HBV-related HCC, the diagnostic sensitivity exceeds 80 percent, and the diagnostic sensitivity is higher than that of other diagnostic modes, so that the probability of HCC being discovered in the early stage is improved to a greater extent, the 5-year survival rate of a tested population is improved, and particularly, the diagnostic efficiency of a new diagnostic mode is further improved under the condition that PIVKA-II and AFP are abnormal. Therefore, the [1.5 XPIVKA-II/(AST × T-Bil) + AFP/(ALT × T-Bil) ] index can be used as a new diagnostic model for early HCC.

According to the definition of early HCC, the data of early HBV-related HCC with the tumor size less than or equal to 5.0cm and less than 3.0cm are analyzed simultaneously. PIVKA-II and AFP are elevated in part of early HCC and benign liver disease, but the elevation degree or proportion in benign liver disease is obviously lower than that in early HCC (Table 1), which should be an important reason that PIVKA-II and AFP can be used as tumor markers of HCC. The diagnosis sensitivity of early HBV related HCC with PIVKA-II diagnosis tumor size less than or equal to 5.0cm and less than 3.0cm is about 70%, the specificity is about 80%, and meanwhile, the diagnosis method also shows that a certain proportion of missed detection and misdiagnosis of benign liver diseases exist in the clinical routine tumor marker and MRI when the early HCC is diagnosed. The sensitivity and specificity of AFP for diagnosing HBV-related HCC in early stage are about 40% and 80%, and the diagnostic value is obviously lower than that of PIVKA-II.

The data of the invention show that the serum levels of AST, ALT and T-Bil are all obviously lower than those of CHB population in early HCC, therefore, the ratio of the serum level with obviously increased index to the serum level with obviously decreased index in early HCC is used as the principle for establishing different diagnosis modes, AUROC for diagnosing early HBV-related HCC in different diagnosis modes is compared, and the optimal diagnosis mode is obtained. The results show that the diagnosis value of the ratio between the serum level in the early HCC and the serum level with obviously reduced liver function index for diagnosing the early HCC is superior to the diagnosis value of PIVKA-II and AFP when the PIVKA-II and the AFP are singly and jointly used (Table 2), and the difference between the HCC related to the early HBV and the benign liver disease can be effectively increased by calculating the ratio, thereby achieving the purposes of diagnosis and differential diagnosis. In addition, the [1.5 times PIVKA-II/(AST times T-Bil) + AFP/(ALT times T-Bil) ] index obtains the optimal diagnostic value when diagnosing early HBV-related HCC with the tumor size less than or equal to 5.0cm and less than 3.0cm, and shows that the [1.5 times PIVKA-II/(AST times T-Bil) + AFP/(ALT times T-Bil) ] index can be used as a new diagnostic model for diagnosing the early HBV-related HCC. In the cases of early HBV-related HCC, the serum levels of PIVKA-II and AFP in diagnosis negative cases are not different from those of CHB control population, and are all lower than those in diagnosis positive cases, while the serum levels of AST, ALT and T-Bil are all higher than those in diagnosis positive cases, which is an important reason for the failure of differential diagnosis between negative cases and CHB cases. Interestingly, the tumor size in the negative cases was significantly smaller than in the positive cases, which may be responsible for the difference between PIVKA-II and AFP, and the negative cases could probably be diagnosed by short follow-up. The tumor size of a diagnosis negative case with the tumor size less than or equal to 5.0cm is 2.70(1.43-3.85) cm, the tumor size of a diagnosis negative case with the tumor size less than 3.0cm is 1.90(1.10-2.50) cm, the tumor size of a case with a certain proportion exceeds 2.0cm, meanwhile, the effect of a new diagnosis mode in the differential diagnosis of HCC and bile duct disease population, hepatic cyst and hepatic hemangioma cases is lower, and the sensitivity of the CT and MRI in the diagnosis of HCC at the early stage of HCC of more than 2.0cm is better. This means that the combination of the new diagnostic model and MRI for the diagnosis of HCC associated with early HBV will further improve the sensitivity and specificity of the diagnosis of HCC associated with early HBV, and improve the diagnostic efficacy of HCC cases.

PIVKA-II ≥ 40mAU/mL and AFP ≥ 20ng/mL are frequently used as cutoff values for the diagnosis of HCC. Generally, the abnormal results of PIVKA-II and AFP detection will not bring sufficient attention to clinicians and the examined population until no clinical symptoms appear, and the early HCC cases with abnormal results of PIVKA-II and/or AFP detection account for about 90% of all early HCC cases (Table 1), so we evaluate the diagnostic value of the new diagnostic model for early HCC when PIVKA-II and AFP are abnormal. In the early HCC case with abnormal PIVKA-II and/or AFP serum level, the diagnosis efficiency of PIVKA-II and AFP is reduced to a certain extent, particularly when the early HCC with the tumor size of less than 3.0cm is diagnosed, the diagnosis efficiency of AFP is reduced most obviously, the result shows that the PIVKA-II and AFP which are used conventionally in clinic have great difficulty in identifying benign liver diseases and malignant tumors, particularly in the early HCC occurrence stage, the increase degree of PIVKA-II and AFP is far less than the increase degree of HCC in the middle and late stages, and the identification and diagnosis difficulty is also greater. However, the efficiency of the new diagnosis mode in diagnosing the early HCC cases with abnormal PIVKA-II and/or AFP serum level is obviously improved compared with the efficiency in diagnosing all early HCC, particularly in the cases with AFP more than or equal to 20ng/mL, the new diagnosis mode has the diagnosis sensitivity of 83.15 percent and the specificity of 95.34 percent in diagnosing the early HCC with the tumor size less than or equal to 5.0cm, and the diagnosis sensitivity of 77.50 percent and the specificity of 90.69 percent in diagnosing the early HCC with the tumor size less than 3.0cm, and the data result shows that the new diagnosis mode is a diagnosis mode which is very worthy of popularization on one hand. On the other hand, also of great concern, a small proportion of cases with normal PIVKA-II and AFP detection should be given more attention, and it is believed that for people with CHB infection, follow-up of PIVKA-II and AFP should be enhanced, while higher sensitivity imaging detection techniques, such as CT or MRI, should be performed periodically to enable more early diagnosis and treatment of HCC, and to improve the survival rate, prognosis and quality of life of HCC patients.

In conclusion, the new diagnosis mode is a diagnosis mode suitable for diagnosing HCC related to HBV in early stage, and the diagnosis value is obviously superior to that when PIVKA-II and AFP are used independently and jointly. For a small part of diagnosis negative cases, follow-up and combined MRI can be adopted to further improve the diagnosis efficiency.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A biomarker for diagnosing early hepatocellular carcinoma, comprising: abnormal prothrombin, alpha-fetoprotein, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase and total bilirubin.

2. The biomarker for diagnosing early hepatocellular carcinoma as claimed in claim 1, wherein the biomarker is derived from serum.

3. The use of the biomarker for diagnosing early hepatocellular carcinoma of claims 1 or 2 in the preparation of a medicament for diagnosing early hepatocellular carcinoma.

4. A kit for diagnosing early hepatocellular carcinoma, comprising the biomarker for diagnosing early hepatocellular carcinoma of claim 1 or 2.

5. The method for constructing the diagnosis mode for diagnosing the early hepatocellular carcinoma by adopting the biomarkers 1 or 2 is characterized by collecting serum, detecting the concentrations of abnormal prothrombin, alpha-fetoprotein, glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase and total bilirubin in the serum, establishing different diagnosis modes according to the concentration difference of the biomarkers in the serum of patients with the early hepatocellular carcinoma and benign liver diseases, and then carrying out ROC curve analysis by using statistical software to construct the diagnosis mode for diagnosing the early hepatocellular carcinoma.

6. The method of construction according to claim 5, wherein the different diagnostic modes are established by a ratio of concentrations of the biomarkers of increased and decreased concentration.

7. The method of claim 5 or 6, wherein the ROC curve is used to analyze the area under the curve of different diagnostic modes, and the diagnostic mode with the largest area under the curve is determined as the diagnostic mode for diagnosing early hepatocellular carcinoma.

8. The method of claim 7, wherein the diagnostic pattern is a [1.5 x PIVKA-II/(AST x T-Bil) + AFP/(ALT x T-Bil) ] index.