The early prediction values of diagnostic markers for hepatocellular carcinoma (HCC) are still unclear at present. This study evaluated the prediction value of ten serum markers in HCC. A total of 109 cases of hepatic cirrhosis patients were followed up for 36 months and the relationship between the lifetime risk of developing HCC and levels of serum markers was analyzed. 31.2 (34/109) percent of hepatic cirrhosis patients developed HCC during the study’s timeframe. Higher alpha-fetoprotein (AFP), alpha-fetoprotein-L3 (AFP-L3), alanine aminotransferase (ALT), and AFP-L3/AFP ratio levels are potential risk factors for malignization in hepatic cirrhosis patients (
Hepatocellular carcinoma (HCC) is the fifth leading cause of cancer death worldwide and about 500,000 people die of it each year [
All study subjects were enrolled at 302 military hospital, Beijing, China, and were followed up during the study period of 36 months, until confirming HCC diagnosis or the date of study end (December 31, 2016). The study population included any hepatic cirrhosis patients over 30 years old who were identified as HBV or HCV infected patients for at least 5 years. Some patients with the following conditions were excluded: patients who were diagnosed with HCC at starting point of this study; patients with other systemic disease such as diabetes and hypertension; patients after surgery, interventional therapy, radiotherapy, chemotherapy, and other invasive treatment; patients suffering from severe complications such as upper gastrointestinal bleeding and hepatic encephalopathy. The final diagnosis was made by liver histopathology or MRI based on guidelines from Ministry of Health of the People’s Republic of China [
A total of ten routine laboratory tests were chosen to be analyzed; they were albumin (ALB), total bilirubin (TBil), alanine transaminase (ALT), platelet count (PLT), prothrombin time (Pt(s)), prothrombin time activity (Pt(a)), AFP, GP73, AFP-L3, and AFP-L3/AFP ratio (L3/AFP). Clinical chemistry tests were applied by an automatic biochemical analyzer (AU5400, Olympus, Japan). PLT was detected using Hematology Analyzer (XE-1800, SYSMEX, Japan). Pt(s) and Pt(a) were measured in automated coagulation instrument (CA-7000, SYSMEX, Japan). AFP and AFP-L3 were measured by Automated Immunoassay Analyzer (COBAS6000, ROCHE, Switzerland). Kits for the enzyme-linked immunosorbent assay for GP73 were obtained from Hotgen Biotech (Beijing, China).
The incidences of HCC during the study period were analyzed by examination of medical records. Ten markers at starting point of this study were compared between patients with abnormal serum biomarkers levels, denoted as the positive groups, and those with normal levels, denoted as negative groups. The judgment criteria are as follows: ALB < 35 g/l, TBIL > 19
A total of 161 cases were diagnosed as hepatic cirrhosis during the study period. Fifty-two cases were excluded (35 were excluded due to history of other systemic diseases, 3 participants were excluded due to excessive missing data, and 14 patients with confirmed severe complications were excluded). Therefore, a total of 109 patients met the inclusion criteria and were analyzed. All participants had a mean age of 53.9 (
The demographic data of the patients.
HCC-group | Non-HCC group |
|
|
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Gender | |||
Male | 22 | 44 | 0.550 |
Female | 12 | 31 | |
Age (years) | 56 (35–83) | 52 (37–74) | 0.118 |
We compared the serum markers levels at starting point between patients who had developed HCC and those who had not developed HCC; the results demonstrated that there were 4 markers, including AFP, AFP-L3, ALT, and AFP-L3/AFP ratio, that were statistically significant (
The statistical analysis of the levels of serum markers between HCC group and non-HCC group.
Markers | HCC group | Non-HCC group |
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Statistical description |
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Statistical description | |||
ALB (g/L) | 34 |
|
75 |
|
0.897 | 0.542 |
TBIL ( |
34 | 24.85 (4.8–385.6) | 75 | 24.1 (6.5–109) | −0.618 | 0.536 |
ALT (IU/L) | 34 | 52 (20–254) | 75 | 35 (9–416) | −3.212 | 0.001 |
PLT (109/L) | 34 | 70.5 (29–165) | 75 | 62 (23–599) | −0.981 | 0.326 |
Pt(s) (s) | 34 | 13.5 (10.5–21.6) | 75 | 14.00 (9.7–21.4) | −1.237 | 0.216 |
Pt(a) (%) | 34 |
|
75 |
|
1.313 | 0.507 |
AFP (ng/mL) | 34 | 30.88 (1.41–1432) | 75 | 5.62 (0.9–617.4) | −3.944 | 0.000 |
GP73 (ng/mL) | 34 | 212.6 (66.12–350) | 75 | 202.8 (65.38–391.9) | −0.654 | 0.513 |
AFP-L3 (ng/mL) | 34 | 2.09 (0.07–77.45) | 75 | 0.273 (0.05–34.4) | −4.068 | 0.000 |
AFP-L3/AFP ratio | 34 | 0.051 (0.03–0.43) | 75 | 0.05 (0.02–0.12) | −2.019 | 0.043 |
Normally distributed data were reported as
The difference of 10 biomarkers levels between patients who had developed HCC within 3 years and those who had not developed HCC.
The risk factors analysis showed that incidence rate of HCC in patients with high AFP, AFP-L3, ALT, and AFP-L3/AFP levels were extremely significantly higher than that those with normal levels (
Analysis of risk factors for the development of HCC based on the levels of serum tumor markers.
Markers | HCC ( |
Non-HCC ( |
Cumulative incidence (CI) | Relative Risk (RR) | Attributable Risk (AR) | Attributable Risk Percent (ARP) | Chi-Square |
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ALB (g/l) | Positive groups | 23 | 54 | 29.87% | 1.06 | 1.75% | 5.84% | 0.033 | 0.855 |
Negative groups | 9 | 23 | 28.13% | ||||||
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TBIL ( |
Positive groups | 21 | 44 | 32.31% | 1.09 | 2.76% | 8.55% | 0.093 | 0.760 |
Negative groups | 13 | 31 | 29.55% | ||||||
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ALT (U/L) | Positive groups | 24 | 30 | 44.44% | 2.72 | 28.08% | 63.18% | 10.178 | 0.001 |
Negative groups | 9 | 46 | 16.36% | ||||||
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PLT (109/L) | Positive groups | 27 | 59 | 31.40% | 1.20 | 5.31% | 16.91% | 0.242 | 0.623 |
Negative groups | 6 | 17 | 26.09% | ||||||
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PT(s) (s) | Positive groups | 20 | 49 | 28.99% | 0.97 | −1.01% | −3.50% | 0.013 | 0.911 |
Negative groups | 12 | 28 | 30.00% | ||||||
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PT(a) (%) | Positive groups | 17 | 52 | 28.81% | 0.77 | −8.69% | −30.15% | 2.020 | 0.155 |
Negative groups | 15 | 25 | 37.50% | ||||||
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AFP (ng/mL) | Positive groups | 24 | 25 | 48.98% | 2.99 | 32.59% | 66.53% | 13.511 | 0.000 |
Negative groups | 10 | 51 | 16.39% | ||||||
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GP73 (ng/mL) | Positive groups | 27 | 64 | 29.67% | 0.89 | −3.66% | −12.35% | 0.096 | 0.757 |
Negative groups | 6 | 12 | 33.33% | ||||||
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AFP-L3 (ng/mL) | Positive groups | 22 | 20 | 52.38% | 2.92 | 34.47% | 65.81% | 14.292 | 0.000 |
Negative groups | 12 | 55 | 17.91% | ||||||
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L3/AFP ratio | Positive groups | 18 | 19 | 48.65% | 2.34 | 27.82% | 57.18% | 8.958 | 0.003 |
Negative groups | 15 | 57 | 20.83% |
HCC: patients who had developed HCC during the study period; non-HCC: patients who had not developed HCC during the study period;
ROC analysis was used to determine whether serum markers are powerful to predict HCC in the cirrhotic population. The results showed that AFP, AFP-L3, and ALT had relatively good predictive power for HCC progression; AUC were 0.736, 0.744, and 0.693, respectively (see Table
The predictive value of all markers and combination of 3 markers for HCC in cirrhotic patients.
Markers | Area under ROC curve | Standard error |
|
95% confidence interval | Cut-off | Sensitivity | Specificity |
---|---|---|---|---|---|---|---|
ALB (g/l) | 0.537 | 0.059 | 0.534 | 0.423–0.652 | 28.5 | 0.765 | 0.373 |
TBIL ( |
0.537 | 0.058 | 0.537 | 0.423–0.651 | 15.15 | 0.912 | 0.293 |
ALT (U/L) | 0.693 | 0.056 | 0.001 | 0.582–0.803 | 44.5 | 0.676 | 0.680 |
PLT (109/L) | 0.559 | 0.057 | 0.327 | 0.447–0.670 | 45.5 | 0.882 | 0.720 |
PT(s) (s) | 0.426 | 0.058 | 0.216 | 0.311–0.540 | 21.5 | 0.029 | 1.000 |
PT(a) (%) | 0.591 | 0.058 | 0.128 | 0.477–0.705 | 75 | 0.529 | 0.693 |
AFP (ng/mL) | 0.736 | 0.052 | 0.000 | 0.634–0.839 | 10.28 | 0.676 | 0.693 |
GP73 (ng/mL) | 0.539 | 0.061 | 0.513 | 0.419–0.659 | 221.85 | 0.471 | 0.693 |
AFP-L3 (ng/mL) | 0.744 | 0.052 | 0.000 | 0.642–0.846 | 0.514 | 0.676 | 0.693 |
L3/AFP ratio | 0.606 | 0.065 | 0.077 | 0.478–0.734 | 0.052 | 0.500 | 0.760 |
ALT + AFP + AFP-L3 | 0.770 | 0.050 | 0.000 | 0.672–0.868 | 0.23 | 0.824 | 0.667 |
ALT + AFP | 0.780 | 0.049 | 0.000 | 0.683–0.877 | 0.24 | 0.824 | 0.693 |
ALT + AFP-L3 | 0.773 | 0.050 | 0.000 | 0.676–0.871 | 0.23 | 0.824 | 0.680 |
AFP + AFP-L3 | 0.740 | 0.052 | 0.000 | 0.638–0.842 | 0.23 | 0.676 | 0.693 |
The ROC curves of 10 biomarkers for prediction of HCC in the cirrhotic patients.
The multiple regression analysis with ALT, AFP, and AFP-L3 for predicting performance.
Among the 34 HCC cases, 17 were excluded due to incomplete data or nonavailable data. We analyzed the dynamic change of the ten markers in other 17 cases during the progression of HCC, and we found that serum GP73 level was significantly decreased (
The dynamic change of serum markers concentrations before and after HCC occurred.
Markers | Starting point (hepatic cirrhosis) | End point (HCC) |
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Statistical description |
|
Statistical description | |||
ALB (g/l) | 17 | 32 (25–43) | 17 | 33 (26–42) | −0.166 | 0.868 |
TBIL ( |
17 | 21 (4.8–49.5) | 17 | 19.6 (9.3–39.2) | −1.154 | 0.248 |
ALT (U/L) | 17 | 58 (20–253) | 17 | 28.5 (16–262) | −1.895 | 0.058 |
PLT (109/L) | 17 | 79 (29–165) | 17 | 61.5 (34–225) | −0.762 | 0.446 |
PT(s) (s) | 17 | 13.2 (11–15) | 17 | 13 (11.3–15.7) | −0.719 | 0.472 |
PT(a) (%) | 17 | 75.3 (64.1–98.7) | 17 | 75.1 (61.7–100.6) | −1.894 | 0.058 |
AFP (ng/mL) | 17 | 74.6 (2.34–469.9) | 17 | 136.2 (2.96–1501) | −0.152 | 0.879 |
GP73 (ng/mL) | 17 | 194.6 (66.12–350) | 17 | 154.2 (13.14–275.4) | 2.212 | 0.041 |
AFP-L3 (ng/mL) | 17 | 8.8 (0.12–77.45) | 17 | 28.8 (0.15–361.5) | −0.544 | 0.586 |
AFP-L3/AFP ratio | 17 | 0.09 (0.03–0.43) | 17 | 0.10 (0.05–0.50) | −0.848 | 0.408 |
Starting point: start time of this study; end point: the time the patients were diagnosed HCC;
The plotting diagram of 10 biomarkers levels of patients who had developed HCC in starting point (hepatic cirrhosis) and end point (HCC).
In the past few decades, many promising candidate biomarkers for HCC had been found, but most of them were not applied to clinical diagnosis due to their limited practicability and high cost [
GP73 is a resident Golgi-specific membrane protein expressed by biliary epithelial cells in normal liver. A meta-analysis reported that GP73 is a valuable serum marker that seems to be superior to AFP and can be useful in the diagnosis and screening of HCC [
Although this study is limited by the small sample size and short study duration, our data suggest that higher serum levels of AFP, AFP-L3, AFP-L3/AFP ratio, and ALT were risk factors associated with the development of HCC and the detection of GP73 has a certain guiding significance to predict the risk of HCC in hepatic cirrhosis patients; regular monitoring of these serum markers in hepatic cirrhosis patients is necessary.
The authors declare that there are no conflicts of interest.
Bo Li and Boan Li contributed equally to this study.
This work was supported by the Capital Medical Development Project of China (2014-2-5031).