4.1. Patient Characteristics
Of all the 134 patients diagnosed as HCC (mean age of 49.6 ± 11.9 (SD) years), a significant number of them (95.5%) were originally diagnosed and the remainders were relapsed after RFA. The male-to-female ratio was 8.6 (120 males and 14 females), with 21.6% of patients (n = 29) under 40 years old. In our series, most patients (90.6%) were developed from chronic hepatitis B. 85.8% (n = 115) of them had cirrhosis, with 95 patients were at uncompensatory stage. As for the controlled groups, patients with cirrhosis (n = 100), patients with severe hepatitis B (n = 94), patients with chronic hepatitis B (n = 103) and elders with positive HBeAg (n = 50) were randomly selected. Apart from these patients with hepatitis B diseases, non-HCC cancers were also selected as controls, including patients with lung carcinoma (n = 50), colorectal cancer (n = 25), prostate cancer (n = 12), esophagus cancer (n = 12) and gastric cancer (n = 6).
The size of the tumors in HCC patients varied a lot, but most were larger than 3 cm (73.9%) and over a half of them were present with huge size (> 10 cm, 53.3%). Of all the tumors, multiple genesis was in dominance (63.4%) and over a half of them presented in both lobes (54.5%). The basic characteristics of these patients are listed in Table 1.
Table 1.
Characteristics of Patients Enrolled in the Study a,b
Variable | HCC (n = 134) | Liver Diseases (n = 347) | Other Cancers (n = 105) | Healthy Controls (n = 53) |
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Age, y | 49.6 ± 11.9 | 46.9 ± 9.8 | 57.7 ± 11.2 | 48.2 ± 11.8 |
Gender | | | | |
Male | 89.6 | 70.1 | 78.1 | 58.5 |
Female | 10.4 | 29.9 | 21.9 | 51.5 |
Tumor number | | NA* | NA | NA |
Single | 36.6 | | | |
Multiple (≥ 2) | 63.4 | | | |
Tumor size (n = 92) | | NA | NA | NA |
< 3 cm | 24 (26.1) | | | |
3 - 6 cm | 12 (13.0) | | | |
6 - 10 cm | 7 (7.6) | | | |
>10 cm | 49 (53.3) | | | |
Localization | | NA | NA | NA |
Right lobe | 36.5 | | | |
Left lobe | 9.0 | | | |
Both lobes | 54.5 | | | |
Etiology | | NA | NA | NA |
HBV | 90.6 | | | |
HCV | 2.4 | | | |
Alcohol | 1.5 | | | |
Others | 5.5 | | | |
Portal vein invasion | 52 (38.8) | NA | NA | NA |
a Abbreviations: HBV, hepatitis B virus; HCC, hepatocellular carcinoma; and HCV, hepatitis C virus.
b Values are presented as mean ± SD, % or No. (%).
4.2. PIVKA-II Levels Elevated in HCC Patients
Serum levels of biomarkers in HCC patients and controlled groups are shown in Figure 1. PIVKA-II levels were significantly elevated compared with controlled groups. The median PIVKA-II levels in HCC, liver related diseases, non-HCC cancers and healthy people were 1012.0 mAU/mL (range: 1.0 - 75000.0 mAU/mL), 29.0 mAU/mL (range: 7.0 - 75000.0 mAU/mL), 38.0 mAU/mL (range: 14.0 - 291.0 mAU/mL) and 26.0 mAU/mL (range: 10.0 - 44.0 mAU/mL), respectively. P value was below 0.0001 comparing HCC with any of the other groups (by Kruskal-Wallis test). In all the controlled groups, serum levels of PIVKA-II in more than 95% of patients were below 200 mAU/mL, while in the HCC group, the serum level had a wide range with the mean level over 200 mAU/mL.
Figure 1.
Levels of AFP and PIVKA-II in Different Types of Liver Diseases
PIVKA-II was measured in mAU/mL and AFP in ng/mL. Five-fold the cut-off value are shown in the figures. PIVKA-II could easily distinguish HCC from severe hepatitis, but AFP could not.
In this study, 40 mAU/mL was set as the cut-off value according to the manufacturer’s instructions based on previous studies. Under this level, 86.6% of patients with HCC were tested positive using the combined biomarkers, while for AFP alone, the positive ratio was 76.9%. (Figure 2) Compared with other non-HCC cancers, HCC groups showed a significant difference by Chi-square test (Pearson χ2 = 19.604, P < 0.001) for PIVKA-II. The same result also occurred in comparison between HCC group and non-HCC liver diseases. We noticed a relatively high positive ratio in severe hepatitis group both with AFP and PIVKA-II as biomarkers. However, Chi-square test found no difference between HCC group and severe hepatitis group with AFP (Pearson χ2 = 0.520, P = 0.471), but a significant difference with PIVKA-II (Pearson χ2 = 8.330, P = 0.004) (Figure 1 B).
Figure 2.
Positive Rate for HCC Patients by AFP and PIVKA-II
The detection rate increased by 9.7% if AFP and PIVKA-II were combined compared to AFP alone.
4.3. Comparisons Between AFP and PIVKA-II Levels in HCC
To figure out the association between AFP and PIVKA-II concentrations, the levels of PIVKA-II and AFP in HCC patients were shown in Figure 3 A after normalization using Spearman test (rs = 0.568, P < 0.001). With the cut-off value of 40 mAU/mL for PIVKA-II and 20 ng/mL for AFP, Chi-square test showed a significant difference between AFP and PIVKA-II (χ2 = 21.167, P < 0.001; Kappa = 0.397, P < 0.001). Compared with patients with hepatitis B diseases, the sensitivity, specificity, positive and negative prediction ratios and Kappa value were 74.6% (95% CI: 0.662 - 0.816), 68.6% (95% CI: 0.634 - 0.734), 47.8% (95% CI: 0.409 - 0.548), 87.5% (95% CI: 0.828 - 0.911), 0.369 (95% CI: 0.289 - 0.449) (P < 0.001) for PIVKA-II and 76.7% (95% CI: 0.684 - 0.834), 65.7% (95% CI: 0.604 - 0.706), 46.2% (95% CI: 0.395 - 0.530), 88.0% (95% CI: 0.833 - 0.916) and 0.352 (95% CI: 0.274 - 0.430) (P < 0.001) for AFP, respectively, while for the combination of the two markers, were 86.6% (95% CI: 0.793-0.916), 53.0% (95% CI: 0.476 - 0.584), 41.6% (95% CI: 0.358 - 0.476), 91.1% (95% CI: 0.861 - 0.945) and 0.297 (95% CI: 0.228 - 0.366) (P < 0.001), respectively (Table 2).
Figure 3.
Scatter Plot for AFP and PIVKA-II. PIVKA-II was Measured in mAU/mL and AFP in ng/mL
A, Scatter plot for all HCC patients; Level of PIVKA-II was independent from level of AFP; B, Scatter plot for all patients with HCC (red) and cirrhosis (blue).
Table 2.
Quantities of Patients of HCC and HBV-Related Diseases Detected by Different Biomarkers in Different Cut-off Values a,b,c
| HCC | HBV-Related Diseases b |
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PIVKA-II (40) c | | |
Positive | 100 | 109 |
Negative | 34 | 238 |
AFP (20) | | |
Positive | 102 | 119 |
Negative | 31 | 228 |
PIVKA-II+AFP | | |
Positive | 116 | 163 |
Negative | 18 | 184 |
PIVKA-II (200) | | |
Positive | 86 | 32 |
Negative | 48 | 315 |
AFP (195.2) | | |
Positive | 81 | 36 |
Negative | 53 | 311 |
PIVKA-II+AFP | | |
Positive | 98 | 58 |
Negative | 36 | 289 |
a Abbreviations: AFP, Alpha-fetoprotein; and PIVKA-II, Protein Induced by Vitamin K Absence or Antagonist-II.
b Patients with HBV-related diseases include patients with chronic HBV infection, severe hepatitis B, HBV-related cirrhosis and old carriers with HBeAg positive.
c PIVKA-II is measured in mAU/mL and AFP in ng/mL.
Different sizes of tumors showed significant difference in the sera concentrations of both AFP and PIVKA-II (Kruskal-Wallis χ2 = 52.473, P < 0.001) and the level elevated with the increase of size, but for the positive ratio of the same size between the two markers, PIVKA-II had no advantages (< 3 cm: 29.2% versus 62.5%; χ2 = 5.371, P = 0.02). The same phenomena also occurred for the number of tumors and different ages. Patients with HCC and cirrhosis displayed higher levels of both AFP and PIVKA-II than those with only cirrhosis (median AFP: 623.25 ng/mL versus 7.78 ng/mL, P < 0.001 by Mann–Whitney test; median PIVKA-II: 1580 mAU/mL versus 36 mAU/mL, P < 0.001 by Mann-Whitney test) (Figures 1 and 3 B). However, AFP levels in non-cirrhotic HCC patients showed no difference with patients with cirrhosis (Mann-Whitney U = 274.00, P = 0.338), while the level of PIVKA-II in non-cirrhotic HCC patients increased significantly (Mann-Whitney U = 184.00, P = 0.036). There was a trend toward higher levels of both AFP and PIVKA-II in HCC patients with portal vein invasion compared to non-invasion controls (median AFP: 1080.08 ng/mL versus 111.24 ng/mL, P < 0.001 by Mann-Whitney test; median PIVKA-II: 5407.5 mAU/mL versus 41.0 mAU/ml, P < 0.001 by Mann-Whitney test), but there was no difference in PIVKA-II between cirrhosis and non-invasion HCC and a significantly higher level of AFP in non-invasion HCC than cirrhosis.
Also in our study, the positive ratio had no difference either using AFP or PIVKA-II as a screening biomarker, when we compared them in the same subgroup as follows: the same tumor number, the same age group, HCC with or without portal vein invasion group and HCC with or without cirrhosis.
4.4. ROC Curve
In our study, we depicted ROC curve to evaluate the diagnostic performance of PIVKA-II or the combination of AFP and PIVKA-II as biomarkers (Figure 4). For the combination procedure, two biomarkers were used as predictors and got every predicting possibility as the combining biomarker value using Logistic regression in SPSS binary logistic session. And then, ROC session in SPSS was applied to depict ROC curve. Finally, our curve showed that the area under the ROC curve (AUROC) for PIVKA-II (0.760, 95% confidence interval, CI: 0.699 - 0.820) was less than the AUROC for AFP (0.826, 95% CI: 0.784 - 0.869) in all HCC patients, but in combination, the AUROC could increase to 0.846 (95% CI: 0.804 - 0.888). Moreover, the best cut-off values indicated by our ROC curve were 195.2 ng/mL (Youden index: 0.501) for AFP and 200.0 mAU/mL (Youden index: 0.550) for PIVKA-II, respectively. Furthermore, these values corresponded to a sensitivity and specificity for PIVKA-II of 64.2% (95% CI: 0.554 - 0.721) and 90.8% (95% CI: 0.871 - 0.935), for AFP of 60.4% (95% CI: 0.516 - 0.687) and 89.6% (95% CI: 0.858 - 0.925) and for combination of 73.1% (95% CI: 0.647 - 0.802) and 83.3% (95% CI: 0.788 - 0.870), respectively (Table 2). Different sensitivities and specificities for different cut-off values of both AFP and PIVKA-II were given in Table 3.
Figure 4.
ROC Curve Evaluating Patients With HCC (n = 134) and Liver Diseases (n = 347)
The area under the ROC curve was shown with its 95% confidence intervals. AFP and PIVKA-II combined showed better performance than alone. The curves showed that the optimal cut-off value for PIVKA-II was 200.0 mAU/mL and that for AFP was 195.2 ng/mL. At the level of recommended cut-off values (40 mAU/mL for PIVKA-II and 20 ng/mL for AFP), the combination of the two markers had a sensitivity, specificity and Youden index of 86.6%, 53.0% and 0.396, respectively. And at the level of our new cut-off values, they were 73.1%, 83.3% and 0.564, respectively.
Table 3.
Sensitivity, Specificity and Youden Index of PIVKA-II and AFP in HCC Cases From Controls at Different Times of Currently Used Cutoff Values a,b,c
Times of Cut-Off c | 1/2 | 1 | 2 | 2.5 | 5 | 10 | 25 |
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PIVKA-II | | | | | | | |
Sensitivity, % | 84.4 | 74.6 | 65.9 | 64.4 | 64.2 | 57.0 | 49.6 |
Specificity, % | 21.4 | 68.6 | 84.0 | 85.2 | 90.8 | 92.9 | 94.9 |
Youden index | 0.058 | 0.432 | 0.499 | 0.496 | 0.550 | 0.499 | 0.445 |
AFP | | | | | | | |
Sensitivity, % | 80.7 | 76.1 | 72.6 | 67.4 | 66.7 | 60.0 | 48.9 |
Specificity, % | 61.0 | 65.7 | 70.9 | 79.5 | 81.8 | 88.9 | 94.3 |
Youden index | 0.417 | 0.418 | 0.435 | 0.469 | 0.485 | 0.489 | 0.432 |
a Abbreviations: AFP, Alpha-fetoprotein; and PIVKA-II, Protein Induced by Vitamin K Absence or Antagonist-II.
b The cut-off value for PIVKA-II is 40 mAU/mL and for AFP is 20 ng/mL.
c PIVKA-II is measured in mAU/mL and AFP in ng/mL.
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