Patient Characteristics
Table 1 demonstrates the clinical and tumor characteristics of the 137 patients with HCC. The mean patient age was 53 ± 12 years (range, 21–74 years). The patients were 89.8% (123/137) male and 10.2% (14/137) female. Hepatitis B surface antigen (HBsAg) was positive in 83.2% (114/137), and six patients were positive for the hepatitis C virus (HCV). 77.4% (106/137) had liver cirrhosis and 29.9% (41/137) had an AFP level > 400 ng/ml. ICG 15R min (%) was 7.2 ± 4.0. The tumor diameter was 5.5± 3.9 cm, twenty-nine (21.2%) was multiple tumor. Portal vein tumor thrombosis was present in 20.4% (28/137) of the patients. Tumor stage was stratified by the Barcelona Clinic Liver Cancer (BCLC) staging system. Of these patients, stage 0+A was 56.9% (78/137). Eight patients (5.8%) had hepatic function of Child-Pugh score B and received short-term liver protective therapy before surgery; the remaining patients were at Child-Pugh score A.
Table 1 Clinical Characteristics of 137 patients
Clinical characteristics
|
No. of patients
|
Age years
|
Mean 53 ± 12
Median 55
|
Sex
|
|
Male
|
123
|
Female
|
14
|
ALT (U/ml)
|
37 ± 30
|
AST (U/ml)
|
30 ± 15
|
TBIL(μmol/l)
|
Mean 15.9± 9.2
Median 13.4
|
Child-Pugh score
|
|
A
|
129
|
B
|
8
|
HBsAg
|
|
Positive
|
114
|
Negative
|
23
|
Liver cirrhosis
|
|
No
|
31
|
Yes
|
106
|
ICG15 min(%)
|
Mean 7.2 ± 4.0
Median 6.6
|
AFP (ng/mL)
|
|
≤ 400
|
96
|
> 400
|
41
|
Tumor diameter(cm)
|
Mean 5.5 ± 3.9
Median 4.5
|
No. of tumor
|
|
Singe
|
108
|
Multiple
|
29
|
Portal vein tumor thrombosis
|
|
No
|
109
|
Yes
|
28
|
BCLC stage
|
|
0+A
|
78
|
B+C
|
59
|
ALT: alanine transaminase; AST: aspartate aminotransferase; TBiL: total bilirubin; HBsAg: hepatitis B surface antigen; ICG R15 min (%): indocyanine green 15 minutes retention rate; AFP: alpha fetoprotein; BCLC: Barcelona Clinic Liver Cancer staging system;
Correlation between MVI and clinicopathological features
Table 2 demonstrates the correlation between MVI and clinical and tumor characteristics of the 137 patients. Univariate and multivariate Regression analysis showed that AFP > 400ng/ml, tumor size, and preoperative CTC count are independent risk factors for the presence of MVI.
Table 2 Factors for microvascular invasion on univariate and multivariate analyses
Variables
|
|
Univariate Analysis
|
|
Multivariate Analysis
|
OR
|
HR (95% CI)
|
P
|
OR
|
HR (95% CI)
|
P
|
Age
|
1.009
|
0.981~1.038
|
0.545
|
|
NA
|
NA
|
Sex, male versus female
|
0.734
|
0.232~2.317
|
0.598
|
|
NA
|
NA
|
HBsAg, positive versus negative
|
1.173
|
0.469~2.930
|
0.733
|
|
NA
|
NA
|
Liver cirrhosis, yes versus no
|
1.738
|
0.747~4.044
|
0.200
|
|
NA
|
NA
|
Child-Pugh score, B versus A
|
1.389
|
0.333~5.800
|
0.652
|
|
NA
|
NA
|
ALT
|
1.016
|
0.993~1.039
|
0.169
|
|
NA
|
NA
|
AFP(ng/ml) ≤ 400 versus > 400
|
14.571
|
5.718~37.135
|
< 0.001
|
6.702
|
2.149~33.354
|
0.002
|
ICG15min(%)
|
0.988
|
0.907~1.076
|
0.781
|
|
NA
|
NA
|
tumor size
|
1.170
|
1.063~1.288
|
0.001
|
1.213
|
1.031~1.427
|
0.020
|
No. of tumors, multiple versus single
|
6.286
|
2.456~16.090
|
<0.001
|
2.059
|
0.470~9.010
|
0.338
|
Tumor encapsulation, yes versus no
|
0.773
|
0.383~1.562
|
0.473
|
|
NA
|
NA
|
Edmondson stage, III-IV versus I-II
|
1.133
|
0.572~2.244
|
0.032
|
1.484
|
0.571~4.259
|
0.463
|
Ki67 (%)
|
1.027
|
1.011~1.044
|
0.003
|
0.994
|
0.968~1.021
|
0.664
|
PVTT, yes versus no
|
7.435
|
2.771~19.950
|
<0.001
|
2.413
|
0.351~16.590
|
0.370
|
Satellite lesion, yes versus no
|
8.437
|
2.673~26.630
|
<0.001
|
2.169
|
0.340~13.826
|
0.413
|
BCLC stage, 0+A versus B+C
|
3.984
|
1.941~8.177
|
<0.001
|
0.683
|
0.163~2.866
|
0.602
|
Preoperative CTC5ml
|
1.935
|
1.500~2.488
|
<0.001
|
1.757
|
1.344~2.295
|
< 0.001
|
ALT: alanine transaminase; AFP: alpha fetoprotein; HBsAg: hepatitis B surface antigen; ICG R15 min (%): indocyanine green 15 minutes retention rate; PVTT: portal vein tumor thrombosis; BCLC: Barcelona Clinic Liver Cancer staging system; CTC: circulating tumor cell
The clinical value of CTCs in predicting the presence of MVI
Preoperative blood sample CTC counts for HCC and benign tumor patients are shown in Figure 1a. Eighteen patients with benign hepatic tumors had 0 CTC (P < 0.001). A comparison of CTC counts between MVI positive and MVI negative patients is shown in Figure 1b. The difference in the mean blood CTC5mL levels between the MVI positive group and the MVI negative group was statistically significant (6.8 ± 5.1 versus 2.9 ± 2.5, P < 0.001). AFP, tumor diameter, and preoperative CTC count were included to predict the presence of MVI. ROC curves of AFP, tumor diameter, preoperative CTC count, and multi-parameter combination were drawn (Figure 1c). The area under the curve (AUC) of the four ROC curves were 0.636, 0.604, 0.856, and 0.900 respectively. The results indicated that a tumor diameter cut-off value of 5 and a preoperative CTC cut-off value of 5 showed the most significant power to predict the presence of MVI. The sensitivity of AFP ≥ 400ng/ml, tumor diameter ≥ 5cm, preoperative CTC ≥ 5 and the multi-parameter combination was 44.8%, 50.0%, 91.4%, and 91.4% respectively; the specificity was 82.3%,70.9%,79.7%, and 79.7% respectively (Table 3). By comparing the ROC curve features and AUC, the results showed that compared with AFP and tumor diameter, the preoperative CTC count had the most significant power to predict the presence of MVI. The diagnostic power of multi-parameter combination is not superior to the single parameter of preoperative CTC.
Table 3 The parameters for predicting the presence of MVI
|
Sensitivity
|
Specificity
|
AUC
|
Cut-off value
|
AFP
|
44.8%
|
82.3%
|
0.636
|
400ng/ml
|
Tumor diameter
|
50.0%
|
70.9%
|
0.604
|
5cm
|
Preoperative CTC
|
91.4%
|
79.7%
|
0.856
|
5
|
Multi-parameter
|
91.4%
|
79.7%
|
0.900
|
-
|
AFP: alpha fetoprotein; CTC: circulating tumor cell
The Impact of the presence of MVI on the prognosis of HCC
Of the 137 patients, 59 were MVI positive. The MVI-positive group showed significantly shorter OS than the MVI-negative group (median survival 19.2 months versus not reached, P = 0.005) (Figure 1d).
Comparison the change of CTC count caused by surgery in the Subgroups
All patients were divided into three groups: no recurrence, early recurrence and non-early recurrence. The CTC counts at each time point for the three groups are shown in Figure 2a. Figure 2b showed that intraoperative CTC did not increase across the three groups compared to pre-surgical values (2.7 versus 3.1, P = 0.233; 4.9 versus 5.5, P = 0.169; 7.3 versus 7.5, P = 0.714). The number of CTC in the non-recurrent group and the non-early recurrent group decreased significantly one week after surgery compared with that in the intraoperative group (1.1 versus 3.1, P < 0.001; 1.2 versus 5.5, P < 0.001), although there was no statistical significance in the group with early recurrence (7.6 versus 7.5, P = 0.95). Mean CTC was defined as the average number of CTC for all time points. Figure 2c demonstrates that the mean CTC count of the three groups was statistically different (1.4 versus 4.0 versus 7.1, P <0.001). Using a mean CTC of 5 as the cut-off value, all patients were divided into two groups. The survival curve showed that the survival time of group CTC ≥ 5 was significantly shorter than that of group CTC count < 5 (14.7 month versus not reached, P < 0.001)(Figure 2d). The proportion of early recurrence for the CTC > 5 group was also higher than that of group CTC count < 5 (84.8% versus 3.3%, P < 0.001). When the mean value of CTC is greater than 5 and continues to be greater than 5 after surgery, it strongly indicates risk of early postoperative recurrence. Extrahepatic metastasis occurred in 12 of the 137 patients. We defined the difference between postoperative CTC 1 week and intraoperative CTC as △CTC. We found that the number of CTC in the non-extrahepatic metastasis group decreased 1 week after surgery, but the number of CTC in the extrahepatic metastasis group increased abnormally 1 week after surgery(-2.4 ± 2.5 versus 2.3 ± 2.8, P < 0.001).