Open Access
Open Peer Review

This article has Open Peer Review reports available.

How does Open Peer Review work?

Hepatitis B virus infection and active replication promote the formation of vascular invasion in hepatocellular carcinoma

  • Xubiao Wei1,
  • Nan Li1,
  • Shanshan Li1,
  • Jie Shi1,
  • Weixing Guo1,
  • Yaxin Zheng1 and
  • Shuqun Cheng1Email author
Contributed equally
BMC Cancer201717:304

https://doi.org/10.1186/s12885-017-3293-6

Received: 20 January 2017

Accepted: 24 April 2017

Published: 2 May 2017

Abstract

Background

Vascular invasion, including microvascular invasion (MVI) and portal vein tumor thrombus (PVTT), is associated with the postoperative recurrence of hepatocellular carcinoma (HCC). We aimed to investigate the potential impact of hepatitis B virus (HBV) activity on the development of vascular invasion.

Methods

Patients with HBV and tumor-related factors of HCC who had undergone hepatectomy were retrospectively enrolled and analyzed to identify the risk factors for developing vascular invasion.

Results

A total of 486 patients were included in this study. The overall proportion of patients with vascular invasion, including MVI and PVTT, was 60.3% (293/486). The incidence of MVI was 58.2% (283/486) whereas PVTT was 22.2% (108/486). Univariate analysis revealed that positive Hepatitis B virus surface Antigen (HBsAg) was significantly associated with the presence of vascular invasion. In a multivariate regression analysis carried out in patients with HBV-related HCC, positive Hepatitis B virus e Antigen (HBeAg)(OR = 1.83, P = 0.019) and a detectable seral HBV DNA load (OR = 1.68, P = 0.027) were independent risk factors of vascular invasion. The patients in the severe MVI group had a significantly higher rate of positive seral HBsAg (P = 0.005), positive seral HBeAg (P = 0.016), a detectable seral HBV DNA load (> 50 IU/ml) (P < 0.001) and a lower rate of anti-viral treatment (P = 0.002) compared with those in the mild MVI group and MVI-negative group. Whereas, HCC with PVTT invading the main trunk showed a significantly higher rate of positive HBsAg (P = 0.007), positive HBeAg (P = 0.04), cirrhosis (P = 0.005) and a lower rate of receiving antiviral treatment (P = 0.009) compared with patients with no PVTT or PVTT invading the ipsilateral portal vein. Patients with vascular invasion also had a significantly higher level of seral HBV DNA load than patients without vascular invasion (P = 0.008).

Conclusions

In HCC patients, HBV infection and active HBV replication were associated with the development of vascular invasion.

Keywords

Hepatitis B virus Hepatocellular carcinoma Vascular invasion Anti-viral treatment Postoperative recurrence

Background

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related death in the world [1]. Although surgical resection and liver transplantation could offer a promising prognosis for selected patients with HCC, the high postoperative recurrence rate has impaired long-time survival. Among various risk factors, vascular invasion, including microvascular and macrovascular invasion, has been proven to be an independent factor predicting high recurrence and poor survival rate [24]. Microvascular invasion (MVI) was defined by most studies as microscopically confirmed tumor cell clusters within a vascular cavity lined with endothelium adjacent to the tumor [5, 6]. Conversely, macrovascular invasion mostly occurs in the portal vein system and is known as a portal vein tumor thrombus (PVTT); a PVTT can be identified during imaging examination or intraoperative exploration. A large tumor size, multinodular lesion, elevated level of desc-carboxy prothrombin (DCP) and certain imaging characteristics were reported to be factors predicting the presence of MVI, whereas the tumor size, Edmondson-Steiner histological grading, number of nodules and α-fetoprotein (AFP) level were associated with PVTT [2, 5, 7, 8].

Chronic hepatitis B virus (HBV) infection is a major risk factor for the development of liver cirrhosis and HCC, especially in East Asia [6]. HBV-related factors, such as seropositivity of hepatitis B e-antigen (HBeAg), high hepatitis B surface antigen (HBsAg) level and high serum HBV DNA load, were found to be significantly related to an increased risk of HBV-associated cirrhosis and HCC [9, 10]. These factors were also reported to be associated with an increased recurrence rate and a decreased survival rate of HCC after surgical resection [11, 12]. Fundamental research has revealed that the HBV-initiated tumorigenic process may play a role in the development of the vascular invasion of HCC [1315]. Recently, Lei Z et al. established a nomogram for preoperative prediction of the presence of MVI in HBV-related HCC, in which a high HBV DNA load (>104 IU/ml) was independently associated with the development of MVI [16]. These findings indicated a potential correlation between active HBV replication and the development of vascular invasion in HCC. To the best of our knowledge, no published study has provided insight into this issue. Therefore, we conducted a clinical study to further explore the impact of HBV-related factors on the formation of vascular invasion in HCC.

Methods

Study population

This was a retrospective study based on a prospectively compiled clinical and pathology database at a treatment center for HCC with PVTT at the Eastern Hepatobiliary Surgery Hospital, Shanghai, China. The study was approved by our Institutional Review Board, and written informed consent was obtained from all patients for their data to be used in this research.

HCC patients who had undergone surgical resection and confirmed by pathological examination at our center were included in this study. Exclusion criteria included hepatitis C virus (HCV)-related HCC, preoperative transarterial chemoembolization (TACE) or radiotherapy, non-curative resection, recurrent lesions, and a lack of complete clinical or pathological information.

For patients included in the study, the following clinical data and pathological results were collected: (1) demographic data, including age and gender and history of anti-viral treatment; (2) results of preoperative laboratory blood tests, including HBsAg, HBeAg, HBV-DNA level, AFP, DCP, albumin, total bilirubin, alanine aminotransferase, and aspartate aminotransferase; and (3) imaging and pathologic findings, including the presence and classification of PVTT, maximal tumor size, tumor number, capsule, presence and classification of MVI, and presence of cirrhosis.

Tests for the viral replication status, including those for HBsAg and its antibody, HBeAg and its antibody, and HBcAb, were performed. The serum HBV-DNA level was quantified by the polymerase chain reaction assay (ABI 7500; Applied Biosystems, Foster City, CA, USA) with a linear range of quantification of 50 to 2,000,000 IU/ml. The lower limit of detection was 50 IU/ml. Patients who had received standard interferon therapy or had been using oral anti-viral drugs for a duration of more than 2 months before surgery were classified as the anti-viral treatment group.

Diagnostic criteria of vascular invasion

The diagnostic criterion of MVI was the presence of a tumor cell nest in the portal vein, hepatic artery, hepatic vein, bile duct or lymph duct in the tumor surrounding the liver tissue under microscopic examination [2, 17]. The number and distribution of invaded vessels were measured to divide the patients with MVI into two groups as follows: patients in the mild MVI group (M1) had 1 to 5 involved vessels distributed within a 1-cm area from the tumor margin, whereas patients in the severe MVI group (M2) had more than 5 vessels invaded or had invaded vessels located more than 1 cm from the tumor margin. Every specimen was reviewed independently by two senior hepatobiliary pathologists to detect MVI. If the two pathologists had an inconsistent diagnosis, the findings were discussed to reach a final decision.

All HCC patients admitted to our center underwent a routine three-phrase dynamic CT or MRI examination before any treatment was carried out. PVTT was diagnosed when there were low-attenuation intraluminal masses that expanded the portal vein, or filling defects in the portal vein system, as presented in CT or MRI imaging. PVTT was confirmed and reassessed by palpation or ultrasound during operation. The final diagnosis was dependent on the intraoperative or pathologic findings. PVTT was classified according to Cheng’s classification, which has been shown to be effective in stratifying the severity of PVTT as follows: type I, invasion of the tumor thrombus into the segmental or sectoral branches of the portal vein or above; type II, involvement of the right or left portal vein; type III, invasion of the main trunk of the portal vein; and type IV, involvement of the superior mesenteric vein.

Statistical analysis

All calculations were performed using Stata 12.0 software (StataCorp, Texas 77,845, USA). Continuous and categorized data were compared using Pearson’s chi-squared test, Fisher’s exact test, or Student’s t test, as appropriate. Binary logistic regression was used to evaluate the relationship between the presence of vascular invasion as the dependent variable and factors that were significant in the univariate analysis as independent variables, using the stepwise backward method (Wald). The enter limit and remove limit were P = 0.05 and P = 0.10, respectively. Because viral factors, including seral HbeAg, the seral HBV DNA load, presence of cirrhosis and usage of antiviral treatment, were only meaningful when patients had HBV infection, only patients with positive seral HbsAg were included in multivariate analysis. A P < 0.05 was considered to indicate statistical significance.

Results

From May 1, 2015 to July 31, 2016, 675 patients with a preoperative diagnosis of HCC who underwent surgical resection at our center were identified. After careful examination, 189 patients were excluded, including 77 for preoperative TACE or radiotherapy, 36 for being diagnosed with a histological type other than HCC, 7 for HCV infection, 27 for recurrent lesions, 21 for non-curative resection, and 21 for failure to obtain detailed clinic information. Finally, 486 HCC patients, 422 men and 64 women, with a median age of 52 years (range, 22–80 years), fulfilled the inclusion criteria and were enrolled in the study.

Most patients (88.5%, 430/486) had HBV-related HCC; the remaining 56 patients (11.5%) had negative serum HBsAg. A total of 297 patients (61.1%) had a detectable seral HBV DNA load (> 50 IU/ml) in which 108 patients (22.2%) had a high HBV DNA load level > 2000 IU/ml. In total, 130 patients (26.7%) were classified into the anti-viral treatment group (interferon, 7; lamivudine, 14 patients; lamivudine + adefovir dipivoxil, 7 patients; lamivudine + entecavir, 11 patients; adefovir dipivoxil, 26 patients; entecavir, 33 patients; entecavir + adefovir dipivoxil, 8 patients; others, 24 patients). The overall proportion of patients with vascular invasion, including MVI and PVTT, was 60.3% (293/486). The incidence of MVI was 58.2% (283/486), whereas that of PVTT was 22.2% (108/486). A total of 98 patients (20.2%) had both MVI and PVTT.

Univariate analysis of viral and tumor factors predicting vascular invasion in HCC

Univariate analysis revealed that virus-related serum markers, including positive HBsAg (P = 0.005), positive HBeAg (P < 0.001) and a detectable HBV DNA load (P < 0.001), were significantly associated with the presence of vascular invasion, whereas vascular invasion was less frequently detected in patients in the anti-viral drug group (P = 0.003). The significant viral factors predicting MVI were the same as those of vascular invasion for patients with vascular invasion and MVI and were mostly overlapping. Similarly, virus-related seral markers, including positive HBsAg (P = 0.003), positive HBeAg (P = 0.005), a detectable HBV DNA load (P = 0.025), and the presence of cirrhosis (P < 0.001), were significantly associated with the presence of PVTT, whereas patients who were undergoing anti-viral treatment (P = 0.015) had a significantly lower risk of developing PVTT (Table 1).
Table 1

Univariate analysis of risk factors for formation of vascular invasion in hepatocellular carcinoma patients who underwent hepatectomy

Parameters

Vascular invasion

P

MVI

P

PVTT

P

yes

no

 

yes

no

 

yes

no

 

Patient demographics

Age

 > 50 years

153

125

0.006*

149

129

0.017*

53

225

0.053

 < = 50 years

140

68

 

134

74

 

55

153

 

Gender

 Male

255

167

0.873

245

177

0.842

99

323

0.092

 Female

38

26

 

38

26

 

9

55

 

Preoperative laboratory test

Total bilirubin

 > 20 μmol/l

48

32

0.954

48

32

0.725

16

64

0.601

 < = 20 umol/l

245

161

 

235

171

 

92

314

 

ALT

 > 42 U/l

128

83

0.882

122

89

0.872

49

162

0.642

 < = 42 U/l

165

110

 

161

114

 

59

216

 

AST,

 > 37 U/l

157

114

0.234

152

119

0.282

62

209

0.696

 < = 37 U/l

136

79

 

131

84

 

46

169

 

Albumin

 > 40 g/l

193

100

0.734

188

135

0.987

67

256

0.270

 < = 40 g/l

130

63

 

95

68

 

41

122

 

DCP

 > 100 mAU/ml

250

147

0.011*

241

156

0.019*

102

295

<0.001*

 < = 100 mAU/ml

43

46

 

42

47

 

6

83

 

Alpha-fetoprotein

 > 20 ng/ml

226

102

<0.001*

219

109

<0.001*

85

243

0.005*

 < = 20 ng/ml

67

91

 

64

94

 

23

135

 

Tumor characteristics

Diameter

 > 3 cm

247

138

0.001*

238

147

0.002*

105

280

<0.001*

 < = 3 cm

46

55

 

45

56

 

3

98

 

Number of lesions

 Multiple

43

15

0.022*

41

17

0.040*

20

38

0.017*

 Single

250

178

 

242

186

 

88

340

 

Encapsulation

 Incomplete/absent

259

145

<0.001*

250

154

<0.001*

101

303

0.001*

 Complete

34

48

 

33

49

 

7

75

 

Edmonson grading

 Grades III/IV

277

151

<0.001*

267

161

<0.001*

101

327

0.047*

 Grades I/II

16

42

 

16

42

 

7

51

 

Virus-related factors

 Seral HBsAg

  Positive

269

161

0.005*

260

170

0.006*

104

326

0.003*

  Negative

24

32

 

23

33

 

4

52

 

 Seral HBeAg

 Positive

93

32

<0.001*

88

37

0.002*

39

86

0.005*

 Negative

200

160

 

195

165

 

69

291

 

HBV DNA load

 Detectable (> 50 IU/ml)

203

94

<0.001*

197

100

<0.001*

76

221

0.025*

 Undetectable (<=50 IU/ml)

90

99

 

86

103

 

32

157

 

 High (> 2000 IU/ml)

110

62

0.222

106

66

0.261

39

133

0.859

 Low (<= 2000 IU/ml)

183

131

 

177

137

 

69

39

 

Presence of cirrhosis

 Yes

101

52

0.08

96

57

0.171

49

104

<0.001*

 No

192

141

 

187

146

 

59

274

 

Anti-virus treatment

 Yes

64

66

0.003*

63

67

0.008*

19

111

0.015*

 No

229

127

 

220

136

 

89

267

 

MVI Microvascular invasion; PVTT Portal vein tumor thrombus; ALT Alanine aminotransferase; AST Aspartate aminotransferase; DCP Des-gamma-carboxy prothrombin; HBsAg Hepatitis B virus s Antigen; HBeAg Hepatitis B virus e Antigen; HBV Hepatitis B virus

*P < 0.05

Multivariate analysis of viral and tumor factors predicting vascular invasion in HCC

Multivariate logistic regression analysis was carried out in patients with positive seral HbsAg, utilizing binary variables that were significant in the univariate analysis. As shown in Table 2, positive seral HBeAg (OR = 1.83, P = 0.019) and a detectable seral HBV DNA load (OR = 1.68, P = 0.027) were independent risk factors of vascular invasion in the multivariate regression analysis. Moreover, tumor-related factors, including a seral AFP level > 20 ng/ml (OR = 2.51, P < 0.001), multiple lesions (OR = 2.18, P = 0.038), tumor size >3 cm (OR = 1.73, P = 0.035), Edmonson grades III/IV (OR = 2.48, P = 0.013) and incomplete/absent tumor capsule (OR = 2.17, P = 0.006), were significantly and independently associated with vascular invasion. Factors predictive of MVI were similar to those predictive of vascular invasion, except that the impact of seral HBeAg on the formation of MVI didn’t reach statistical significance (OR = 1.59, P = 0.059). Regarding the risk factors of PVTT, the impact of the seropositivity of HBeAg (OR = 1.67, P = 0.046), tumor diameter > 3 cm (OR = 8.86, P < 0.001), incomplete or absent encapsulation (OR = 3.59, P = 0.003) and DCP > 100 mAU/ml (OR = 2.90, P = 0.022) were significant in the multivariable analysis.
Table 2

Multivariate logistic regression analysis of factors predictive of vascular invasion in patients with positive seral HbsAg

Variables

Odds ratio

95% CI

P value

Risk of vascular invasion

 Age (>50 years vs < =50 years)

0.68

0.44–1.04

0.078

 Alpha-fetoprotein (> 20 ng/ml vs < = 20 ng/ml)

2.51

1.59–3.96

<0.01*

 Tumor number (Multiple vs Single)

2.18

1.04–4.55

0.038*

 Diameter (> 3 cm vs < = 3 cm)

1.73

1.04–2.88

0.035*

 Edmonson grading (Grades III/IV vs Grades I/II)

2.48

1.21–5.05

0.013*

 Tumor capsule (Incomplete/absent vs Complete)

2.17

1.25–3.77

0.006*

 Seral HBeAg (Positive vs Negative)

1.83

1.10–3.03

0.019*

 Seral HBV DNA load (> 50 IU/ml vs < = 50 IU/ml)

1.68

1.06–2.65

0.027*

Risk of microscopic vascular invasion

 Alpha-fetoprotein (> 20 ng/ml vs < = 20 ng/ml)

2.59

1.65–4.05

<0.01*

 Tumor number (Multiple vs Single)

2.21

1.09–4.51

0.028*

 Diameter (> 3 cm vs < = 3 cm)

1.58

0.96–2.61

0.074

 Edmonson grading (Grades III/IV vs Grades I/II)

2.24

1.11–4.54

0.024*

 Tumor capsule (Incomplete/absent vs Complete)

2.04

1.18–3.51

0.011*

 Seral HBeAg (Positive vs Negative)

1.59

0.98–2.57

0.059

 Seral HBV DNA load (> 50 IU/ml vs < = 50 IU/ml)

1.76

1.12–2.76

0.013*

Risk of portal vein tumor thrombus

 DCP (>100 mAU/ml vs < = 100 mAU/ml)

2.90

1.17–7.12

0.022*

 Tumor diameter (>3 cm vs < =3 cm)

8.86

2.67–29.39

<0.01*

 Tumor capsule (Incomplete/absence vs Complete)

3.59

1.56–8.25

0.003*

 Seral HBeAg (Positive vs Negative)

1.67

1.01–2.75

0.046*

 Anti-virus treatment (Yes vs No)

0.59

0.33–1.05

0.075

CI Confidential Interval; HBeAg Hepatitis B virus e Antigen; HBV Hepatitis B virus; DCP Des-gamma-carboxy prothrombin; HBsAg Hepatitis B virus s Antigen; HBeAg Hepatitis B virus e Antigen

*P < 0.05

Correlation between the features of vascular invasion and HBV-related factors

Table 3 shows that the severe MVI group had a significantly higher rate of positive seral HBsAg, positive seral HBeAg, a detectable seral HBV DNA load (> 50 IU/ml), as well as a lower rate of antiviral treatment, compared with the mild MVI group and negative group. By contrast, for the classification of PVTT, HCC with type III/IV PVTT had a significantly higher rate of positive seral HBsAg, positive seral HBeAg, and cirrhosis, as well as a lower rate of receiving antiviral treatment compared with the type I/II group and PVTT-negative group. Patients with vascular invasion had a significantly higher seral HBV DNA load than patients without vascular invasion (Table 4).
Table 3

Correlations between the severity of microvascular invasion or portal vein tumor thrombus and viral features in hepatocellular carcinomaa

HBV-related factors

Severity of MVI

P

Classification of PVTT

P

None (%)

Mild (%)

Severe (%)

None (%)

I/II (%)

III/IV (%)

Seral HBsAg

 Positive

171 (83.8)

127 (89.4)

131 (94.9)

0.005*

326 (86.2)

74 (94.9)

30 (100)

0.007*

 Negative

33 (16.2)

15 (10.6)

7 (5.1)

 

52 (13.8)

4 (5.1)

0 (0)

 

Seral HBeAg

 Positive

37 (21.6)

38 (29.9)

48 (36.6)

0.016*

84 (25.8)

30 (40.5)

9 (30)

0.04*

 Negative

134 (78.4)

89 (60.1)

83 (63.4)

 

242 (74.2)

44 (59.5)

21 (70)

 

Seral HBV DNA load

  > 50 IU/ml

99 (57.9)

90 (70.9)

106 (80.9)

<0.001*

219 (67.2)

52 (70.3)

24 (80)

0.355

  < = 50 IU/ml)

72 (42.1)

37 (29.1)

25 (19.1)

 

107 (32.8)

22 (29.7)

6 (20)

 

Presence of cirrhosis

 Yes

52 (30.4)

48 (37.8)

44 (33.6)

0.418

96 (29.4)

32 (43.2)

16 (53.3)

0.005*

 No

119 (69.6)

79 (62.2)

87 (66.4)

 

230 (60.6)

42 (56.8)

14 (46.7)

 

Antivirus treatment

 Yes

66 (38.6)

36 (28.3)

26 (19.8)

0.002*

109 (33.4)

15 (20.3)

4 (13.3)

0.009*

 No

105 (61.4)

91 (61.7)

105 (80.2)

 

217 (66.6)

59 (79.7)

26 (86.7)

 

HBV Hepatitis B virus; HBsAg Hepatitis B virus s Antigen; HBeAg Hepatitis B virus e Antigen

aanalysis was only carried out in patients with positive HBsAg except the “HBsAg” row

*P < 0.05

Table 4

Difference in the seral HBV DNA load between HBV-related hepatocellular carcinoma patients with or without vascular invasiona

Variable

HBV DNA load, log 10 IU/ml (mean ± SD)

P

Vascular invasion

 Yes

3.28 ± 0.14

0.008*

 No

2.64 ± 0.20

 

Microvascular invasion

 Yes

3.29 ± 0.14

0.008*

 No

2.66 ± 0.19

 

Portal vein tumor thrombus

 Yes

3.13 ± 0.22

0.694

 No

3.02 ± 0.14

 

HBV Hepatitis B virus; SD Standard Deviation

aanalysis was only carried out in patients with positive HBsAg

*P < 0.05

Discussion

The presence of vascular invasion, including MVI and PVTT, was significantly associated with a high risk of postoperative recurrence, which is a major obstacle to improving the prognosis of HCC [6, 17, 18]. However, the risk factors and underlying mechanism leading to the formation of vascular invasion remain largely unknown. In East Asia, the majority of HCC develops within an environment of chronic inflammation caused by HBV infection. Recently, the results of fundamental studies have indicated that the HBV status is a potent etiological factor predisposing HCC patients to develop vascular invasion. HBV X protein (HBx), a key regulatory multifunctional protein of the virus, has been reported to be involved in the development of MVI and is associated with postoperative recurrence [14, 19, 20]. Yang et al. found that the seropositivity of HBsAg was associated with a high risk of developing PVTT, and the activity of the TGF-β-miR-34a-CCL22 axis induced by the change in the liver microenvironment caused by HBV infection may play an important role in the development of PVTT [15]. The potential correlation between HBV replication and the formation of MVI in HCC have also been studied in some preliminary clinical studies. Chen et al. retrospectively studied the impact of ascites, as well as tumor- and HBV-related factors, on the formation of vascular invasion and found negative results concerning the impact of viral factors; however, it is worth noting that the limited number of cases with MVI (n = 12) and incomplete data concerning the status of HBV infection may limit the power of their results [21]. To establish a preoperative prediction model for MVI, a large cohort of HBV-related HCC patients (n = 1004) was analyzed by Lei et al., revealing that a high seral HBV DNA load (> 104 IU/ml) was an independent factor predicting the presence of MVI. The other predictive variables were well-known tumor-related factors, including a large tumor diameter, multiple nodules, an incomplete capsule and an AFP level > 20 ng/ml [16]. Nevertheless, the relationship between HBV infection and vascular invasion has rarely been intentionally researched in a well-designed clinical study.

Our study was based on a prospectively collected database with comprehensive data indicating the status of HBV infection and vascular invasion. The results showed that compared with patients without HBV infection, the incidence of vascular invasion, including MVI and PVTT, was significantly increased in HBV-related HCC. In the multivariate analysis carried out in positive HBsAg patients, positive HBeAg and a detectable seral HBV DNA load (> 50 IU/ml) were significantly associated with development of vascular invasion. In addition, our results revealed that in HBV-related HCC patients, a more severe level of vascular invasion was associated with a higher rate of active HBV replication, as reflected in positive HBeAg or a detectable HBV DNA load. These findings provided promising clinical evidence to demonstrate that in addition to tumor-related factors, the activity of HBV infection plays a key role in the development of vascular invasion in HCC patients.

The postoperative recurrence of HBV-related HCC was categorized into two groups, early and late recurrence, with a cut-off time at 2 years [22]. Late recurrence (> 2 years after resection) usually presented as a metachronous tumor with different genetic and histological features from the primary HCC [22, 23]. It was revealed that HBV-related factors, including a high hepatic inflammatory activity score and high HBV DNA load, were significantly associated with late recurrence, whereas sustained suppression of HBV replication by anti-viral drugs achieved a lower rate of late recurrence [12, 24, 25]. Tumors occurring within 2 years after surgery were classified as early recurrence, which was strongly associated with tumor-related factors, including tumor size and the presence of nodules, vascular invasion and resection margin [22]. A randomized controlled trial by Lin et al. revealed that patients receiving anti-viral treatment showed a significantly better 2-year overall (93.8% vs 62.2%) and recurrence-free (55.6% vs 19.5%) survival [26]. However, it is difficult to understand the effect of anti-viral drugs on inhibiting early postoperative recurrence (< 2 years after resection), which was considered the result of the regrowth of micro-metastases in the liver that were not detected and resected during the operation. Our result demonstrates that active HBV replication is associated with a high rate of vascular invasion in HCC patients, which may partially explain the anti-tumor effect of antiviral treatment. We could speculate that the suppression of HBV replication via anti-viral treatment might decrease the invasiveness and metastatic potential of HCC to reduce the risk of early postoperative recurrence.

The seral HBV DNA load is usually divided into high and low levels at a cut-off value of 2000 IU/ml. In this study, the impact of the seral HBV DNA load on the formation of vascular invasion was not significant if the cut-off value was set at this point. This result implies that the correlation between the HBV DNA load and occurrence of vascular invasion is not linear. Additionally, this result may also be caused by a proportion of patients with a high HBV DNA load being in the “immune tolerant” phase with no or mild substantial liver injury [10]. According to the current guidelines, anti-viral drugs should be prescribed in chronic hepatitis B patients with a serum HBV DNA load above 2000 IU/ml and elevated ALT levels, in the absence of sufficient evidence of cirrhosis [10]. However, for patients with a low HBV DNA level (< 2000 IU/ml) without advanced liver disease, the benefit of anti-viral treatment has not been well clarified. In this study, HCC patients with an undetectable HBV DNA load (≤ 50 IU/ml) had a lower incidence of vascular invasion than patients with a detectable HBV DNA load (> 50 IU/ml). These results suggested that it may also be beneficial to receive anti-viral drugs for patients who do not meet the current treatment indication.

The suppressed HBV replication by anti-viral treatment was supposed to correlate with a lower rate of vascular invasion. However, the inhibitory effect of anti-viral treatment on the development of vascular invasion was overshadowed by tumor-related factors in the multivariate analysis. The following reasons may explain this phenomenon. First, patients who have undergone anti-viral treatment usually have no or mild cirrhosis with normal liver function [27, 28]. Surgeons are more likely to apply hepatectomy with a wider resection margin to these patients. Theoretically, a wide surgical margin will lead to a higher detection rate of MVI during pathological evaluation. Second, patients who have undergone anti-viral treatment have enjoyed good health care and regular surveillance, leading to early detection of HCC. Thus, the inhibitory effect of anti-viral treatment tended to be overshadowed by the early tumor features.

It is worth noting that MVI and PVTT have different risk factor profiles in our research. For HBV-related factors, only detectable HBV load was associated MVI, while only positive HBeAg was associated with PVTT. Although MVI and PVTT were two common types of vascular invasion of HCC, there was no evidence indicating potential causal relationship between them. Previous clinic studies also revealed that MVI and PVTT had inconsistent predicting factors [2, 5, 7, 8]. Further fundamental and clinic studies are needed to clarify the relationship between MVI and PVTT.

This study might not be able to reveal the full landscape of the relationship between HBV activity and the occurrence of vascular invasion in HCC. In particular, because of the limited follow-up time, we failed to carry out a survival analysis to determine significant factors contributing to recurrence or survival. However, the main finding of this study is the association between HBV infection status and presence of vascular invasion in HCC, lack of survival information may have a less impact on our conclusion. Additionally, inconsistency existed between the protocol of anti-viral treatment and surgical procedures because of the retrospective nature of this research. Furthermore, the surgical margin varied in patients with different levels of cirrhosis, a finding that might affect the detection rate of MVI. At last, only HBV-related HCC was studied in this research, the conclusion isn’t applicable for HCC caused by other hepatic virus. Despite these limitations, we first found the interesting phenomenon that HBV infection and replication status were independently associated with the formation of vascular invasion in HCC, which may partially explain the inhibitory effect of anti-viral treatment on early HCC recurrence.

Conclusions

In addition to characteristics of the tumor itself, HBV infection and active replication were independently associated with the development of vascular invasion in HCC. In patients with HBV-related HCC with positive HBeAg or a detectable HBV DNA load, an increased risk of vascular invasion should be recognized.

Abbreviations

AFP: 

α-fetoprotein

DCP: 

Desc-carboxy Prothrombin

HBeAg: 

Hepatitis B e-antigen

HBsAg: 

Hepatitis B surface-antigen

HBV: 

Hepatitis B Virus

HBx: 

HBV X Protein

HCC: 

Hepatocellular Carcinoma

HCV: 

Hepatitis C Virus

MVI: 

Microvascular Invasion

PVTT: 

Portal Vein Tumor Thrombus

TACE: 

Transarterial Chemoembolization

Declarations

Acknowledgement

Not applicable.

Funding

Study design: The National Key Basic Research Program “973 project”(2015CB554000); Shanghai Shenkang Project (SHDC12015106).

Data collection and analysis: the Science Fund for Creative Research Groups (81521091).

Shanghai Science and Technology Committee (134119a0200);

Manuscript drafting and revision: 2012 SMMU Innovation Alliance for Liver Cancer Diagnosis and Treatment; Collaborative Innovation Center for Cancer Medicine.

Availability of data and materials

The datasets generated and analyzed during the current study are not publicly available because further fundamental and clinical research will be carried out based on this cohort; however, they are available from the corresponding author upon reasonable request.

Authors’ contributions

XBW and NLi: contributed equally to this article. collected and analyzed data, drafted and revised the manuscript. SSL: collected and analyzed data. JS, WXG and YXZ: collected data, revised the manuscript. SQC: designed the study, collected data, revised the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

The study was approved by the Institutional Review Board of Eastern Hepatobiliary Surgery Hospital, and written informed consent was obtained from all patients for their data to be used in this research.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Hepatic Surgery VI, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University

References

  1. McGlynn KA, London WT. The global epidemiology of hepatocellular carcinoma: present and future. Clin Liver Dis. 2011;15(2):223–43. vii-xView ArticlePubMedPubMed CentralGoogle Scholar
  2. Rodriguez-Peralvarez M, Luong TV, Andreana L, Meyer T, Dhillon AP, Burroughs AK. A systematic review of microvascular invasion in hepatocellular carcinoma: diagnostic and prognostic variability. Ann Surg Oncol. 2013;20(1):325–39.View ArticlePubMedGoogle Scholar
  3. Llovet JM, Bustamante J, Castells A, Vilana R, Ayuso Mdel C, Sala M, Bru C, Rodes J, Bruix J. Natural history of untreated nonsurgical hepatocellular carcinoma: rationale for the design and evaluation of therapeutic trials. Hepatology. 1999;29(1):62–7.View ArticlePubMedGoogle Scholar
  4. Shi J, Lai EC, Li N, Guo WX, Xue J, Lau WY, Wu MC, Cheng SQ. Surgical treatment of hepatocellular carcinoma with portal vein tumor thrombus. Ann Surg Oncol. 2010;17(8):2073–80.View ArticlePubMedGoogle Scholar
  5. Fujita N, Aishima S, Iguchi T, Mano Y, Taketomi A, Shirabe K, Honda H, Tsuneyoshi M, Oda Y. Histologic classification of microscopic portal venous invasion to predict prognosis in hepatocellular carcinoma. Hum Pathol. 2011;42(10):1531–8.View ArticlePubMedGoogle Scholar
  6. Iguchi T, Shirabe K, Aishima S, Wang H, Fujita N, Ninomiya M, Yamashita Y, Ikegami T, Uchiyama H, Yoshizumi T, et al. New pathologic stratification of microvascular invasion in hepatocellular carcinoma: predicting prognosis after living-donor liver transplantation. Transplantation. 2015;99(6):1236–42.View ArticlePubMedGoogle Scholar
  7. Zhou L, Rui JA, Wang SB, Chen SG, Qu Q. Risk factors of microvascular invasion, portal vein tumor thrombosis and poor post-resectional survival in HBV-related hepatocellular carcinoma. Hepato-Gastroenterology. 2014;61(134):1696–703.PubMedGoogle Scholar
  8. Shirabe K, Toshima T, Kimura K, Yamashita Y, Ikeda T, Ikegami T, Yoshizumi T, Abe K, Aishima S, Maehara Y. New scoring system for prediction of microvascular invasion in patients with hepatocellular carcinoma. Liver international : official journal of the International Association for the Study of the Liver. 2014;34(6):937–41.View ArticleGoogle Scholar
  9. Qu LS, Zhang HF. Significance of viral status on prognosis of hepatitis B-related hepatocellular carcinoma after curative resection in East Asia. Hepatology research : the official journal of the Japan Society of Hepatology. 2015;Google Scholar
  10. European Association For The Study Of The L. EASL clinical practice guidelines: management of chronic hepatitis B virus infection. J Hepatol. 2012;57(1):167–85.View ArticleGoogle Scholar
  11. Yu LH, Li N, Shi J, Guo WX, Wu MC, Cheng SQ. Does anti-HBV therapy benefit the prognosis of HBV-related hepatocellular carcinoma following hepatectomy? Ann Surg Oncol. 2014;21(3):1010–5.View ArticlePubMedGoogle Scholar
  12. Wu CY, Chen YJ, Ho HJ, Hsu YC, Kuo KN, Wu MS, Lin JT. Association between nucleoside analogues and risk of hepatitis B virus-related hepatocellular carcinoma recurrence following liver resection. JAMA. 2012;308(18):1906–14.View ArticlePubMedGoogle Scholar
  13. B. H, G. F, L. R, C. D, M. S, J. C, P. B-S, R. H, M.-A. B, D. S et al: hepatocellular carcinoma replicating hepatitis B virus: a clinical, virological and transcriptional entity. Hepatology 2014, 60(SUPPL 1):995A-996A.Google Scholar
  14. Chen L, Zhang Q, Chang W, Du Y, Zhang H, Cao G. Viral and host inflammation-related factors that can predict the prognosis of hepatocellular carcinoma. Eur J Cancer. 2012;48(13):1977–87.View ArticlePubMedGoogle Scholar
  15. Yang P, Li QJ, Feng Y, Zhang Y, Markowitz GJ, Ning S, Deng Y, Zhao J, Jiang S, Yuan Y, et al. TGF-beta-miR-34a-CCL22 signaling-induced Treg cell recruitment promotes venous metastases of HBV-positive hepatocellular carcinoma. Cancer Cell. 2012;22(3):291–303.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Lei Z, Li J, Wu D, Xia Y, Wang Q, Si A, Wang K, Wan X, Lau WY, Wu M, et al. Nomogram for preoperative estimation of microvascular invasion risk in hepatitis B virus-related hepatocellular carcinoma within the Milan criteria. JAMA surgery. 2015:1–8.Google Scholar
  17. Sumie S, Nakashima O, Okuda K, Kuromatsu R, Kawaguchi A, Nakano M, Satani M, Yamada S, Okamura S, Hori M, et al. The significance of classifying microvascular invasion in patients with hepatocellular carcinoma. Ann Surg Oncol. 2014;21(3):1002–9.View ArticlePubMedGoogle Scholar
  18. Chen JS, Wang Q, Chen XL, Huang XH, Liang LJ, Lei J, Huang JQ, Li DM, Cheng ZX. Clinicopathologic characteristics and surgical outcomes of hepatocellular carcinoma with portal vein tumor thrombosis. J Surg Res. 2012;175(2):243–50.View ArticlePubMedGoogle Scholar
  19. Ryu SH, Chung YH, Lee H, Kim JA, Shin HD, Min HJ, Seo DD, Jang MK, Yu E, Kim KW. Metastatic tumor antigen 1 is closely associated with frequent postoperative recurrence and poor survival in patients with hepatocellular carcinoma. Hepatology. 2008;47(3):929–36.View ArticlePubMedGoogle Scholar
  20. Xu J, Liu H, Chen L, Wang S, Zhou L, Yun X, Sun L, Wen Y, Gu J. Hepatitis B virus X protein confers resistance of hepatoma cells to anoikis by up-regulating and activating p21-activated kinase 1. Gastroenterology. 2012;143(1):199–212. e194View ArticlePubMedGoogle Scholar
  21. Chen C, Chen DP, Gu YY, Hu LH, Wang D, Lin JH, Li ZS, Xu J, Wang G. Vascular invasion in hepatitis B virus-related hepatocellular carcinoma with underlying cirrhosis: possible associations with ascites and hepatitis B viral factors? Tumour Biol. 2015;36(8):6255–63.View ArticlePubMedGoogle Scholar
  22. Wu JC, Huang YH, Chau GY, Su CW, Lai CR, Lee PC, Huo TI, Sheen IJ, Lee SD, Lui WY. Risk factors for early and late recurrence in hepatitis B-related hepatocellular carcinoma. J Hepatol. 2009;51(5):890–7.View ArticlePubMedGoogle Scholar
  23. Kim WR, Gores GJ. Recurrent hepatocellular carcinoma: it's the virus! J Clin Oncol. 2013;31(29):3621–2.View ArticlePubMedPubMed CentralGoogle Scholar
  24. Kim BK, Park JY, Kim do Y, Kim JK, Kim KS, Choi JS, Moon BS, Han KH, Chon CY, Moon YM et al: Persistent hepatitis B viral replication affects recurrence of hepatocellular carcinoma after curative resection. Liver international : official journal of the International Association for the Study of the Liver 2008, 28(3):393–401.Google Scholar
  25. Huang G, Lai EC, Lau WY, Zhou WP, Shen F, Pan ZY, Fu SY, Wu MC. Posthepatectomy HBV reactivation in hepatitis B-related hepatocellular carcinoma influences postoperative survival in patients with preoperative low HBV-DNA levels. Ann Surg. 2013;257(3):490–505.View ArticlePubMedGoogle Scholar
  26. Yin J, Li N, Han Y, Xue J, Deng Y, Shi J, Guo W, Zhang H, Wang H, Cheng S, et al. Effect of antiviral treatment with nucleotide/nucleoside analogs on postoperative prognosis of hepatitis B virus-related hepatocellular carcinoma: a two-stage longitudinal clinical study. J Clin Oncol. 2013;31(29):3647–55.View ArticlePubMedGoogle Scholar
  27. Li N, Lai EC, Shi J, Guo WX, Xue J, Huang B, Lau WY, Wu MC, Cheng SQ. A comparative study of antiviral therapy after resection of hepatocellular carcinoma in the immune-active phase of hepatitis B virus infection. Ann Surg Oncol. 2010;17(1):179–85.View ArticlePubMedGoogle Scholar
  28. Yu LH, Li N, Cheng SQ: the role of antiviral therapy for HBV-related hepatocellular carcinoma. Int J Hepatol 2011, 2011:416459.Google Scholar

Copyright

© The Author(s). 2017