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Naturally occurring basal core promoter A1762T/G1764A dual mutations increase the risk of HBV-related hepatocellular carcinoma: a meta-analysis

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Oncotarget. 2016; 7:12525-12536. https://doi.org/10.18632/oncotarget.7123

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Zongguo Yang, Liping Zhuang, Yunfei Lu, Qingnian Xu, Bozong Tang, Xiaorong Chen _

Abstract

Zongguo Yang1,*, Liping Zhuang2,3,*, Yunfei Lu1, Qingnian Xu1, Bozong Tang1 and Xiaorong Chen1

1 Shanghai Public Health Clinical Center, Fudan University, Shanghai, China

2 Fudan University Shanghai Cancer Center, Shanghai, China

3 Shanghai Medical College, Fudan University, Shanghai, China

* These authors have contributed equally to this work

Correspondence to:

Xiaorong Chen, email:

Zongguo Yang, email:

Keywords: hepatitis B virus X protein, hepatocellular carcinoma, basal core promoter, mutation, A1762T/G1764A

Received: October 05, 2015 Accepted: January 23, 2016 Published: Febraury 02, 2016

Abstract

Basal core promoter (BCP) A1762T/G1764A dual mutations in hepatocarcinogenesis remain controversial. Published studies up to June 1, 2015 investigating the frequency of A1762T/G1764A dual mutations from chronic hepatitis B virus (HBV) infection, including hepatocellular carcinoma (HCC), were systematically identified. A total of 10,240 patients with chronic HBV infection, including 3729 HCC cases, were included in 52 identified studies. HCC patients had a higher frequency of BCP A1762T/G1764A dual mutations compared with asymptomatic HBsAg carriers (ASC) and patients with chronic hepatitis B (CHB) and liver cirrhosis (LC) (OR = 5.59, P < 0.00001; OR = 2.87, P < 0.00001; OR = 1.55, P = 0.02, respectively). No statistically significant difference was observed in the frequency of A1762T/G1764A dual mutations in cirrhotic HCC versus non-cirrhotic HCC patients (OR = 2.06, P = 0.05). Chronic HBV-infected patients and HCC patients with genotype B had a significantly lower risk of A1762T/G1764A dual mutations compared with patients with genotype C (OR = 0.30, P < 0.0001 and OR = 0.34, P = 0.04, respectively). In HBV genotype C subjects, A1762T/G1764A dual mutations contributed to significantly higher risk for HCC developing compared with non-mutation ones (OR = 3.47, P < 0.00001). In conclusion, A1762T/G1764A dual mutations increase the risk of HBV-related hepatocellular carcinoma, particularly in an HBV genotype C population, even without progression to cirrhosis.


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INTRODUCTION

Viral genomic mutations may contribute to HCC development. HBV nonstructural X protein (HBx) is a key regulatory protein of the virus and is at the intersection of HBV infection, replication, pathogenesis, and possibly carcinogenesis. HBx has different consequences for hepatocyte physiology because HBV-infected cells are targeted by the immune system or as hepatocytes, in which HBx is expressed, and undergo transformation and progression to HCC. In addition, HBx can influence apoptotic and cell cycle regulatory pathways [1].

However, the roles that HBx mutations play in hepatocarcinogenesis remain controversial, particularly for the basal core promoter (BCP) A1762T/G1764A dual mutation. Several prospective studies have demonstrated that patients with an A1762T/G1764A dual mutation were more predisposed to HCC than those with the wild type and that HBV mutations, including A1762T/G1764A, are associated with an increased risk of HCC [2]. However, a recent study using global data found no significant difference in BCP mutations between HCC and non-HCC patients with HBV genotype C, and the differences between chronic hepatitis B (CHB) and liver cirrhosis (LC) and between LC and HCC were not significant, although the mutant ratio increased with disease progression. [3].

In this meta-analysis, we summarized the prevalence of A1762T/G1764A mutations from ASC, CHB, LC and HCC patients. We also analyzed the BCP mutation rates in chronic HBV infection, including HCC patients grouped by HBV genotype and HBeAg status, hoping that the results might provide useful insights into the risk of HCC occurrence.

RESULTS

Study and patient characteristics

A total of 1883 abstracts were reviewed. From these articles, 163 that were closely related to the current subject were retrieved. The study selection process is summarized in Figure 1. Finally, 52 case-control or cohort studies [4-55] were included in the meta-analysis. A total of 10,240 individuals with chronic HBV infection were included, 3729 of whom had HCC. The baseline characteristics and quality scores of the studies examined in this meta-analysis are listed in Table 2.

Table 1: Quality criteria for the included studies*

Quality parameter

Score

2

1

0

Study design

Cohort study or nested case-control study

Incidence case-control study. Prevalence case-control study

-

No. of case subjects

>100

50-100

<50

Source of population

Community-based or from two or more countries

≥2 hospitals

1 hospitals

Mutation detection method

DNA direct sequencing

Innogenetics line-probe assay

Single-strand conformation polymorphism; Mass spectrometer

Matching of case and control subjects

Confounder group 1

Age and sex

Age or sex

None

Confounder group 2

HBeAg status and HBV genotype

HBeAg status or HBV genotype

None

* -, designs other than cohort or nested, incidence, or prevalence case–control not included in meta-analysis; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus.

Flow program of study selection.

Figure 1: Flow program of study selection.

Prevalence of A1762T/G1764A mutations from ASC, CHB, LC and HCC

Heterogeneity was significant among the included studies [4-38, 40-55] when comparing the prevalence of A1762T/G1764A dual mutations between HCC and non-HCC patients (P < 0.00001, I2 = 82%). Thus, a random-effects model was used. Our data revealed that more HCC patients had A1762T/G1764A dual mutations compared with non-HCC patients (66.5% vs. 39.8%, OR = 3.05, 95% CI = 2.35-3.95, P < 0.00001, Table 3 and Supplementary Figure S1).

Table 2: Baseline characteristics of studies included in the meta-analysis

Study

Design

Country or area

No. of cases

No. of controls

Detection method

HBV genotype

Matching factors

Quality score

Age

Sex

HbeAg status

HBV genotype

Asim 2010

PCC

India

150

136

SSCP

A, D

-

-

-

-

≤4

Bahramali 2008

Cohort

Iran

7

55

Sequencing

D

-

-

-

-

≤4

Bai 2011

Cohort

China

152

136

Sequencing

B, C, others

-

-

-

-

7

Baptista 1999

PCC

South Africa

59

52

Sequencing

NA

+

-

-

-

5-7

Blackberg 2003

PCC

Sweden, others

16

19

Sequencing

A, B, C, D

-

+

+

+

≥8

Chen 2006

PCC

Taiwan

50

102

INNO-LiPA

B, C

+

-

-

-

5-7

Chen 2008

PCC

Taiwan

80

160

Sequencing

B, C, D

+

+

+

-

5-7

Chen 2009

PCC

Taiwan

222

300

Sequencing

B, C

+

+

-

-

5-7

Chen 2012

PCC

China

156

310

Sequencing

B, C

+

+

-

-

5-7

Cho 2011

PCC

Korea

69

125

Sequencing

C

-

-

-

-

≤4

Choi 2009

PCC

Korea

42

46

Sequencing

C

-

-

-

-

≤4

Chou 2008

NCC

Taiwan

154

316

Sequencing

A,B,C

+

-

-

-

≥8

Chu 2012

PCC

Taiwan

80

120

Sequencing

B, C

+

+

+

-

4-6

Constantinescu 2014

Cohort

Romania

94

390

Sequencing

A, D

-

-

-

-

5

Datta 2014

PCC

India

22

46

Sequencing

A, C, D

-

-

-

-

≤4

Elkady 2008

PCC

Mongolia

23

25

Sequencing

A, D

-

-

-

-

≤4

Fan 2011

PCC

China

34

38

Sequencing

B, C

+

+

+

-

5-7

Guo 2008

NCC

China

58

71

Sequencing

B, C

+

+

-

-

≥8

Ito 2006

ICC

Japan, United States, Hong Kong

40

80

Sequencing

C

+

+

+

+

≥8

Jang 2007

Cohort

Korea

6

23

Sequencing

C

-

-

+

+

≥8

Kao 2012

PCC

Taiwan

56

112

Sequencing

B, C

+

-

-

-

4-5

Kim 2008

PCC

Korea

60

124

Sequencing

C

-

-

-

+

5-7

Kim 2009

PCC

Korea

135

135

Sequencing

C

+

+

+

-

5-7

Kuang 2005

PCC

Thailand

34

68

Mass spectrometer

NA

+

+

-

-

≤4

Lee 2011

PCC

Korea

31

65

Sequencing

C

-

-

-

-

≤4

Li 2013

Cohort

China

102

105

Sequencing

C

-

-

-

-

6

Lin 2005

PCC

Taiwan

32

142

Sequencing

B, C, Others

-

-

+

-

≤4

Liu 2006

PCC

Taiwan

200

160

INNO-LiPA

B, C

-

-

-

-

≤4

Livingston 2007

ICC

United States

47

1129

INNO-LiPA

A, C, D, F

+

+

-

-

≤4

Lyu 2013

ICC

Korea

318

234

Sequencing

C

-

-

-

-

5

Malik 2012

PCC

India

118

294

Sequencing

A, D

-

-

-

-

5

Muroyama 2006

PCC

Japan

39

36

Sequencing

C

+

+

-

+

5-7

Panigrahi 2012

PCC

India

20

132

Sequencing

A, C, D

-

-

-

-

≤4

Park 2014

PCC

Korea

132

310

Sequencing

C

-

-

-

-

5

Qu 2014

ICC

China

152

131

Sequencing

B, C

+

-

-

-

5-7

Sakamoto 2006

PCC

Japan, Philippines

31

69

Sequencing

A, B, C

-

+

-

-

5-7

Shinkai 2007

PCC

Japan

80

80

Sequencing

C

+

+

+

+

≥8

Song 2005

PCC

Vietnam

48

74

Sequencing

NA

-

-

-

-

≤4

Tanaka 2006

PCC

Japan, Hong Kong

148

180

Sequencing

C

+

+

+

-

≥8

Tangkijvanich 2010

ICC

Thailand

60

60

Sequencing

B, C

+

+

+

+

6

Tong 2007

ICC

United States

101

67

Sequencing

A, B, C, D

-

-

-

-

5-7

Tong 2013

ICC

United States

173

240

Sequencing

A, B, C, D, E, F

+

-

-

-

5-7

Truong 2007

PCC

Japan, Vietnam

32

88

Sequencing

C

-

-

+

+

5-7

Utama 2009

PCC

Indonesia

48

123

Sequencing

B, C

-

-

-

-

5

Wang 2007

PCC

China

47

164

Sequencing

B, C

-

+

-

-

5-7

Xu 2010

PCC

China

60

120

Sequencing

A, B, C

+

+

-

-

4-6

Yin 2011

PCC

China

190

1269

Sequencing

B, C

+

+

-

-

≥8

Yuan 2007

PCC

China

34

207

Sequencing

B, C

-

+

-

+

5-7

Yuan 2009

ICC

China

49

97

Sequencing

NA

+

-

-

-

5-7

Zhang 2007

Cohort

China

32

32

Sequencing

NA

+

+

-

-

6-8

Zheng 2011

PCC

China

156

185

Sequencing

B, C, E

-

-

-

-

7

Zhu 2008

NCC

China

20

83

Sequencing

C

+

+

+

+

≥8

Abbreviations: NA, not available; PCC, prevalence case-control; NCC, nested case-control; ICC, incidence case-control; SSCP, Single-strand conformation polymorphism; INNO-LiPA, Innogenetics line-probe assay.

In addition, we performed a subgroup analysis to compare A1762T/G1764A dual mutations between ASC, CHB, LC and HCC patients. Compared to LC patients, HCC patients had a higher risk of A1762T/G1764A dual mutations (OR = 1.55, 95% CI = 1.06-2.26, P = 0.02, Table 3 and Supplementary Figure S2). Moreover, a subgroup analysis of four studies [12, 16, 47, 54] with a random-effects model revealed no statistically significant difference between cirrhotic HCC (LC-HCC) and non-cirrhotic HCC (non-LC-HCC) patients in terms of the frequency of A1762T/G1764A dual mutations (OR = 2.06, 95% CI = 0.99-4.28, P = 0.05, Table 3 and Supplementary Figure S3). This result should be reevaluated in studies with larger samples sizes. We performed another comparison of BCP dual mutations between non-LC-HCC and non-LC patients, including CHB and ASC patients. Using a random-effects model, a meta-analysis of four studies [12, 16, 47, 54] showed that the non-LC-HCC patients had a significantly higher frequency of A1762T/G1764A dual mutations compared with the CHB and ASC patients (OR = 2.16, 95% CI = 1.08-4.32, P = 0.03, Table 3 and Supplementary Figure S4). Given the above results, we assumed that BCP A1762T/G1764A dual mutations might promote hepatocarcinogenesis even without progression to cirrhosis.

Table 3: Frequency of BCP A1762T/G1764A dual mutations from chronic HBV-infected patients, including HCC

Comparisons

Studies

A1762T/G1764A dual mutation, n (%)

Heterogeneity

OR

95% CI

P value

Chi2

df

P

I2 (%)

HCC

[4-38, 40-55]

2480/3729 (66.5)

270.4

50

< 0.00001

82

3.05

2.35-3.95

< 0.00001

Non-HCC

2594/6511 (39.8)

HCC

[5, 9, 12-14, 16, 18, 23, 25, 28,

30, 34, 36, 41, 46-48, 51, 55]

544/808 (67.3)

33.38

18

0.02

46

1.55

1.06-2.26

0.02

LC

441/708 (62.3)

HCC

[5, 9, 12, 13, 20-23, 25, 28-30,

33, 34, 36, 41, 47, 48, 51, 53, 55]

927/1300 (71.3)

72.49

20

< 0.00001

72

2.87

1.96-4.20

< 0.00001

CHB

796/1566 (50.8)

HCC

[5, 7-9, 16, 17, 22-25, 30, 31,

36, 41, 44, 51]

591/924 (64.0)

70.33

15

< 0.00001

79

5.59

3.17-9.83

< 0.00001

ASC

246/1068 (23.0)

LC-HCC

[12, 16, 47, 54]

327/476 (68.7)

6.57

3

0.09

54

2.06

0.99-4.28

0.05

Non-LC-HCC

93/165 (56.4)

Non-LC-HCC

[12, 16, 47, 54]

93/165 (56.4)

7.60

3

0.05

61

2.16

1.08-4.32

0.03

CHB/ASC

225/466 (48.3)

HCC, hepatocellular carcinoma; LC, liver cirrhosis; CHB, chronic hepatitis B; ASC, asymptomatic HBsAg carriers; LC-HCC, cirrhotic hepatocellular carcinoma; Non-LC-HCC, non-cirrhotic hepatocellular carcinoma.

Additionally, A1762T/G1764A dual mutations contributed to a higher risk of HCC occurrence compared with CHB patients (OR = 2.87, 95% CI = 1.96-4.20, P < 0.00001, Table 3 and Supplementary Figure S5). A similar trend in A1762T/G1764A dual mutations was found in HCC and ASC patients. HCC patients had a significantly higher frequency of A1762T/G1764A dual mutations than the ASC patients (OR = 5.59, 95% CI = 3.17-9.83, P < 0.00001, Table 3 and Supplementary Figure S6). In summary, the frequency of A1762T/G1764A dual mutations increased in this manner: from ASC to chronic disease, and then to HCC (Supplementary Table S1).

BCP A1762T/G1764A dual mutations in HBV genotype B and C from chronic HBV infection patients, including HCC.

Figure 2: BCP A1762T/G1764A dual mutations in HBV genotype B and C from chronic HBV infection patients, including HCC.

A1762T/G1764A mutations in different HBV genotypes

Heterogeneity was significant among the included studies [12, 20, 30, 31, 39, 47], which described A1762T/G1764A dual mutations from chronic HBV infection in HBV genotypes B and C. Chronic HBV-infected patients with genotype B had a significant lower incidence of A1762T/G1764A dual mutations compared with those with genotype C (OR = 0.30, 95% CI = 0.18-0.52, P < 0.0001, Figure 2.1); no heterogeneity was found when we compared BCP dual mutations between HBV genotypes B and C in HCC patients (P = 0.27, I2 = 24%). Similarly, HCC patients with HBV genotype B had lower levels of A1762T/G1764A dual mutations compared with those with genotype C (OR = 0.34, 95% CI = 0.13-0.94, P = 0.04, Figure 2.2). In HBV genotype C subjects, BCP A1762T/G1764A dual mutations contributed to significantly higher risk for HCC developing compared with non-mutation ones (OR = 3.47, 95% CI = 2.28-5.27, P < 0.00001, Figure 3.1). Heterogeneity was significant between studies comparing HCC occurrence in HBV genotype B (P < 0.00001, I2 = 93%), no difference was found (P = 0.34, Figure 3.2).

HCC occurrence between BCP A1762T/G1764A dual mutation and non-BCP dual mutation patients with HBV genotype C and genotype B.

Figure 3: HCC occurrence between BCP A1762T/G1764A dual mutation and non-BCP dual mutation patients with HBV genotype C and genotype B.

A1762T/G1764A mutations from HBeAg-positive and HBeAg-negative patients

Nine studies [7, 12-14, 29, 37, 40, 42, 54] reported A1762T/G1764A dual mutations based on HBeAg status in chronic HBV infection. Heterogeneity was significant among these studies (P < 0.00001, I2 = 81%), and no significance in BCP A1762T/G1764A mutations was observed in either HBeAg-positive or HBeAg-negative chronic HBV-infected patients (OR = 1.06, 95% CI = 0.63-1.78, P = 0.83, Figure 4.1). In HCC, there was no difference of A1762T/G1764A mutations between HBeAg-positive and HBeAg-negative patients (OR = 1.38, 95% CI = 0.84-2.27, P = 0.20, Figure 4.2).

Comparison of BCP A1762T/G1764A dual mutations grouped by HBeAg status from chronic HBV infection patients, including HCC.

Figure 4: Comparison of BCP A1762T/G1764A dual mutations grouped by HBeAg status from chronic HBV infection patients, including HCC.

DISCUSSION

HCC is one of the most lethal cancers [56, 57]. The most common naturally occurring mutations in the BCP A1762T/G1764A dual mutation has been associated with hepatocarcinogenesis, but conflicts still exist [1,3]. Our meta-analysis showed that the A1762T/G1764A dual mutation was present at statistically significantly higher frequencies in HCC patients than in non-HCC controls, including LC, CHB and ASC patients. As the disease progressed, the risk of the A1762T/G1764A dual mutation in ASC, CHB, LC and HCC patients increased. Therefore, we hypothesized that A1762T/G1764A dual mutation might play a vital role in promoting disease progression in chronic HBV infection. Several previous case series have also shown a higher frequency of BCP T1762/A1764 mutation in patients with cirrhosis than in inactive carriers or patients with chronic hepatitis [16, 30, 58]. Our results confirmed this observation. However, the BCP A1762T/G1764A dual mutation is increasingly more prevalent as chronic HBV infection progresses from the asymptomatic HBsAg carrier state to liver cirrhosis or HCC, indicating that these mutations accumulate before the diagnosis of HCC. This finding suggests that BCP dual mutation may be a promising biomarker for predicting the clinical outcomes of patients with chronic HBV infection, particularly in terms of predicting whether they will develop HCC. Additionally, we performed a subgroup analysis that revealed no statistically significant difference in terms of the frequency of A1762T/G1764A dual mutations in liver cirrhotic HCC and non-cirrhotic HCC patients. Thus, we assumed that BCP A1762T/G1764A dual mutations might promote hepatocarcinogenesis even without progression to cirrhosis. Because the samples of non-cirrhotic patients with HCC in this investigation were relatively small, this hypothesis must be proven based on future studies with larger patient cohorts.

HBV genotype is associated with the risk of HCC. One possible reason for this association is that HBV mutations may be more common in some HBV genotypes than in others [2]. In this research, we found that chronic HBV-infected patients with genotype B had a significantly lower risk of A1762T/G1764A dual mutations compared with those with genotype C. In HCC, patients with HBV genotype C suffered higher A1762T/G1764A dual mutations compared with those with genotype B. Previous reports have shown that patients with genotype C infection have a higher positive rate of HBeAg, more severe liver disease and higher risk of developing HCC [30, 59-61]. On the basis of previous research and our results, we suggested that HBV genotype C patients tend to have a higher proportion of BCP T1762/A1764 mutations in conjunction with chronic HBV infection, including HCC [31, 58].

It has been suggested that the A1762T/G1764A double mutation in the BCP region can reduce the synthesis of HBeAg and enhance viral replication. However, this analysis found no significance of BCP A1762T/G1764A mutations from HBeAg-positive and HBeAg-negative chronic HBV-infected patients. The same finding was true for HCC patients. When introduced into wild-type HBV genomes, the BCP dual mutation indeed decreased HBeAg expression and enhanced viral genome replication of about twofold [60,62]. Transfection studies have shown that the T1762A/G1764A dual mutations decrease the level of pre-C mRNA by 50% to 70%, thereby inhibiting HBeAg production. A previous report showed that HBeAg titers are closely correlated with BCP mutation, and HBeAg-positive patients of genotype C infection had a higher prevalence of the A1762T/G1764A mutations [63]. Although no difference of BCP dual mutations was found in our meta-analysis, further research focusing on the mechanisms and the relationship between BCP mutation and HBeAg status is needed.

This meta-analysis had the following limitations. First, patients in the included studies had different HBV genotypes, different ethnicities, leading to significant heterogeneity; second, most of the included studies had small samples, with mid- to low-quality designs. In the future, high-quality, well-designed research focused on the mechanisms of BCP dual mutation in the progression of chronic HBV infection and HCC must be performed.

Based on our results, we could conclude that BCP A1762T/G1764A dual mutations are associated with disease progression and HCC occurrence in chronic HBV-infected patients. Additionally, this mutation might promote hepatocarcinogenesis even without progression to cirrhosis. Considered the frequency ratio of A1762T/G1764A dual mutation from ASC, CHB, LC and HCC, we suggested that BCP dual mutation should be screened in the CHB and LC patients.

MATERIALS AND METHODS

Search strategy and study selection

We searched PubMed, Ovid, Web of Science, and Cochrane Library databases for studies published to June 1, 2015. The following medical subject headings were used: “hepatocellular carcinoma;” “hepatitis B virus X protein;” “mutation;” “basal core promoter;” “A1762T;” “G1764A;” and “variation.” Electronic searches were supplemented with manual searches of reference lists used in all retrieved review articles, primary studies, and abstracts from meetings to identify other studies not found in the electronic searches. The literature was searched by two authors (Z Yang and L Zhuang) independently.

Two authors independently selected studies and discussed them with each other when inconsistencies were found. Articles that satisfied the following criteria were included: (1) case-control or cohort studies, (2) HCC and control subjects, including ASC, CHB or LC patients, (3) BCP A1762T/G1764A dual mutations (for HBV mutation), (4) HCC outcomes, and (5) available full texts. If the duration and sources of the study population recruitment overlapped by more than 30% in two or more papers by the same authors, we only included the most recent study or the study with the larger number of HCC patients. The following exclusion criteria were applied: (1) studies that included patients who were coinfected with hepatitis A, C, D, E virus or human immunodeficiency virus, had alcohol-related liver diseases, or had previously received antiviral treatments, (2) studies without control subjects, and (3) studies that only investigated A1762T or G1764A single mutations.

Data extraction and methodological quality assessment

In our evaluation of the BCP A1762T/G1764A dual mutant, subjects with either a single mutation or a deletion at either site were not included in the analysis. Two researchers independently read the full texts and extracted the following information: publication data; study design; sample size; HBV genotypes; country or area; and study matching factors. The methodological qualities of the included studies were assessed according to the report by Liu et al. [64]. A 12-point scoring system was used that was based on factors that indicated good-quality observational studies, as shown in Table 1. Two authors (ZG Yang and LP Zhuang) independently assessed the study quality, and inconsistency was discussed with another reviewer-author (X Chen), who acted as an arbiter.

Definitions

All diagnoses should be made according to the guidelines. ASC were defined as being seropositive for HBsAg for at least 6 months, having no evidence of LC or HCC based on the clinical criteria and undergoing ultrasound examination, with normal alanine aminotransferase (ALT). CHB was defined as being seropositive for HBsAg for at least 6 months, having persistent or intermittently elevated ALT levels or a liver biopsy showing chronic hepatitis with moderate or severe necroinflammation, with no clinical evidence of LC. The patients with LC were diagnosed by histologic analysis of liver biopsy specimens or by findings of repeated ultrasonography that were suggestive of cirrhosis and supplemented with clinical criteria indicating portal hypertension (i.e., the presence of ascites, thrombocytopenia, and esophageal varices). HCC was defined by at least one of the following criteria: (1) liver biopsy or (2) elevated alpha-fetoprotein levels and sonography, computed tomography or magnetic resonance imaging evidence.

Statistical methods

The effect measures of interest were odds ratios (ORs) and the corresponding 95% confidence intervals (CIs). Heterogeneity across studies was informally assessed by visually inspecting forest plots and then formally estimated by Cochran’s Q test in which chi-squared distribution was used to make inferences regarding the null hypothesis of homogeneity (considered significant at P < 0.10). A rough guide to our interpretation of I2 follows:

• 0% to 40% shows that heterogeneity may not be important

• 30% to 60% corresponds to moderate heterogeneity

• 50% to 90% indicates substantial heterogeneity

• 75% to 100% indicates considerable heterogeneity [65, 66]

If the eligibility of some studies in the meta-analysis was uncertain because of missing information, a sensitivity analysis was performed by conducting the meta-analysis twice: in the first meta-analysis, all studies were included; in the second meta-analysis, only studies that were definitely eligible were included. A fixed-effects model was initially used for our meta-analyses; a random-effects model was then used in the presence of heterogeneity. Description analysis was performed when the quantitative data could not be pooled. Review Manager software, version 5.1, was used for data analysis. All statistical tests were two-tailed, and differences with P < 0.05 were considered statistically significant.

ACKNOWLEDGMENTS

This work was not supported by any pharmaceutical company or government agency or grants from other sources.

CONFLICTS OF INTEREST

There is no conflict of interest.

Editorial note

This paper has been accepted based in part on peer-review conducted by another journal and the authors’ response and revisions as well as expedited peer-review in Oncotarget.

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