Distinct clinical characteristics and poor prognosis for diffuse large B-cell

lymphoma patients with hepatitis B virus infection in China: a systematic review

and meta-analysis

Minyue Zhang1*, Fei Gao2*, Guiqi Zhu3, Yanwei Zhao4, Beiwen Ni1, Honghui Huang1,

Jian Hou1

1. Division of Hematology, Renji Hospital, School of Medicine, Shanghai Jiaotong

University, 160 Pu Jian Road, Shanghai, 200127, China

2. College of Pharmacy, Chengdu University of Traditional Chinese Medicine,

Chengdu, 611730, China

3. Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai 200032,

China; State Key Laboratory of Genetic Engineering, Fudan University, Shanghai,

200032, China

4. Division of Hospitalist, Shanghai Jiahui International Hospital, 689 Quiping Road,

Shanghai, 200233, China

*Co-first authors, these authors contributed equally to this work

Corresponding author: Honghui Huang, Email: honghui_huang@163.com

Jian,Hou, Email: houjian@medmail.com.cn

Abstract

Background: China is an endemic area of hepatitis B virus (HBV) infection. Plenty

of studies showed that HBV infection was associated with diffuse large B-cell

lymphoma (DLBCL). However, no clear consensus was reached on the impact of

HBV infection on DLBCL patients’ clinical features and outcomes. We therefore

performed a systematic review and meta-analysis to explore the clinical

characteristics and survival of DLBCL patients with HBV infection (Hepatitis B virus

surface antigen positive, HBsAg+) in the Chinese population.

Methods: PubMed, Embase and Cochrane Library database were searched for

retrospective study up to Apr 30, 2018. Hazard ratio (HR) or odds ratio (OR)

corresponding to 95% confidence interval (CI) were calculated to estimate the

outcomes. Publication biases were assessed by Egger's test, Begg's test and funnel

plots. Statistical analyses were performed by the software RevMan 5.2 and Stata

version 10.0.

Results: Total 8 studies were included in the current review, comprising of 493

HBsAg+ DLBCL patients and 1779 HBsAg– DLBCL patients. Compared with

HBsAg– DLBCL patients, significantly shorter overall survival (HR 1.62; 95% CI

1.36~1.93, P<0.00001) and progress-free survival (HR 2.04; 95% CI 1.64~2.53,

P<0.00001) were demonstrated in HBsAg+ DLBCL patients. The treatment response

in HBsAg– DLBCL patients was slightly better than that in HBsAg+ DLBCL

patients. Furthermore, HBsAg+ patients were characterized by a younger age of

disease onset (OR 3.35; 95% CI 2.23~5.02, P<0.00001), advanced disease stages (OR

1.88; 95% CI 1.50~2.36, P<0.00001), higher level of LDH (OR 1.46; 95% CI

1.18~1.80, P=0.0005) and more frequent involvement of spleen (OR 3.40; 95% CI

2.40~4.80, P<0.00001) at diagnosis in comparison to HBsAg– DLBCL patients.

However, no significant difference was found in International Prognostic Index scores

between two groups.

Conclusions: The current study showed that DLBCL patients with HBV infection

displayed distinct clinical features and inferior prognosis thereby indicating HBV-

associated DLBCL can be categorized as a separated subtype of DLBCLs.

Key words: diffuse large B-cell lymphoma, HBV infection, prognosis, meta-analysis

Background

Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoid malignancy

characterized by heterogeneous clinical and genetic features, representing about 30-

40% of non-Hodgkin’s lymphoma (NHL) worldwide [1]. Increasing evidences

suggested that viral infection contributed to the pathogenesis of different subtypes of

lymphoma, such as Epstein-Barr virus (EBV) in Hodgkin disease or Burkitt’s

lymphoma, human T-cell leukemia virus type 1 (HTLV-1) in adult T cell leukemia

and lymphoma, hepatitis C virus (HCV) in marginal zone lymphoma [2-4]. According

to the 2016 revision of the World Health Organization classification of lymphoid

neoplasms, EBV-positive DLBCL, human herpesvirus type-8 (HHV-8)-positive

DLBCL, have been classified as separated subtypes of DLBCL[5]. These new

categories strongly suggested that DLBCL patients with specific infection displayed a

distinct clinical manifestations and prognosis.

Hepatitis B virus (HBV) is primary hepatotrophic virus and is well known for

contributing to the development of hepatic cirrhosis and hepatocellular carcinoma.

Moreover, HBV was also found to infect peripheral blood mononuclear cells and

lymphoblastoid cell lines[6]. High level of HBV-DNA could be detected in tumour

biopsies from B-cell lymphoma patients with HBV infection [7], hence indicating that

HBV was also lymphotropic. A recent meta-analysis has also provided quantitative

evidence that HBV infection can lead to a 2.06-fold increased risk of developing

DLBCL[8]. Nevertheless, the pathogenesis and clinical characteristic of HBV-

associated lymphoma is still undefined.

China is an endemic area of HBV infection where the prevalence of Hepatitis B

virus surface antigen (HBsAg) seropositive is 7.2% nationwide [9]. Therefore, arising

clinical attention to the association of HBV infection and the clinical outcomes of

DLBCL patients in China was paid[10-15]. However, no clear consensus has been

reached about the impact of HBV infection on DLBCL patients’ clinical

manifestation and survival based on existing retrospective studies with small sample

size. In view of the limitations of previous studies, we hereby performed a systematic

review and meta-analysis aiming to evaluate the clinical features and prognosis in

DLBCL patients with HBV infection.

Materials and methods

Identification of relevant studies

To identify all studies that explored the impact of HBV infection on the clinical

characteristics and outcome of DLBCL patients, a literature search of PubMed,

Embase and Cochrane Library database up to Apr 30, 2018 was conducted. The

search strategy was based on combinations of the following keywords and search

terms: (“lymphoma” [MeSH] or “Lymphoma, Non-Hodgkin” [MeSH] or

“Lymphoma, Large B-Cell, Diffuse” [MeSH] or Non-Hodgkin lymphoma or diffuse

large B-cell lymphoma or NHL or DLBCL) AND (“hepatitis B” [MeSH] or hepatitis

B virus or HBV or Hep B virus or Hep B). The language of publication was limited to

English. We also screened the references of retrieved studies, meeting abstracts,

relevant meta-analyses and systematic reviews. Case reports, editorials and review

articles were excluded. When a publication overlapped with other publication of the

same trial, only the article with more details or the most recent article was retained.

Selection criteria

The studies included in the meta-analysis should satisfy all the following criteria: (1)

the study population was Chinese DLBCL patients; (2) two groups (i.e. HBsAg+ and

HBsAg–) of DLBCL patients were included; (3) clinical features and survivals

between two groups of patients were compared. Exclusion criteria were: (1) patients

had co-infection with human immunodeficiency virus (HIV) or other hepatitis viruses

(HCV, hepatitis D virus [HDV]); (2) patients with an occult HBV infection [HBsAg

negative and hepatitis B core antibody (HBcAb) positive] as case group; (3) patients

with other types of lymphoma. When the relevant data was not reported in paper, we

contacted the author to get the relevant information by e-mail or telephone.

The primary outcome was survival outcome, including overall survival (OS) and

progress-free survival (PFS). OS was defined as the time from diagnosis to death from

any causes or last follow-up. PFS was defined as the time from the date of diagnosis

to the date of first progression, death from any causes or last follow-up. The

secondary outcome measured treatment response, including rate of complete response

(CR) and overall response (ORR), and clinical characteristic including age of onset,

advanced disease stage (Ann Arbor staging III/IV), elevated LDH level, involvement

of spleen and International Prognostic Index (IPI) score (3~5 points).

Data extraction and study quality

Two reviewers (Zhang M., and Gao F.) independently extracted data and outcomes

using an electronic standard form. The following information from each study was

summarized: (1) first author, (2) year of publication, (3) use of rituximab, (4)

frequency of HBV reactivation, (5) prophylactic interventions for HBV reactivation,

(6) follow-up duration, (7) number of patients with HBsAg seropositivity and

seronegativity, (8) patient’s characteristics including age of onset (<60 years old),

advanced disease stage (Ann Arbor staging III/IV), elevated LDH level, involvement

of spleen, IPI score (3~5 points), treatment response (CR and ORR), OS and PFS.

Any discrepancies amongst the two reviewers were resolved by an additional

investigator, Zhu G.

Quality assessment

Newcastle-Ottawa Quality Assessment Scale (NOS) was adopted to assess the

methodological quality of the included studies[16]. The following three items were

evaluated including: (1) patient selection, (2) comparability of interventions and

observations group, and (3) assessment of outcome.

Statistical analysis

Hazard ratio (HR) corresponding to 95% confidence interval (CI) were used to

appraise OS and PFS. If HRs and 95% CIs were not obtained from the original article,

Kaplan–Meier curves of the included studies were read and re-analyzed by software

Engauge digitizer which HRs and 95% CIs were indirectly calculated from Kaplan–

Meier curve using Tierney’s methods [17]. Odds ratio (OR) corresponding to 95% CI

were calculated to estimate other outcomes. Publication biases were assessed by

Egger's test, Begg's test and funnel plots. The methods of meta-analysis and

publication biases tests were detailed in our previous publication[18-21].

Statistical analysis was performed by software ReviewManager 5.2 (The Cochrane

Collaboration, Oxford, UK) and Stata version 10 (Stata Corp, College Station, Texas,

USA). All P-values were both-sided. P-value of <0.05 was considered significance.

Results

Characteristics of studies

Through the comprehensive search and selection based on the criteria above, 1703

articles were identified as potentially relevant publications. Upon further assessment

of the full text, 13 publications didn’t meet the inclusion criteria and were excluded.

Eventually, 8 articles[10-15, 22, 23] with a total of 2272 DLBCL patients, including

493 patients with HBsAg seropositive and 1779 patients with HBsAg seronegative,

were deemed suitable for the meta-analysis (Figure 1). The studies were published

between 2008 and 2018. Sample size was ranged from 81 to 587. The HRs with 95%

CIs could be directly obtained in 2 original studies. Detailed characteristics of the

eligible studies were outlined in Table 1. With regards to the methodological quality,

all the included studies had reliable quality as indicated by NOS scores > 6 points

(Table 1).

OS

A total of 8 studies, comprising of 493 DLBCL patients with HBsAg seropositive and

1779 DLBCL patients with HBsAg seronegative, were included for the assessment of

OS. The fixed-effects model was used to calculate the result due to non-significant

heterogeneity (P = 0.11). The result indicated that HBsAg+ patients had significantly

worse OS than HBsAg–patients (HR 1.62; 95% CI 1.36~1.93, P<0.00001) (Figure.

2A)

PFS

Among all 8 studies, only 4 studies (i.e. 250 DLBCL patients with HBsAg

seropositivity and 953 DLBCL patients with HBsAg seronegativity), were available

for PFS analysis. The fixed-effects model was used to calculate the result as there was

no significant heterogeneity (P = 0.33). Meta-analysis revealed that HBsAg+ patients

showed significantly reduced PFS as compared with the HBsAg– patients (HR 2.04;

95% CI 1.64~2.53, P<0.00001) (Figure. 2B).

Treatment response

Treatment responses including CR and ORR rate were analyzed by using random-

effects model as the heterogeneity tests suggested significant heterogeneity (P CR =

0.04, P ORR = 0.02). The combined results showed that there was a trend that HBsAg–

DLBCL patients achieved a slightly better treatment response than HBsAg+ DLBCL

patients (OR CR 0.60, 95% CI 0.34~1.04, P=0.07; OR ORR 0.60, 95% CI 0.34~1.05,

P=0.07) (Figure. 3A, B).

Clinical characteristics

Clinical characteristics of DLBCL patients, including age of disease onset (<60 years

old), advanced disease stage (Ann Arbor staging III/IV), elevated LDH level,

involvement of spleen and IPI score (3~5 points) at diagnosis were also

comprehensively investigated between two groups of patients. The results of meta-

analysis were summarized in Figure. 4 A-E. DLBCL patients with HBsAg

seropositivity were associated younger age of onset (OR 3.35; 95% CI 2.23~5.02,

P<0.00001), more advanced disease stage (OR 1.88; 95% CI 1.50~2.36, P<0.00001),

higher level of LDH (OR 1.46; 95% CI 1.18~1.80, P=0.0005) and more frequent

involvement of spleen (OR 3.40; 95% CI 2.40~4.80, P<0.00001). However, there was

no significant difference in IPI score (OR 0.97; 95% CI 0.55~1.69, P=0.91) between

the two groups.

Sensitivity analysis

Due to the significant heterogeneity observed among the included studies in meta-

analysis of treatment response, sensitivity analysis was performed by a sequential

exclusion of individual studies to check the origin of the heterogeneity and specific

sensitivity of the findings. Sensitivity analysis showed the study of Deng et al[10]

was responsible for such heterogeneity, removal of which could not affect the overall

ORs. For IPI score outcome, the heterogeneity still existed even though studies were

excluded one by one.

Publication bias

We conducted Begg’s test and Egger's test to assess the publication bias. As was

shown in Table 2, no significant publication bias was observed for all the outcomes.

Furthermore, funnel plots of publication bias were symmetrical (figure not shown)

which also suggested the absence of publication bias.

Discussion

In this study, we performed a systematic review and meta-analysis to evaluate the

impact of HBV infection on Chinese DLBCL patients. The results indicated that in

comparison with HBsAg– DLBCL patients, HBsAg+ DLBCL patients had

significantly inferior survival, younger age of onset, more advanced disease stage,

higher level of LDH, more frequent involvement of spleen and slightly worse

treatment response.

Although substantial evidence has demonstrated the association between DLBCL

and HBV infection [8, 24-28], the pathogenic mechanism of HBV contributing to

DLBCL was still unclear. Two different models of HBV infection-driven DLBCL

were proposed [10, 13]. Deng et al suggested HBV-associated antigen stimulation as a

likely mechanism of HBV-associated DLBCL. It was evident by long history of

chronic HBV infection, frequent involvement spleen and retroperitoneal lymph nodes,

strongly biased usage of both Ig heavy and light chain genes, and high homology of

CDR3 sequence to specific antibodies for HBsAg [10]. On the other hand, Ren et al

considered HBV-driven mutagenesis in a hit-and-run manner as an alternative

mechanism of HBV-driven DLBCL [13], as supported by a distinctive molecular

profile of HBV-associated DLBCLs via whole-genome/exome and transcriptomic

sequencing. They have found that HBsAg+ DLBCL patients have enhanced mutation

activity. Several genes, such as BCL6, KLF2 and ZFP36L1 were preferentially

mutated in HBsAg+ DLBCL patients, which mainly affected p53 signaling, FOXO

signaling and immune evasion signaling pathway.

In our study, we found that HBsAg+ DLBCL patients showed a poorer outcome,

which was consistent with the results form two individual retrospective studies[10,

29]. The poor outcome of HBsAg+ DLBCL patients might be mainly due to early

disease progress[10]. In the era of rituximab, the treatment response and prognosis of

DLBCL patients have been significantly improved[30, 31]. However, can DLBCL

patients with HBV infection benefit from rituximab treatment? Based on the existing

studies, it revealed that rituximab-containing chemotherapy (R-CHOP) did not seem

to overcome the inferior outcome conferred by HBV infection when compared with

rituximab-absent chemotherapy (CHOP)[10, 29], as evidenced by no significant

difference of OS and PFS between HBsAg + DLBCL patients receiving R-CHOP and

CHOP chemotherapy. Therefore, new therapeutic strategies specific for these patient

subgroups is of crucial demand. On the other hand, BCL6 genetic alternations were

frequently observed in HBsAg+ tumors[13], and BCL-6 inhibitor, FX1, was

demonstrated to suppress the growth of DLBCL cells in vitro and in vivo[32].

Therefore, targeted therapy against BCL6 might serve as adjuvant therapy to improve

the clinical prognosis in HBV-infected DLBCL patients.

To the best of our current knowledge, this is the first study which systematically

assessing the clinical features of HBV-associated DLBCL. Hepatitis B virus is

endemic in China while DLBCLs display high genetic and ethnic heterogeneity,

leading us to focus on the Chinese DLBCL population. There have been in total eight

studies included for meta-analysis. The methodological quality of included studies

was moderate to high by NOS. Although the sample size in analysis was not large

enough, low heterogeneity and no publication bias were observed, supporting the

reliability of our results. However, there were three major limitations in this study.

Firstly, during the evaluation of OS and PFS, most HR and 95% CI of individual

studies could not be obtained from the original article hence they were indirectly

calculated from Kaplan–Meier curve. The results calculated by this method only

reflected the association between HBV infection and DLBCL patients’ survival. Other

risk factors (e.g. Ki-67 expression, MYC/BCL2 protein coexpression, usage of

rituximab) which could also predict DLBCL patient prognosis [33-35], could not be

taken into account for in-depth analysis. Wei et al reported that Therefore, based on

our results, whether HBV infection was an independent poor predictor of survival

remained elusive. Further Cox multivariate analysis with large sample size was

preferred for justification.

Secondly, hepatic dysfunction is a common complication of chemotherapy in

HBsAg+ DLBCL patients due to HBV reactivation. Hepatic dysfunction during

chemotherapy may terminate or delay chemotherapy. But whether it could lead to

inferior prognosis in HBsAg+ DLBCL patients remained unknown. Based on the

exiting study, no relevant data was available for analysis to clarify this issue.

Lastly, the impact of HBV occult infection on DLBCL failed to be evaluated in our

meta-analysis despite high incidence of occult HBV infection was found in B-NHL

patients[36]. Deng et al reported that there was a trend that occult HBV-infection

DLBCL patients had inferior survival compared with those without HBV

infection[10]. Therefore, the association study of occult HBV-infection and DLBCL

was required in the future.

Conclusions

This meta-analysis provided the compelling evidence about a direct link between

HBV infection and DLBCL, which led to a better understanding of the clinical

characteristic and survival of HBsAg+ DLBCL patients. Due to the unique clinical

and molecular features, HBV-associated DLBCL could be categorized as a subtype of

DLBCL.

Abbreviations

HBV: hepatitis B virus; DLBCL: diffuse large B cell lymphoma; NHL: non-

Hodgkin’s lymphoma; HBsAg: Hepatitis B virus surface antigen; HR: hazard ratio;

OR: odds ratio; CI: confidence interval; EBV: Epstein-Barr virus; HTLV-1: human T-

cell leukemia virus type 1; HCV: hepatitis C virus; HIV: human immunodeficiency

virus; HDV: hepatitis D virus; HBcAb: hepatitis B core antibody; OS: overall

survival; PFS: progress-free survival; CR: complete response; ORR: overall response;

IPI: International Prognostic Index; NOS: Newcastle-Ottawa Quality Assessment

Scale

Authors’ contributions

Ni B and Zhu G performed literature research. Zhang M, Gao F and Zhu G extracted

and analyzed the data, Zhang M, Zhao Y and Gao F wrote the manuscript; Huang H

and Hou J conceived and designed this study. All authors reviewed and approved the

final manuscript.

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

This work was supported by Health industry research projects of Pudong New Area

health and Family Planning Commission (PW2015E-1) and Project of Further

Accelerating Development of Traditional Chinese Medicine in Shanghai

(ZY3-CCCX-3-3037).

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Table 1. Baseline characteristics of included studies

Author Year Number of patients Median follow-up

month (range)

Use of rituximab % HBV reactivation % HBV reactivation

prophylaxis

NOS Hazard ratio

HBsAg+ HBsAg– HBsAg+ HBsAg- HBsAg+ HBsAg-

Deng LJ 2015 81 506 41 (1-135) 40.7 53.6 6.2 0 Yes 8 Extrapolated

Law MF 2015 16 65 47.8 (1.1–188) 50 48 - - LAM 9 Reported

Liu WP 2016 46 61 24.8 (2.57-62.8) 63 90.2 21.7 0 LAM, ENT, TDF, ADV 8 Extrapolated

Liu ZH 2017 30 51 26.4 (3-65) 43.3 64.7 10 0 LAM, ENT 9 Reported

Ren WC 2018 56 219 - - - - - - 7 Extrapolated

Wang F 2008 81 181 - <10 <10 - - LAM 7 Extrapolated

Xie WZ 2013 90 361 27 (1-76) 54.4 68.1 - - LAM 8 Extrapolated

Zhao XY 2018 93 335 - 53.8 64.8 - - - 8 Extrapolated

LAM = lamivudine; ETV = entecavir; ADV = adefovir; TDF = tenofovir;

Table 2. Results of Begg’s test and Egger’s test

Outcome P Begg’s test P Egger’s test

OS 0.900 0.455

PFS 1.000 0.319

CR 0.734 0.604

ORR 0.806 0.467

Age of onset 1.000 0.970

Advanced disease stage 0.711 0.380

Elevated LDH level 1.000 0.947

Spleen involvement 0.734 0.394

IPI score 0.452 0.529

Figure legend:

Figure 1. Literature search and selection.

Figure 2. Meta-analysis of the association between status of HBsAg and survival of

DLBCL patients. (A) OS and (B) PFS.

Figure 3. Meta-analysis of the association between status of HBsAg and treatment

response of DLBCL patients. (A) CR rate and (B) ORR rate.

Figure 4. Meta-analysis of the association between status of HBsAg and clinical

characteristic of DLBCL patients. (A) Age of disease onset (< 60 years old); (B) IPI

score (3~5); (C) Involvement of spleen; (D) Advanced disease stage (Ann Arbor

staging III/IV); and (E) Elevated LDH level.

Figure 1. Literature search and selection.

Figure 2. Meta-analysis of the association between status of HBsAg and survival of

DLBCL patients. (A) OS and (B) PFS.

Figure 3. Meta-analysis of the association between status of HBsAg and treatment

response of DLBCL patients. (A) CR rate and (B) ORR rate.

Figure 4. Meta-analysis of the association between status of HBsAg and clinical

characteristic of DLBCL patients. (A) Age of disease onset (< 60 years old); (B) IPI

score (3~5); (C) Involvement of spleen; (D) Advanced disease stage (Ann Arbor

staging III/IV); and (E) Elevated LDH level.