Epstein–Barr Virus Status and Its Prognostic Value in Lymphoma

Research Article

Ann Hematol Oncol. 2016; 3(5): 1094.

Epstein–Barr Virus Status and Its Prognostic Value in Lymphoma

Wang BJ, Cen XN*, Ren HY*, Liu W, Liang ZY, Qiu ZX, Ou JP, Wang WS, Li Y, Dong YJ, Wang MJ, Wang LH and Wang Q

Department of Hematology, Peking University First Hospital, China

*Corresponding author: Xi-Nan Cen, Department of Hematology, Peking University First Hospital, No.8 Xi Shi ku Street, Xi Cheng District, Beijing, China

Han-Yun Ren, Department of Hematology, Peking University First Hospital, No.8 Xi Shi Ku Street, Xi Cheng District, Beijing, China

Received: June 14, 2016; Accepted: July 25, 2016; Published: July 27, 2016


Aim: EBV status is closely associated with the oncogenesis and clinical course of lymphoma, but the significance of EBV status discords. We conducted our study to investigate the EBV status among different types of lymphoma and evaluate the prognostic value of different detections of EBV.

Methods: 263 lymphoma patients who received EBER-ISH, LMP-1 staining or peripheral blood EBV-DNA quantification were recruited. EBV status was demonstrated according to different lymphoma types and the correlations were analyzed between different detections of EBV status and lymphoma clinical courses.

Results: EBV status varies among different types of lymphoma, in which Extra nodal NK/T cell lymphoma, nasal type and angioimmunoblastic T cell lymphoma had relatively high EBER and EBV-DNA positive rate while diffuse large B cell lymphoma(unspecified) and follicular lymphoma often only has concomitant EBV virosis. EBV-DNA positive in EBER/LMP-1 positive lymphoma was associated with worse IPI scores (P=0.030) and initial treatment outcome (P=0.027), furthermore, the EBV-DNA positive did markedly influence OS (P=0.005) and PFS (P=0.003). With regard to the different subtypes, EBER/ LMP-1 positive did not make differences to the OS and PFS of T-NHL, B-NHL and HL. Whereas, T-NHL and B-NHL with positive EBV-DNA demonstrated substantially poorer OS and PFS.

Conclusion: Although EBV status varies among different lymphoma types, positivity of peripheral blood EBV-DNA is often associated with the presence of B symptoms, elevated LDH level and high risk IPI scores. These patients are likely to pursue a more deteriorating clinical course with poorer treatment response, OS and PFS.

Keywords: Lymphoma; Epstein - Barr virus status; EBV-DNA; EBER; LMP- 1; Prognosis


Since it was first identified in 1964, the Epstein–Barr virus (EBV) has been a very successful member of the herpes virus family and it is the most common virus closely associated with human lymphoma [1]. About 90-95% adults have been infected by EBV and majority of these people become persistent carriers as a result of latent infection. In which, about 10% resting B lymphocytes will transform into permanent and activated lymphoblastoid cell lines which are mostly mediated by the restricted expression of EBV-encoded latent genes such as latent membranous protein 1(LMP-1) [2,3].

EBV status differs in different types of lymphoma. Up till now, several lymphoma types, such as extra nodal NK/T-cell lymphoma nasal type, angioimmunoblastic T-cell lymphoma, have been recognized as EBV-associated lymphoma because of the close correlation with EBV infection. However, the oncogenesis of some lymphoma types is weakly associated with EBV status, such as Mantle lymphoma, Follicular lymphoma and etc. In addition, EBV status of certain lymphoma type has regional and racial differences.

In the meantime, EBV status is recognized to be a prognostic biomark for the clinical course of lymphoma but the best detection of EBV status is still uncertain. Dupuis J, et al. reported that EBVencoded small nuclear RNAs (EBER) was associated with a worse overall survival (OS) in elderly population of nodal peripheral T-cell lymphoma, unspecified [4]. As to Extra nodal NK/T cell lymphoma, nasal type, detectable plasma EBV-DNA was found to be associated with clinical characteristics and it served as a good indicator for the OS rate [5]. Furthermore, the OS rate of Hodgkin’s lymphoma patients was shown to be closely linked to positive EBER /LMP or plasma EBV-DNA [6-8].

Given that EBV status differs among different lymphoma types, regions and the prognostic value are not well recognized, the EBV status of lymphoma cases should be identified for large population size and the prognostic value of different types of EBV detections should be analyzed and verified by more studies. Therefore, we conducted our study to gain a better insight of the EBV significance in lymphoma.

Materials and Methods

Patients selection

The criteria for case inclusion were as follows: (1) Pathologically confirmed diagnosis of lymphoma according to the World Health Organization classification [9]. (2) Detection of EBV status was available including EBER in situ/LMP-1 and/or peripheral blood EBV-DNA quantification. (3) No prior history of immunodeficiency diseases including AIDS. (4) No prior history of chemotherapy or radiotherapy. The research was in compliance of the Declaration of Helsinki and approved by the ethics committee of the Peking University First Hospital. Written informed consent was obtained from each adult participant. As for the children, written informed consents were obtained from their guardians on behalf of them.


A complete set of clinical information in this study included the following: patient demographics, lymphoma type, tumor stage and group according to Ann-Arbor stage [10], International Prognostic Index (IPI) score [11], treatment outcome and vital status. Treatment outcome evaluated by repeated CT or PET/CT was defined as complete response (CR), partial response (PR), stable disease (SD), relapsed disease (RD) or progressive disease (PD) according to the revised response criteria [12].

EBV-encoded small nuclear RNAs (EBER) in situ hybridization and/or immunohistochemistry for latent membrane protein 1(LMP- 1) was done to identify EBV-tissue status. EBER-1 oligonucleotide probes were provided by Zhong Shan Biological Technology Company of China and were operated in accordance with the instruction. A known case of EBV-positive post transplant lymphoproliferative disorder was used as a positive control and the sense RNA probes served as negative control. Nuclear staining was interpreted as EBER expression. A positive reaction was defined as more than 5% nuclear positive of examined cells. The anti-LMP IgG1 (Dako, Glostrup, Denmark) was applied to detect the presence of LMP-1. The staining of tumor cells was defined as positive reactivity.

Peripheral blood sample prior to treatment and/or during treatment was collected into an EDTA-containing tube and centrifuged to isolate mononuclear cells and plasma using a Ficoll- Hypaque gradient method for EBV-DNA quantification. The EBV polymerase chain reaction fluorescence quantitative commercial kit was provided by Da An Gene Company of SUN YAT-SEN University and performed by skilled lab technician in accordance with the instruction. The EBV-DNA greater than 500 copies/ml was regarded as EBV-DNA positive.

Statistical analysis

Patient characteristics and remission rates were compared using the chi-squared test or Fisher exact test, as appropriate. Overall survival (OS) was calculated from the date of diagnosis to the date of death from any cause or the last follow-up. Progression-free survival (PFS) was calculated from the date of diagnosis to the date of first progression or relapse, death from any cause or the last follow-up visit. Patient survival data were analyzed with the method of Kaplan- Meier and compared using the log-rank test. Data were analyzed with SPSS13, P values less than 0.05 were considered statistically significant and all P values correspond to 2-sided significance tests.


In total, 263 cases dated from January 2008 to April 2014 have been included in the analysis. The age of the patients ranged from 5 to 91 years old with the median age around 54 years old. The ratio between male and female patients is 160/103. All the patients were Chinese yellow people. There were 135 B-NHL cases, 94 T-NHL cases and 34 Hodgkin’s lymphoma cases.

Correlation between different types of detections of EBV

In summary, 100 cases received EBER-ISH test, 65 cases received LMP-1 test and 4 received both. Among these, 70 cases were identified to be EBER positive and 14 cases were LMP-1 positive. However, there were two cases with discordant EBER and LMP-1 stains but both of these were considered to be positive (EBER/LMP-1 positive). Peripheral blood mononuclear cells (PBMCs) EBV-DNA quantification assay was conducted for 233 cases, of which 104 cases tested plasma EBV-DNA at the same time. We defined either plasma or PBMC’s EBV-DNA positive as EBV-DNA positive (peripheral blood EBV-DNA positive). The results showed 90 cases were EBV positive, furthermore, 24 cases among them were both plasma and PBMC’s EBV positive. Significant correlation was observed between PBMCs with plasma EBV-DNA positive (r=0.408, P=0.000) and EBER/LMP-1 positive with PBMCs EBV-DNA positive (r=0.223, p=0.008).

EBV status among different types of lymphoma

We evaluated the EBV status by EBER-ISH, LMP-1 and peripheral blood EBV-DNA according to pathological type (Table 1). Extra nodal NK/T cell lymphoma, nasal type had the highest EBERISH positive rate of 97.4% and the peripheral blood EBV-DNA had a positive rate of 74.3%. The positive rate of EBER and EBV-DNA in angioimmunoblastic T cell lymphoma cases was relatively high, which is 82.3% and 56.3%, respectively. In total, 12 out of 13 peripheral T cell lymphoma, unspecified cases were available for EBER-ISH and the positive rate was 33.3%, which was lower than the positive rate of EBV-DNA (54.5%). However, other T cell lymphoma such as T Lymphoblastic cell lymphoma, anaplastic large cell lymphoma and hepatosplenic T cell lymphoma had a relatively low EBV positive rate. Both of the two EBV-associated DLBCL cases had positive peripheral blood EBV-DNA. However, other DLBCL had a lower EBV-DNA positive rate of 19.1%.Both of the 2 cases of grey zone lymohoma (DLBCL/Burkitt’s) had positive EBER staining and peripheral blood EBV-DNA. The positive rate of peripheral EBV-DNA in Follicular lymphoma was 25% while the rate of mantle cell lymphoma was 75%. Among the 34 Hodgkin’s lymphoma cases, 8 cases conducted EBER staining and 7 of them were positive, 11 out of 16 LMP-1 staining cases were positive. Furthermore, the positive rate of peripheral blood EBV-DNA was almost 35%.