Abstract

To define prognostic impact of Epstein-Barr virus (EBV) infection in diffuse large B-cell lymphoma (DLBCL), we investigated EBV status in patients with DLBCL. In all, 380 slides from paraffin-embedded tissue were available for analysis by EBV-encoded RNA-1 (EBER) in situ hybridization, and 34 cases (9.0%) were identified as EBER-positive. EBER positivity was significantly associated with age greater than 60 years (P = .005), more advanced stage (P < .001), more than one extranodal involvement (P = .009), higher International Prognostic Index (IPI) risk group (P = .015), presence of B symptom (P = .004), and poorer outcome to initial treatment (P = .006). The EBER+ patients with DLBCL demonstrated substantially poorer overall survival (EBER+ vs EBER 35.8 months [95% confidence interval (CI), 0-114.1 months] vs not reached, P = .026) and progression-free survival (EBER+ vs EBER 12.8 months [95% CI, 0-31.8 months] vs 35.8 months [95% CI, 0-114.1 months], respectively (P = .018). In nongerminal center B-cell–like subtype, EBER in situ hybridization positivity retained its statistical significance at the multivariate level (P = .045). Nongerminal center B-cell–like patients with DLBCL with EBER positivity showed substantially poorer overall survival with 2.9-fold (95% CI, 1.1-8.1) risk for death. Taken together, DLBCL patients with EBER in situ hybridization+ pursued more rapidly deteriorating clinical course with poorer treatment response, survival, and progression-free survival.

Introduction

Epstein-Barr virus (EBV) preferentially infects B lymphocytes by binding the major viral envelope glycoprotein gp350 to the CD21 receptor on the surface of B cells1  and a second glycoprotein, gp42, to human leukocyte antigen class II molecules.2  Moreover, EBV has the unique ability to transform resting B cells into permanent, latently infected lymphoblastoid cell lines.3,4  In immunocompetent hosts, EBV infection has been implicated in several lymphoid malignancies, including Burkitt lymphoma,5  extranodal natural killer (NK)–T-cell lymphomas,6  aggressive NK leukemia/lymphoma,7  lymphomatoid granulomatosis,7  angioimmunoblastic T-cell lymphoma,8  and a proportion of Hodgkin lymphoma.9  The prognostic significance of EBV infection in lymphoid malignancies has not been established, however. A recent population-based study on 437 classic Hodgkin lymphoma cases showed that EBV positivity was an independent adverse factor for survival.10  Yet another study reported that EBV positivity was associated with better survival in young patients and poorer survival in older patients with nodular sclerosis type, but not in other subtypes.11  In support of these findings, our recent study also demonstrated a varying impact of EBV infection on survival by different age groups.12 

Diffuse large B-cell lymphoma (DLBCL) is the most common type of nonHodgkin lymphoma accounting for 30% to 40% of new lymphoma cases,13  and only 40% to 50% of patients achieve durable remissions. Hence, an acquisition of prognostic parameters at initial diagnosis may contribute to implementation of risk-based stratification of therapy in these patients and may facilitate identification of those who may benefit from early intensive therapy. It was not until recently that DLBCL was recognized as a clinically and morphologically heterogeneous disease entity with the advent of cDNA microarray.14  Using a cDNA microarray and immunohistochemical studies of CD10, bcl-6, or MUM-1, DLBCL can be reclassified into prognostically distinct subgroups with germinal center B-cell–like (GCB), and non-GCB phenotypes.15,16 

The negative impact of EBV positivity on treatment outcome has been previously observed in peripheral T-cell lymphoma, unspecified,17,18  senile B cell lymphomas,19,20  and DLBCL,21  although the small number of cases makes conclusive attribution difficult. Interestingly, one group observed that 50% of the gastric patients with DLBCL, who did not initially respond or relapsed after chemoradiotherapy, were EBV-positive, suggesting that EBV status might have a role as a predictive factor for resistance to treatment.22  Nevertheless, an independent and systematic approach on the incidence of EBV infection and the impact of EBV status on treatment outcome has not been undertaken in DLBCL. To define the prognostic impact of EBV infection in DLBCL, we retrospectively investigated 380 patients with DLBCL in whom biopsy specimens were available for further pathologic examination.

Patients, materials, and methods

Patients

The criteria for case inclusion were as follows: (1) pathologically confirmed diagnosis of DLBCL, according to the World Health Organization classification23 ; (2) complete set of clinical data; (3) adequate paraffin-embedded biopsy specimen or unstained slides for EBV-encoded RNA-1 (EBER-1) in situ hybridization; and (4) age ≥ 18 years. A complete set of clinical information in this analysis included the following: patient demographics, type of treatment, treatment outcome, and vital status. This study was approved by the Institutional Review Board at Samsung Medical Center, Seoul, Korea, in accordance with the Declaration of Helsinki.

Histology

All pathologic specimens were reviewed by one pathologist with expertise and reclassified in accordance with the World Health Organization criteria for pathologic diagnosis. Immunohistochemical analysis was performed on paraffin sections using monoclonal and polyclonal antibodies for the detection of lineage-specific or lineage-characteristic antigens. Cases of any confirmed follicular architecture or transformed lymphoma were excluded from this study. Immunophenotyping was performed using a panel of monoclonal antibodies, including antibodies against CD10 (Dakopatts, Copenhagen, Denmark), bcl-6 (Dakopatts), and MUM-1 (Dakopatts). Using these markers, DLBCL was categorized into 2 subgroups, GCB and non-GCB type, using the algorithm proposed by Hans et al (n = 296).15 

EBV RNA was detected by an in situ hybridization (ISH) technique. The paraffin-embedded sections (5 μm) were dewaxed with xylene followed by treatment with proteinases K and hybridized with fluorescein isothiocyanate conjugated EBER-1 and -2 oligonucleotide probes (Novocastra, Newcastle upon Tyne, United Kingdom). After incubation with antifluorescein isothiocyanate–conjugated antibody tagged with alkaline phosphatase, slides were covered with nitrobluetetrazolium, 5-bromo-4-chloro-3-indolyl phosphates, and 1 M levamisole. We used EBV-negative lymphoid tissues and the hybridization mixture without EBV oligonucleotides as negative controls. To identify cases with strong pathogenic association with EBV, a positive reaction was defined as more than 20% nuclear positivity of examined cells (Figure 1).

Figure 1

Representative results of Epstein-Barr virus–encoded RNA-1 in situ hybridization (EBER ISH). (A) positive EBER ISH; (B) negative EBER ISH. Images were captured using a Polaroid DMC2 digital microscope camera (Polaroid, Tokyo, Japan) and processed using Adobe Photoshop 7.0 (Adobe Systems, Seattle, WA). Original magnification, ×200.

Figure 1

Representative results of Epstein-Barr virus–encoded RNA-1 in situ hybridization (EBER ISH). (A) positive EBER ISH; (B) negative EBER ISH. Images were captured using a Polaroid DMC2 digital microscope camera (Polaroid, Tokyo, Japan) and processed using Adobe Photoshop 7.0 (Adobe Systems, Seattle, WA). Original magnification, ×200.

Treatment

Patients received one of the following initial treatment modalities: (1) an anthracycline-containing chemotherapeutic regimen; (2) a nonanthracycline-containing chemotherapeutic regimen; and (3) supportive care only. The anthracycline-based regimens used were as follows: cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP; n = 216); rituximab-CHOP (n = 63); doxorubicin, bleomycin, vinblastine, dacarbazine (n = 1); ProMACE-CytaBOM (prednisolone, doxorubicin, cyclophosphamide, etoposide, cytarabine, bleomycin, vincristine, methotrexate, and leucovorin, n = 1); and others (n = 6). The nonanthracycline-containing regimens used were methotrexate/cytarabine (n = 37); ifosfamide, methotrexate, etoposide (n = 3); cyclophosphamide, vincristine, prednisone (n = 3); and others (n = 2). Forty-eight patients received best supportive care only. The treatment response was assessed according to standard response criteria.24 

Statistical analysis

Overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier product-limit method. Overall survival was calculated from the date of diagnosis to the date of death from any cause or the last follow up. PFS was measured from the date of diagnosis to the date of the first documented progression, death, or the last follow-up visit. Survival rates were compared for statistical differences by using log-rank analysis. Continuous biologic variables were dichotomized. A backward stepwise Cox regression analysis was performed to delineate prognostic factors at multivariate level and all hazard ratios (HRs) were adjusted for age. P values less than .05 were considered statistically significant and all P values correspond to 2-sided significance tests.

Results

Patient characteristics and Epstein-Barr virus–encoded RNA-1 in situ hybridization results

A total of 380 cases, which were pathologically diagnosed with DLBCL between September 1994 and December 2005, were included in this analysis. All patients were Korean, immunocompetent, and negative for antihuman immunodeficiency virus antibody. Baseline characteristics are provided in Table 1. In all, 380 slides from paraffin-embedded tissue were available for EBV analysis by EBER ISH, and 34 (9.0%) cases were identified as EBER-positive. EBER positivity was significantly associated with age (> 60 years, P = .005), more advanced stage (P < .001), more than one extranodal involvement (P = .009), higher International Prognostic Index (IPI) risk group (P = .015), the presence of B symptom (P = .004), and poorer response to initial treatment (P = .006). There were no significant differences in distribution of primary treatment modalities between the EBER+ and EBER groups (P = .825). Moreover, proportions of GCB were similar between the 2 groups (EBER+ vs EBER; 28.6% vs 34.4%, P = .253). Of the 232 (95.4%) patients in whom the treatment responses were available (EBER, n = 207; EBER+, n = 25), 209 responded to the initial therapy (overall response rate, 90.1%). The overall response rate to initial treatment was significantly lower in the EBER+ patients with DLBCL (19 of 25 [72.0%]) compared with that in the EBER patients with DLBCL (191 of 207 [92.3%], P = .006). The distributions of primary lesions were similar between the 2 groups (data not shown) except for a low incidence of gastrointestinal involvement in the EBER+ group (8.8% vs 25.2%, P = .026).

Table 1

Baseline characteristics of patients with diffuse large B-cell lymphoma according to EBV-encoded RNA-1 (EBER) status

Characteristics and parameters All patients EBER+ EBER P 
Total cases, no. (%) 380 (100) 34 (9.0) 346 (91.0)  
Median age, y (range) 56 (18–95) 65 (20–95) 56 (18–89) .009 
Age, no. (%)     
    60 y or less 241 (63.4) 14 (41.2) 227 (65.6) .005 
    Older than 60 y 139 (36.6) 20 (58.8) 119 (34.4)  
Sex, no. (%)     
    Male 221 (58.2) 18 (52.9) 203 (58.7) .518 
    Female 159 (41.8) 16 (47.1) 143 (41.3)  
Performance status, no. (%)     
    ECOG 0–1* 293 (80.1) 24 (70.6) 269 (81.0) .147 
    ECOG 2–4 73 (19.9) 10 (29.4) 63 (19.0)  
Initial presentation, no. (%)     
    Nodal 96 (25.3) 9 (26.5) 87 (26.1) .339 
    Extranodal 100 (26.3) 6 (17.6) 94 (28.2)  
    Nodal and extranodal 171 (45.0) 19 (55.9) 152 (45.6)  
Ann Arbor stage, no. (%)     
    Limited, I-II 254 (66.8) 14 (42.4) 240 (72.1) <.001 
    Advanced, III-IV 112 (29.5) 19 (57.6) 93 (27.9)  
No. of extranodal involvement, no. (%)     
    0 or 1 312 (82.1) 23 (67.6) 289 (83.5) .009 
    2 or more 58 (15.3) 11 (32.4) 47 (13.6)  
Lactic dehydrogenase, no. (%)     
    Upper limit of normal or below 203 (56.2) 20 (60.6) 183 (55.8) .071 
    Over the upper limit of normal 158 (43.8) 13 (39.4) 145 (44.2)  
International Prognostic Index risk groups, no. (%)     
    Low/low intermediate 279 (73.4) 20 (62.5) 259 (79.9) .015 
    High intermediate/high 77 (20.3) 12 (37.5) 65 (20.1)  
B symptom, no. (%)     
    Positive 74 (20.4) 13 (39.4) 61 (18.5) .004 
    Negative 289 (79.6) 20 (60.6) 269 (81.5)  
Bone marrow involvement, no. (%)     
    Positive 74 (20.4) 13 (39.4) 61 (18.5) .104 
    Negative 289 (79.6) 20 (60.6) 269 (81.5)  
Primary treatment, no. (%)     
    Anthracycline-based chemotherapy with or without radiotherapy 224 (58.9) 25 (73.5) 199 (57.5)  
    Rituximab with cyclophosphamide, doxorubicin, vincristine, prednisone chemotherapy with or without radiotherapy 63 (16.6) 2 (5.9) 61 (17.6) .825* 
    Nonanthracycline-based chemotherapy 45 (11.8) 3 (8.8) 42 (12.1)  
    Best supportive care only 48 (12.6) 4 (11.8) 44 (12.7)  
Response to front-line, no. (%)     
    Chemotherapy, n = 232†     
    Complete remission or partial remission 209 (90.1) 18 (72.0) 191 (92.3) .006 
    Stable disease or progressive disease 23 (9.9) 7 (28.0) 16 (7.7)  
Histologic subtype, no. (%); n = 296     
    Germinal center B cell–like 125 (42.2) 6 (28.6) 119 (34.4) .253 
    Nongerminal center B cell–like 171 (57.8) 15 (71.4) 156 (45.1)  
Characteristics and parameters All patients EBER+ EBER P 
Total cases, no. (%) 380 (100) 34 (9.0) 346 (91.0)  
Median age, y (range) 56 (18–95) 65 (20–95) 56 (18–89) .009 
Age, no. (%)     
    60 y or less 241 (63.4) 14 (41.2) 227 (65.6) .005 
    Older than 60 y 139 (36.6) 20 (58.8) 119 (34.4)  
Sex, no. (%)     
    Male 221 (58.2) 18 (52.9) 203 (58.7) .518 
    Female 159 (41.8) 16 (47.1) 143 (41.3)  
Performance status, no. (%)     
    ECOG 0–1* 293 (80.1) 24 (70.6) 269 (81.0) .147 
    ECOG 2–4 73 (19.9) 10 (29.4) 63 (19.0)  
Initial presentation, no. (%)     
    Nodal 96 (25.3) 9 (26.5) 87 (26.1) .339 
    Extranodal 100 (26.3) 6 (17.6) 94 (28.2)  
    Nodal and extranodal 171 (45.0) 19 (55.9) 152 (45.6)  
Ann Arbor stage, no. (%)     
    Limited, I-II 254 (66.8) 14 (42.4) 240 (72.1) <.001 
    Advanced, III-IV 112 (29.5) 19 (57.6) 93 (27.9)  
No. of extranodal involvement, no. (%)     
    0 or 1 312 (82.1) 23 (67.6) 289 (83.5) .009 
    2 or more 58 (15.3) 11 (32.4) 47 (13.6)  
Lactic dehydrogenase, no. (%)     
    Upper limit of normal or below 203 (56.2) 20 (60.6) 183 (55.8) .071 
    Over the upper limit of normal 158 (43.8) 13 (39.4) 145 (44.2)  
International Prognostic Index risk groups, no. (%)     
    Low/low intermediate 279 (73.4) 20 (62.5) 259 (79.9) .015 
    High intermediate/high 77 (20.3) 12 (37.5) 65 (20.1)  
B symptom, no. (%)     
    Positive 74 (20.4) 13 (39.4) 61 (18.5) .004 
    Negative 289 (79.6) 20 (60.6) 269 (81.5)  
Bone marrow involvement, no. (%)     
    Positive 74 (20.4) 13 (39.4) 61 (18.5) .104 
    Negative 289 (79.6) 20 (60.6) 269 (81.5)  
Primary treatment, no. (%)     
    Anthracycline-based chemotherapy with or without radiotherapy 224 (58.9) 25 (73.5) 199 (57.5)  
    Rituximab with cyclophosphamide, doxorubicin, vincristine, prednisone chemotherapy with or without radiotherapy 63 (16.6) 2 (5.9) 61 (17.6) .825* 
    Nonanthracycline-based chemotherapy 45 (11.8) 3 (8.8) 42 (12.1)  
    Best supportive care only 48 (12.6) 4 (11.8) 44 (12.7)  
Response to front-line, no. (%)     
    Chemotherapy, n = 232†     
    Complete remission or partial remission 209 (90.1) 18 (72.0) 191 (92.3) .006 
    Stable disease or progressive disease 23 (9.9) 7 (28.0) 16 (7.7)  
Histologic subtype, no. (%); n = 296     
    Germinal center B cell–like 125 (42.2) 6 (28.6) 119 (34.4) .253 
    Nongerminal center B cell–like 171 (57.8) 15 (71.4) 156 (45.1)  

Data were missing as follows: performance status (n = 14), initial disease presentation (n = 13), Ann Arbor stage (n = 14), lactic dehydrogenase level (n = 19), no. of extranodal involvement (n = 10), International Prognostic Index score (n = 24), B symptoms (n = 17), bone marrow involvement (n = 15), and histologic subtype (n = 84).

*

ECOG indicates Eastern Cooperative Oncology Group.

Two hundred forty-three patients received chemotherapy as a front-line treatment and 232 patients were assessable for response.

Impact of Epstein-Barr virus–encoded RNA-1 in situ hybridization positivity on survival

After a median follow-up duration of 40.5 months (range, 1-165.7 months), the 5-year OS and 5-year PFS rates were 58.9% and 48.6%, respectively (Figure 2). The EBER+ patients with DLBCL demonstrated substantially poorer OS (EBER+ vs EBER; 35.8 months [95% confidence interval {CI}, 0-114.1 months] vs median OS not reached, respectively, P = .026) and PFS (EBER+ vs EBER; 12.8 [95% CI, 0-31.8 months] vs 35.8 months [95% CI, 0-114.1 months], P = .018).

Figure 2

Overall survival and progression-free survival according to Epstein-Barr virus–encoded RNA-1 status.

Figure 2

Overall survival and progression-free survival according to Epstein-Barr virus–encoded RNA-1 status.

Prognostic significance of Epstein-Barr virus–encoded RNA-1 in situ hybridization positivity in subgroup analyses

We further performed subgroup analyses according to IPI risk groups (low/low intermediate risk vs high intermediate/high risk), age groups (18-50 years vs 51+), and GCB vs non-GCB (Figures 3,4). In low- to low-intermediate risk groups, there was no significant difference in survival (Figure 3A) or progression-free survival according to EBER status (Figure 3B). In high-intermediate to high-risk groups, however, EBER+ patients with DLBCL pursued a more rapidly deteriorating clinical course (median OS, 10.5 months [95% CI, 0.0–22.7]) compared with EBER patients with DLBCL (median OS, 20.8 months [95% CI, 15.6-26.2]) in terms of survival with statistical significance (P = .003) (Figure 3C).

Figure 3

The impact of Epstein-Barr virus–encoded RNA-1 status on overall survival and progression-free survival according to International Prognostic Index risk groups.

Figure 3

The impact of Epstein-Barr virus–encoded RNA-1 status on overall survival and progression-free survival according to International Prognostic Index risk groups.

Figure 4

The impact of Epstein-Barr virus–encoded RNA-1 status on overall survival and progression-free survival according to histologic subtypes.

Figure 4

The impact of Epstein-Barr virus–encoded RNA-1 status on overall survival and progression-free survival according to histologic subtypes.

Next, the prognostic value of EBER ISH positivity was evaluated in subgroup analysis according to age groups (data not shown). EBER positivity did not markedly influence OS or PFS in patients younger than 50 years of age; however, there was a trend toward poorer OS and PFS for EBER+ in patients older than 50 years. In non-GCB patients with DLBCL, EBER positivity had an adverse impact on OS (EBER+ vs EBER, 13.3 months [95% CI, 10.0-16.5] vs median OS not reached; P > .001) and PFS (7.6 months [95% CI, 1.6-13.7] vs 53.2 months [95% CI, 16.1-90.3]; P > .001) with statistical significance (Figure 4C,D). However, the EBER positivity did not influence survival or PFS in GCB DLBCL (Figure 4A,B).

Prognostic factor analyses

The clinical factors predicting poor survival at univariate analysis were as follows: EBER ISH positivity (P = .026), age greater than 60 years (P = .001), poor performance status (Eastern Cooperative Oncology Group 2-4, P<.001), advanced Ann Arbor stage (stage III/IV, P < .001), elevated lactic dehydrogenase level (P < .001), more than one extranodal involvement (P < .001), the presence of B symptom (P > .001), bone marrow involvement (P > .001), and no use of rituximab as part of primary treatment (P = .022) (Table 2). Clinical parameters that were included in the multivariate analysis were EBER ISH status, bone marrow involvement, B symptom, performance status (0-1 vs ≥ 2), age (≤60 vs <60), lactic dehydrogenase level (normal vs elevated), Ann Arbor stage, extranodal involvement (0-1 vs ≥2), histologic subtype (GCB vs non-GCB), and the use of rituximab. The backward conditional Cox regression model was used. Prognostic factors for survival in all patients were performance status (P > .001; HR, 3.4; 95% CI, 2.0-5.9), lactic dehydrogenase (P > .001; HR, 3.2; 95% CI, 1.8-5.5), number of extranodal sites (P > .001; HR, 2.9; 95% CI, 1.7-5.0), and age (P > .001; HR, 2.7; 95% CI, 1.6-5.1) (Table 3). In the non-GCB subtype, however, EBER ISH positivity retained its statistical significance at the multivariate level (P = .045). Non-GCB patients with DLBCL with EBER positivity showed substantially poorer OS with 2.9-fold (95% CI, 1.1-8.1) risk for death. In contrast, the EBER status did not affect survival in the GCB DLBCL with statistical significance (P = .091).

Table 2

Univariate analysis of prognostic factors for survival in patients with diffuse large B-cell lymphoma

Parameters Overall survival
 
Median, mo (95% CI) P 
Epstein-Barr virus status  .026 
    EBER-positive 35.8 (0-114.1)  
    EBER-negative NR  
Age, y  <.001 
    60 y old or younger NR  
    Older than 60 y 44.7 (28.5-60.9)  
Performance status  <.001 
    ECOG, 0 to 1 NR  
    ECOG, 2 to 4 20.9 (11.1-30.7)  
Ann Arbor stage  <.001 
    Limited (I/II) NR  
    Advanced (III/IV) 28.5 (1.8-55.1)  
Lactic dehydrogenase  <.001 
    ULN or below NR  
    Over ULN 58.8 (20.7-96.9)  
No. of extranodal involvement  <.001 
    0 or 1 NR  
    2 or more 21.1 (12.0-29.1)  
B symptom  <.001 
    Positive 54.7 (11.0-98.4)  
    Negative NR  
Bone marrow involvement  <.001 
    Positive 20.4 (8.2-32.5)  
    Negative NR  
Histologic subtype  .990 
    Nongerminal center B cell–like NR  
    Germinal center B cell–like NR  
Primary treatment  <.001 
    Anthracycline-based NR  
    Non-anthracycline-based 44.7 (22.9-66.4)  
The use of rituximab  .022 
    Rituximab NR  
    No rituximab 101.9  
Parameters Overall survival
 
Median, mo (95% CI) P 
Epstein-Barr virus status  .026 
    EBER-positive 35.8 (0-114.1)  
    EBER-negative NR  
Age, y  <.001 
    60 y old or younger NR  
    Older than 60 y 44.7 (28.5-60.9)  
Performance status  <.001 
    ECOG, 0 to 1 NR  
    ECOG, 2 to 4 20.9 (11.1-30.7)  
Ann Arbor stage  <.001 
    Limited (I/II) NR  
    Advanced (III/IV) 28.5 (1.8-55.1)  
Lactic dehydrogenase  <.001 
    ULN or below NR  
    Over ULN 58.8 (20.7-96.9)  
No. of extranodal involvement  <.001 
    0 or 1 NR  
    2 or more 21.1 (12.0-29.1)  
B symptom  <.001 
    Positive 54.7 (11.0-98.4)  
    Negative NR  
Bone marrow involvement  <.001 
    Positive 20.4 (8.2-32.5)  
    Negative NR  
Histologic subtype  .990 
    Nongerminal center B cell–like NR  
    Germinal center B cell–like NR  
Primary treatment  <.001 
    Anthracycline-based NR  
    Non-anthracycline-based 44.7 (22.9-66.4)  
The use of rituximab  .022 
    Rituximab NR  
    No rituximab 101.9  

ULN indicates upper limit normal; NR, not reached.

Table 3

Cox proportional hazards model hazard ratios for death from all causes in patients with diffuse large B-cell lymphoma

Parameters All patients
 
Non-GCB subtype
 
GCB subtype
 
HR (95% CI) P HR (95% CI) P HR (95% CI) P 
EBV status  .544  .045  .091 
    EBER ISH  —  1.0  —  
    EBER ISH + —  2.9 (1.1-8.1)  —  
Performance status  <.001  .003  <.001 
    ECOG, 0 to 1 1.0  1.0  18.1 (7.0-47.0)  
    ECOG, 2 to 4 3.4 (2.0-5.9)  3.3 (1.5-7.0)    
Lactic dehydrogenase  <.001  .006  .154 
    ULN or below 1.00  1.0  —  
    Over ULN 3.2 (1.8-5.5)  3.1 (1.3-7.3)  —  
No. of extranodal sites  <.001  .083  .060 
    0 or 1 1.0  —  —  
    1 or more 2.9 (1.7-5.0)  —  —  
Age, years  <.001  .059  .198 
    60 years old or less 1.0  —  —  
    Over 60 years old 2.7 (1.6-5.1)  —  —  
B symptom  .246  .006  .474 
    Absent —  1.0  —  
    Present —  3.3 (1.5-7.0)  —  
Ann Arbor stage  .161  .085  .020 
    Stage I/II —  —  1.0  
    Stage III/IV —  —  3.0 (1.2-7.5)  
Parameters All patients
 
Non-GCB subtype
 
GCB subtype
 
HR (95% CI) P HR (95% CI) P HR (95% CI) P 
EBV status  .544  .045  .091 
    EBER ISH  —  1.0  —  
    EBER ISH + —  2.9 (1.1-8.1)  —  
Performance status  <.001  .003  <.001 
    ECOG, 0 to 1 1.0  1.0  18.1 (7.0-47.0)  
    ECOG, 2 to 4 3.4 (2.0-5.9)  3.3 (1.5-7.0)    
Lactic dehydrogenase  <.001  .006  .154 
    ULN or below 1.00  1.0  —  
    Over ULN 3.2 (1.8-5.5)  3.1 (1.3-7.3)  —  
No. of extranodal sites  <.001  .083  .060 
    0 or 1 1.0  —  —  
    1 or more 2.9 (1.7-5.0)  —  —  
Age, years  <.001  .059  .198 
    60 years old or less 1.0  —  —  
    Over 60 years old 2.7 (1.6-5.1)  —  —  
B symptom  .246  .006  .474 
    Absent —  1.0  —  
    Present —  3.3 (1.5-7.0)  —  
Ann Arbor stage  .161  .085  .020 
    Stage I/II —  —  1.0  
    Stage III/IV —  —  3.0 (1.2-7.5)  

— indicates not applicable; HR, hazard ratio.

Discussion

To the best of our knowledge, this study represents the largest one to evaluate the significance of EBV positivity on treatment outcome and survival of patients with DLBCL. Only a few other studies have speculated a negative correlation between EBV status and prognosis in DLBCL based on a limited number of cases.19-22  To elucidate the clinical implication of detecting EBV status by EBER-1 ISH in patients with DLBCL, we systematically performed EBER-1 ISH in 380 tumor specimens of DLBCL. The incidence of EBER+ DLBCL was 9% (34 of 380) in this series, which is comparable with those reported in other studies (8% to 11%). In agreement with previous studies, the EBER+ patients with DLBCL were more likely to be diagnosed older than 60 years, at Ann Arbor stage III/IV, and to present with more than one extranodal involvement and high-intermediate/high risk according to IPI.

Despite a similar distribution of treatment modalities between EBER+ and EBER groups (anthracycline-based chemotherapy; 76% vs 79%, respectively), EBER+ DLBCL showed substantially poorer response to front-line chemotherapy compared with EBER DLBCL (72.0% vs 92.3%, P = .006). It is generally known that most EBV-associated tumors respond poorly to intensive chemotherapy regimens or have a significant relapse rate. Moreover, a small Japanese study investigated the role of EBV infection in primary refractory gastric DLBCL to CHOP chemotherapy and observed that 50% (4 of 8) of refractory patients were EBV-positive.22  Attributable to the retrospective nature of this study, the stratification of therapy according to EBV status in DLBCL may not be firmly established.

Univariate analysis of survival indicated that EBER+ patients had a significantly worse OS and PFS compared with EBER patients. In subgroup analyses, the EBV status adversely influenced only patients with DLBCL with high-intermediate/high IPI risk groups or non-GCB subtype, but not those with low/low-intermediate risk groups or GCB subtype. Furthermore, only in the cohort of patients with non-GCB subtype was this impact still retained in multivariate analysis of survival (HR, 2.9; 95% CI, 1.1-8.9 P = .045). These findings may have clinical implications that are noteworthy. The IPI score is a well-known prognostic index, which effectively separates DLBCL into 4 risk groups.25,26  Nevertheless, patients in each risk group may not pursue a consistent clinical course with a uniform response to treatment. In addition, a recently developed subclassification of DLBCL into GCB and non-GCB phenotypes has shown distinct prognostication.14-16  Yet, the EBV status further categorized non-GCB subtypes into 2 groups with considerably different prognosis (Table 3). A small study reported a substantial correlation between EBV positivity and non-GCB phenotype in HIV-positive patients with DLBCL.27  Taken together, EBER ISH status may be a useful tool in further identifying high-risk patients who may benefit from early intensive treatment such as hematopoietic stem cell transplantation in addition to IPI and immunohistochemical studies. An issue of whether the EBV-associated DLBCL represents an independent disease entity remains unanswered and needs to be clarified in future studies. In addition, because the number of EBER ISH+ patients was only 29 in our series, the impact of the EBER status in DLBCL should be validated in a larger number of patients.

Recently, Kwong et al demonstrated that the level of circulating EBV DNA is correlated with stage and survival, reflecting the tumor load, leading to a conclusion that plasma EBV DNA can be used as a tumor biomarker to monitor treatment response in EBV-positive NK/T-cell lymphoma.28  They hypothesized that the increase of EBV DNA was attributable to tumor release of EBV fragments rather than reactivation of latent EBV infection because patients with NK/T-cell lymphoma are immunocompetent. Thus, the relationship between the quantification of EBV viral load in blood and EBER ISH in tumor specimens should be confirmed in DLBCL.

The role of EBV infection in pathogenesis of DLBCL is not known. EBV can infect resting B lymphocytes efficiently and drive it out of the resting state to become an activated lymphoblastoid cell lines.3,4,29  These transforming effects are associated with the restricted expression of the EBV-encoded latent genes such as latent infection membrane protein 1.30  Latent infection membrane protein 1 is an integral membrane protein that up-regulates antiapoptotic proteins Bcl-231  and functions as a constitutively activated member of the tumor necrosis factor receptor superfamily activating several signaling pathways,32  including nuclear factor kappa-B transcription factor pathway,33  MAP kinase cascade,34  and the phosphatidylinositol 3-kinase/Akt pathway.35  Constitutively activated proteins in these pathways may contribute to the clinical characteristics of EBV-positive tumors. Thus, correlative analyses with EBER ISH and key proteins of these cascades may be interesting to investigate to further clarify the pathogenic role of EBV in DLBCL.

Based on our data, patients with DLBCL who are EBER ISH+ pursue a more rapidly deteriorating clinical course with poorer treatment response, survival, and PFS. Thus, more effective treatment should be adopted in this particular subset of patients with possible addition of EBV-targeted therapy to conventional chemotherapy.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Authorship

Contribution: S.P. collected and analyzed data; J.L. analyzed data, wrote and revised the manuscript; Y.H.K. designed the research and performed pathologic examinations; A.H., H.J.J., S.C.L., and I.G.H. performed data collection; J.S.A., C.W.J., K.K., Y.C.A., W.K.K., and K.P. performed patient provision; and W.S.K. designed the research, analyzed the data, and approved the final manuscript.

S.P. and J.L. contributed equally to this study.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Won Seog Kim, Division of Hematology–Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong Kangnam-Gu, Seoul, Korea, 135-710; e-mail: wskimsmc@smc.samsung.co.kr; or Young Hyeh Ko, Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong Kangnam-Gu, Seoul, Korea, 135–710; e-mail: yhko@smc.samsung.co.kr.

References

References
1
Nemerow
GR
Mold
C
Schwend
VK
Tollefson
V
Cooper
NR
Identification of gp350 as the viral glycoprotein mediating attachment of Epstein-Barr virus (EBV) to the EBV/C3d receptor of B cells: sequence homology of gp350 and C3 complement fragment C3d.
J Virol
1987
, vol. 
61
 (pg. 
1416
-
1420
)
2
Borza
CM
Hutt-Fletcher
LM
Alternate replication in B cells and epithelial cells switches tropism of Epstein-Barr virus.
Nat Med
2002
, vol. 
8
 (pg. 
594
-
599
)
3
Pope
JH
Horne
MK
Scott
W
Transformation of foetal human leukocytes in vitro by filtrates of a human leukaemic cell line containing herpes-like virus.
Int J Cancer
1968
, vol. 
3
 (pg. 
857
-
866
)
4
Pattengale
PK
Smith
RW
Gerber
P
Selective transformation of B lymphocytes by E.B. virus.
Lancet
1973
, vol. 
2
 (pg. 
93
-
94
)
5
Kieff
EaR
AB
In Fields Virology.
2001
Philadelphia
Lippincott Williams & Wilkins
6
Siu
LL
Chan
JK
Kwong
YL
Natural killer cell malignancies: clinicopathologic and molecular features.
Histol Histopathol
2002
, vol. 
17
 (pg. 
539
-
554
)
7
Heslop
HE
Biology and treatment of Epstein-Barr virus-associated non-Hodgkin lymphomas.
Hematol Am Soc Hematol Educ Program
2005
(pg. 
260
-
266
)
8
Anagnostopoulos
I
Hummel
M
Finn
T
, et al. 
Heterogeneous Epstein-Barr virus infection patterns in peripheral T-cell lymphoma of angioimmunoblastic lymphadenopathy type.
Blood
1992
, vol. 
80
 (pg. 
1804
-
1812
)
9
Pallesen
G
Hamilton-Dutoit
SJ
Rowe
M
Young
LS
Expression of Epstein-Barr virus latent gene products in tumour cells of Hodgkin's disease.
Lancet
1991
, vol. 
337
 (pg. 
320
-
322
)
10
Jarrett
RF
Stark
GL
White
J
, et al. 
Impact of tumor Epstein-Barr virus status on presenting features and outcome in age-defined subgroups of patients with classic Hodgkin lymphoma: a population-based study.
Blood
2005
, vol. 
106
 (pg. 
2444
-
2451
)
11
Keegan
TH
Glaser
SL
Clarke
CA
, et al. 
Epstein-Barr virus as a marker of survival after Hodgkin's lymphoma: a population-based study.
J Clin Oncol
2005
, vol. 
23
 (pg. 
7604
-
7613
)
12
Kwon
JM
Park
YH
Kang
JH
, et al. 
The effect of Epstein-Barr virus status on clinical outcome in Hodgkin's lymphoma.
Ann Hematol
2006
, vol. 
85
 (pg. 
463
-
468
)
13
A clinical evaluation of the International Lymphoma Study Group classification of non-Hodgkin's lymphoma. The Non-Hodgkin's Lymphoma Classification Project.
Blood
1997
, vol. 
89
 (pg. 
3909
-
3918
)
14
Rosenwald
A
Wright
G
Chan
WC
, et al. 
The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.
N Engl J Med
2002
, vol. 
346
 (pg. 
1937
-
1947
)
15
Hans
CP
Weisenburger
DD
Greiner
TC
, et al. 
Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray.
Blood
2004
, vol. 
103
 (pg. 
275
-
282
)
16
Poulsen
CB
Borup
R
Nielsen
FC
, et al. 
Microarray-based classification of diffuse large B-cell lymphoma.
Eur J Haematol
2005
, vol. 
74
 (pg. 
453
-
465
)
17
d'Amore
F
Johansen
P
Houmand
A
Weisenburger
DD
Mortensen
LS
Epstein-Barr virus genome in non-Hodgkin's lymphomas occurring in immunocompetent patients: highest prevalence in nonlymphoblastic T-cell lymphoma and correlation with a poor prognosis. Danish Lymphoma Study Group, LYFO.
Blood
1996
, vol. 
87
 (pg. 
1045
-
1055
)
18
Dupuis
J
Emile
JF
Mounier
N
, et al. 
Prognostic significance of Epstein-Barr virus in nodal peripheral T-cell lymphoma, unspecified: A Groupe d'Etude des Lymphomes de l'Adulte (GELA) study.
Blood
2006
, vol. 
108
 (pg. 
4163
-
4169
)
19
Shimoyama
Y
Oyama
T
Asano
N
, et al. 
Senile Epstein-Barr virus-associated B-cell lymphoproliferative disorders: a mini review.
J Clin Exp Hematop
2006
, vol. 
46
 (pg. 
1
-
4
)
20
Oyama
T
Ichimura
K
Suzuki
R
, et al. 
Senile EBV+ B-cell lymphoproliferative disorders: a clinicopathologic study of 22 patients.
Am J Surg Pathol
2003
, vol. 
27
 (pg. 
16
-
26
)
21
Kuze
T
Nakamura
N
Hashimoto
Y
Sasaki
Y
Abe
M
The characteristics of Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma: comparison between EBV(+) and EBV(−) cases in Japanese population.
Jpn J Cancer Res
2000
, vol. 
91
 (pg. 
1233
-
1240
)
22
Yoshino
T
Nakamura
S
Matsuno
Y
, et al. 
Epstein-Barr virus involvement is a predictive factor for the resistance to chemoradiotherapy of gastric diffuse large B-cell lymphoma.
Cancer Sci
2006
, vol. 
97
 (pg. 
163
-
166
)
23
Jaffe
ES
Harris
NL
SH
Vardiman
JW
World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Hematopoietic and Lymphoid Tissues.
Lyon, France
IARC Press
pg. 
2001
 
24
Cheson
BD
Horning
SJ
Coiffier
B
, et al. 
Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas. NCI Sponsored International Working Group.
J Clin Oncol
1999
, vol. 
17
 (pg. 
1244
-
1253
)
25
A predictive model for aggressive non-Hodgkin's lymphoma. The International Non-Hodgkin's Lymphoma Prognostic Factors Project.
N Engl J Med
1993
, vol. 
329
 (pg. 
987
-
994
)
26
Lopez-Guillermo
A
Montserrat
E
Bosch
F
Terol
MJ
Campo
E
Rozman
C
Applicability of the International Index for aggressive lymphomas to patients with low-grade lymphoma.
J Clin Oncol
1994
, vol. 
12
 (pg. 
1343
-
1348
)
27
Vilchez
RA
Lopez-Terrada
D
Middleton
JR
, et al. 
Simian virus 40 tumor antigen expression and immunophenotypic profile of AIDS-related non-Hodgkin's lymphoma.
Virology
2005
, vol. 
342
 (pg. 
38
-
46
)
28
Au
WY
Pang
A
Choy
C
Chim
CS
Kwong
YL
Quantification of circulating Epstein-Barr virus (EBV) DNA in the diagnosis and monitoring of natural killer cell and EBV-positive lymphomas in immunocompetent patients.
Blood
2004
, vol. 
104
 (pg. 
243
-
249
)
29
Thorley-Lawson
DA
Epstein-Barr virus: exploiting the immune system.
Nat Rev Immunol
2001
, vol. 
1
 (pg. 
75
-
82
)
30
Kaye
KM
Izumi
KM
Kieff
E
Epstein-Barr virus latent membrane protein 1 is essential for B-lymphocyte growth transformation.
Proc Natl Acad Sci USA
1993
, vol. 
90
 (pg. 
9150
-
9154
)
31
Henderson
S
Rowe
M
Gregory
C
, et al. 
Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death.
Cell
1991
, vol. 
65
 (pg. 
1107
-
1115
)
32
Kilger
E
Kieser
A
Baumann
M
Hammerschmidt
W
Epstein-Barr virus-mediated B-cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor.
EMBO J
1998
, vol. 
17
 (pg. 
1700
-
1709
)
33
Huen
DS
Henderson
SA
Croom-Carter
D
Rowe
M
The Epstein-Barr virus latent membrane protein-1 (LMP1) mediates activation of NF-kappa B and cell surface phenotype via two effector regions in its carboxy-terminal cytoplasmic domain.
Oncogene
1995
, vol. 
10
 (pg. 
549
-
560
)
34
Roberts
ML
Cooper
NR
Activation of a ras-MAPK-dependent pathway by Epstein-Barr virus latent membrane protein 1 is essential for cellular transformation.
Virology
1998
, vol. 
240
 (pg. 
93
-
99
)
35
Dawson
CW
Tramountanis
G
Eliopoulos
AG
Young
LS
Epstein-Barr virus latent membrane protein 1 (LMP1) activates the phosphatidylinositol 3-kinase/Akt pathway to promote cell survival and induce actin filament remodeling.
J Biol Chem
2003
, vol. 
278
 (pg. 
3694
-
3704
)