EBV-positive diffuse large B-cell lymphoma of the elderly: A case series from Peru

doi:10.1182/blood-2013-03-489708Prepublished online May 6, 2013;

Chi Young Ok, Thomas G. Papathomas, L. Jeffrey Medeiros and Ken H. Young EBV-positive diffuse large B-cell lymphoma of the elderly

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EBV-Positive Diffuse Large B-Cell Lymphoma of the Elderly

- A New Provisional Entity of Lymphoma in the WHO Classification

Chi Young Ok1, Thomas G. Papathomas2, L. Jeffrey Medeiros1, Ken H. Young1¶

1Department of Hematopathology, The University of Texas MD Anderson Cancer Center,

Houston, Texas, USA; 2Department of Pathology, Erasmus MC University Medical Center

Rotterdam, Rotterdam, The Netherlands

Running title: EBV-positive DLBCL of the elderly

Key words: Epstein-Barr virus, diffuse large B-cell lymphoma, elderly, CD30, NF-κB,

LMP1, EBNA1, EBNA2, Akt, PI3K, Jak-Stat

¶Correspondence:

Ken H. Young, MD, PhD

The University of Texas MD Anderson Cancer Center

Department of Hematopathology

1515 Holcombe Boulevard

Houston, Texas 77030-4009

Phone: 1-713-745-2598

Email: [email protected]

Blood First Edition Paper, prepublished online May 6, 2013; DOI 10.1182/blood-2013-03-489708

Copyright © 2013 American Society of Hematology


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Abstract

Epstein-Barr virus (EBV) positive diffuse large B-cell lymphoma (DLBCL) of the elderly,

initially described in 2003, is a provisional entity in the 2008 World Health Organization

classification system, and is defined as an EBV-positive monoclonal large B-cell proliferation

that occurs in patients > 50 years of age and in whom there is no known immunodeficiency or

history of lymphoma. These tumors are more common in Asia, but also occur in North

America and Europe at a low frequency. These neoplasms exhibit a morphologic continuum,

from polymorphous to monomorphous, but morphologic features do not correlate with

prognosis as all patients have a clinically aggressive course. Most EBV-positive DLBCL of the

elderly patients have an activated B-cell immunophenotype and are characterized by prominent

NF-κB activation. Cytogenetic complexity is usually low. In this review, we comprehensively

delineate the data emerging from analyses of EBV latency program, microRNA-mediated EBV

viral oncogenesis, functional genomics of EBV and its biology, as well as differential diagnosis

challenge for EBV-positive DLBCL of the elderly. It is hoped that the improved understanding

of these tumors will lead to development of novel therapeutic approaches, enhance the

effective clinical trials and improve the prognosis.


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Introduction

The 2008 World Health Organization (WHO) classification of tumors of hematopoietic and

lymphoid tissues has recognized a new provisional entity designated as Epstein-Barr virus-

positive diffuse large B-cell lymphoma (DLBCL) of the elderly. This tumor is defined as an

EBV-positive monoclonal large B-cell lymphoproliferative disorder arising in

immunocompetent patients > 50 years.1 To denote patient age in the definition was a means

of emphasizing the tendency of these tumors to arise in patients of advanced age, but this age

cutoff is clearly arbitrary, and EBV-positive DLBCL of the elderly has been reported rarely in

younger patients.

Since the initial description of 22 patients with EBV-positive DLBCL of the elderly in 2003

by Oyama et al,2 knowledge of EBV-mediated mechanisms of oncogenesis, microRNA

profiles in EBV-positive lymphomas and novel efficient therapeutics for patients with these

neoplasms have been developed. In this review, we discuss the epidemiology,

clinicopathologic features, and differential diagnoses of EBV-positive DLBCL of the elderly.

We also review novel molecular findings including host and virus-induced miRNAs, and

some novel therapeutic approaches.

Epidemiology

The median age of patients with EBV-positive DLBCL of the elderly is 71 years (range, 50-

91), however, younger patients can be affected.3 There is a slight male predominance with a

male to female ratio of 1.4 to 1.4-10 There is a higher prevalence of EBV-positive DLBCL

among East Asians (8.7-11.4%)11-13 compared with less than 5% in Western countries5,6

(Table 1). Meanwhile, Beltran et al4 has reported the highest frequency of EBV-positive

DLBCL in a Peruvian population, 14.9%, using a criterion of ≥ 20% cells with positive EBV-


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encoded RNA (EBER) expression. Of note, there are no uniform criteria for the percentage of

EBV-positive cells in EBV-positive DLBCL and this is a limitation when assessing disease

prevalence. The fact that differences in cutoffs for EBER positivity clearly impact the

frequency of EBV positive DLBCL was respectively shown by Wada et al and others.13-15

These authors emphasize the need for establishing uniform criteria for EBV positivity, either

>20%, >50%, or almost all tumor cells. Hofscheier et al interpreted EBER-positive DLBCL

with <20% EBER as clonally unrelated EBV-transformed B-cells or secondary EBV infection

in an established B-cell clone.7 The definitive criterion for EBV positivity in EBV-positive

DLBCL is remained to be agreed.

Epidemiologic features correlate with geographical differences in the literature. In contrast

with East Asian series, cases in Mexico display a relatively younger age at presentation, with

predominantly nodal disease and rare expression of the EBNA2.7 Recent evidence casts a

doubt on geographic and/or ethnic variation in the prevalence of EBV-positive DLBCL of the

elderly. Contrary to earlier reports from the Far East, a report from Northern China showed a

frequency of EBV positivity in only 8 of 212 (3.8%) in patients >50 years old with DLBCL

(using a cutoff of >50% EBER positivity).9

Pathogenetic aspects

From an immunologic viewpoint, aging can be characterized as a dysregulated relationship

between inflammatory and inflammation-neutralizing processes, resulting in a low-grade

chronic pro-inflammatory state.16 This imbalance might exert other potential effects promoting

lymphomagenesis. Inflammation induces radical oxygen species which can cause

dysregulation of critical oncogenic pathways, such as p53, retinoblastoma (Rb), NF-κB, and

mitogen-activated protein (MAP) kinases.17


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It has been postulated that EBV-positive DLBCL of the elderly might be caused by the

senescence of the immune system, as a part of the normal aging process, based largely on

shared features with immunodeficiency-associated lymphoproliferative disorders (LPDs).

These shared features include EBV infection, similar EBV latency pattern, morphologic

similarities, and presence of monoclonal T-cell populations.8,12,18,19 Of note,

immunosenescence is not simply the result of a progressive decline of all immune functions,

but rather refers to a continuous remodeling process, whereby the adaptive immunity is

preferentially affected compared with innate immunity.20,21 In this context, B-cell diversity

decreases with age and is characterized by clonal expansions of B cells in vivo.22

Concurrently, the T-cell compartment shows characteristic modifications, such as: (i) a

reduction of the absolute number of total T lymphocytes, including helper/inducer (CD4+) and

suppressor/cytotoxic (CD8+) cell counts; (ii) a progressive decline of the näive T-cell

repertoire with a parallel decline in T-cell receptor repertoire diversity; and (iii) a progressive

expansion of oligoclonal CD28- T cells, particularly among the CD8+ T-cell subset.20,21

Particularly, Ouyang et al23 showed a significantly greater frequency of EBV-specific

receptor-carrying cells within the CD8+ T-cell subset as well as a lower frequency of EBV

antigen-specific interferon (IFN)-gamma-producing T cells in the elderly, suggesting clonal

expansion of dysfunctional EBV-specific cells to age. Whether oligoclonal or monoclonal T-

cell populations in patients with EBV-positive DLBCL of the elderly reflects a physiological

T-cell response to EBV infection8 or antedate the development of the LPD stemming from an

underlying deficiency in T-cell function18 is unknown.

EBV is a gamma-herpes virus that has a tropism for lymphocytes. The virus infects and

replicates within epithelial cells and most viral particles are cleared by cytotoxic T cells.


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However, EBV survives in a host cell by establishing latency as an episome (a double-

stranded circular form of the genome) in memory B-cells. In latent infection, EBV uses only a

limited number of genes to maintain its genome and evades the host immune reaction. Based

on different patterns of viral gene usage, latency can be classified as type III, type II, or type I.

Type III is characterized by the expression of all six EBV nuclear antigens (EBNA1, 2, 3A,

3B, 3C, and LP), three latent membrane proteins (LMP1, 2A and 2B), and EBER (Figure 1A-

B). Type III latency is observed in patients with infectious mononucleosis or a subset of post-

transplant lymphoproliferative disorders (PTLD). Type II latency is associated with

expression of EBNA1, LMP1, LMP2A, and EBER and is commonly seen in classical

Hodgkin lymphoma and a subset of PTLD. In type I latency, only EBNA1 and EBER are

expressed; this pattern is characteristic of Burkitt lymphoma.

EBV-positive DLBCL of the elderly shares a similar EBV latency patterns (type II or III) with

PTLD.24 Using reverse transcription polymerase chain reaction (RT-PCR) method, Nguyen-

Van et al19 showed that many EBV-positive DLBCL of the elderly and EBV-positive PTLD

resembling DLBCL shared features of EBV latency type III. A relatively high frequency of

EBNA2 expression (28% to 32%) has been observed in several studies.2,12 However,

Hofscheier et al found history of immunosuppression or immunodysfunction in 4/17 cases

with EBNA2 expression, emphasizing that care should be taken in the diagnosis of EBNA2-

positive DLBCL cases.7

EBV-positive DLBCL of the elderly is characterized by prominent classical and alternative

NF-κB pathway activation, with most cases displaying an activated B-cell (ABC)

immunophenotype.8 Whereas NF-κB activation has been known in DLBCL, mostly in the

ABC subtype,25 the association between NF-κB activation and the presence of EBV infection


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in DLBCL is much stronger.8 Interestingly, Montes-Moreno et al found NF-κB activation not

only in the ABC but also in the germinal center B-cell (GCB) subtype of EBV-positive

DLBCL, suggesting an additional role for EBV in NF-κB activation independent of the cell of

origin. This finding could be related to c-Rel (a subunit of NF-κB transcription factor)

amplification, which has been observed exclusively in the GCB immunophenotype of DLBCL

on the basis of gene expression profiling.

The oncogenic mechanisms of EBV are thought to be attributable predominantly to LMP1

(Figure 2). LMP1 (MW 63kDa) is an integral membrane protein that consists of three different

domains. The N-terminus cytoplasmic domain orientates LMP1 protein and binds to the

plasma membrane. The long C-terminus cytoplasmic tail contains signaling activity. A six-

transmembrane loop between the C- and N-terminus domains provides a platform for self-

aggregation and oligomerization. LMP1 functionally mimics CD40, which is expressed

constitutively on the B-cell membrane involving in B-cell activation and proliferation. Of

note, the function of LMP1 is ligand-independent and is constitutively activated as a result of

self-aggregation and oligomerization.26 The C-terminal tail has two distinct domains, C-

terminal activation regions 1 and 2 (CTAR1 and 2), which activate the NF-κB pathway.27

LMP1 aggregates provide a platform for CTAR1 and CTAR2 to interact with downstream

molecules (Figure 1-C).28-30 LMP1 can also activate the c-Jun N-terminal kinase (JNK)/AP-1,

MAPK, and phosphatidylinositol 3-kinase (PI3K)-AKT pathways.31-33

LMP1 also deregulates the cell cycle. Everly et al showed that LMP1 increases expression of

cyclin-dependent kinase 2 (Cdk2), phosphorylates retinoblastoma (Rb) protein, and inhibits

expression of p27Kip1.34 The p16INK4a-Rb pathway has been shown to be hindered by LMP1.35

LMP1 also increases expression of Bcl-2 to prevent latently EBV-infected cells from


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undergoing apoptosis.36 Of note, virus and host transcriptome analyses have revealed an

unexpected coordination between viral lytic reactivation and cellular pathways involved in B-

cell expansion as well as tumor promotion,37 supporting a role for the lytic cycle in virus-

mediated oncogenesis while further highlighting the complexity of interactions between EBV

and host.38

Clinical features

EBV-positive DLBCL of the elderly is characterized by higher age distribution and an

aggressive clinical course with a median survival of two years in Asian patients (Figure 3A-

B).4,7,8,10,12,13,18 Initial reports emphasized that EBV-positive DLBCL of the elderly commonly

involved extranodal sites.2,13 Site of primary extranodal involvement include the skin, soft

tissue, bones, nasal cavity, pharynx/hypopharynx, tonsils, tongue, lung, pleura, stomach, liver,

spleen, peritoneum, cecum and bone marrow.4-8,10,39 Subsequently, several studies showed that

lymph node involvement is very common, in up to 70% of patients.4-8,10,39

EBV-positive DLBCLs, including EBV-positive DLBCL of the elderly, respond more poorly

to treatment with a poorer outcome compared with patients who have EBV-negative DLBCL.

Therefore, novel therapeutic agents are needed to treat patients with EBV-positive DLBCL of

the elderly.13 Montes-Moreno et al8 compared the survival distributions of age-related EBV-

positive DLBCL cases and a control cohort either selecting ABC-type DLBCL cases or

patients > 60 years of age and subsequently confirmed that patients with EBV-positive

DLBCL of the elderly have a poorer overall survival and progression-free survival than

patients with ABC-type EBV-negative DLBCL in older European patients (Figure 3C-D). In

comparison, a negative impact of EBV infection in DLBCL survival was not observed in the

North America patients. There was a trend toward higher percentage of stage III/IV disease


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and bone marrow involvement (85% vs 57% and 46% vs 16%, p = 0.052), but no unique

clinical features were found in EBV-positive DLBCL patients (Figure 3E-F). However,

significantly worse survival is observed in EBV-positive DLBCL patients if CD30 expression

is also present (Figure 3G-H).

Recently, Dojcinov et al18 expanded the spectrum of EBV-positive DLBCL. They analyzed

122 cases of EBV-positive B-cell LPDs in adults with no other identifiable cause of

immunosuppression and classified them into four diagnostic categories: (1) reactive lymphoid

hyperplasia; (2) polymorphic extranodal lymphoproliferative disease; (3) polymorphic nodal

lymphoproliferative disease; and (4) DLBCL. In these four groups the five-year disease-

specific survival decreases progressively (100%, 93%, 57%, and 25%, respectively) while the

frequency of monoclonality increases (16%, 33%, 63%, and 56%, respectively). It needs to be

emphasized that these categories are not fixed as patients can progress from reactive

hyperplasia to polymorphic or monomorphic EBV-positive LPD.40 With respect to

morphology, some EBV-positive DLBCL of the elderly display a mixed pattern with

polymorphic and monomorphic areas8 or polymorphic cases eventually progress to the

monomorphic subtype of EBV-positive DLBCL of the elderly5,41.

Two additional entities have been described that further add to the spectrum of EBV-positive

LPDs. EBV-positive mucocutaneous ulcer is a term used recently to designate small-volume

extranodal polymorphic lymphoproliferative disease, particularly involving mucosal sites and

skin, and characterized by an indolent clinical course and good prognosis.18,42 Plasmablastic

lymphoma of the elderly is another newly recognized entity with a relatively indolent clinical

course and better prognosis compared with other age-related EBV-positive B-cell LPDs24.

Plasmablastic lymphoma of the elderly shares features with age-related EBV-positive B-cell


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LPDs, including older age, immunosenescence, EBV infection, HIV negativity, and an ABC

immunophenotype, although contrasting features include a higher of extranodal infection and

MYC translocations and EBV type I latency pattern.24

Histopathologic Features

Two morphologic subtypes of EBV-positive DLBCL of the elderly have been recognized,

polymorphic and monomorphic. Both subtypes may include large transformed cells or

immunoblasts as well as Hodgkin and Reed-Sternberg (HRS)-like giant cells and may

demonstrate increased mitotic activity and areas of geographic necrosis.1 The polymorphic

subtype displays a broad range of B-cell maturation, and lesions are composed of centroblasts,

immunoblasts and plasmablasts with a variable component of admixed reactive cells,

including small lymphocytes, plasma cells, histiocytes and epithelioid histiocytes.1 Montes-

Moreno et al8 subdivided the polymorphic EBV-positive DLBCL of the elderly into three

subgroups based on the relative proportion of large neoplastic cells and presence of HRS-like

cells: (1) canonical large B-cell neoplasm (high density of large neoplastic cells and scattered

cells with HRS-like features); (2) DLBCL with Hodgkin lymphoma-like features (lower

density of neoplastic cells with HRS-like features); and (3) DLBCL with polymorphic LPD-

like features (low density of neoplastic cells without HL-like features). The monomorphic

subtype of EBV-positive DLBCL of the elderly is composed of monotonous sheets of large

transformed B cells (Figure 4).1 Although this distinction is helpful for recognizing the

spectrum, these morphologic variants do not impart clinical or prognostic significance.1 Cases

of EBV-positive DLBCL of the elderly also can have a mixed pattern with intermingled

polymorphic/and monomorphic areas8, suggesting that the subtypes represent two ends of a

morphologic spectrum.


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Immunophenotype

The neoplastic cells are of B-cell lineage as they express the pan B-cell antigens CD19, CD20,

CD22, CD79a and PAX5 and are negative for pan T-cell antigents. Immunoglobulin light

chain restriction may be difficult to demonstrate, except in cases with immunoblastic or

plasmablastic features in which cytoplasmic Ig can be assessed. Plasmacytoid cases can be

weakly positive or negative CD20.1 EBV-positive DLBCL of the elderly usually has an ABC

immunophenotype being MUM1/IRF4+ and CD10- and usually BCL6-. BCL-2 and CD30 are

usually positive; CD15 is negative.1,8 Ki-67 generally shows a high proliferation index.8 By

definition, the neoplastic cells are EBV-positive and accordingly display EBER, LMP1 in >

90% and EBNA2 in approximately 15% and 30% of cases.1,43 The concordance rate between

EBER and LMP1 can reach 80% to 90%, but is not uniformly consistent.

The proportion of EBV-positive DLBCL cases among all DLBCL patients increases with

age,12,44 which is analogous to the ABC subtype that also increases with age.45 These findings

are in accord with recent data indicating that aging is an important determinant of lymphoma

biology.46 In an effort to explain the proportional increase of ABC DLBCL with age,

Mareschal et al45 speculated that either a change in B-cell population during aging or on the

putative pathological specificity of EBV-positive DLBCL of the elderly. Not surprisingly,

most cases of EBV-positive DLBCL of the elderly displayed a striking shift to an ABC

immunophenotype with prominent activation of NF-κB pathway.8

Differential Diagnosis

The major entities in the differential diagnosis of EBV-positive DLBCL of the elderly are

summarized in Table 2. In part, EBV-positive DLBCL of the elderly is a diagnosis of


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exclusion. If there is a known cause of immunodeficiency, these tumors are better classified as

post-transplant LPDs, other iatrogenic immunodeficiency-associated LPDs, etc. Other forms of

EBV-positive DLBCL such as lymphomatoid granulomatosis, DLBCL associated with chronic

inflammation, primary effusion lymphoma, and plasmablastic lymphoma are also considered in

the differential diagnosis. Reactive diseases such as infectious mononucleosis of the elderly

and chronic active EBV infection also may be considered based on the morphologic findings

but clinical history and serologic testing are helpful.

Genetic and miRNA profile

Only a few genetic studies on cases of EBV-positive DLBCL of the elderly have been

performed.8,18 The immunoglobulin genes are monoclonally rearranged in most cases, with

clonality of EBV also usually detectable using EBV terminal repeat regions probes and

molecular techniques.1,5,7,8,18,47 IgH-mediated translocations are uncommon (~15%).8,18

However, these analyses were restricted to single loci (IGH, IGK, IGL, PAX5, C-MYC, BCL2

and BCL6). A single case with a t(9;14)(p13;q32) translocation involving the PAX5 gene has

been reported.48 Given the presence of a low number of genomic aberrations in EBV-positive

DLBCL of the elderly, it has been suggested that immunosenescence coupled with the EBV

oncogenic properties are sufficient and additional chromosomal alterations are therefore

usually not needed for lymphomagenesis.8

EBV was the first virus in which miRNAs were identified and 44 mature EBV-miRNAs are

currently known.49 A detailed list of EBV-miRNAs is summarized in Table 3. EBV-miRNAs

can regulate viral and cellular targets, and they generally target host transcripts during latent

infection (Table 3).50,51 Although viral miRNA constitutes only ~2% of all miRNA in EBV-

positive DLBCL, the viral miRNAs share seed sequence homology with cellular miRNA. For


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example, EBV-miR-BART5 and EBV-miR-BART1-3p share similarity with cellular miR-18

(miR-17-92 cluster) and miR-29a/b, respectively.52,53 Furthermore, viral miRNAs share seed

sequence homology among themselves.50 Moreover, individual EBV-miRNAs can target

multiple mRNAs, suggesting that many other targets are actually affected and await further

identification.54

Several studies have shown that EBV-miRNAs contribute to EBV-induced oncogenesis.

EBV-miR-BART5 targets and degrades p53-up-regulated modulator of apoptosis, a pro-

apoptotic protein.55 EBV-miR-BHRF1 promotes B-cell transformation and proliferation and

prevents apoptosis.56 EBV-miR-BART9 and BART17-5p can downregulate BCL6

expression.57 Considering the fact that BCL6 represses NF-κB, downregulation of BCL6 by

EBV-miRNA might indirectly contribute to NF-κB activation.57,58 Riley et al59 showed that

numerous EBV-miR-Bam HI-A region rightward transcripts (BARTs) likely target at least

132 apoptotic mRNAs in latency type III infection, highlighting their collective influence on

apoptosis regulation.

It also becomes evident that EBV-miRNAs have evolved to target multiple cellular pathways

rather than a single pathway. Pathway analysis has predicted that there are several EBV-

miRNA targeted genes in at least 20 specific pathways, including p53 feedback loops, B-cell

activation, oxidative stress response, inflammation mediated by chemokines or the cytokine

signaling pathway, the PI3K pathway and apoptosis (Figure 6).52

Interestingly, cellular miRNAs are modulated by viral proteins. Mir-155 has been shown in

DLBCL, especially in ABC subtype, and can be induced by LMP1 via the NF-κB pathway.60

Of note, miR-155 has been shown to co-target INPP5D (SHIP1), a key target implicated in


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lymphomagenesis, with EBV-miRNAs.59,61 Mir-34a is induced by LMP1 via the NF-κB

pathway and promotes the growth of EBV-transformed cells.62 EBNA2 has been shown to

induce miR-21 and downregulate miR-146a.63 Overexpression of miR-21 has been found to

promote tumor progression and invasion in solid tumors via targeting PTEN.64,65 Mir-146a has

ben shown to inhibit the NF-κB pathway in the NK/T-cell lymphoma cell lines.66 Therefore,

EBNA2 can provide extra signals via miRNAs to enhance the NF-κB and Akt pathways,

thereby providing survival/proliferation benefits.

Novel therapeutic approaches

Novel therapeutic approaches need to be considered for patients with EBV-positive DLBCL

of the elderly. Possible therapeutic approaches include: (1) EBV-specific adoptive

immunotherapy; (2) microRNA-targeted therapy; (3) combination therapy based on EBV lytic

phase induction followed by exposure of the tumor cells to anti-herpesvirus drugs; and (4)

targeting specific signaling pathways, including the NF-κB pathway (Table 4 and Figure 6).

EBV-specific adoptive immunotherapy, in which EBV-specific cytotoxic T-lymphocytes

(CTLs) are ex vivo activated and expanded and then reinfused seem promising.67 Using this

approach, EBV-specific CTLs could be engineered to recognize and lyse tumor cell targets via

chimeric receptors, while maintaining their ability to proliferate in response to EBV target

antigens and accordingly destroy virus-infected cells.68 Optimal tumor eradication appears to

require proper target antigen selection, co-stimulatory signalling and the ability of chimeric

antigen receptor-modified T-cells to traffic, persist, and retain function following adoptive

transfer.69 Recent evidence has suggested that both EBV-positive monomorphic PTLD that

resemble DLBCL and EBV-positive DLBCL of the elderly share features consistent with type


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III EBV latency, including immunodominant EBNA3A protein expression, indicating that

such an approach may be feasible.19

Given the aforementioned complex interplay between host and viral-encoded miRNAs in

EBV-mediated oncogenesis, targeting miRNA as a therapeutic approach appears to be

promising. Various approaches have been proposed: (1) anti-miRNA oligonucleotides are

inhibitory molecules blocking the interactions between miRNAs and their target mRNAs by

interacting directly to the target miRNA; (2) miRNA mask is a sequence with perfect

complementarity to the binding site for a miRNA in the target mRNA, which can form a

duplex with the target mRNA with higher affinity and block the access of the miRNA; (3)

miRNA sponges are synthetic decoys that contain multiple binding sites for a miRNA of

interest, preventing the interaction between miRNA and its targets; (4) small molecule

inhibitors against specific miRNAs can specifically inhibit miRNA synthesis; (5) restoring

activity of miRNAs with oncosuppressive functions; and (6) using miRNA to sensitize tumors

to known and efficient cancer therapeutic modalities.70

Another potential approach could be combining induction of EBV lytic phase with subsequent

exposure to anti-herpesvirus drugs.71 Lytic phase inducers may refer to phorbol esters, DNA

methylase transferase inhibitors, histone deacetylase (HDAC) inhibitors, proteasome

inhibitors, B-cell receptor-blocking antibodies, chemotherapeutic drugs and cellular

miRNAs.71,72 Although proof of principle of a combination therapy approach has been

documented in a clinical trial,73 the optimal pharmacological lytic phase inducer remains to be

determined. It has been recently shown that other HDAC inhibitors such as panobinostat

appear to be efficient and potent for the treatment of EBV-associated lymphomas.74


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Given that the EBV-positive DLBCL of the elderly is an aggressive post-germinal center B-

cell neoplasm characterized by prominent NF-κB activation8, targeting the NF-κB pathway

constitutes a rational therapeutic approach (Table 4). Bortezomib, a proteasome inhibitor, has

been found to induce apoptosis in EBV lymphoblastoid cell lines and to reduce canonical and

non-canonical activities of NF-κB pathway.75 A recent phase I/II clinical trial with bortezomib

plus R-CHOP in DLBCL abrogated the adverse outcome in non-GCB and showed similar

survival between GCB and non-GCB patients.76 Bay 11-7085, a pharmacological inhibitor of

NF-κB activity, or NF-κB/p65 small interfering RNA (siRNA), can inhibit DLBCL growth in

vitro via induction of apoptosis.25 In contrast with bortezomib, the latter strategies of NF-κB

inhibition down-regulate expression of key NF-κB regulated gene products, including Bcl-2

and Bcl-XL.25,75 NF-κB essential modulator-binding domain peptide has been shown to inhibit

NF-κB target gene expression and reduce tumor burden in an in vivo model as well.77

Recent evidence indicates a critical function of PI3K-PDK1 signaling upstream of NF-κB in

ABC-DLBCL cells.78 PI3K inhibition reduced NF-κB activity and decreased the expression of

NF-κB target genes in ABC-DLBCL cell lines, providing a rationale for the pharmacological

use of PI3K inhibitors for treating patients with ABC-type DLBCL.78 In addition, LMP1 has

been implicated in the activation of the PI3K/Akt signaling pathway, which lies upstream of

mTOR.79 Experimental evidence stemming either from dual PI3K/mTOR inhibitors or a single

mTOR inhibitor further reinforces the putative exploitation of LMP1/LMP2A-activated

PI3K/Akt/mTOR signaling pathway.80 Another attractive target for developing antiviral or

antitumor strategies is EBNA1 owing to its essential role in maintaining the EBV genome and

the consistent expression of EBNA1 in all proliferating EBV-positive cells.81 Sun et al showed

that Hsp90 inhibitors block the outgrowth of EBV-infected malignant cells in vitro and in vivo

through an EBNA1-dependent mechanism.81 Notably, Hsp90 inhibitors have been found to


- 17 -

suppress NF-κB activity.82 A CD30-directed antibody-drug conjugate could be a good option

given frequent CD30 expression in EBV-positive DLBCL patients. A recent trial Brentuximab

Vedotin in 2 cases of EBV-positive DLBCL did not show promising result, but small size trial

might be not representative.83

Conclusions

We have provided a comprehensive review of EBV-positive DLBCL of the elderly discussing

its epidemiologic, pathogenetic and clinicopathologic features, differential diagnosis, and

proposed some comments about potential novel therapeutic approaches for patients with this

disease. Currently, an arbitrary cutoff of 50 years is used to define this entity in the current

WHO classification, but patients younger than 50 years can also be affected. Although these

tumors were originally reported as usually being extranodal, it is clear that nodal presentation

is also seen. Most cases of EBV-positive DLBCL of the elderly have an activated B-cell

immunophenotype. Assessment for EBV infection in tissue biopsy specimen can be easily

performed by in situ hybridization analysis for EBER, but other methods of EBV detection

including PCR can be used. Caution should be exercised before rendering this particular

diagnosis as other causes of EBV-positive DLBCL must be excluded and there can be no

known explanation for immunodeficiency. Clearly, a better understanding of the molecular

genetic basis of EBV-positive DLBCL of the elderly is needed. Further studies are required to

address the heterogeneous EBV latency status and miRNA expression profile of EBV-positive

DLBCL of the elderly, as well as the role of the NF-κB pathway.


- 18 -

Acknowledgments

This work was supported by an MD Anderson Pathology Fellowship Award (C.Y.O); The

University of Texas MD Anderson Cancer Center Institutional R & D Fund, an Institutional

Research Grant Award, an MD Anderson Lymphoma SPORE Research Development

Program Award, an MD Anderson Myeloma SPORE Research Development Program Award,

and MD Anderson Collaborative Research Funds with Daiichi Sankyo Pharmaceuticals, High-

Throughput Molecular Diagnostics and Roche Molecular System (K.H.Y.). This work was

also partially supported by National Cancer Institute and National Institute of Health

(R01CA138688, 1RC1CA146299, P50CA136411 and P50CA142509).

Authorship Contributions:

C.Y.O., T.P., L.J.M., and K.H.Y. designed the study, analyzed data, and wrote the paper.

CONFLICTS OF INTEREST DISCLOSURE

The authors declare no conflicts of interest.


- 19 -

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- 25 -

Table 1. Frequency of EBV positivity with different cutoffs in B-cell lymphoproliferative

disorder among different countries

Country EBER+ cases %

EBER cutoff

20%

EBER cutoff

50% Diagnosis Ref

Japan 13/114 11.4 not specified not specified DLBCL 11

Japan 156/1,792 8.7 n/a 156/1,792 (8.7%) B-LPD* 12

Japan 5/460* 1.1 11/460 (2.4%) 5/460 (1.1%) DLBCL 14

Korea 34/380 8.9 34/380 (8.9%) n/a DLBCL 13

Korea 14/468 3.0 n/a 14/468 (3.0%) DLBCL 15

China 10/212 4.7 > 50% DLBCL DLBCL 9

Turkey 12/178* 6.7 not specified not specified DLBCL 10

Peru 28/188* 14.9 28/188 (14.9%) 17/188 (9.0%) DLBCL 4

Mexico 9/134 6.7 9/134 (6.7%) 9/134 (6.7%) DLBCL 7

Germany 4/167 2.4 4/167 (2.4%) 4/167 (2.4%) DLBCL 7

S/I/A 8/258 3.1 7/258 (2.7%) 5/258 (1.9%) DLBCL 6

USA

5 patients

(2002-2007) n/a not specified not specified DLBCL 5

Saudi

Arabia 16/217 7.4 not specified not specified DLBCL 25

*EBER positive rate was re-calculated in cases with an asterisk after excluding

immunodeficiency-associated LPD (including PTLD and HIV-associated lymphoma),

autoimmune-associated LPD, and non-DLBCL patients from each study.

** EBER: EBV-encoded RNA; LPD: lymphoproliferative disorder; S/I/A: Switzerland, Italy,

and Austria; Ref: reference; n/a: not applicable; DLBCL: diffuse large B-cell lymphoma


26

Tabl

e 2.

Diff

eren

tial d

iagn

ostic

con

side

ratio

ns o

f EB

V-p

ositi

ve D

LBC

L of

the

elde

rly

Entit

y Si

te

Arc

hite

ctur

e Im

mun

ophe

noty

pe

Spec

ific

findi

ngs

Ligh

t cha

in

Clo

nalit

y

EBV

-pos

itive

DLB

CL,

eld

erly

N

(70%

)

E (3

0%)

Effa

ced

Pan

B m

arke

rs+

CD

10- /B

CL6

- /MU

M1+ ,

EBER

+ ,CD

30+/

-

Geo

grap

hic

necr

osis

M

onot

ypic

M

onoc

lona

l

EBV

-ass

ocia

ted

reac

tive

atyp

ical

lym

phop

rolif

erat

ions

/infe

ctio

us

mon

onuc

leos

is in

eld

erly

N

Not

effa

ced

Mix

ed B

- and

T-c

ell n

atur

e of

HR

S-lik

e ce

lls (C

D3+ /2

0+ /30+ )

EBER

+

n/a

Poly

typi

c Po

lycl

onal

EBV

-pos

itive

cla

ssic

al H

odgk

in

lym

phom

a

N

Effa

ced

CD

20- /C

D79

a- /Pax

5w+

CD

30+ /C

D15

+ , EB

ER+/

-

HR

S ce

lls in

reac

tive

back

grou

nd

Poly

typi

c Po

lycl

onal

DLB

CL,

NO

S, a

napl

astic

var

iant

N

Ef

face

d Pa

n B

mar

kers

+

EBER

- , CD

30+

Sinu

soda

l or c

ohes

ive

patte

rn

Mon

otyp

ic

Mon

oclo

nal

T-ce

ll/hi

stio

cyte

-rich

larg

e B

-cel

l

lym

phom

a

N

Effa

ced

Pan

B m

arke

rs+

EBER

- , CD

30+/

- , CD

15-

Scat

tere

d B

cel

ls in

ben

ign

T-ce

lls/h

istio

cyte

s

back

grou

nd

Mon

otyp

ic

Mon

oclo

nal

Prim

ary

cuta

neou

s DLB

CL,

leg

type

E (S

kin)

D

erm

al

infil

tratio

n

Pan

B-c

ell m

arke

rs+ , E

BER

-

BC

L2+ /M

UM

1+ /FO

XP1

+

Diff

use

shee

ts o

f

cent

robl

asts

and

Mon

otyp

ic

Mon

oclo

nal


27

imm

unob

last

s

Lym

phom

atoi

d gr

anul

omat

osis

E po

lym

orph

ic

infil

tratio

n

EBER

+ , LM

P1-

Ang

ioin

vasi

on, H

RS-

like

cells

in re

activ

e ba

ckgr

ound

Non

-

info

rmat

ive

Mon

oclo

nal

Plas

mab

last

ic ly

mph

oma

of th

e

elde

rly

N (5

0%)

E (5

0%)

Effa

ced

CD

38+ , C

D13

8+ , MU

M1+ , C

D20

-

EBER

+ , LM

P1- , E

BN

A2-

Plas

mab

last

ic m

orph

olog

y

EBV

late

ncy

I

Mon

otyp

ic

n/a

Ang

ioim

mun

obla

stic

T-c

ell

lym

phom

a

N

Effa

ced

CD

10+ , C

XC

L13+ , P

D1+

FDC

exp

ansi

on

Poly

typi

c M

onoc

lona

l

in T

-cel

ls

Perip

hera

l T-c

ell l

ymph

oma,

not

othe

rwis

e sp

ecifi

ed

N

Effa

ced

CD

3+/- w

ith p

artia

l los

s of T

-

mar

kers

, CD

30+/

- , CD

15-/+

EBER

-/+

Larg

e at

ypic

al c

ells

clea

r cyt

opla

sm+/

-

Poly

typi

c M

onoc

lona

l

in T

-cel

ls

* Pa

n B

-cel

l mar

kers

: CD

19, C

D20

, CD

22; S

ite N

: nod

al; S

ite E

: ext

rano

dal;

EBV

: Eps

tein

Bar

r viru

s; E

BER

: EB

V-e

ncod

ed R

NA

; DLB

CL:

diff

use

larg

e B

-

cell

lym

phom

a; H

RS:

Hod

gkin

Ree

d-St

ernb

erg;

n/a

: non

-ava

ilabl

e

** R

efer

ence

s 1,

25,4

2-44

,46,

82,8

4-88


- 28 -

Table 3. Viral and cellular targets and functions of ebv-miRNAs

EBV-miRNA Function of ebv-miR Reference

Viral targets

BALF5 EBV-miR-BART2 Inhibition of lytic cycle 89

EBNA2 EBV-miR-BART6 Maintenance of viral latency 90

LMP1EBV-miR-BART-Cluster1 Growth control or escape from

immunosurveillance 59,91

EBV-miR-BART19-5p Prevent apoptosis 59

LMP2A EBV-miR-BART22 Growth control or escape from immunosurveillance

92

Cellular targets

Dicer EBV-miR-BART6 Promote latency 90

Bim EBV-miR-BART-Cluster 1 Anti-apoptosis 93

EBV-miR-BART-Cluster 2 Anti-apoptosis 93

CXCL-11/I-TAC EBV-miR-BHRF1-3 Anti-apoptosis 94

PUMA EBV-miR-BART5 Anti-apoptosis 55,93

IPO7 EBV-miR-BART3-3p Anti-inflammation 59,95

TOMM22 EBV-miR-BART16-5p Anti-apoptosis 59,95

MICB EBV-miR-BART2-5p Immune evasion 96

BCLAF1 EBV-miR-BART17-5p Anti-apoptosis 59

CLEC2D (LLT1) EBV-miR-BART3-3p Immune evasion 52

EBV-miR-BART1-3p Immune evasion 52

LY75 (DEC205) EBV-miR-BART1-5p Immune evasion 52

CLEC7A (Dectin-1) EBV-miR-BART4 Immune evasion 52

CLEC2B (AICL) EBV-miR-BART2-5p Immune evasion 52

NLRP3 EBV-miR-BART15 Immune dysregulation 97


- 29 -

Table 4. Differentially expressed genes in EBV-positive ABC DLBCL vs. EBV-negative ABC DLBCL

A. Genes upregulated in EBV-positive ABC-DLBCL

Gene functional categories

No. of genes

Representative genes

NF-κB target 11 PRDM1, CYLD, FYN, TNFAIP3, FTH1, LITAF

Cell cycle regulation 4 CYTIP, PPM1A, TMEM30A, MRPL41

Anti-apoptosis 4 MDM2, DNAJC5, SERPINB9, GSTO1

Tumor progression 5 LAMP1, PTP4A2, DYNLRB1, ASAP1, ERLEC1

Cell proliferation 3 TXN, PDXK, GLUL

Cell adhesion and cytoskeleton

6 GABARAP, WIPF, AOC3, DST, SLAMF7, C1orf38

Intracellular trafficking 4 TMED2, RAB27A, M6PR, FLOT1

Immune response 2 SAMHD1, GBP2

Cell metabolism 12 GPD2, P4HB, GNS, RPN2, APOL6, LASS6

Transcription factors 2 ARNTL, TLE3

Others and unknown 17 RPN2, SLC31A1, M6PR, SETD3, KIAA2013, TPCN2

B. Genes downregulated in EBV-positive ABC-DLBCL

Gene functional categories

No. of genes

Representative genes

Pro-apopototic 2 TBX5, CUL3

Immune response 2 ENPP3, CD79B

Cell metabolism 3 GATM, MOCOS, GALNT6

ECM, adhesion, cytoskeleton

10 CDHR1, CDHR3, CDH11, TRIP6, CLDN10, SMAGP

Signaling and transcription factors

3 CHN2, STYXL1, SPIB

Others and unknown 49 SPINK5, MYO3A, PHF14, LRRC39, MAGEB3, MGP

* ECM: extracellular matrix


- 30 -

Figure legends

Figure 1. Schematic diagram of linear EBV genome (~172 Kb).

(A), In proportion to their size, largely unique (U1-U5), internal repeat (IR1-IR4) and terminal repeat

(TR) sequence domains are demonstrated. Ori P (indicated in red), the origin for latent infection EBV

episome replication, has plasmid maintenance and DNA replication activity. Location of exons for

latent EBV-induced membrane proteins and nuclear proteins and the size of each gene are shown. Of

note, LMP1 transcription has an opposite direction to the other gene transcripts. LMP2 can only be

transcribed when EBV exists as episome because the gene spans across the TR. LMP2A is transcribed

from the exon 1, whereas LMP2B is transcribed from the exon 2. EBNAs are transcribed from

differently spliced primary EBNA transcript. In latency III, EBNA1 transcription begins from Cp or

Wp, whereas it starts from Qp in latency I or II. EBNA-LP has variable sizes due to variable numbers

of repetitive exons.98

(B), Schematic diagram of circular EBV genome. The U1-U5, IR1-IR4, TR, and Ori P sequence

domains are indicated. Open reading frames for latent proteins are shown. Of note, the size of each

open reading frame does not illustrate the size of each latent protein.98

(C), Amino acid sequence of LMP1. C-terminal activating regions (CTARs) are indicated in red.

TRAF-binding motif in CTAR1 and TRADD-binding motif are indicated in blue with an underbar in

the CTAR1 and CTAR2 domains, respectively. Numbers on the right denote the order of the amino

acid sequence.99 Cp: C promoter, Qp: Q promoter, Wp: W promoter, LMP: latent membrane protein,

EBNA: Epstein-Barr virus nuclear antigen, EBNA-LP: EBNA leader protein, EBER: Epstein-Barr

virus-encoded RNA

Figure 2. Schematic diagram of EBV-mediated oncogenic signalling pathway activation in EBV-

positive diffuse large B-cell lymphoma of the elderly. LMP1 has a six-transmembrane domain with a

long cytoplasmic C-terminal chain. It can self-aggregate to activate itself constitutively and provide a

platform to interact with downstream molecules. LMP1 provides a proliferation signal via activating


- 31 -

NF-κB, PI3K/Akt, MEK-ERK, and JNK-AP-1 MAP kinase pathways. It also gives signal for cell

cycle progression by enhancing cyclin-dependent kinase 2 and phosphorylation of retinoblastoma

protein and by inhibiting p16 and p27. LMP1 can also activate bcl-2 to provide an anti-apoptotic

signal. CTAR1 can directly interact with TRAF1, 2, 3, and 5 to activate NF-κB, PI3K/Akt, MEK-

ERK, and JNK-AP-1 MAP kinase pathways. CTAR2 needs TRADD to interact with TRAF2 to

activate NF-κB. Both canonical and non-canonical pathways of NF-κB are activated to give

proliferation signal to the nucleus. There are 3 putative JAK3-binding motifs between CTAR1 and

CTAR2. However, this finding was not reproduced by others.26-36 AP-1: activator protein 1, CTAR:

C-terminal activation region, ERK: extracellular signal-regulated kinases, JAK3: Janus kinase 3, JNK:

Jun amino-terminal kinases, MEK: MAPK (mitogen-activated protein kinase)/ERK kinase, PI3K:

phosphatidylinositol 3-kinase, TRADD: tumor necrosis factor receptor type 1-associated DEATH

domain, TRAF: tumor necrosis factor receptor-associated factor

Figure 3. Geographical variations of overall survival and progression-free survival in EBV positive

diffuse large B-cell lymphoma of the elderly. (A and B), EBV positive DLBCL of the elderly showed

worse overall survival and progression-free survival in Korean patients13; (C and D), EBV positive

DLBCL of the elderly were more aggressive compared to > 60 year-old patients with ABC subtype

and GCB subtype in European patients8. (E and F), In North America, EBV positive DLBCL of the

elderly showed no difference in overall survival and progression-free survival in comparison to EBV

negative DLBCL patients100. (G and H), In North America, EBV+CD30+ DLBCL of the elderly

showed significant difference in overall survival and progression-free survival in comparison to either

EBV+CD30- or EBV-D30+ DLBCL patients.

Figure 4. Morphologic variants and immunophenotypic profiling in EBV-positive diffuse large B-cell

lymphoma of the elderly. (A-F), The monomorphic subtype is characterized by monotonous sheets of

large transformed B cells. (G-L), Polymorphic DLBCL-like shows canonical large B-cell neoplasm

morphology, characterized by a high density of large neoplastic cells and scattered cells with (Reed-


- 32 -

Sternberg) RS-like and Hodgkin-like features. (M-R), The polymorphic HL-like image, the image

with DLBCL with Hodgkin lymphoma-like features, shows a lower density of neoplastic cells with

RS-like and Hodgkin-like features. (S-X), The polymorphic PLPD-like subtype is DLBCL with

polymorphic lymphoproliferative disorder (LPD)-like features. It is characterized by a low density of

neoplastic cells without HL-like features. EBV-positive DLBCL of the elderly is predominantly the

ABC subtype. Interestingly, the monomorphic case presented in this figure shows the GCB subtype

(A-F). All polymorphic subtypes show the ABC-DLBCL molecular phenotype.

Figure 5. Gene expression profiling in the DLBCL ABC subtype was performed according to EBV

status. The EBV-positive case shows a distinct gene expression pattern compared to cases with EBV

negativity, with overexpression of multiple components of the NF-κB pathway.

Figure 6. Therapeutic modulation of EBV infection and associated signaling pathways. Strategies to

inactivate EBV infection or EBV associated oncogenic pathways have been explored in lymphoma

cells. Many therapeutic targets have been identified. (1) Conventional chemotherapeutic agents or

radiation target DNA. (2) Different strategies disrupting miRNAs can be offered. (3) EBV-specific

cytotoxic T-cells are engineered and re-infused to patients as immunoregulatory therapy approach. (4)

Lytic cycle of EBV is induced by a couple of agents and anti-herpetic agents targeting virus in lytic

cycle are added. Agents targeting (5) NF-κB pathway, (6), PI3K/Akt pathway, (7) PKC or (8) MAPK

pathway can be tried. Monoclonal antibodies, Brentuximab vedotin and Rituximab, are available

targeting, (9) CD30 and (10) CD20, respectively.


EB

V G

enom

e, 1

72 k

b

Figu

re-1

Figu

re-2

x

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 0

A

C

E

G

B

D

F

H

Survival Time (months)

Surv

ival

Tim

e (%

)

EBV-CD30+ (n=65)

EBV+CD30+ (n=14)

EBV-CD30+ (n=65)

EBV+CD30+ (n=14)

EBV+ (n=13)

EBV- (n=171)

EBV+ (n=13)

EBV- (n=171)

EBV- (n=346)

EBV+ (n=34)

EBV- (n=346)

EBV+ (n=34)

EBV-, GCB (n=162)

EBV-, ABC (n=78)

EBV+ DLBCL (n=38)

EBV-, GCB (n=162)

EBV-, ABC (n=78)

EBV+ DLBCL (n=38)

P=0.026 P=0.018

P < 0.001 P < 0.001

P = 0.14 P = 0.14

P < 0.0001 P < 0.0001

Overall Survival Progression Free Survival

Figure-3

A B C

G

H I

M

N

O

S T U

Mon

omor

phic

Po

lym

orph

ic-

DLBC

L-lik

e Po

lym

orph

ic-

HL-li

ke

Poly

mor

phic

-PL

PD-li

ke

H &

E

CD10

BCL6

Figu

re-4

D E F

J K L

P Q

R

V W

X

MU

M1

FOXP

1

EBER

Mon

omor

phic

Po

lym

orph

ic-

DLBC

L-lik

e Po

lym

orph

ic-

HL-li

ke

Poly

mor

phic

-PL

PD-li

ke

Figu

re-4

Figu

re-5

EB

V+D

LBC

L EB

V- DLB

CL

Figu

re-6

EBV-positive diffuse large B-cell lymphoma of the elderly: A case series from Peru

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Transcript of EBV-positive diffuse large B-cell lymphoma of the elderly: A case series from Peru