The

Oncologist ®

Lymphoma
EBV-Associated Lymphoproliferative Disorders: Classification
and Treatment

ANTONINO CARBONE,a ANNUNZIATA GLOGHINI,a GIAMPIETRO DOTTIb

a
Department of Pathology, Fondazione IRCCS Istituto Nazionale Tumori, Milano, Italy; bCenter for Cell and

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Gene Therapy, Baylor College of Medicine, The Methodist Hospital, Houston, Texas, USA

Key Words. EBV • EBV-associated lymphomas • Post-transplant–associated lymphoproliferative disorders •

HIV-associated lymphoproliferative disorders • EBV cell– based immunotherapy

Disclosure: No potential conflicts of interest were reported by the authors, planners, reviewers, or staff managers of this article.

LEARNING OBJECTIVES
After completing this course, the reader will be able to:
1. Assess patients with EBV-associated lymphoproliferative disorders.
2. Describe the pathogenesis of the lymphoproliferative disorders linked to EBV infection.
3. Evaluate EBV cell– based immunotherapy for use in patients with EBV-associated lymphoproliferative disorders.
CME This article is available for continuing medical education credit at CME.TheOncologist.com.

ABSTRACT
Since its discovery as the first human tumor virus,
Epstein-Barr virus (EBV) has been implicated in the
development of a wide range of B-cell lymphoprolif-
erative disorders, including Burkitt’s lymphoma,
classic Hodgkin’s lymphoma, and lymphomas arising
in immunocompromised individuals (post-transplant
and HIV-associated lymphoproliferative disorders).
T-cell lymphoproliferative disorders that have been
reported to be EBV associated include a subset of pe-
ripheral T-cell lymphomas, angioimmunoblastic T-
cell lymphoma, extranodal nasal type natural killer/
T-cell lymphoma, and other rare histotypes. EBV
encodes a series of products interacting with or exhib-
iting homology to a wide variety of antiapoptotic mol-
ecules, cytokines, and signal transducers, hence
promoting EBV infection, immortalization, and
transformation. However, the exact mechanism by
which EBV promotes oncogenesis is an area of active
debate. The focus of this review is on the pathology,
diagnosis, classification, and pathogenesis of EBV-
associated lymphomas. Recent advances in EBV cell–
based immunotherapy, which is beginning to show
promise in the treatment of EBV-related disorders,
are discussed. The Oncologist 2008;13:577–585

Correspondence: A. Carbone, M.D., Department of Pathology, Fondazione IRCCS Istituto Nazionale Tumori, via Venezian 1, Milano
I-20133, Italy. Telephone: 39-02-2390-2876; Fax: 39-02-2390-2877; e-mail: antonino.carbone@istitutotumori.mi.it Received Febru-
ary 13, 2008; accepted for publication March 18, 2008. ©AlphaMed Press 1083-7159/2008/$30.00/0 doi: 10.1634/theoncologist.2008-
0036

The Oncologist 2008;13:577–585 www.TheOncologist.com

578 EBV-Associated Lymphomas

INTRODUCTION
Epstein-Barr virus (EBV) is a member of the herpesvirus Table 1. Latent EBV-encoded genes
family. As with other herpesviruses, EBV is an enveloped Latency
EBV-encoded genes Location type
virus that contains a DNA core surrounded by an icosahe-
dral nucleocapsid and a tegument. Family members include EBNA-1 Nucleus I, II, III
herpes simplex I and II and varicella zoster virus (␣-herpes- EBNA-2 Nucleus III
virus subfamily), cytomegalovirus and human herpesvirus EBNA-3 Nucleus III
(HHV)-6 and HHV-7 (␤-herpesvirus subfamily), and LMP-1 Membrane II, III
HHV-8 and EBV (␥-herpesvirus subfamily) [1]. LMP-2 Membrane II, III
Human tumors have been attributed to both HHV-8 EBER-1 and EBER-2 Nucleus I, II, III
(Kaposi’s sarcoma, primary effusion lymphoma [PEL], and Abbreviations: EBV, Epstein–Barr virus; EBNA,
Epstein–Barr nuclear antigen; LMP, latent membrane
multicentric Castleman’s disease) and EBV (Burkitt’s lym- protein; EBER, Epstein–Barr RNA.
phoma, nasopharyngeal carcinoma, and Hodgkin’s and

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non-Hodgkin’s lymphomas) [2–5].
In this review we discuss only the EBV-associated lym-
phoproliferative disorders, dividing them into B-cell and
T/natural killer (NK)-cell lymphomas. The focus is on the
pathology, diagnosis, and classification, pathogenesis, and
treatment of EBV-associated lymphomas.
EBV PRODUCTS AND PATTERNS OF EBV
GENE EXPRESSION
The EBV genome encodes a series of products interacting
with or exhibiting homology to a wide variety of antiapo-
ptotic molecules, cytokines, and signal transducers, hence
promoting EBV infection, immortalization, and transfor-
mation [6 – 8].
Based on patterns of expression of the EBV genome,
three types of latent gene expression have been described:
latency I, II, and III (Table 1). During latency I, Epstein–
Barr nuclear antigen 1 (EBNA-1) and the two small non-
coding Epstein–Barr RNAs (EBERs) are expressed [9].
EBV gene expression in latency II is usually limited to
EBNA-1, the EBERs, latent membrane protein (LMP)-1,
and LMP-2A and LMP-2B [10]. Latency III usually in-
volves the unrestricted expression of all EBNAs, EBERs,
and LMPs [10]. Latency I is generally associated with the
EBV-related Burkitt’s lymphoma [9], latency II has been
associated with classic Hodgkin’s lymphoma (cHL) and T-
cell non-Hodgkin’s lymphoma (NHL) [11], and latency III
occurs mainly in immunocompromised individuals suffer-
ing from post-transplant lymphoproliferative disorders
(PTLDs) and HIV-associated lymphoproliferative disor-
ders and in lymphoblastoid cell lines [12].
Furthermore, EBV encodes the following important
proteins that show sequence and functional homology to di-
verse human proteins: BCRF-1 and interleukin (IL)-10,
BHRF-1 and BCL-2, BARF-1 and intracellular adhesion
molecule 1 [13].
Table 2. EBV-associated lymphoproliferative disorders
EBV-associated B-cell lymphoproliferative disorders
Burkitt’s lymphoma
Classic Hodgkin’s lymphoma
Post-transplant lymphoproliferative disorders
HIV-associated lymphoproliferative disorders
Primary central nervous system lymphoma
Diffuse large B-cell lymphoma, immunoblastic
HHV-8–positive primary effusion lymphoma and its
solid variant
Plasmablastic lymphoma
Other histotypes (rare)a
aOther histotypes include: lymphomatoid granulomatosis,
pyothorax-associated lymphoma, senile EBV-associated
B-cell lymphoproliferative disorders.
Abbreviations: EBV, Epstein–Barr virus; HHV-8, human
herpesvirus 8.
EBV-ASSOCIATED B-CELL
LYMPHOPROLIFERATIVE DISORDERS
Since its discovery as the first human tumor virus, EBV has
been implicated in the development of a wide range of B-
cell lymphoproliferative disorders (Table 2).
Burkitt’s Lymphoma
Burkitt’s lymphoma is a highly proliferative B-cell tumor
that includes three variants: endemic (affecting children in
equatorial Africa and New Guinea), sporadic (affecting
children and young adults throughout the world) (Fig. 1),
and immunodeficiency related (primarily in association
with HIV infection). EBV has been detected in virtually all
cases of the endemic variant, 15%–20% of cases of the spo-
radic variant, and 30%– 40% of cases of the immunodefi-
ciency-related variant [1]. In all variants, irrespective of
EBV status, constitutive activation of the c-myc oncogene
through its translocation into one of the immunoglobulin
Carbone, Gloghini, Dotti
Figure 1. Burkitt’s lymphoma. EBV is detected in almost all
tumor cells. (Inset): Tumor consists of a homogeneous prolif-
eration of medium-sized cells displaying a cohesive pattern.
Tumor cells have round nuclei, multiple centrally located nu-
cleoli, and limited cytoplasm. (EBER in situ hybridization, he-
matoxylin counterstain, original magnification ⫻200; inset,
hematoxylin-eosin, original magnification ⫻400.)
Abbreviations: EBER, Epstein–Barr RNA; EBV, Epstein–
Barr virus.
loci is clearly the key factor in the oncogenesis of Burkitt’s
lymphoma [1]. The detection of somatic hypermutations in
the V region of the immunoglobulin genes and the pheno-
type of the Burkitt’s lymphoma cells indicate a germinal
center (GC) cell origin of the lymphoma [14]. The tumors
isolated from nonendemic Burkitt’s lymphoma patients are
usually from different stages of B-cell development than
those isolated from patients with endemic Burkitt’s lym-
phoma [15, 16].
Most EBV-positive cases exhibit a highly restrictive
pattern of expression of latent encoded proteins, only ex-
pressing EBNA-1 and the EBERs (latency I) [1]. However,
it was recently reported that some cases, in addition to
EBNA-1 and the EBERs, express EBNA-3A, EBNA-3B,
EBNA-3C, and EBNA leader protein but still lack EBNA-2
and the latent membrane proteins [17]. EBNA-1 plays a
crucial role in the maintenance and replication of the viral
genome, but its oncogenic potential is highly controversial
[1, 18]. Conversely, as the EBERs are believed to possess
antiapoptotic activity, it has been postulated that they may
play an essential role in the oncogenesis of Burkitt’s lym-
phoma [19].
cHL
Not all subtypes of cHL harbor EBV to the same degree
[20 –22]. There are also data that suggest that the incidence
of EBV-positive cHL is age related [23].
Hodgkin’s Reed-Sternberg (RS) cells of cHL represent
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579
transformed B cells. The detection of destructive somatic
mutations in the rearranged immunoglobulin genes of RS
cells of cHL indicates that they originate from preapoptotic
GC B cells [24]. In RS cells, constitutive nuclear factor
(NF)-␬B activation, which is essential for RS cell survival,
seems to result from various mechanisms. Several recep-
tors, such as CD30, CD40, receptor activator of nuclear fac-
tor ␬B (RANK), and RANK ligand, which activate the
classic NF-␬B pathway, are expressed by RS cells, and li-
gands for these receptors are frequently expressed on acti-
vated CD4⫹ T cells and other bystander cells surrounding
RS cells, including eosinophils, neutrophils, and B-cell
subsets. In EBV-positive cHL cases, the LMP-1 gene also
contributes to NF-␬B activation because it mimics acti-
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vated CD40 receptors [25–27].
In HIV/AIDS persons with severe immunosuppression,
the acquisition of an antiapoptotic phenotype by RS cells is
not a result of CD40 –CD40L interactions, which are lack-
ing, but rather is induced by EBV-encoded LMP-1, which is
functionally homologous to activated CD40. Thus, EBV is
thought to play a pivotal role in the pathogenesis of lym-
phoma in patients with immunosuppression [28].
EBV-Associated Lymphomas in
Immunocompromised Individuals
There exist several distinct classes of EBV-associated lym-
phoproliferative disorders in immunocompromised indi-
viduals. First, there is a disorder resulting from an inherited
immunodeficiency known as X-linked lymphoproliferative
disorder. Second, there are lymphomas associated with
immunosuppressive drugs given to transplant recipients.
Finally, there are AIDS-related lymphoproliferative dis-
orders. The most common gene-expression pattern in these
disorders is latency III.
PTLDs
Since the original report in 1969 [29, 30], it has been well
established that there is a higher incidence of lymphopro-
liferative disorders in transplant recipients of both a solid
organ and bone marrow. According to the World Health Or-
ganization (WHO) classification [31], PTLDs may be clas-
sified into: (a) early lesions, generally represented by EBV-
driven polyclonal lymphoproliferations and (b) true
monoclonal diseases, including polymorphic PTLD and
monomorphic PTLD; the latter is further distinguished into
Burkitt’s lymphoma/Burkitt’s-like lymphoma, diffuse
large B-cell lymphoma (DLBCL), and cHL.
Early-onset PTLDs are mainly regarded as EBV-driven
lymphoproliferations that are frequently, though not al-
ways, polyclonal or oligoclonal, whereas most late-onset
PTLDs are true monoclonal lymphoid malignancies that are
580
not necessarily associated with EBV infection. Correlative
studies of the morphologic and molecular features of
PTLDs have contributed to the recognition of specific dis-
ease categories and have provided prognostic indicators for
these disorders [32]. Among monoclonal B-cell PTLD, der-
ivation from GC-related B cells occurs independently of the
type of organ transplanted, the interval between transplant
and lymphoma, the histology, and the site of origin of the
lymphoma.
Oncogenic viruses known to be involved in PTLD
pathogenesis include EBV and HHV-8. Both EBV and
HHV-8 act predominantly through direct mechanisms, that
is, the virus is able to directly infect the tumor clone and
exerts a transforming effect upon B cells. Viral infection in
PTLD exploits several strategies to ensure persistent infec-
tion, namely, prevention of death of infected cells, enhance-
ment of their proliferation to maintain the infected
reservoir, and evasion of the immune system [33–35]. Sev-
eral lines of evidence suggest that EBV infection has a ma-
jor pathogenetic role in PTLDs: (a) EBV infects ⬃60%–
80% PTLD patients, including 100% of early-onset PTLD
patients [6]; (b) in many cases of monomorphic PTLD,
EBV infection is monoclonal, consistent with the hypothe-
sis that the virus has been present in the tumor progenitor
cell since the early phases of clonal expansion; (c) a de-
crease in EBV-specific cytotoxic T lymphocytes (CTLs)
and an increase in the EBV viral load are strongly associ-
ated with PTLD development [36]; and (d) treatment of
PTLDs with autologous EBV-specific CTLs results in viral
load control and tumor size reduction.
HIV-Associated Lymphoproliferative Disorders
HIV-associated lymphoproliferative disorders are a het-
erogeneous group of diseases that arise in the presence of
HIV-associated immunosuppression, a state that permits
the unchecked proliferation of EBV-infected lympho-
cytes. Traditionally, these aggressive disorders include
both central nervous system and systemic lymphomas.
PEL also occurs and often involves EBV in addition to
HHV-8.
The categories of HIV-associated NHL (HIV-NHL) in-
cluded in the latest WHO proposal [37] are grouped as fol-
lows. (a) Lymphomas also occurring in immunocompetent
patients. The vast majority of these HIV-NHLs belong to
three high-grade B-cell lymphomas: Burkitt’s lymphoma,
DLBCL with centroblastic features, and DLBCL with im-
munoblastic features. According to the site of involvement,
the present spectrum of HIV-NHL includes extranodal/
nodal lymphomas and primary central nervous system
lymphomas. (b) Unusual lymphomas occurring more
specifically in HIV-positive patients. These lymphomas in-
EBV-Associated Lymphomas
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Figure 2. HIV-associated diffuse large B-cell lymphoma
with immunoblastic features. Most large tumor cells express
EBV infection, the signal is nuclear. (Inset): Tumor cells con-
tain abundant cytoplasm and round, oval or ovoid nuclei com-
monly containing prominent nucleoli. (EBER in situ
hybridization, hematoxylin counterstain, original magnifica-
tion ⫻400; inset, hematoxylin-eosin, original magnification
⫻400.)
Abbreviations: EBER, Epstein–Barr RNA; EBV, Epstein–
Barr virus.
clude two rare entities, namely, PEL and plasmablastic
lymphoma of the oral cavity [38, 39]. (c) Lymphomas also
occurring in other immunodeficiency states.
EBV-associated HIV-NHL includes primary central
nervous system lymphoma, systemic lymphomas having a
DLBCL immunoblastic morphology (Fig. 2), HHV-8 –
positive PEL and its solid variant with and without serous
effusions (Fig. 3), and plasmablastic lymphoma [7, 40 – 46]
(Table 2).
EBV-ASSOCIATED T/NK-CELL
LYMPHOPROLIFERATIVE DISORDERS
EBV is known primarily for its ability to infect B cells, but
it can also infect other cells. Several types of non-B-cell
NHL are associated with EBV (Table 3). T-cell lymphopro-
liferative disorders that have been reported to be EBV as-
sociated include a subset of peripheral T-cell lymphomas,
angioimmunoblastic T-cell lymphoma (AILT), extranodal
nasal type NK/T-cell lymphoma, enteropathy-type T-cell
lymphoma, ␥␦ T-cell lymphomas (hepatosplenic and non-
hepatosplenic), T-cell lymphoproliferative disorders after
chronic EBV infection, EBV-associated cutaneous T-cell
lymphoproliferative disorders (especially in Asia), and ag-
gressive NK-cell leukemia/lymphoma [8]. This section fo-
cuses on the two types in which EBV has been most directly
implicated: AILT and nasal T/NK-cell lymphoma.
Carbone, Gloghini, Dotti
Figure 3. HHV-8-associated extracavitary solid lymphoma
without serous effusion. Large tumor cells express EBV infec-
tion as detected by EBER in situ hybridization. Tumor cells are
pleomorphic and of heterogeneous size. (EBER in situ hybrid-
ization, hematoxylin counterstain, original magnification
⫻400.)
Abbreviations: EBER, Epstein–Barr RNA; EBV, Epstein–
Barr virus; HHV-8, human herpesvirus 8.
Table 3. EBV-associated lymphoproliferative disorders
EBV-associated T/NK-cell lymphoproliferative
disorders
Peripheral T-cell lymphoma, unspecified
Angioimmunoblastic T-cell lymphoma
Extranodal nasal T/NK-cell lymphoma
Other histotypes (rare)a
aOther histotypes include hepatosplenic T-cell lymphoma,
nonhepatosplenic ␥␦ T-cell lymphomas, enteropathy-type
T-cell lymphoma.
Abbreviations: EBV, Epstein–Barr virus; NK, natural
killer.
Peripheral T-Cell Lymphomas
Peripheral T-cell lymphomas (PTCLs) represent 10%–15%
of all lymphoid neoplasms [47]. They are a heterogeneous
group of tumors that in the Revised European-American
Lymphoma/WHO classification are subdivided into speci-
fied and unspecified forms [47, 48].
AILT
AILT is a PTCL characterized by systemic disease, a poly-
morphous infiltrate primarily involving lymph nodes, and
prominent proliferation of high endothelial venules and fol-
licular dendritic cells [49]. AILT is the second most com-
mon PTCL subtype, accounting for 15%–20% of cases
[49]. Laboratory findings include polyclonal hyper-␥-
globulinemia, circulating immunocomplexes, cold aggluti-
nins with hemolytic anemia, positive rheumatoid factor,
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581
and anti–smooth muscle antibodies. The clinical behavior
is very aggressive, the response to therapy is scarce, and the
long-term outcome is dismal. The differential diagnosis
with other PTCLs, and in particular with unspecified PTCL,
is puzzling and mainly based on different features of the re-
active components (i.e., prominent vascular structures and
abundant follicular dendritic cells) and phenotype (CD10
and BCL-6 expression) [50]. Notably, no unique markers
can reliably discriminate between these two entities. Re-
cently, chemokine (C-X-C motif) ligand 13 was proposed
as a possible candidate to distinguish the two diseases [51].
The molecular pathogenesis of AILT, as in general for
all peripheral T-cell neoplasms, is poorly understood. The
karyotype is often characterized by complex abnormalities,
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but specific alterations have not been identified, and only
few reports focused on the gene-expression profile of nodal
PTCLs [51, 52]. In one study, an AILT molecular signature
was identified together with a molecular link between AILT
and T follicular helper lymphocytes [52].
AILT is a lymphoma in which expanding B-cell clones
are often present beside the T-cell clones. EBV infection is
seen mainly in the B lymphocytes and B immunoblasts, al-
though the virus also occurs in rare neoplastic and non-
neoplastic T cells. The presence of EBV in only a
subpopulation of cells suggests that EBV infection is sec-
ondary to malignancy or that the viral genome has been lost
from the malignant cell [6].
Nasal T/NK-Cell Lymphoma
Nasal T/NK-cell lymphoma cells exhibit several unique ge-
notypic and phenotypic features. These features include an
absence of T-cell antigens, the expression of the NK cell
marker CD56, and the absence of T-cell receptor gene re-
arrangement [15]. Clinically, these tumors occur in the na-
sal and upper aerodigestive area. EBV is consistently
associated with these lymphomas, regardless of geograph-
ical location [53, 54] (Fig. 4).
TREATMENT
This section focuses on the treatment of patients with EBV-
associated lymphomas in which EBV cell– based immuno-
therapy has been most directly applied.
EBV Cell–Based Immunotherapy
Treatment of EBV-Related PTLDs
Because the great majority of PTLDs occurring in patients
receiving a solid organ transplant (SOT) or allogeneic stem
cell transplant are a result of EBV infection/reactivation
that follows the impairment of the immune system, modu-
lation of the immunosuppressive treatment is recom-
582
Figure 4. Nasal T/NK-cell lymphoma. EBV-infected tumor
cells display an angiocentric pattern of growth. The figure
shows several vascular structures of heterogeneous size.
(EBER in situ hybridization, hematoxylin counterstain, origi-
nal magnification ⫻400.)
Abbreviations: EBER, Epstein–Barr RNA; EBV, Epstein–
Barr virus; NK, natural killer.
mended as a first-line approach in patients with PTLDs
[55]. This approach induces tumor regression in 25%–50%
of patients [55]. Importantly, modulation of immunosup-
pression is currently applied by many transplant groups in
patients with a high EBV-DNA viral load in order to pre-
vent the occurrence of a PTLD [56].
The management of patients with PTLDs who fail the
reduction in immunosuppression is complex and remains
controversial. Chemotherapy and monoclonal antibodies
are the therapeutic options most frequently offered to these
patients. The use of rituximab has significantly changed the
therapeutic approach to PTLDs. The administration of the
antibody is generally well tolerated and rapidly induces de-
pletion of mature B lymphocytes in the peripheral blood,
thus reducing the compartment of EBV-infected cells, with
an associated normalization of the viral load [57]. In the
case of PTLDs occurring in SOT patients, very enthusiastic
results were reported in small uncontrolled studies, with a
complete remission rate in the range of 30%–70% [58, 59].
However, more recent phase II studies including larger co-
horts of patients have shown an overall median survival
time of 15 months, suggesting that rituximab should be in-
tegrated with chemotherapy in patients with poor prognos-
tic factors, such as a high tumor burden, an EBV-negative
PTLD, and PTLD occurring late after transplantation [60].
In our own experience, the duration of remission in SOT
patients with EBV-related PTLD treated with rituximab can
be relatively short, because the recovery of B cells in the
presence of impaired T-cell immunity is often accompanied
EBV-Associated Lymphomas
by a new increase in the EBV viral load and eventually re-
lapse of the PTLD [61].
Chemotherapy still remains the only option for many
patients with aggressive PTLDs. The response rate and
overall survival time were widely variable on retrospective
analysis, and few prospective studies have been reported so
far [62– 64]. The best time and the best chemotherapy
schedule for these patients remain controversial. Low-dose
chemotherapy has been prospectively used in order to re-
duce the morbidity and mortality associated with standard
protocols. Although this approach significantly reduces
toxicities, it is also associated with a high relapse rate, sug-
gesting that more conventional chemotherapy regimens are
indicated in patients with aggressive PTLD [65]. CHOP-
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like regimens are currently recommended by the European
guidelines at least for renal transplantation [66].
Is there a role for cellular immunotherapy in patients
with EBV-related PTLD? Adoptive transfer of donor-de-
rived EBV-specific CTLs (EBV-CTLs) has been successful
when used as prophylaxis and treatment of EBV-related
PTLDs in the context of allogeneic stem cell transplantation
[67]. Analogously, adoptive transfer of EBV-CTLs may
help to restore the balance of the immune compartment in
patients receiving a SOT, while avoiding the risk for graft
rejection that is frequently associated with the withdrawal
of immunosuppression.
Several groups recently reported their preliminary ex-
periences with adoptive EBV-CTL transfer in SOT recipi-
ents as pre-emptive treatment in cases of high EBV DNA
viral load or as treatment in the presence of clinical evi-
dence of a PTLD [68, 69]. Overall, these studies indicate
that CTL infusions are well tolerated. No graft toxicity was
reported. Although EBV DNA did not decrease uniformly
in all patients, in several studies EBV-CTLs prevented the
development of PTLDs, and in small series tumor regres-
sion after CTL therapy was also reported.
The main limitations to the extensive application of
CTL therapies in these patients remain the cost and the time
required for EBV-CTL line generation. PTLDs can be very
aggressive and rapidly fatal, while the generation of autol-
ogous EBV-CTL lines requires several weeks. The use of
partly HLA-matched EBV-CTLs seems a promising alter-
native to the generation of autologous EBV-CTLs [70]. In
our first experience, we generated EBV-CTL lines for pa-
tients with a high EBV DNA viral load in the peripheral
blood (⬎4,000 DNA copies). However, because the EBV
DNA viral load is not always predictive of PTLD occur-
rence in SOT recipients, we are currently generating EBV-
CTL lines only for patients who have received previous
treatment for a PTLD. Because the relapse rate remains
high in these patients, we are proposing the generation and
Carbone, Gloghini, Dotti
infusion of EBV-CTLs as a salvage treatment, thus reduc-
ing the cost of manufacturing and decreasing the risk–
benefit ratio for this procedure.
Treatment of EBV-Associated cHL
Modern radiotherapy and/or chemotherapy regimens have
dramatically improved the cure rate of patients with cHL
[71]. However, despite the identification of clinical prog-
nostic factors and the optimal use of primary and secondary
treatments, cHL remains fatal for ⬎15% of patients [71].
Therefore, new therapeutic agents are required for primary
refractory patients and biological strategies are also desir-
able to maintain remission in high-risk patients after con-
ventional treatment. In addition, biological treatments may
reduce many of the serious long-term side effects correlated
with radiation and chemotherapy [71].
Therapy with monoclonal antibodies, and in particular
with anti-CD30 monoclonal antibodies, has been used in
patients with cHL. Although the great majority of patients
enrolled in these studies were heavily resistant to previous
therapies, clinical responses were minimal and not compa-
rable with the response rate obtained with rituximab in B-
cell– derived lymphomas [72].
The association between EBV and a subset of cHL and
the expression of the latent proteins LMP-1 and LMP-2 by
tumor cells constitute the rationale to assess the feasibility
of EBV-CTL adoptive transfer for patients with EBV-re-
lated cHL. We have conducted several phase I clinical trials
in patients with cHL using both polyclonal EBV-CTLs and
CTLs enriched in precursors targeting LMP-2 [73]. CTL in-
fusions were well tolerated, with objective and sustained
clinical responses in several patients.
EBV-CTL THERAPIES AND FUTURE DIRECTIONS
These studies demonstrate both the feasibility and efficacy
of CTL therapies in patients with PTLDs after SOT and in
patients with EBV-associated malignancies like cHL.
However, other important factors need to be considered to
improve the clinical benefit of these approaches.
SOT patients require continuous immunosuppression to
prevent graft rejection. Although adoptive transfer of EBV-
CTLs may help in restoring immunocompetence, CTL
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3 Delecluse HJ, Feederle R, O’Sullivan B et al. Epstein Barr virus-associated
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583
function is still partially impaired by the immunosuppres-
sive drugs [73]. We and others are currently evaluating
strategies to genetically modify EBV-CTLs to make them
resistant to the effects of immunosuppressive drugs.
A more robust in vivo expansion of adoptively trans-
ferred CTLs may help to increase the antitumor effects.
This concept was proven in previous clinical trials in mel-
anoma patients in which adoptive T-cell transfer followed
the administration of nonmyeloablative chemotherapy reg-
imens to create a lymphodepleted environment that would
favor the expansion of CTLs [74]. The tumor microenvi-
ronment may be particularly hostile for antitumor immu-
nity. Indeed, many tumor cells, such as HL tumor cells,
produce factors, including transforming growth factor ␤,
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thymus and activation-regulated chemokine, IL-10 and Fas
ligand, that significantly impair the function or survival of
antitumor CTLs. Genetic modification of CTLs to over-
come tumor escape mechanisms and increase their survival
in the tumor microenvironment may help in arming CTLs
and increasing their antitumor effects [75]. Finally, the ca-
pacity of tumor cells to downregulate tumor-associated an-
tigens needs to be carefully evaluated. Modern technology
allows the genetic modification of CTLs to expand their an-
tigen specificity. In particular, gene transfer of chimeric an-
tigen receptors targeting crucial molecules expressed on the
cell surface of cHL cells, such as CD30, may improve their
antitumor effects. Phase I clinical studies will help in clar-
ifying the role of these mechanisms to improve the treat-
ment of these malignancies.
ACKNOWLEDGMENTS
This work was supported in part by a Grant from the Istituto
Superiore Sanità, VI Programma Nazionale di Ricerche
sull’AIDS, Rome, Italy.
The authors thank Maria Morelli for help in the prepa-
ration of the review and Gianni Roncato for photographic
assistance.
AUTHOR CONTRIBUTIONS
Conception/design: Antonino Carbone
Financial support: Antonino Carbone
Manuscript writing: Antonino Carbone, Annunziata Gloghini, Giampietro
Dotti
Final approval of manuscript: Antonino Carbone, Annunziata Gloghini,
Giampietro Dotti
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