The Role of Infectious Agents in B-cell Lymphomas
Moderators: Dr. Miguel A. Piris and Dr. Steven H. Swerdlow
Section 2 -
Epstein-Barr Virus and Lymphoproliferative Disorders in Iatrogenic Immunodeficiency
Steven H. Swerdlow
Post-Transplant Lymphoproliferative Disorders (PTLD)
PTLD represent lymphoid/plasmacytic proliferations that follow solid organ/bone
marrow/stem cell transplantation and that are not due to some other definable cause.  Their
incidence and clinical spectrum vary greatly probably related to many different factors. Most cases
present within one year following transplant but "late" cases occur after many years. Presentations
include infectious mono-like, localized tumor masses, disseminated disease, allograft dysfunction, and
subtle disease with non-specific symptoms. Therapy for PTLD varies widely based on the clinical
situation, available therapeutic options, and institution. Decreasing patients' levels of
immunosuppression, anti-viral agents, rituxan, novel therapies and conventional lymphoma therapy are
among the varied therapeutic modalities that have been utilized. Outcomes vary widely depending on many
features; however, overall a mortality of up to 50-80% is reported. Some studies report much better
Pathology of PTLD
PTLD have a wide pathologic spectrum. Some biopsies show architectural preservation
of the underlying tissues, whereas most show architectural destruction. Many of the former cases are not
considered PTLD by many. PTLD also vary cytologically. Some are composed of very heterogeneous cell
populations including small and large lymphocytes with round to irregular nuclear contours and plasma
cells whereas other cases are composed of a much more homogeneous population of B-cells, T-cells or
plasma cells. A minority of PTLD are polyclonal but most have variably dominant monoclonal
lymphoid/plasma cells. Clonal B-cell populations are much more frequent than clonal T-cell populations.
A subset of those with monoclonal populations also demonstrate additional genotypic abnormalities such as
mutations in oncogenes.
Post-tranplant lympho(plasmacytic) lesions according to the WHO 
Oncogene/tumor suprressor gene abnormalities occur in
PTLD at least of monomorphic type but their overall frequency is low. C-MYC rearrangements are
associated with a minority of monomorphic B-cell PTLD.
P53 mutations are also uncommon.
 More frequent cases have p53 expression (wild-type). N-RAS mutations have not been as
widely studied but have been reported in 2/2 myeloma type PTLD and 1/3 immunoblastic lymphoma type PTLD
but in 0/23 PH/P-PTLD. 
- Early lesions
- Reactive plasmacytic hyperplasia
- Infectious-mononucleosis-like PTLD
- Polymorphic PTLD
- Monomorphic PTLD (classify according to lymphoma classification)
- B-cell neoplasms
- T-cell neoplasms
- Hodgkin lymphoma & Hodgkin-like PTLD
In contrast, BCL-6 mutations are more common, being reported in 43% of P-PTLD and
90% M-PTLD.  Although somewhat controversial, the presence of BCL-6 mutations are reported
to be an adverse prognostic indicator in terms of survival and response to decreased
Most PTLD are associated with latent EBV infection with
some reporting all cases to be positive. The EBV is usually of type A rather than the mix of types A and
B as seen in HIV-associated neoplasms. Although expression of different latency associated proteins
varies and not all cells fall neatly into a standard category, most cases appear to have latency pattern
type III with some of type II (HL-like) and others of type I. There is also often evidence of a lytic
infection as well.
However, EBV negative PTLD must be recognized and their frequency appears
to be increasing. LeBlond, et al reported 29% B-PTLD are EBV-.  In our experience, from
1982-1990 only 1/58 biopsies with PTLD were EBV negative, whereas from 1991-1996 17/75 (23%) were EBV
negative and, in unreported data from 1997- 2004, 34/109 patients (31%) had EBV negative PTLD.
EBV negative PTLD have distinctive features.
They occur later following transplantation, have an increased frequency of monomorphic
(lymphoma-like) PTLD (81-100% M-PTLD) and are reported by some to be associated with an adverse
prognosis. T-cell PTLD also have a higher relative incidence of EBV negativity with only about 20-30%
T-cell PTLD EBV positive (series vary). The etiology for the majority of the EBV negative cases is
uncertain. Other viruses such as HHV-8/KSHV have been identified in only rare cases. Whether some
unidentified virus is responsible or whether they have a pathogenesis like lymphomas in immunocompetent
hosts remains to be established (see below). EBV- PTLD can respond to decreased immunosuppression. In
our series, there were 7/17 patients treated with decreased immunosuppression without chemo- or radiation
therapy.  Of those 7 patients, 5 had non-surgical complete remissions and were alive at
3-26 months, 1 had a surgical complete remission and was alive at 68 months and 1 had a partial remission
but died at 15 months. Other patients who failed this conservative therapy would have received more
Further Dissecting the Monomorphic B-cell PTLD and the Impact of EBV
The monomorphic PTLD are one of the most common types of PTLD with the majority of
the cases resembling a diffuse large B-cell lymphoma (DLBCL). As noted above, this is also the type of
B-cell PTLD most likely to be EBV-. DLBCL are also well known to be a very heterogeneous "entity."
These observations raise a number of questions. How homogeneous are the monomorphic B-cell PTLD and how
do they compare to transformed B-cell lymphomas seen in the normal host? What is the impact of EBV on
the histogenetic spectrum of the monomorphic B-cell PTLD?
Histogenetic Spectrum of B-cell PTLD and the Impact of EBV
Monomorphic B-cell PTLD are a heterogeneous entity in terms of their histogenesis,
just like DLBCL in general. Johnson, et al (AJSP, in press) demonstrated that they range from having a
germinal center (GC) to late GC/early post-GC to post-GC phenotype. The rather scanty literature in this
area has shown conflicting results.
The EBV+ cases were less likely to be of GC type
than the EBV- cases which is consistent with many observations of the effect of EBV on B-cells. Some
examples include the finding in transgenic mice that EBV LMP1 induces extrafollicular B-cell
differentiation and blocks GC formation, the observation that EBV infection induces many cytokines
including IL-6 which downregulates bcl-6 and leads to plasma cell differentiation, gene expression
profiling of EBV infected cells clusters them with IgM-stimulated B-cells rather than with GC cells and
the observation that EBV infection of B-cells leads to downregulation of CD10. Statistically significant
survival differences between the phenotypic groups could not be identified; however, the groups are small
and there are other confounding variables. Comparison of our non-Burkitt monomorphic PTLD with DLBCL in
presumably immunocompetent hosts  and HIV-associated DLBCL (including immunoblastic,
plasmablastic and primary effusion lymphoma cases)  suggests that the EBV negative cases are
more like lymphomas in immunocompetent hosts than the EBV positive cases and that the HIV-associated
cases are intermediate between the two groups.
The Contribution of Gene Profiling Studies
We also undertood a small pilot gene profiling study of monomorphic B-cell PTLD to
further investigate the impact of EBV on these cases. Unsupervised clustering separated the EBV+ from
EBV- cases. 54 transcripts were over-expressed in EBV+ PTLD relative to EBV- PTLD which included several
that have been associated with a virally induced immune response. 269 transcripts were underexpressed in
EBV+ relative to the EBV- cases including 31 signaling molecules with the largest subset representing the
CD79a/CD79b B-cell receptor and its downstream signaling molecules. Comparison with literature on in
vitro EBV modulated gene expression in B-cell lines showed a much greater concordance than would be
expected by chance alone.
These results, which must be interpreted with great caution,
provide additional evidence that EBV positive and EBV negative PTLD are distinctive and that the former
show changes known to be associated with in vitro EBV infection of B-cells. This highlights the impact
of EBV on the PTLD and suggests that the EBV- cases may not be associated with any virus.
Methotrexate-Associated Lymphoproliferative Disorder (MLPD)
The best known of the other iatrogenic associated lymphoproliferative disorders is
that related to methotrexate.  About 85% of the patients have received the methotrexate as
therapy for rheumatoid arthritis. The disorder occurs after the patients have been on methotrexate for
an average of 3 years. The impact of the rheumatoid arthritis itself on lymphoma risk also must be taken
into account here. It is controversial if there is an increased incidence of non-Hodgkin lymphomas in
rheumatoid arthritis but there does appear to be an increased incidence of Hodgkin lymphoma.
The pathologic spectrum of MLPD is similar to the PTLD; however, the distribution of
cases differs. Many MLPD are of monomorphic type with many more DLBCL-like (~35%) than of Burkitt
type. Geographic necrosis is one of the pathologic features of these cases. Only rare T-cell cases are
reported. A small proportion of cases resemble polymorphic PTLD or "lymphoplasmacytic" proliferations.
About 25% are of Hodgkin lymphoma type (or Hodgkin-like) which is more common than what is seen in the
post-transplant setting. Some small B-cell lymphomas are also reported. Up to about 50% are EBV
After discontinuing methotrexate, up to about 50% of patients with MLPD have a
complete response with others showing either a partial response or no response at all. Responses are
reported in some of the Hodgkin and Hodgkin-like cases. Some responders do relapse and some series
report fewer responses. Many reported patients have received chemotherapy without a trial of
methotrexate withdrawal. The patients with EBV positive disease are more likely to respond than the EBV
negative cases, although, as with the PTLD, the latter may also go into complete remission.
Other Iatrogenic-Asssociated EBV+ LPD
In addition to occasional LPD reported in other settings, a series of five patients
with an EBV-associated B-cell LPD in patients with CLL related to presumptive iatrogenic
immunosuppression related to Fludarabine therapy has been reported.  The LPD
occurred 2-12 months after completion of the Fludarabine therapy. Three of four were clonally distinct
from the CLL. Two received no therapy and spontaneously regressed (one recurred), one had a surgical
excision only and was alive with no evidence of disease, one received anti-viral therapy and died without
disease. Pathologically, three biopsies resembled a polymorphic PTLD (one with a Hodgkin lymphoma-like
area), two biopsies resembled a monomorphic PTLD, one a Hodgkin type PTLD and one relapse biopsy
resembled "lymphomatoid granulomatosis." Prospective identification of these cases is problematic as
oftentimes these patients have received more than one drug, plus one has to exclude the possibility of
transformation of the CLL or a coexistent lymphoma (Richter's syndrome).
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