Posttransplant Lymphoproliferative Disorders Not Associated
With Epstein-Barr Virus: A Distinct Entity?
By V6ronique Leblond, Fr6d6ric Davi, Fr6d6ric Charlotte, Richard Dorent, Marc-Olivier Bitker, Laurent Sutton,
Iradj Gandjbakhch, Jacques-Louis Binet, and Martine Raphael
Purpose: Organ recipients are at a high risk of post-
transplant lymphoproliferative disorders (PTLD) as a
result of immunosuppressive therapy. Most B-cell lym-
phomas are associated with Epstein-Barr virus (EBV)
infection. We describe a morphologically and clinically
distinct group of PTLD in 11 patients that occurred late
after organ transplantation and were not associated
with EBV.
Patients and Methods: There were seven kidney,three
heart, and one liver transplant recipients (group I). The
clinical manifestations, pathologic findings, treatment,
and outcome were compared with those in 21 patients
with EBV-associated PTLD treated in our institution (group
II). EBV was detected with at least two techniques:
Epstein-Barr-encoded RNA (EBER) in situ hybridization
with EBER 1 + 2 probes, Southern blotting, and detection
of latent membrane protein 1 (LMPi) expression by
immunohistochemistry.
Results: The time between transplantation and the
diagnosis of lymphoma ranged from 180 to 10,220
days in group I (mean, 2,324; median, 1,800) and from
60 to 2,100 days in group II (mean, 546; median, 180),
and was significantly shorter in group II (P = .02).
PREVENTION OF ORGAN REJECTION requires long-
term immunosuppression, which places recipients at
an increased risk of both infections and neoplastic diseases
such as Kaposi's sarcoma and posttransplant lymphoprolifer-
ative disorders (PTLDs).1,2 Patients who have received
solid-organ transplants have a 20- to 120-fold higher inci-
dence of non-Hodgkin's lymphoma, depending on the
degree and duration of immunosuppression. 13 PTLDs have
been reported after transplantation of kidneys,4 bone mar-
row,5 heart,6, 7 heart and lungs, 8 and liver.9 They are mostly
of B-cell origin and are often associated with active infec-
tion by Epstein-Barr virus (EBV), which is found in virtually
all posttransplant lymphomas.10 These lymphomas character-
istically have a rapid onset, aggressive behavior, and a
From the Departements d'Himatologie, de Chirurgie Cardio-
thoracique,de TransplantationRdnale, d'Anatomo-pathologie,HIpital
Pitid-Salpetribre,Paris; and Service d'Hematologie Biologique, Hcpi-
talAvicenne, Bobigny, France.
Submitted October20, 1997; acceptedMarch 16, 1998.
Address reprint requests to VWronique Leblond, MD, Dipartement
d'Himatologie, Hdpital Pitig-Salpjtribre,47 Bd de l'H6pital, 75013
Paris,France; Email veronique.leblond@psl.ap-hp-paris.fr.
©1998 by American Society of ClinicalOncology.
0732-183X/98/1606-0028$3.00/0
2052
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Among 19 tumors diagnosed within 2 years after the
graft, 16 were associated with EBV; among 13 tumors
diagnosed after more than 2 years, only five were
associated with EBV. All of the B-cell PTLDs in group I
were classified as monomorphic, meeting the criteria of
B diffuse large-cell lymphoma (B-DLCL) with a compo-
nent of immunoblasts, and genotyping confirmed their
monoclonality. Three tumors were T-cell pleomorphic
lymphomas. Tumor sites were mainly bone marrow and
lymph nodes. Overall median survival was 1 month in
group I and 37 months in group II, with two patients still
alive in group I and nine in group II. The survival time
was significantly longer in group II (P < .01).
Conclusion: EBV-negative PTLD may be a late serious
complication of organ transplantation. Half the tumors
observed after kidney transplantation in our center
were not associated with EBV and emerged after more
than 5 years, which suggests the number of EBV-
negative PTLDs in organ recipients might increase with
time.
J Clin Oncol 16:2052-2059. o 1998 by American
Society of Clinical Oncology.
tropism for extranodal sites; they show partial or complete
regression after reduction or withdrawal of immunosuppres-
sive therapy.
In 1995, we reported the clinical manifestations, patho-
logic features, and response to therapy of 24 cases of
PTLD. 11 Of 34 cases that have now been diagnosed, we failed to
detect EBV in 11. We thus compared the clinical characteristics,
pathologic findings, clonality, and outcome between EBV-
negative and EBV-positive PTLD to determine if the former
is a distinct entity in organ transplant recipients.
PATIENTS AND METHODS
Patients
We studied 34 patients who developed a lymphoproliferative disorder
after kidney (14 patients), heart (15), lung (three patients), or liver (two)
transplantation in our institution between July 1986 and January 1997.
DiagnosticEvaluation
All patients in whom PTLD was diagnosed before death underwent a
thorough work-up to detect lymphoproliferative sites, with computed
tomographic (CT) scans of the chest, abdomen, and CNS, together with
bone marrow biopsy and aspiration.
Treatment
The treatment modalities varied between 1980 and 1997. The
immunosuppressive regimen was modified in 27 patients after the
20 52 2 0 59
Joumol of ClinicalOncology,Vol 16, No 6 (June), 1998: pp -
EBV-NEGATIVE PTLD
diagnosis of PTLD. From 1988 through 1992, treatment consisted of
anti-B-cell monoclonal antibody infusions (specific for CD21 and
CD24) every day at a dose of 0.2 mg/kg intravenously (10 patients) or
by the intraventricular route at a daily dose of 2.5 mg (two patients) for
CNS PTLD, as described by Stephan et a112 and Fischer et al, 13 and/or
polychemotherapy (16 patients). Chemotherapy was the cyclophospha-
mide, doxorubicin, vincristine, and prednisone (CHOP) regimen in
eight patients; lomustine (CCNU), high-dose methotrexate (5 g/m 2),
and holoxan in three patients with CNS involvement; etoposide,
cisplatin, high-dose cytarabine, and prednisolone (ESHAP) in three
patients"5 ; vincristine, melphalan, cyclophosphamide, and prednisone
(VMCP) in one patient' 6 ; and mechlorethamine, vincristine, procarba-
zine, prednisone, doxorubicin, bleomycin, and vinblastine (MOP-
PABV) in one patient.' 7 Two patients with localized PTLD were treated
surgically. One patient was recently treated with anti-interleukin-6
(IL-6) monoclonal antibody.' 8
Methods
Tissues
Surgical biopsies were fixed in formalin or Bouin's fixative, partly
processed, embedded in paraffin, and stained with hematoxylin-eosin
and Giemsa for conventional histologic examination. All of the slides
were classified morphologically according to Locker and Nalesnik.19
Two types were distinguished: polymorphic PTLD, with a spectrum of
lymphoid cells, and monomorphic PTLD, mostly consisting of centro-
blasts or immunoblasts. This material was available for intracytoplas-
mic immunoglobulin (CIg) detection with monoclonal antibodies
(anti-K and anti-A Ig light chains) and polyclonal antibodies (anti-a,
anti-y, and anti-p heavy chains) using a commercial kit (Dakopatts,
Trappes, France)) for immunoperoxidase staining. The other material
was snap-frozen in liquid nitrogen and stored at - 80'C for immunophe-
notyping and genotyping. Immunophenotyping studies, including B-
cell and T-cell differentiation antigens, were performed on cryostat
sections using the avidine-biotine peroxidase method 20 or the alkaline
2
antialkaline phosphatase technique (APAAP). 1
EBV Detection
In situ hybridization. Epstein-Barr-encoded RNA (EBER) in situ
hybridization was performed on routinely processed sections with
fluorescein isothiocyanate (FITC)-labeled EBER 1 + 2 specific oligo-
nucleotides (Dakopatts) according to the manufacturer's instructions.
22
The quality of RNA was confirmed by the rarity of nonlymphomatous
positive cells (one or two per slide).
Southern blot analysis. EBV DNA was detected by Southern blot
using BamHI digested DNA extracted from frozen material and a
phosphorus-32-labeled probe specific for the BamHI W internal repeats
of the virus. Positive and negative controls consisted of the EBV-
positive Raji cell line and placental DNA, respectively.
Immunohistochemistry. Latent membrane protein 1 (LMP 1) expres-
sion was detected by immunochemistry using the immunoperoxidase
technique and monoclonal antibody CS.1-4 (Dakopatts) on paraffin-
embedded sections.
Ig gene and T-Cell Receptor Rearrangement Studies
Ig gene and T-cell receptor (TCR) rearrangements were detected by
Southern blotting with a heavy-chain joining region (JH) and the TCR
p-chain constant region (C pl and C R2) complementary probe labeled
with 32P by random priming. A clonal rearrangement was considered to
be present if additional bands were seen on blots prepared from DNA
treated with at least two restriction enzymes.
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2053
StatisticalAnalysis
We used the modified X test (Yates test) to compute statistical
significance of intergroup differences. Survival curves were drawn with
the Kaplan-Meier method. A nonparametric Wilcoxon rank-sum test
was used to compare the survival time in the two groups.
RESULTS
EBV Studies
The EBV genome and/or EBER 1 + 2 mRNAs were
detected in 21 of 32 patients tested (group II) (Table 1). In
the remaining 11 cases, we failed to detect EBV despite the
use of in situ hybridization, Southern blot, and LMPI
detection by immunocytochemistry in eight cases, and in
situ hybridization and LMP 1 detection by immunocytochem-
istry in the other three cases (group I) (Tables 2 and 3).
Time to Tumor Diagnosis
The time between organ allografting and tumor diagnosis
ranged from 180 to 10,220 days in group I, with a mean of
2,324 ± 2,905 days and a median of 1,800 days. It ranged
from 180 to 1,380 days (median, 900) in heart recipients and
from 360 to 10,220 days in kidney recipients (median,
1,800). The time between organ allografting and tumor
diagnosis ranged from 60 to 2,100 days in group II, with a
mean of 546 ± 615 days and a median of 180 days; these
periods were significantly shorter than in group I (P = .02).
The interval ranged from 90 to 1,660 days (median, 165) in
heart recipients and from 180 to 1,800 days in kidney
recipients (median, 435). Among 19 tumors diagnosed
within 2 years of the graft, 16 were associated with EBV;
among 13 tumors diagnosed more than 2 years after the
graft, only five were associated with EBV (Fig 1).
PathologicFindings and Clonality
Diffuse proliferation of lymphoid cells was observed in all
cases. All of the B-cell PTLDs in group I were monomor-
phic, meeting the criteria of diffuse large B-cell lymphoma
(B-DLCL) as in nonimmunosuppressed patients, according
to the Revised European-American Lymphoma (REAL)
classification 23 and were composed of uniform, large, non-
cleaved lymphocytes (Table 3). A mixture of predominantly
centroblast-like cells or immunoblast-like cells that showed
plasmacytic differentiation was observed. In one patient, the
tumor was a multiple myeloma characterized by plasmacytic
cells with cytologic atypia. In group II, only eight cases were
classified as monomorphic with the features of DLCL
(P = .005); the other lesions were composed of small
lymphocytes, small and large noncleaved cells, and a large
proportion of immunoblasts, which often showed plasma-
cytic differentiation (Table 1). In group I, eight tumors
2054 LEBLOND ET AL

Table 1. Patient Population, Pathologic Findings, Clonality, and EBV Studies of PTLDs Associated With EBV
EBV
Age Organ Time Between Graft Genotypic
UPN (years)/Sex Transplanted and PTLD (days) Morphology Immunophenotype CIg/SIg Clonality HIS Southern
1 45/m Heart 150 Polymorphic B M ND + ND
2 43/m Heart 750 Polymorphic B M JHR + +
3 50/m Heart 90 Polymorphic B M ND + ND
4 38/f Heart 150 Polymorphic B M JHR + +
5 68/f Heart 210 Polymorphic B M JHR + +
7 39/m Kidney 180 Polymorphic B M ND + ND
10 20/m Heart 150 Polymorphic B NM ND + ND
11 52/m Heart 150 Polymorphic B NM GL + +
12 46/m Heart 120 Polymorphic B NM GL + +
13 33/f Lung 60 Polymorphic B NM ND + ND
14 51/m Lung 180 Polymorphic B NM GL + +
16 40/m Kidney 1,800 Monomorphic B M ND + ND
19 54/m Kidney 910 Monomorphic B M JHR + +
21 32/m Kidney 420 Monomorphic B M JHR + +
22 57/m Kidney 450 Monomorphic B M JHR + +
23 51/m Kidney 180 Monomorphic B M ND + ND
24 63/m Kidney 680 Monomorphic B M JHR + +
25 31/m Heart 1,660 Monomorphic B M JHR + +
29 15/m Heart 120 Polymorphic T (CD8) - TCR R + +
32 40/m Heart 960 HD B - JHR + +
34 59/m Lung 2,100 Monomorphic B M JHR + +
Abbreviations: UPN, unique patient number; JHR, rearrangement of immunoglobulin gene; TCR R, rearrangement of T-cell receptor; M, monotypic; NM,
nonmonotypic; ND, not done; GL, germ line; HD, Hodgkin's disease; HIS, histochemistry; m,male; f, female.

expressed immunologic markers of the B-cell lineage (20 in observed in six cases. In group II, four patients had lymph
group II), as assessed by immunophenotyping of B-cell node and/or bone marrow involvement and 18 had extra-
differentiation antigens and/or surface Ig (SIg) analysis; nodal sites (Tables 5 and 6). All of the primary CNS PTLDs
three tumors expressed T-cell markers (one in group II). were associated with EBV.
Clonality was assessed by the detection of CIg and SIg and
genotypic studies. All tumors in group I were monotypic/ Clinical Outcome
monoclonal, while five of 21 tumors in group II were
In group I, immunosuppressive therapy was reduced in
polytypic/polyclonal (difference not significant).
seven patients (azathioprine withdrawal), but this failed to
ClinicalManifestations prevent disease progression. The disease progressed within
1 month in all patients except patient no. 15, who died of
In group I, six patients had lymph node and/or bone
infection after nephrectomy. One patient (no. 6) received
marrow involvement (Table 4). Extranodal tumor sites were
anti-B-cell antibodies without success and died after salvage
chemotherapy. Seven patients were initially treated with
Table 2. Methods Used for Detection of EBV in Group ITumors
chemotherapy: complete or partial remission was achieved
UPN LMPi ISH Southern Blot
in three patients (no. 8, 18, and 31), while four patients did
6 - - -
not respond (no. 20, 26, 30, and 33). Both patients treated
surgically died, of sepsis (no. 15) or relapse (no. 27). One
15 - - ND
18 - - - patient (no. 28) died before any treatment. None of the
20 - - ND patients with T-cell PTLD survived. Eight patients (72%) in
26 - - ND group I died of PTLD or its treatment (Table 4). The
27 - - -
probability of survival was 14% at 44 months, with a median
28 - - -
- - survival time of 1 month (Fig 2).
30 -
31 - - - In group II, two tumors were diagnosed after death
33 - - - (patients no. 16 and 22). Immunosuppressive therapy was
Abbreviations: LMPI, detection of LMP 1 by immunochemistry; ISH, detection
reduced in 18 patients and was considered successful in two
of EBERs by in situ hybridization; Southern blot, Southern blotting with BomHI patients (no. 1 and 27), one of whom died of infection after 3
probe; -, performed; ND, not performed. months (no. 27). Eleven patients received anti-B-cell anti-

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EBV-NEGATIVE PTLD 2055
Table 3. Patient Population, Pathologic Findings, and Clonality of PTLDs Not Associated With EBV
Age Organ Time Between Grafting Genotypic
UPN (years)/Sex Transplanted and PTLD(days) Morphology Immunophenotype CIg/Sig Clonality

6 50/f Heart 180 Monomorphic B M JHR

8 43/f Liver 900 Monomorphic B M JHR
15 62/m Kidney 3,450 Monomorphic B M ND
18 59/m Heart 900 Monomorphic B M JHR
20 35/f Kidney 360 Monomorphic B M ND
26 49/m Kidney 440 Monomorphic B M ND
27 58/f Kidney 4,140 Polymorphic T (CD4) - TCR R
28 67/m Heart 1,380 Polymorphic T (CD8) - TCR R
30 49/m Kidney 10,220 Polymorphic T (CD3) - TCR R
31 31/m Kidney 1,800 Monomorphic (myeloma) B M JHR
33 46/m Kidney 2,100 Monomorphic B M JHR

bodies either by the intravenous route (nine patients) or by DISCUSSION

the intraventricular route via an Ommaya reservoir (two
patients with CNS PTLDs), as previously described' 2 ;
complete remission was obtained in eight patients treated by
the intravenous route, while both patients treated by the
intraventricular route died of disease progression. No re-
lapses were observed in responders, but one patient (no. 12)
died in complete remission of a second malignancy at 37
months. Five patients were treated initially with chemother-
apy (no. 2, 3, 24, 25, and 32); four died and the fifth is alive
in complete remission (no. 32). One patient (no. 34) was
treated with anti-IL-6 monoclonal antibodies; the response
was good, but a relapse occurred and the patient died of
disease progression at 6 months. Deaths related to PTLD or
its treatment occurred in nine (48%) of 19 patients in group
II (Tables 5 and 6). The probability of survival was 41% at
96 months, with a median survival time of 37 months. The
difference in survival time between the two groups was
significant (P < .01) (Fig 2).
EBV has long been implicated in the pathogenesis of
postransplant lymphoproliferative disorders. 24-27 Marked im-
munosuppression may allow EBV-infected cells to express
all latency proteins and still expand without selective
pressure from cytotoxic T cells.2 8-30 EBV, which acts as a
continuously present oncogenic agent in these patients,
permits the expansion of multiple (polyclonal) EBV-infected
and immortalized B-cell clones. The cell growth rate alone is
sufficient to explain the monoclonal/oligoclonal nature of
tumor foci: the proliferative advantage of some clones leads
to the selection of a few (oligoclonal) or a single (monoclo-
nal) EBV-infected B-cell clone.
We found 11 EBV-negative cases (34%) among 32
allograft recipients with lymphoproliferative disorders. Few
investigators have observed EBV-negative B-cell
PTLDs. 26,31-33 We used several sensitive methods to detect
EBV in the tumors. RNA detection in situ is becoming a

18

16
SEBV+ PTLDs (21)
14
NUMBER OEBV- PTLDs (11)
TV PA TIEN•T 12
12

10
Fig 1. Time between grafting and
PTLD in EBV-positive (n = 21) and 8
EBV-negative (n = 11) PTLD.

4
4

42 2 2
1
m--
S2 2
3

1*1 , , I
2 3 4 5 6 >6
YEARS

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Copyright © 2018 American Society of Clinical Oncology. All rights reserved.
2056 LEBLOND ET AL

Table 4. Clinical Features, Treatment, and Outcome of Patients With PTLDs Not Associated With EBV
Change in Monoclonal Chemotherapy
UPN Tumor Sites Surgical Treatment Immunosuppression Antibodies (no. of cycles) Outcome
6 Large bowel, bladder, peritoneum Large bowel resection Azathioprine Anti-B ESHAP (1) Died of infection at 1 month
8 Lymph nodes Biopsy Azathioprine - CHOP (6) CR 44+ months
15 Native kidney Nephrectomy Azathioprine - - Died of infection at 1 month
18 Skin, testis, breast Orchidectomy Azathioprine - ESHAP (3) Died in CR of heart failure at
30 months
20 Brain, CSF, bone marrow - - - MTX, Holoxan, CCNU Died of infection at 1 month
26 Brain, lung Biopsy Azathioprine - ESHAP (1) Died of infection at 1 month
27 Small bowel Small bowel excision Azathioprine - - Died of relapse at 6 months
28 Lymph nodes, blood, spleen - Died of infection at 1 month
30 Bone marrow, spleen, liver Biopsy Azathioprine - CHOP (3), ESHAP (2) Died of progression 6+ months
31 Bone marrow Biopsy - - VMCP (6) + ABMT Alive in PR 12+ months
33 Lymph nodes Biopsy - - CHOP (1) Died of progression at 1 month
Abbreviations: ABMT, autologous bone marrow transplantation; CR, complete remission; PR, partial remission; MTX, high-dose methotrexate; Anti-B, anti-B
monoclonal antibodies.

standard approach to the detection and localization of latent
viral infection: EBER expression has been detected in
malignant cells of all EBV-associated tumors, but there is
some variation in the pattern. 22,34,35 The sensitivity, specific-
ity, and rapidity of this technique make it useful for
diagnostic detection of EBV in latently infected clinical
specimens. Lytic viral infection not detected by EBER in
situ hybridization is readily detected by other tests, as many
targets (DNA, RNA, or proteins) are expressed at high levels
in lytic infection. Furthermore, few posttransplant lympho-
mas are associated with lytic gene expression in the tu-
mors. 29 The sensitivity and specificity of in situ hybridiza-
tion for the detection of EBV genomes approaches that of
Southern blotting, even when using routinely processed
archival, paraffin-embedded material.36 Southern blot hybrid-
ization with cloned probes has contributed substantially to
the identification of EBV-associated diseases and also
allows recognition of lytic infection. 3 False negativity in
Southern blot analysis can be ruled out, as all experiments
included positive and negative controls, and all membranes
were rehybridized with Ig gene probes, thereby ensuring
DNA integrity.
Immunocytochemistry with a monoclonal antibody to
detect LMP1 in tissue sections, which is expressed only in
tumors associated with EBV, can be negative in tumors that
express only EBNA-1, and so is not universally applicable
as a screening tool.35
We used the three methods on eight tumors, and at least
two methods on three tumors.
All but three of the 11 EBV-negative PTLDs in this series
occurred more than 2 years after the graft, and half were
diagnosed after 4 years. The interval between grafting and
the diagnosis of EBV-negative PTLDs was significantly
longer than that of EBV-positive PTLDs. In principle,
EBV-derived PTLDs emerge early after the graft: the
interval between transplantation and tumor diagnosis falls
with increasingly intense immunosuppression, and is shorter
in heart and lung recipients. Half of the tumors observed

Table 5. Clinical Features, Treatment, and Outcome of Patients With PTLDs Associated With EBV
Change in
Immunosuppression Monoclonal Chemotherapy
UPN Tumor Sites Surgical Treatment (drug stopped) Antibodies (no. of cycles) Outcome
1 Lung, liver Lung biopsy Azathioprine - - CR 96+ months
2 Lymph nodes, brain, kidney Lymph node biopsy Azathioprine - MTX, Holoxan (1), CCNU Died of infection at 1 month
3 Lung, liver, lymph nodes, small bowel Lymph node biopsy Azathioprine - CHOP (2) Died of progression at 3 months
4 Liver, lung, uterus Biopsy Azathioprine Anti-B - CR 72+ months
5 Lung Biopsy Azathioprine Anti-B - CR 72+ months
7 Brain Biopsy I CSA Anti-B (IT) - Died of progression at 4 months
10 Liver Liver biopsy Azathioprine Anti-B - CR 96+ months
11 Lung Biopsy Azathioprine Anti-B - CR 72+ months
12 Large bowel Biopsy Azathioprine Anti-B - Died in CR of lung carcinoma at
37 months
13 Transplanted lung Biopsy Azathioprine Anti-B - CR 72+ months
14 Transplanted lung Biopsy Azathioprine Anti-B - CR 60+ months
Abbreviations: CSA, cyclosporine; IT,intrathecal.

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EBV-NEGATIVE PTLD 2057
Table 6. Clinical Features, Treatment, and Outcome of Patients With PTLDs Associated With EBV
Change in
Surgical Immunosuppression Monoclonal Chemotherapy
UPN Tumor Sites Treatment (drug stopped) Antibodies (no. of cycles) Outcome

16 Brain - - - - Autopsy findings

19 Native kidney Nephrectomy CSA Anti-B CHOP (6), ESHAP + ABMT Relapse, died of infection in CR
at 48 months
21 Bladder Biopsy Azathioprine Anti-B - CR 72+ months
22 Small bowel, large bowel Biopsy - - - Autopsy findings
23 Brain Biopsy - Anti-B (IT) MTX, Holoxan, CCNU Died of progression at 1 month
24 Large bowel, lymph nodes, spleen Biopsy Azathioprine - CHOP (4) Died of infection at 4 months
25 Skin Biopsy Azathioprine - CHOP (1) Died of infection at 1 month
27 Blood - Azathioprine - - Died of infection at 3 months
32 Lymph nodes Biopsy Azathioprine - MOPP-ABV (6) Alive inCR at 12 months
34 Lymph nodes, bone Biopsy CSA Anti-lL-6 (2) CHOP (1) Relapse at 4 months, died of
progression at 6 months

after kidney transplantation in our center were not associated phic to monoclonal monomorphic, as extensively described
with EBV and occurred after 5 years, which suggests the in the literature. 38,39 However, all of the EBV-negative
number of EBV-negative PTLDs in organ recipients might tumors (28% of B-cell PTLDS) met the criteria of B-DLCL,
increase with time. with centroblasts and/or immunoblasts with plasmacytic
The entire morphologic spectrum of B-cell differentia- differentiation. Ig and TCR gene rearrangement studies
tion, from nonspecific reactive hyperplasia to large-cell showed that all had clonal rearrangements, which mimicked
lymphoma, can be observed in PTLD lesions, and a broad the morphology and clonality found in immunocompetent
spectrum of morphologic and genotypic aspects was also hosts.
observed in this series, ranging from polyclonal polymor- The frequency of T-cell PTLD in our series was similar to
that reported in the literature. 2 Most tumors in transplant
recipients are monoclonal or oligoclonal B-cell lym-
phomas. 38 In this series of 32 PTLDs, we found four tumors
that expressed T-cell surface markers and lacked surface
expression of Ig and CD20; all of these tumors had TCR
p gene rearrangements and one had EBV DNA sequences,
as first described by Waller et al. 40 The T-cell PTLD
associated with EBV emerged early after the graft and was
sensitive to a reduction in immunosuppression. The other
T-cell PTLDS were similar to those described by Hanson et
al,41 and we found no evidence of EBV infection. All of the
4,
C
C) PTLDs emerged late after grafting. The sites of involvement
oa-
12t were chiefly the bone marrow, blood, and spleen. Outcome
was poor, with all of the patients dying of disease progres-
sion.
Armitage et a17 have reported different clinical presenta-
".Eu
Ng.tti-l
tions and outcomes between early (< 1 year) and late (> 1
year) PTLDs in heart and lung transplant recipients. Late-
II4X
onset PTLD, as in our group I, was characterized by
disseminated disease with a poor outcome and no response
to a milder immunosuppressive regimen. Unfortunately,
tests for EBV infection were based on serum antibody titers
and not on EBV genome detection in the tumor.
Months The precise effects of EBV infection in immunosup-
Fig 2. Overall survival rate of patients with PTLD not associated with EBV pressed patients, such as those infected by human immuno-
(...... ) and associated with EBV (---). deficiency virus (HIV), are controversial. Between 40% and

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Copyright © 2018 American Society of Clinical Oncology. All rights reserved.
2058
60% of patients with systemic AIDS-related lymphoma do
not have EBV sequences within their tumor cells. Multiple
cytogenetic aberrations appear necessary for lymphoma to
occur, and it is possible that the causes of lymphoma in
transplant recipients are multiple, as in HIV-infected pa-
tients.42 The ongoing B-cell proliferation induced by chronic
antigenic stimulation (by the graft and/or another transform-
REFERENCES
1. Penn I: Cancers after cyclosporine therapy. Transplant Proc
20:276-279, 1988 (suppl 1)
2. Penn I: Cancers complicating organ transplantation. N Engl J Med
323:1767-1769, 1990
3. Opelz G, Henderson R: Incidence of non-Hodgkin lymphoma in
kidney and heart transplant recipients. Lancet 342:1514-1516, 1993
4. Cockfield SM, Preiksaitis JK, Jewell LD, et al: Post-transplant
lymphoproliferative disorder in renal allograft recipients: Clinical
experience and risk factor analysis in a single center. Transplantation
56:88-95, 1993
5. Zutter MM, Martin JP, Sale GE, et al: Epstein-Barr virus
lymphoproliferation after bone marrow transplantation. Blood 72:520-
529, 1988
6. Cleary ML, Sklar J: Lymphoproliferative disorders in cardiac
transplant recipients are multiclonal lymphomas. Lancet 2:489-493,
1984
7. Armitage JM, Kormos RL, Stuart RS, et al: Postransplant
lymphoproliferative disease in thoracic organ transplant patients: Ten
years of cyclosporine-based immunosuppression. J Heart Lung Trans-
plant 10:877-887, 1991
8. Randhawa PS, Yousem SA, Paradis IL, et al: The clinical
spectrum, pathology, and clonal analysis of Epstein-Barr virus-
associated lymphoproliferative disorders in heart-lung transplant recipi-
ents. Am J Clin Pathol 92:177-185, 1989
9. Poison RJ, Neuberger J, Forman D, et al: De novo malignancies
after liver transplantation. Transplant Proc 20:94-97, 1988
10. Shapiro RS: Epstein-Barr virus-associated B-cell lymphoprolif-
erative disorders in immunodeficiency: Meeting the challenge. J Clin
Oncol 8:371-373, 1990
11. Leblond V, Sutton L, Dorent R, et al: Lymphoproliferative
disorders after organ transplantation: A report of 24 cases in a single
center. J Clin Oncol 13:961-968, 1995
12. Stephan JL, Le Deist F, Blanche S, et al: Treatment of central
nervous system B lymphoproliferative syndrome by local infusion of a
B cell-specific monoclonal antibody. Transplantation 54:246-249, 1992
13. Fischer A, Blanche S, Le Bidois J, et al: Anti-B-cell monoclonal
antibodies in the treatment of severe B-cell lymphoproliferative syn-
drome following bone marrow and organ transplantation. N Engl J Med
324:1451-1456, 1991
14. Elias L, Portlock CS, Rosenberg SA: Combination chemother-
apy of diffuse histiocytic lymphoma with cyclophosphamide, Adriamy-
cin, vincristine and prednisone (CHOP). Cancer 42:1705-1710, 1978
15. Velasquez WS, McLaughlin P, Tucker S, et al: ESHAP-An
effective chemotherapy regimen in refractory and relapsing lymphoma:
A 4-year follow-up study. J Clin Oncol 12:1169-1176, 1994
16. Oken MM: Standard treatment of multiple myeloma. Mayo Clin
Proc 69:781-786, 1994
17. Klimo P, Connors JM: MOPP/ABV hybrid program. Combina-
Downloaded from ascopubs.org by Fairview Health Services on May 27, 2018 from 162.096.053.037
Copyright © 2018 American Society of Clinical Oncology. All rights reserved.
LEBLOND ET AL
ing virus), associated with deficient immune surveillance
and lengthy use of immunosuppressive drugs, creates an
environment in which mutations in critical oncongenes
and/or suppressor genes may occur, which leads to clonal
selection and B-cell lymphoma. Further work is required to
identify all of the potential causes of lymphoma in immuno-
depressed transplantation patients.
tion chemotherapy based on early introduction of seven effective drugs
for advanced Hodgkin's disease. J Clin Oncol 3:1174-1182, 1985
18. Durandy A, Emilie D, Peuchmaur M, et al: Role of IL-6 in
promoting growth of human EBV-induced B-cell tumors in severe
combined immunodeficient mice. J Immunol 52:5361-5367, 1994
19. Locker J, Nalesnik M: Molecular genetic analysis of lymphoid
tumors arising after organ transplantation. Am J Pathol 135:977-987,
1989
20. Hsu SM, Raine L, Fanger H: Use of avidin biotin peroxidase
complex (ABC) in immunoperoxidase technique: A comparison be-
tween ABC and unlabelled (PAP) procedures. J Histochem Cytochem
29:577-580, 1980
21. Cordell JL, Falini B, Erber WN: Immunoenzymatic labeling of
monoclonal antibodies using immune complexes of alkaline phosphate
anti alkaline phosphatase (APAAP complex). J Histochem Cytochem
32:219-229, 1984
22. Barletta JM, Kingma DW, Ling Y, et al: Rapid in situ hybridiza-
tion for the diagnosis of latent Epstein-Barr virus infection. Mol Cell
Probes 7:105-109, 1993
23. Harris NI, Jaffe ES, Stein H, et al: A revised European-American
classification of lymphoid neoplasms: A proposal from the International
Lymphoma Study Group. Blood 84:1361-1392, 1994
24. Cleary ML, Nalesnik MA, Shearer WT, et al: Clonal analysis of
transplant-associated lymphoproliferations based on the structure of the
genomic termini of the Epstein-Barr virus. Blood 72:349-352, 1988
25. Young L, Alfieri C, Henessy K, et al: Expression of Epstein-Barr
virus transformation associated genes in tissues of patients with EBV
lymphoproliferative disease. N Eng] J Med 321:1080-1085, 1989
26. Knowles DM, Cesarman E, Chadburn A, et al: Correlative
morphologic and molecular genetic analysis demonstrates three distinct
categories of posttransplantation lymphoproliferative disorders. Blood
85:552-565, 1995
27. List AF, Greco A, Vogler LB: Lymphoproliferative diseases in
immunocompromised hosts; The role of Epstein-Barr virus. J Clin
Oncol 5:1673-1689, 1987
28. Farrell PJ: Epstein-Barr virus immortalizing genes. Trends
Microbiol 3:105-109, 1995
29. Rea D, Fourcade C, Leblond V, et al: Patterns of EBV latent and
replicative gene expression in EBV B-cell lymphoproliferative disor-
ders after organ transplantation. Transplantation 58:317-324, 1994
30. Rowe M, Young LS, Crocker J, et al: Epstein-Barr virus
(EBV)-associated lymphoproliferative disease in the SCID mouse
model: Implications for the pathogenesis of EBV-positive lymphomas
in man. J Exp Med 173:147-158, 1991
31. Garcia RC, Brown NA, Schreck R, et al: B-cell lymphoma in
severe combined immunodeficiency not associated with Epstein-Barr
virus. Cancer 60:2941-2947, 1987
32. Ho M, Jaffe R, Miller G, et al: The frequency of Epstein-Barr
EBV-NEGATIVE PTLD
virus infection and associated lymphoproliferative syndrome after
transplantation and its manifestations in children. Transplantation
45:719-727, 1988
33. Case records of the Massachussetts General Hospital (case
4-1985). N Engl J Med 312:226-237, 1985
34. Ambinder RF, Mann RB: Epstein-Barr-encoded RNA in situ
hybridization: diagnostic applications. Hum Pathol 25:602-605, 1994
35. Ambinder RF, Mann RB: Detection and characterization of
Epstein-Barr virus in clinical specimens. Am J Pathol 145:239-252,
1994
36. Halmiton-Dutoit SJ, Delecluse HJ, Raphael M, et al: Detection
of Epstein-Barr virus genomes in AIDS related lymphomas: Sensitivity
and specificity of in situ hybridization compared with Southern blotting.
J Clin Pathol44:676-680, 1991
37. Katz BZ, Raab-Traub N, Miller G: Latent and replicating forms
of Epstein-Barr virus DNA in lymphomas and lymphoproliferative
diseases; J Infect Dis 160:589-597, 1989
Downloaded from ascopubs.org by Fairview Health Services on May 27, 2018 from 162.096.053.037
Copyright © 2018 American Society of Clinical Oncology. All rights reserved.
2059
38. Nalesnik MA, Jaffe R, Starzl TE, et al: The pathology of
posttransplant lymphoproliferative disorders occurring in the setting of
cyclosporine A-prednisone immunosuppression. Am J Pathol 133:173-
192, 1988
39. Hanto DW, Frizzera G, Galj-Peczalska, et al: Epstein-Barr virus,
immunodeficiency, and B-cell lymphoproliferation. Transplantation
39:461-472, 1985
40. Waller EK, Ziemianska M, Bangs CD, et al: Characterization of
posttransplant lymphomas that express T-cell-associated markers: Immu-
nophenotypes, molecular genetics, cytogenetics, and heterotransplanta-
tion in severe combined immunodeficient mice. Blood 82:247-261,
1993
41. Hanson MN, Morrison VA, Peterson BA, et al: Posttransplant
T-cell lymphoproliferative disorders. An aggressive, late complication
of solid-organ transplantation. Blood 88:3626-3633, 1996
42. Levine AM: Acquired immunodeficiency syndrome-related lym-
phoma. Blood 80:8-20, 1992