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Semiquantitative Epstein-Barr Virus (EBV) Polymerase Chain Reaction
for the Determination of Patients at Risk for EBV-Induced
Lymphoproliferative Disease After Stem Cell Transplantation
By Kenneth G. Lucas, Robert L. Burton, Sarah E. Zimmerman, Jinghong Wang, Kenneth G. Cornetta,
Kent A. Robertson, Chao H. Lee, and David J. Emanuel
Epstein-Barr virus–induced lymphoproliferative disease (EBVLPD) is a serious and potentially fatal complication after
allogeneic stem cell transplantation (SCT). To evaluate levels
of EBV DNA in SCT patients, a semiquantitative polymerase
chain reaction (PCR) assay was developed. DNA was extracted from peripheral blood leukocytes and diluted, and
PCR was performed by using a primer set specific for a
well-conserved sequence of the internal repeat 1 region of
the EBV genome. Forty-one SCT patients were screened
with this method. Thirty-seven patients received allogeneic
transplants, of which 18 were T-cell–depleted marrow. Four
additional patients received autologous SCT, one of which
was T-cell depleted. The mean time of follow-up by EBV PCR
was 147 days (range, 47 to 328 days) posttransplant. The
range of EBV copies/mg DNA from normal EBV sero-positive
donors was 40 to 4,000. Seven patients had H40,000 copies
of EBV DNA/mg DNA, all of whom were recipients of
T-cell–depleted SCT. Five of the seven patients with elevated
levels of EBV DNA developed EBV-LPD. Four of these five
patients with EBV-LPD had elevated levels of EBV DNA from
1 to 8 weeks before diagnosis. Two patients with EBV-LPD
had normal levels of EBV DNA, and two patients with
H40,000 copies EBV/mg DNA did not develop EBV-LPD. In
one patient, clinical resolution of disease correlated with a
decrease in EBV DNA and an increase in the level of EBVspecific cytotoxic T-cell precursors. These data indicate that
the measurement of EBV viral load with semiquantitative
PCR is useful in detecting EBV-LPD in high-risk patients
before the onset of clinical symptoms. Because not all
patients with elevated levels of EBV DNA develop EBV-LPD,
semiquantitative PCR results cannot substitute for clinical,
radiographic, and pathological confirmation of this diagnosis.
r 1998 by The American Society of Hematology.
E
cytotoxic T cells (EBV-CTLs) has been shown to be curative for
SCT patients with EBV-LPD.2,12
Rooney et al12 have shown that patients receiving T-cell–
depleted (TCD) SCTs can be prevented from developing
EBV-LPD by using prophylactic EBV-CTL infusions. Institutions performing TCD transplants are more frequently using
strategies to prevent EBV-LPD in high-risk patients by using
either donor PBMCs or EBV-CTLs. The differential susceptibility to EBV-LPD among recipients of TCD marrow grafts is not
understood and may represent a combination of factors, such as
viral burden and level of cellular immunity to EBV. Differences
in levels of EBV DNA found in the peripheral blood posttransplant may distinguish which patients are at highest risk to
develop EBV-LPD and, therefore, could permit earlier intervention in high-risk patients. Previous studies have shown a
correlation between levels of EBV DNA and the occurrence of
EBV-LPD in organ transplant patients13-15 and in recipients of
SCTs.16 However, it is unclear to what extent levels of EBV
DNA correlate with the presence of EBV-LPD, disease outcome, and cellular immunity to EBV in SCT recipients. The
purpose of this study was to examine the correlation between
levels of EBV DNA in the peripheral blood of SCT patients
posttransplant with (1) the type of transplant received, (2) the
development of EBV-LPD, (3) clinical outcome of adoptive
immunotherapy, and (4) the cellular immunity to EBV in
recipients of donor lymphocytes.
PSTEIN-BARR VIRUS–induced lymphoproliferative disease (EBV-LPD) is a serious complication because of the
profound immune deficiency that occurs after allogeneic stem
cell transplantation (SCT).1-5 EBV infects most individuals by
adulthood and latently infects B lymphocytes.6 In the absence of
adequate cellular immunity to protect against EBV, latently
infected B cells undergo transformation into lymphoblastoid
cells. After SCT, these lymphoproliferations are generally of
donor B-cell origin.2-4,7,8 The clinical spectrum of EBV-LPD
varies from polyclonal lymphoproliferations to clonal malignancies; the latter being frequently fatal.3,4,8 Among recipients of
allogeneic SCT, the incidence of EBV-LPD is highest for those
receiving grafts that have been manipulated to reduce the
number of T cells or who have received aggressive immunosuppressive regimens to facilitate engraftment.3-5,7,8 Attempts to
treat these patients with interferon, acyclovir, and anti–B-cell
monoclonal antibodies are generally not successful.3,4,9-11 However, adoptive immunotherapy with unprimed peripheral blood
mononuclear cells (PBMCs) or donor-derived, EBV-specific
From the Department of Pediatrics, Stem Cell Transplantation
Program, Riley Hospital for Children; The Department of Medicine,
Bone Marrow Transplantation Program; and the Department of
Pathology and Laboratory Medicine, Indiana University Medical
Center, Indianapolis, IN.
Submitted October 2, 1997; accepted January 12, 1998.
Supported by Grant No. 97-043-01-EDT from the American Cancer
Society.
Address reprint requests to David J. Emanuel, MD, Stem Cell
Transplantation Program, Riley Hospital for Children, 702 Barnhill
Drive, Indianapolis, IN 46202-5225.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9110-0034$3.00/0
3654
MATERIALS AND METHODS
Patients. Patient characteristics are summarized in Table 1. Both
adult and pediatric stem cell transplant patients had 5 mL of whole
blood collected starting at approximately 6 to 8 weeks posttransplant.
This test was performed every 2 to 4 weeks thereafter. Forty-one
patients had levels of EBV DNA measured. The mean time of follow-up
was 147 days posttransplant (range, 47 to 328 days). Some patients
were followed for a shorter time than others because of relapse,
EBV-LPD, death, or distance from the hospital precluding frequent
follow-up. Thirty-seven patients (90%) received allogeneic SCT, and 4
(10%) received autologous transplants. One of the autologous SCT
Blood, Vol 91, No 10 (May 15), 1998: pp 3654-3661
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EBV-DNA LEVELS AND POST-TRANSPLANT EBV-LYMPHOMA
Table 1. Patient Characteristics
Mean age (yrs)
Sex (male:female)
Pretransplant diagnosis
Acute lymphoblastic leukemia
Acute myelogenous leukemia
Chronic myelogenous leukemia
Myelodysplastic syndrome
Other*
Allogeneic T-cell–depleted marrow grafts†
Unmodified marrow
Unrelated cord blood
Autologous, T-cell–depleted transplant
Autologous peripheral blood
24 (1-56)
20:21
11
9
6
7
8
18
16
3
1
3
The clinical characteristics of patients evaluated by semiquantitative EBV PCR are summarized above.
*Includes 2 patients with non-Hodgkin’s lymphoma, 3 patients with
Ewing’s sarcoma, 1 patient with neuroblastoma, 1 patient with juvenile myelomonocytic leukemia, and 1 patient with multiple sclerosis.
†T-cell depletion by sheep erythrocyte and soybean agglutination
(15) or CD34 selection device.3
patients received a TCD graft as treatment for multiple sclerosis. Of the
37 allogeneic transplant recipients, 16 patients received unmodified
marrow grafts from a related donor, 3 patients received unrelated cord
blood transplants, and 18 patients received TCD transplants. Three of
the TCD SCT patients received CD34-selected marrow grafts (Baxter
Healthcare, Santa Ana, CA) and 15 received marrow that was TCD with
sheep red blood cells and soybean agglutination as previously described.17 These studies were performed under protocols approved by
the Institutional Review Board of the Indiana University School of
Medicine, Indianapolis, IN.
Transplant regimens. The preparative regimens varied depending
on the clinical protocol. Recipients of TCD marrow grafts received
hyperfractionated total body irradiation (TBI; 1,375 cGy) with 60
mg/kg cyclophosphamide over 2 days and 5 mg/kg/d thiotepa for 2
days. These patients received antithymocyte globulin (ATG) at total
cumulative doses of 75 mg/kg posttransplant (6 patients) or 120 mg/kg
pretransplant (12 patients). The patient receiving an autologous TCD
bone marrow transplant as treatment for multiple sclerosis received a
total of 90 mg/kg ATG pretransplant with 120 mg/kg cyclophosphamide
Fig 1. The five lanes on the right, labeled ‘‘B 95-8
Cell Line’’, represent the electrophoresis of DNA from
this cell line, which is used as a control to determine
the sensitivity of the semiquantitative EBV PCR
assay. DNA from a known number of cells is run in
each lane, and the lowest dilution at which a band is
still visible corresponds to 10 cells. Because each B
95-8 cell has two copies of the EBV genome, the
sensitivity of this assay is 20 copies EBV DNA. The
first five lanes represent different amounts of DNA
per lane from a patient. The last visible band corresponds to the 0.05-ng dilution. The number of copies
of EBV genome per microgram DNA (in this case
400,000 copies/mg DNA) is calculated by dividing the
sensitivity of the assay (20 copies/mg DNA) by the
concentration of DNA for the last visible band (0.05 3
1023 mg).
3655
and 1,200 cGy hyperfractionated TBI. Recipients of unmodified
marrow grafts received TBI (1,375 cGy), 500 mg/m2/d etoposide for 2
days, and 60 mg/kg/d cyclophosphamide for 2 days. Autologous
transplant recipients received 1,800 mg/m2/d etoposide for 1 day, 90
mg/m2/d melphalan for 2 days, and hyperfractionated TBI (1,375 cGy).
The cord blood transplant patients received hyperfractionated TBI
(1,375 cGy), 60 mg/kg/d cyclophosphamide for 2 days, and 30 mg/d
ATG for 2 days. Recipients of unmodified allogeneic stem cell
transplants received cyclosporin A and methotrexate for graft-versushost disease (GVHD) prophylaxis.18
Diagnosis of EBV-LPD. Biopsy specimens were evaluated morphologically, and immunostains were used against CD19, CD20, and latent
membrane protein 1 (LMP-1). Clonality was determined by k and l
immunoglobulin light chain restriction or by immunoglobulin gene
rearrangement by polymerase chain reaction (PCR).
Semiquantitative PCR. DNA from the B95-8 cell line, which
contains two copies of the EBV genome per cell, was used as the
positive control and to determine the sensitivity of the assay. DNA was
isolated from a known number of B95-8 cells by phenol/chloroform
extraction followed by alcohol precipitation. The DNA was quantified
on a spectrophotometer at OD260, and then a 10-fold dilution series was
prepared, ranging from 500 ng to 0.05 ng. These titrations were
performed for all patient studies. These template concentrations were
used in a PCR containing 10 µL DNA template, 4 µL of 10 µmol/L each
of deoxynucleoside triphosphate (Oncor, Gaithersburg, MD), 2.5 units
Taq polymerase (Oncor), and 20 pmol each of the primers EBV-1 and
EBV-2.19 The final volume of 60 µL was placed in a thermal cycler
(Perkin Elmer, Foster City, CA) for a 35-cycle PCR. The formation of a
PCR product was detected by gel electrophoresis followed by ethidium
bromide staining of the gel. The lowest dilution at which a band was
visible corresponded to 10 cells. Because there are two copies of the
EBV genome per cell, the sensitivity of this assay was determined to be
20 copies of the EBV genome (Fig 1).
Patient DNA was isolated from the white blood cells of whole blood
using the Puregene DNA isolation kit (Gentra, Minneapolis, MN)
according to the manufacturer’s directions. PCR was performed on
patient specimens as outlined previously, with known amounts of DNA
per lane, ranging from 0.05 to 500 ng. The following formula was used
to calculate the number of EBV copies per microgram DNA: sensitivity
of the assay/DNA concentration of last visible band 5 number of copies
EBV genome/µg DNA.
For the example in Fig 1, the lowest visible band was from the 0.05
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3656
LUCAS ET AL
Table 2. Characteristics of Patients With EBV-LPD
Patient
No.
361
379
380
381
426
429*
440
Age/
Sex
48/F
6/M
23/F
9/F
45/M
44/F
46/F
Pretransplant
Diagnosis
Weeks
Post-transplant
MDS
MDS
CML
MDS
AML
ALL
AML
26
9
8
11
26
25
9
Clonality
ND
ND
Monoclonal
Monoclonal
Monoclonal
Monoclonal
Monoclonal
Peak
EBV DNA
(copies/µg)
4,000
40,000
400,000
400,000
400
400,000
40,000
Days With
.40,000
Copies
Before Dx.
Treatment
(CD31
cells/kg)
Response
Outcome
—
12
7
8
—
58
3
13
1 3 106
1.2 3 105
1.0 3 106
excision
2 3 105
7 3 105
NR
NR
NR
NR
CR
CR
CR
D-LPD/ARDS
D-LPD
D-LPD/GVH
D-LPD/ARDS
A-NED
A-NED
D-GVH
106
The clinical features of the 7 patients with EBV-LPD are summarized above.
Abbreviations: AML, acute myelogenous leukemia; ALL, acute lymphoblastic leukemia; CML, chronic myelogenous leukemia; MDS,
myelodysplastic syndrome; ND, not done; GVHD, graft versus host disease; ARDS, adult respiratory distress syndrome; LPD, lymphoproliferative
disease; Dx, diagnosis; NED, no evidence of disease; CR, complete remission; NR, no response; A, alive; D, dead.
*UPN 429 received donor-derived EBV-specific T cells.
ng dilution. Therefore, 20 copies (sensitivity of the assay)/0.05 3 1023
µg DNA 5 400,000 copies EBV genome/µg DNA.
Cell culture and chromium-51 release assays. Blood was obtained
on patients at study intervals and PBMCs were isolated by using
Ficoll-Hypaque (Accurate Chemical Co, Westbury, NY) density gradient centrifugation. Bulk and limiting dilution cultures with patient
PBMCs and irradiated donor B lymphoblastoid cell lines (BLCLs) were
set up as previously described,5 and chromium release assays were
performed on day 12 of culture by using an effector:target ratio of
12.5:1. Donor phytohemagglutinin blasts (EBV negative, autologous
control), donor EBV BLCLs, and allogeneic BLCLs were used as
targets cells. Spontaneous and total release for each target was used to
calculate percent-specific release by using the following formula: %
specific release 5 (experimental cpm 2 spontaneous cpm)/(total
cpm 2 spontaneous cpm). Limiting dilution wells were scored as
positive if release exceeded 10%. EBV-specific cytotoxic T-cell precursor (CTLp) frequencies were calculated by the method of Taswell20 by
using a computer program provided by Dr Y. Kawanishi, Medical
College of Wisconsin (Milwaukee, WI).
RESULTS
The clinical characteristics of the patients who developed
EBV-LPD during this study period are presented in Table 2. Six
of the seven patients with EBV-LPD were recipients of TCD
unrelated-donor marrow grafts, and one patient (unique patient
number [UPN] 361) received a TCD related-donor transplant.
Five of these patients had $40,000 copies EBV genome/µg
DNA. The time from transplant to the development of EBVLPD ranged from 8 to 26 weeks. All patients had a diagnosis of
EBV-LPD confirmed immunohistologically. Clonality was determined in five cases of EBV-LPD and all were monoclonal.
Table 3 presents the peak levels of EBV DNA for all patients.
Ten EBV sero-positive normal adult donors were evaluated for
levels of EBV DNA, and all had #4,000 copies EBV/µg DNA
(data not shown). Patients receiving TCD transplants had a
much higher prevalence of elevated levels of EBV DNA
compared with recipients of unmodified transplants (P , .001,
Fisher’s exact test). Five of the seven patients with EBV-LPD
had levels of EBV DNA $40,000 copies/µg DNA. Two of the
34 patients not developing EBV-LPD had levels of EBV DNA
$40,000 copies/µg DNA. The positive predictive value of this
test for developing EBV-LPD based on having $40,000 copies
EBV genome/µg DNA was 71%, and the negative predictive
value was 94%.
The immunologic and viral response to donor leukocyte
infusions are presented for two patients in Figs 2 and 3. UPN
440 (Fig 2) received a donor leukocyte infusion for EBV-LPD 9
weeks posttransplant. This patient had elevated levels of EBV
DNA concomitant with the development of lymphadenopathy
and pulmonary infiltrates. After biopsy confirmation of EBVLPD, this patient received a donor leukocyte infusion. This
patient had resolution of fevers and lymphadenopathy 3 weeks
postinfusion with a persistent decrease in the level of EBV DNA
by 4 weeks postinfusion. There was a progressive increase in
EBV-specific CTLp frequencies (Fig 2C) ranging from undetectable at the time of the infusion and 1 week postinfusion,
1/267,980 2 weeks postinfusion, to 1/5,160 at 24 days postinfusion (13 weeks posttransplant). There was also an increase in
cytotoxicity from bulk cultures 2 weeks after the donor
leukocyte infusion (Fig 2B). This patient developed grade-4
GVHD involving the liver and skin, required systemic corticosteroids, and died because of complications of GVHD. UPN
381 (Fig 3) developed fever and hepatomegaly 11 weeks after
an unrelated-donor TCD transplant. One week before developing clinical symptoms there was an elevation in the level of
EBV DNA. A liver biopsy specimen showed a diffuse, monoclonal, immunoblastic B-cell lymphoma. After a donor leukocyte
infusion, this patient’s level of EBV DNA continued to be
elevated, and at week 14 she died. This patient had an increase
in the level of cytotoxicity against the donor BLCLs 2 weeks
after the donor leukocyte infusion (Fig 3B). Postmortem
examination showed a diffuse infiltration of the lungs, liver, and
Table 3. Levels of EBV DNA in Study Patients
EBV DNA Level (copies/ µg DNA)
Type of transplant
T-cell depleted*
Unmanipulated
EBV-LPD
Present
Absent
$40,000
7
0
#4,000
12
22
5
2
2
32
Fisher’s
Exact Test
P , .001
P , .001
Patient levels of EBV DNA in relation to type of transplant (T-cell
depleted or unmanipulated) and the presence or absence of EBV-LPD.
*T-cell depletion by soybean agglutinin and sheep erythrocyte
rosette formation or by a CD34 selection device.
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EBV-DNA LEVELS AND POST-TRANSPLANT EBV-LYMPHOMA
A
B
C
3657
persistence of lymphoblastoid cells and the patient died of
progressive EBV-LPD. UPN 426 developed a monoclonal,
polymorphic B-cell lymphoma of a cervical lymph node 26
weeks posttransplant, which was CD201 and LMP-11 and was
strongly positive for EBV DNA by PCR. UPN 426 underwent
an excisional biopsy of the involved node, did not receive donor
leukocytes, and remained in remission 4 months after diagnosis
without any evidence of recurrent EBV-LPD. Levels of EBV
DNA in the peripheral blood did not exceed 400 copies/µg
DNA. This patient had no significant EBV-specific cytotoxicity
against the donor BLCLs at the time of diagnosis or 3 months
after diagnosis and continues to be in remission with 400 copies
EBV/µg DNA.
Two patients in this study had elevated levels of EBV DNA
but did not develop EBV-LPD. UPN 386 was noted to have
fever of undetermined origin and elevated levels of EBV DNA 3
months after an unrelated-donor TCD transplant (Fig 4). This
patient had computerized tomography scans performed of the
head, chest, abdomen, and pelvis, which showed no evidence of
lymphoma. A bone marrow aspirate showed recurrent acute
myelogenous leukemia, and this patient received 1 3 106 CD31
donor leukocytes/kg as treatment for relapsed leukemia. Two
weeks after the donor leukocyte infusion, UPN 386 had a 2-log
decrease in the level of EBV DNA. Another patient had 40,000
copies EBV/µg DNA 6 months after a TCD unrelated-donor
SCT, which decreased to 400 copies 1 month later. This patient
did not have any symptoms of EBV-LPD and did not receive
donor leukocytes.
Fig 2. UPN 440 developed EBV-LPD 9 weeks after an unrelated,
T-cell–depleted SCT. Levels of EBV DNA determined by semiquantitative PCR are presented on the top panel (A). Two weeks after
receiving a donor leukocyte infusion, this patient had a decrease in
levels of EBV DNA (B). There was a progressive increase in the
amount of EBV-specific cytotoxicity against the donor BLCL, which
had reached normal levels at 2 weeks postinfusion (C). This correlated with the development of normal levels of EBV-specific, cytotoxic T-cell precursor frequencies. This patient died from graft-versushost disease.
kidneys with CD191 lymphoblastoid cells consistent with
disseminated EBV-LPD. A CD31 cellular infiltrate was present
among the lymphoblastoid cells.
Two patients in this study with EBV-LPD (UPN 361 and 426)
did not have elevations in their levels of EBV DNA at the time
of diagnosis. UPN 361 developed a diffuse CD201, LMP-11,
B-cell lymphoma of the lungs 26 weeks after a TCD relateddonor SCT. The patient’s levels of EBV DNA were persistently
#4,000 copies EBV/µg DNA. This patient received 1 3 106
donor CD31 cells/kg but developed worsening respiratory
insufficiency. Two weeks postinfusion, pleural fluid showed
Fig 3. UPN 381 developed fevers and hepatosplenomegaly 11
weeks after an unrelated T-cell–depleted SCT (A). This patient received a donor leukocyte infusion at the time of diagnosis, at which
time there were 40,000 copies of EBV/mg DNA in the peripheral
blood. After the infusion, there was a progressive increase in the
amount of EBV-specific cytotoxicity against the donor BLCL (B). This
patient died secondary to pulmonary failure, complicated by persistent EBV-LPD in the liver and lungs.
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3658
To assess whether the stem cell donors of patients with
EBV-LPD had deficient EBV-specific cytotoxicity, donor
PBMCs were assayed for lysis of autologous BLCLs. Table 4
presents cytotoxicity data from bulk culture PBMCs and
EBV-CTLp frequencies for four stem cell donors of patients
who developed EBV-LPD. Three of four donors (UPN 361,
380, and 429) had $25% specific lysis of autologous EBV
BLCLs at day 12 of culture, and the donor for UPN 426 (who
did not receive a donor leukocyte infusion) had 15% EBVspecific cytotoxicity at this time, which increased to 50% at day
21 of culture. Analysis of EBV-CTLp frequencies for these
donors shows that three of the four had low levels of EBVspecific CTLs (1/41,000 to 1/129,000).
Table 5 summarizes all cases of EBV-LPD in stem cell
transplant patients at Indiana University from January 1991 to
June 1997. The preparative regimens varied during this time
period, and patients received TCD transplants with sheep red
blood cell and soybean agglutination17 or a device to selectively
deplete T-cells (Applied Immunoscience, Menlo Park, CA) or
enrich for CD341 stem cells (Baxter Healthcare, Santa Ana,
CA). Of the 165 patients receiving TCD transplants during this
time period, 23 (13.9%) developed EBV-LPD. Of the 89
patients receiving TCD SCT from a related donor, there were 3
(3.4%) cases of EBV-LPD. Twenty of 76 patients (26.3%) who
received unrelated TCD SCT during this time developed
EBV-LPD. T-cell doses for the patients receiving donor leukocytes ranged from 1 3 105 to 1 3 106 CD31 cells/kg. Of the 13
patients treated with donor leukocytes, 4 patients had a complete response; however, 1 of these patients succumbed from
GVHD and another patient died from aspergillosis. The patients
receiving no specific therapy and those receiving immunoglobulin, interferon, or chemotherapy did not have a clinical response. Five of the 13 patients receiving donor leukocytes who
did not have a clinical response died from progressive EBVLPD 2 to 10 days after the donor leukocyte infusion.
LUCAS ET AL
Table 4. EBV-Specific Cytotoxicity of Four Stem Cell Donors
of Patients With EBV-LPD
UPN Donor
Percent Specific Lysis
of Autologous BLCL*
EBV CTLp
Frequency
361
426
429
440
31
15
41
25
1/129,000
1/81,000
1/25,000
1/41,000
EBV-specific cytotoxicity from bulk culture peripheral blood mononuclear cells and EBV-CTL precursor frequency analysis from donors
of patients who developed EBV-induced lymphoproliferative disease.
Abbreviations: BLCL, B lymphoblastoid cell line; CTLp, cytotoxic T
cell precursor.
*From bulk culture peripheral blood mononuclear cells.
DISCUSSION
There are several risk factors for the development of EBVLPD after SCT, the most significant of which are T-cell
depletion, the use of unrelated or mismatched transplants, and
the use of antithymocyte globulin.2-5,7,8 Differences in EpsteinBarr viral load and cellular immunity to EBV may account for
the differential susceptibility to EBV-LPD in these patients. A
reliable method of predicting which patients are likely to
develop EBV-LPD would permit intervention at a time point
earlier in lymphomagenesis and could possibly reduce the
morbidity associated with this complication. From the present
study there is a strong correlation between developing EBVLPD and having an elevated level of EBV DNA after TCD SCT,
with some patients noted to have high levels of EBV DNA
weeks before the onset of symptoms. However, despite this
correlation two of seven patients with EBV-LPD had levels of
EBV DNA within the normal range, and two patients without
EBV-LPD had $40,000 copies EBV/µg DNA. Elevated levels
of EBV were only seen in recipients of TCD transplants, and
none of the ‘‘low-risk’’ patients had elevated levels of EBV
DNA or developed EBV-LPD.
Other groups have examined levels of EBV DNA to determine risk for developing EBV-LPD in both solid organ and SCT
patients.12,14-16 Previous studies in SCT patients indicate a
strong correlation between levels of EBV DNA and the
occurrence of EBV-LPD.12,16 Rooney et al16 reported elevated
Table 5. Past Cases of EBV-LPD in Stem Cell Transplant Patients
at Indiana University
Fig 4. UPN 386 received a T-cell–depleted, unrelated donor bone
marrow transplant for acute myelogenous leukemia and developed
fever 12 weeks posttransplant. There was no evidence of EBV-LPD on
computerized tomography, but levels of EBV DNA were 40,000
copies/mg DNA. This patient was noted to have relapsed leukemia at
this time, received a donor leukocyte infusion, and had a decrease in
EBV DNA to the normal range.
Treatment
Total
Patients
Complete
Remission
(%)
Disease
Progression
(%)
GVHD
(%)
Patients
Surviving
(%)
Observation
Interferon, IVIg*
Chemotherapy
Excision
Donor leukocytes
6
2
1
1
13
0 (0)
0 (0)
0 (0)
1 (100)
4 (31)
6 (100)
2 (100)
1 (100)
0 (0)
9 (69)†
0 (0)
0 (0)
0 (0)
0 (0)
4 (31)
0 (0)
0 (0)
0 (0)
1 (100)
2 (15)‡
Treatment outcomes in 23 stem cell transplant patients with EBVLPD at Indiana University.
*Intravenous immunoglobulin.
†Five of these nine patients died 2 to 10 days after receiving donor
leukocytes and had advanced EBV-LPD at the time of diagnosis and
treatment.
‡One of these patients received EBV-specific CTL and one patient
unprimed donor leukocytes.
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EBV-DNA LEVELS AND POST-TRANSPLANT EBV-LYMPHOMA
levels of EBV DNA ($20,000 copies EBV/µg DNA) in four
SCT patients with EBV-LPD with normal levels (2 to 2,000
copies EBV/ µg DNA) in all unaffected patients. Although there
was also a strong correlation between EBV-LPD and elevated
levels of EBV DNA in the present study, some patients with
$40,000 copies/µg DNA did not develop this complication.
UPN 386, for example, had 40,000 copies EBV/µg DNA
concurrently with fevers and leukemic relapse, with no evidence of EBV-LPD by computerized tomography. However,
there was a sharp decline in this patient’s level of EBV DNA
after the donor leukocyte infusion, raising the possibility that
this patient may have developed EBV-LPD if donor leukocytes
were not administered. Also of interest is UPN 426, who had a
monoclonal B-cell lymphoma but did not have elevated levels
of EBV DNA at the time of diagnosis. UPN 361 in the present
study had normal levels of EBV DNA, despite a clinically
aggressive, disseminated form of EBV-LPD, which caused this
patient’s death. Rooney et al12 also reported one patient with
EBV-LPD without elevated levels of EBV DNA. It is known
that some BLCLs do not produce EBV, whereas others are
associated with virus production.21-23 Katz et al24 showed that
only 40% of BLCLs grown from EBV-LPD lesions contain
replicating EBV DNA. Therefore, it is possible that in vivo
some transformed B cells do not produce virus, resulting in
normal levels of EBV DNA in some patients with EBV-LPD.
A review of past cases of EBV-LPD at Indiana University
shows a high incidence in recipients of TCD stem cell
transplants (13.9%) with a striking difference in the incidence of
EBV-LPD between recipients of related and unrelated TCD
transplants (3.4% v 26.3%). Other groups have noted a similar
distribution in cases of EBV-LPD among recipients of TCD
transplants.4,5 Differences in levels of cellular immunity to EBV
among recipients of TCD SCT patients may account for the fact
that some patients with viral reactivation do not develop
EBV-LPD. EBV-specific CTL precursor frequency analysis of
SCT peripheral blood shows that many of these patients have
deficient or emerging cellular immunity to EBV during the first
3 to 6 months posttransplant,5,25 the time period during which
the majority of these disorders occur. T-cell depletion of marrow
from a patient with a low level of EBV-CTL could result in a
lower dose of these cells given at the time of transplant.
However, it is unclear whether these differentiated precursors in
the stem cell inoculum given at the time of transplant are the
cells that result in the restoration of EBV-specific cellular
immunity several months later. PBMCs in bulk culture from
four stem cell donors of patients with EBV-LPD had in vitro
capacity to lyse autologous BLCLs. However, three of these
four stem cell donors had low levels of EBV-specific CTL
precursors. Three of the four PBMCs assayed from these donors
were used in leukocyte infusions, which resulted in resolution
of disease in two patients with EBV-LPD (UPN 429 and 440).
UPN 361, who had progressive EBV-LPD despite a donor
leukocyte infusion, received PBMCs from a donor whose CTLp
frequency was within the range previously reported in EBV
sero-negative donors.5
In acute EBV infection in the normal host, the cellular
immune response to EBV is mediated by human leukocyte
antigen (HLA)-unrestricted and HLA-restricted cytotoxic lymphocytes.26,27 Protection against recurrent EBV-induced illness
3659
is largely mediated by CD81, major histocompatibility complex
class I–restricted, EBV-specific cytotoxic T cells.28-31 In the
present study, donor leukocyte infusions resulted in the development of EBV-specific cytotoxicity in two patients studied. UPN
440 had a dramatic increase in levels of EBV-specific CTL
precursors, which correlated with an increase in EBV-specific
cytotoxicity against the donor BLCLs from bulk culture lymphocytes. There was a gradual decrease in levels of EBV DNA after
the T-cell infusion. However, in one patient, increasing EBVspecific cytotoxicity after donor leukocytes was not accompanied by a clinical response. Despite an increase in EBV-specific
cytotoxicity after a donor leukocyte infusion, UPN 381 continued to have elevated levels of EBV DNA, and at autopsy 3
weeks postinfusion had disseminated EBV-LPD. At the time of
death, this patient had a CD31 infiltrate in the lymphomatous
lesions, which may have been a result of the donor leukocyte
infusions. Papadopoulos et al2 also reported a T-cell infiltrate in
the lymphomatous lesions of SCT patients treated with donor
leukocytes; however the patients in this latter study had
complete resolution of their EBV-LPD. Although donor T-cell
infusions generally result in resolution of these disorders,2,13
there may be a subset of patients with disease that is more
aggressive and does not respond as quickly to this form of
therapy. UPN 381’s elevated levels of EBV DNA 3 weeks
postinfusion may be indicative of failure of adoptive immunotherapy, and monitoring levels of EBV DNA may serve as a
sensitive measure of response to therapy.
Overall, the patients reported here had a substantially higher
morbidity compared with patients in other series who received
donor leukocytes2 or EBV-CTLs.12 Of the five patients who
died, one death was a result of severe GVHD, most likely
caused by the donor leukocyte infusion. Another patient (UPN
379) had disseminated EBV-LPD 9 weeks posttransplant and
had a rapidly progressive course, dying 2 days after the donor
leukocyte infusion. These data contrasted with the report from
Papadopoulos et al2 in which all five patients with EBV-LPD
receiving donor leukocyte infusions had a complete response.
Three of these patients developed chronic GVHD, and two
patients, although with resolution of EBV-LPD on autopsy,
expired from respiratory insufficiency possibly related to the
T-cell infusion. Of interest is that two patients in the present
study who received donor leukocytes developed GVHD, one
patient with grade 2 GVHD of the skin (UPN 361) and another
with grade 3 GVHD of the skin, gut, and liver (UPN 380), but
neither patient had resolution of their EBV-LPD. Recently,
Imashuku et al32 have also reported an SCT patient with
EBV-LPD who failed to respond to infusions of donor-derived
EBV-CTLs (albeit at low doses of 9.2 3 106 total cells). In
applying adoptive immunotherapy with donor leukocytes for
relapsed leukemia, there is a direct correlation between the
development of GVHD and disease response.33-36 The relationship between GVHD and a graft-versus-leukemia effect is
especially poignant in chronic myelogenous leukemia,35-36 with
a 91% response rate to donor leukocyte infusions if there was
evidence of GVHD or myelosuppression and a 42% response
rate in patients without these complications.36 Although GVHD
and graft-versus-leukemia appear to be related in adoptive
immunotherapy for relapsed leukemia, GVHD- and EBVspecific cytotoxicity are likely mediated by different effector
From www.bloodjournal.org by guest on August 9, 2017. For personal use only.
3660
LUCAS ET AL
cell populations. Therefore, the presence of GVHD in patients
without a clinical response to donor leukocytes for EBV-LPD
would be possible, especially if these donors have low levels of
EBV-CTL precursors.
In comparing clinical features of patients in the present series
with that reported by Papadopoulos et al,2 of note is the fact that
the majority of patients in the latter study had nodular multiorgan involvement. Three of the four patients who died with
EBV-LPD in the present series (Table 2) had diffuse infiltration
of affected organs without distinct nodules identified radiographically. Three of the four patients with nodal disease had a
complete response to donor leukocytes (UPN 429 and 440) or
surgical excision (UPN 426). Another possible explanation for
the clinically aggressive nature of some cases of EBV-LPD is
that they are genetically different from other lymphoproliferations. The cases in the present study in which adoptive
immunotherapy failed to induce remission of disease histopathologically resemble the more aggressive variant of EBV-LPD
reported by Knowles et al,37 with an immunoblastic morphology and presenting with widely disseminated disease. Another
possible explanation for poor response to donor leukocytes may
be a lack of tumor cell immunogenicity as has been noted in a
subset of EBV-related lymphomas in human immunodeficiency
virus–infected patients lacking expression of EBV peptides
important in immune recognition.38-40
Although the overall survival and success of therapy for the
13 patients receiving donor leukocytes is less than that previously reported,2 five of the nine ‘‘nonresponders’’ in the present
series presented with advanced disease or were moribund at the
time of diagnosis, surviving less than 2 weeks after the T-cell
infusion. Therefore, this poor response rate may be in part
caused by late detection of EBV-LPD, at a point when these
patients were unable to survive the time period necessary to
achieve an immunologic response, as well as by GVHD as a
result of unprimed donor leukocyte infusions. These findings
emphasize the importance of early detection of EBV-LPD in
high-risk patients. Additionally, the high incidence of severe
GVHD after infusion of unprimed donor leukocytes to treat
either EBV-LPD or relapsed leukemia highlights a potential
advantage of using EBV or leukemia-specific CTLs. Several
patients in the present study had elevated levels of EBV DNA
before the development of symptoms or signs of EBV-LPD.
Semiquantitative EBV PCR offers the possibility for screening
high-risk patients at a time before the development of overt
disease. Patients who are found to have elevated levels on
screening would be candidates for a more thorough clinical
evaluation, such as computerized tomography of potentially
affected areas and biopsies. With this assay, patients may be
identified and treated at an earlier time point, and response to
therapy can be assessed by measuring viral burden. This may be
especially advantageous in patients with diffuse aggressive
lymphomas that, from our series, have a poor outcome.
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1998 91: 3654-3661
Semiquantitative Epstein-Barr Virus (EBV) Polymerase Chain Reaction for
the Determination of Patients at Risk for EBV-Induced Lymphoproliferative
Disease After Stem Cell Transplantation
Kenneth G. Lucas, Robert L. Burton, Sarah E. Zimmerman, Jinghong Wang, Kenneth G. Cornetta, Kent
A. Robertson, Chao H. Lee and David J. Emanuel
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