Abstract

We evaluated HLA-compatible donor leukocyte infusions (DLIs) and HLA-compatible or HLA-disparate EBV-specific T cells (EBV-CTLs) in 49 hematopoietic cell transplantation recipients with biopsy-proven EBV-lymphoproliferative disease (EBV-LPD). DLIs and EBV-CTLs each induced durable complete or partial remissions in 73% and 68% of treated patients including 74% and 72% of patients surviving ≥ 8 days after infusion, respectively. Reversible acute GVHD occurred in recipients of DLIs (17%) but not EBV-CTLs. The probability of complete response was significantly lower among patients with multiorgan involvement. In responders, DLIs and EBV-CTLs regularly induced exponential increases in EBV-specific CTL precursor (EBV-CTLp) frequencies within 7-14 days, with subsequent clearance of EBV viremia and resolution of disease. In nonresponders, EBV-CTLps did not increase and EBV viremia persisted. Treatment failures were correlated with impaired T-cell recognition of tumor targets. Either donor-derived EBV-CTLs that had been sensitized with autologous BLCLs transformed by EBV strain B95.8 could not lyse spontaneous donor-derived EBV-transformed BLCLs expanded from the patient's blood or biopsied tumor or they failed to lyse their targets because they were selectively restricted by HLA alleles not shared by the EBV-LPD. Therefore, either unselected DLIs or EBV-specific CTLs can eradicate both untreated and Rituxan-resistant lymphomatous EBV-LPD, with failures ascribable to impaired T-cell recognition of tumor-associated viral antigens or their presenting HLA alleles.

Introduction

EBV-induced lymphoproliferative diseases (EBV-LPDs) are a significant cause of morbidity and mortality for recipients of allogeneic hematopoietic cell transplantation (HCT), particularly in those who have received certain T-cell reactive Abs to prevent or treat GVHD,1,2  T cell–depleted HCT,3,4  or cord blood transplantation (CBT).5  EBV-LPDs in HCT recipients present as rapidly progressive, monoclonal, diffuse large B-cell lymphomas (DLBCLs).6  Combination chemotherapies can induce sustained remissions in 40%-50% of cases but there is a risk for suppression of the HCT.7,8  The CD20-specific mAb rituximab administered preemptively can induce sustained reversal of EBV viremia in up to 93% of patients.9  However, only 50%-60% of patients with clinically and radiologically established disease achieve durable remissions.10 

In 1994, our group reported 5 patients with monoclonal EBV lymphomas who achieved durable complete remission (CR) after adoptive transfer of PBMCs containing unselected T cells from their EBV-seropositive transplantation donors (donor leukocyte infusions or DLIs).11  Other studies subsequently confirmed these results.12-14  However, a recent review of reported cases suggested that only 41% of patients with established disease achieve sustained CR.14  Rooney et al introduced the use of EBV-specific cytotoxic T cells (EBV-CTLs) generated in vitro to reconstitute EBV-specific immunity without GVHD.15  To date, they have administered EBV-CTLs as prophylaxis to 101 patients at risk, and none of the patients has developed EBV-LPD or GVHD. Rooney et al also successfully treated 11 of 13 patients with EBV viremia and clinical evidence of EBV-LPD. Of these patients, 8 were biopsy proven and of these, 6 achieved CR.16  Prior cumulative reported experience for treatment of established EBV-LPD (reviewed in Merlo et al17 ) comprises only 11 additional cases. Although this experience suggests that EBV-CTLs can induce regression of biopsy-proven EBV-LPD in 50%-70% of patients, investigators have still not adequately defined the clinical, virologic, and immunologic parameters associated with treatment response or failure. Furthermore, studies have neither compared the relative efficacy of DLIs and EBV-CTLs nor analyzed their activity in patients who have failed to respond to rituximab.

In the present study, we report a single-center experience with a cohort of 49 patients who were treated with DLI, EBV-CTL, or both for biopsy-proven EBV-LPD emerging after allogeneic HCT. We also analyze attributes of the disease, its prior treatment, and the T cells used for adoptive therapy that contribute to tumor response or continued progression of disease.

Methods

Patients

A total of 49 patients received either DLI or EBV-CTL or both between 1991 and 2009 as treatment for biopsy-proven EBV-LPD that developed after an allogeneic HCT. All patients and donors gave consent and were treated according to protocols approved by the Institutional Review/Privacy Board at Memorial Sloan Kettering Cancer Center and, for recipients of EBV-CTLs, the Food and Drug Administration and the National Marrow Donor Program. Of 49 patients, 17 received EBV-CTLs alone, 27 DLIs alone, 3 DLIs followed by EBV-CTLs, and 2 EBV-CTLs followed by DLIs. Twenty-one patients (46.7%) had received rituximab before T-cell therapies with no or short-lived responses followed by disease progression. Thirty-five patients (73%) were receiving no immunosuppressive drugs at the initiation of T-cell therapy; 14 were receiving cyclosporine (n = 3), sirolimus (n = 5), and/or steroids (n = 9) as treatment for GVHD or to reduce edema complicating a lymphoma of the CNS. In this analysis of our complete series, we have included the 5 patients treated with DLIs reported in 199411  (patients 38, 46, 47, 48, and 49) and 2 recently reported CBT recipients who received third party EBV-CTLs18  (patients 16 and 17).

Diagnosis and characterization of EBV-LPD

All patients had at least 1 diagnostic biopsy. Tumors were classified according to the histologic criteria of Knowles et al.19  Biopsy specimens were tested for EBV by immunohistochemical stains for Latent Membrane Protein-1 (LMP-1) and/or by chromogenic in situ hybridization for EBV-encoded early RNAs as described previously.20,21 

Whenever possible, we examined the EBV+ tumor cells for clonality of the B cells, clonality of the EBV genome, and host or donor origin. DNA was extracted from snap-frozen tissue using an Oncor Probe-Tech vacuum blotting device (Oncor). The genetic origin of the lymphoma was analyzed by ascertainment of HLA type or definition of donor or host unique autosomal or sex chromatin polymorphisms by PCR-amplified restriction fragment length polymorphisms derived from a variable number of tandem repeats as described previously.22  B-cell and EBV clonality were determined as described previously.23,24 

Treatment with DLIs or EBV-CTLs

Except as noted in Table 3, patients received 1 × 106 CD3+ EBV-CTLs/kg IV weekly for 3 weeks or a one-time infusion of 0.2-1 × 106 unselected CD3+ T cells/kg. DLIs and EBV-CTLs were obtained from HCT donors; EBV-CTLs were generated from HCT donors (n = 14) or third-party donors (n = 5) who specifically consented to donate for this purpose.

EBV-CTLs were generated under Good Manufacturing Practice conditions using a modification of methods described previously.3,25  Briefly, T cells were enriched from PBMCs by depletion of monocytes by adherence to plastic and natural killer (NK) cells by adsorption to anti-CD56 immunomagnetic beads (Miltenyi Biotec).25  T cells were sensitized in vitro at a 20:1 responder:stimulator ratio with irradiated autologous EBV transformed B cells (EBV-BLCLs) generated previously by transformation with the B95.8 strain of EBV26  (kindly provided by C. Rooney, Baylor College of Medicine). T cells were then cultured in Yssel medium (Gemini Bioproducts) supplemented with 5% heat-inactivated pooled normal human serum and restimulated with the same EBV-BLCLs weekly at a 4:1 responder:stimulator ratio.

Beginning on day 16, IL2 (Novartis) was added at 10-50 IU/mL 3 times/wk. After 28-35 days of culture, the T cells were characterized by flow cytometry using mAbs against CD3, CD4, CD8, CD56, CD19, TCRa/b, CD28, and CD45RA (BD Biosciences).25  The EBV-specific cytotoxicity, lack of alloreactivity, and HLA restrictions of the EBV-CTLs were identified by assessing their cytotoxicity against autologous donor- and patient-derived EBV+ BLCL and EBV phytohemagglutinin (PHA) blasts and thereafter against a panel of allogeneic EBV-BLCLs, each matching one of the HLA alleles expressed by the T cells.27 

The frequency of EBV-specific T cells in DLIs and EBV-CTLs was measured by limiting dilution analysis for EBV-specific CTL precursors (CTLps) and by FACS-based quantitation of T cells producing intracellular IFN-γ in response to secondary stimulation with autologous EBV-BLCLs, compared with autologous PHA blasts.3,27,28 

The ability of EBV-CTLs to recognize endogenous EBV derived from a patient was assessed by measuring their cytotoxic activity against spontaneously transformed EBV+ B cells cultured from either a tumor biopsy or the PBMCs of the patient with EBV-LPD.26,29 

Monitoring of patients

All patients were monitored sequentially for response by clinical assessments; imaging with CT, PET/CT, and/or MRI if clinically indicated; quantitation of peripheral blood EBV DNA copy numbers; and measurements of EBV-specific T-cell frequencies before and at intervals after the EBV-CTL and/or DLI therapy. Patients were also monitored closely for toxicities using the National Cancer Institute common toxicity grading criteria30  and for acute GVHD as graded by the Glucksberg criteria.31 

EBV DNA copy numbers in the blood were monitored from 1995-2003 with a semiquantitative PCR-amplified assay32  and since 2003 with a quantitative real-time PCR assay of the EBV-BNRF 1-p 143 locus (Roche Applied Science).33 

EBV-CTLps in the blood were quantified before adoptive transfer of the DLIs or EBV-specific T cells and thereafter on days 1, 7, 14, 21, and 28, and then monthly for 4 months. EBV-CTLps frequencies were determined by limiting dilution analysis.3,25,28 

Statistical methods

The Fisher exact test and the Wilcoxon rank sum test were used to examine covariate differences between treatment groups and between responses and in subgroup analyses to examine associations between clinical factors and response outcomes according to treatment groups. The cumulative incidence function was used to estimate the probabilities for time from primary T-cell therapy to death because of EBV, development of acute GVHD, and or development of chronic GVHD.34  Deaths from other causes were considered as competing events. The Gray test was used to determine whether the cumulative incidence curves differed by therapy administered.35  The software packages SAS Version 9.1 and R Version 2.3.1 were used to compute the test statistics.

Results

Patient characteristics

Forty-nine patients were treated primarily with DLIs (n = 30) or EBV-CTLs (n = 19). Patients who received both cell therapies were included in the treatment group of the initial cell therapy that they received for comparisons of the 2 therapy modalities. The characteristics of the patients and the clinical and radiologic presentation of EBV-LPD for all patients in each treatment group are summarized in Table 1. These groups differed significantly in 2 respects: (1) the median age at time of treatment for the DLI group was 33 years versus 13 years for the EBV-CTL group (P = .01), and (2) the DLI group consisted predominantly of recipients of HLA-matched transplantations (90%), whereas the EBV-CTL group mainly comprised patients receiving HLA-disparate grafts (68%; P < .03), who would have been at high risk of severe GVHD if treated with unselected DLIs. Patients treated with DLIs were somewhat less likely to have received rituximab before cell therapy (P = .08). There was no difference in sex (P = .13) or number of sites involved with disease (P = .08) between the 2 treatment groups. All but 6 patients had received T cell–depleted HCT. One patient in each group had received an unmodified PBSC graft; 3 patients in the EBV-CTL group had received an umbilical cord blood (UCB) transplantation.

Table 1

Patient general characteristics and clinical features

Variable All (n = 49) DLI (n = 30) EBV-CTL (n = 19) P 
Median age at T-cell treatment, y (IQR) 29 (16-47) 33 (24-52) 13 (8-44) < .01 
Sex 
    Female 18 (36) 14 (47) 4 (21) .13 
    Male 31 (64) 16 (53) 15 (79)  
HLA match 
    MMREL or MMUREL 16 (33) 3 (10) 13 (68) < .01 
    MREL 21 (43) 18 (60) 3 (16)  
    MUR 12 (24) 9 (30) 3 (16)  
BMT type 
    T-cell depleted 43 (88) 29 (97) 14 (74) .03 
    Others 6 (12) 1 (3) 5 (26)  
Immunosuppressive medication* 14 (28.6) 6 (20) 8 (42) .32 
Presenting symptoms and/or PE findings 
    Fever 30 (61) 18 (60) 12 (63)  
    Lymphoadenopathy 21 (43) 15 (50) 6 (31)  
    Enlarged tonsils/sore throat 15 (31) 11 (37) 4 (21)  
    EBV PCR positivity 12 (24) 6 (20) 6 (31)  
    Abdominal pain 8 (16) 5 (16) 3 (16)  
    Nausea/vomiting 6 (12) 4 (13) 2 (11)  
    Neurologic symptoms† 4 (9) 2 (6) 2 (11)  
    Hepatomegaly and/or splenomegaly 4 (9) 1 (3) 3 (16)  
    Jaundice 3 (6) 3 (10) 0 (0)  
    Diarrhea 3 (6) 1 (3) 2 (11)  
    None 2 (4) 1 (3) 1 (5)  
Sites involved in disease (clinical and/or radiologic) 
    Lymph nodes 38 (78) 23 (77) 15 (79)  
    Waldeyer ring 21 (43) 16 (53) 5 (26)  
    Liver 16 (33) 9 (30) 7 (37)  
    Lung 18 (36) 14 (47) 4 (21)  
    Gastrointestinal tract 14 (29) 8 (26) 6 (31)  
    Spleen 7 (14) 4 (13) 3 (16)  
    CNS/spine 6 (12) 4 (13) 2 (11)  
    Pancreas 1 (2) 1 (3) 0 (0)  
    Thyroid 1 (2) 1 (3) 0 (0)  
    Larynx 1 (2) 1 (3) 0 (0)  
Median time to EBV-LPD after HSCT, d (IQR) 102.5 (64-247) 113 (84-138) 91 (64-247)  
Variable All (n = 49) DLI (n = 30) EBV-CTL (n = 19) P 
Median age at T-cell treatment, y (IQR) 29 (16-47) 33 (24-52) 13 (8-44) < .01 
Sex 
    Female 18 (36) 14 (47) 4 (21) .13 
    Male 31 (64) 16 (53) 15 (79)  
HLA match 
    MMREL or MMUREL 16 (33) 3 (10) 13 (68) < .01 
    MREL 21 (43) 18 (60) 3 (16)  
    MUR 12 (24) 9 (30) 3 (16)  
BMT type 
    T-cell depleted 43 (88) 29 (97) 14 (74) .03 
    Others 6 (12) 1 (3) 5 (26)  
Immunosuppressive medication* 14 (28.6) 6 (20) 8 (42) .32 
Presenting symptoms and/or PE findings 
    Fever 30 (61) 18 (60) 12 (63)  
    Lymphoadenopathy 21 (43) 15 (50) 6 (31)  
    Enlarged tonsils/sore throat 15 (31) 11 (37) 4 (21)  
    EBV PCR positivity 12 (24) 6 (20) 6 (31)  
    Abdominal pain 8 (16) 5 (16) 3 (16)  
    Nausea/vomiting 6 (12) 4 (13) 2 (11)  
    Neurologic symptoms† 4 (9) 2 (6) 2 (11)  
    Hepatomegaly and/or splenomegaly 4 (9) 1 (3) 3 (16)  
    Jaundice 3 (6) 3 (10) 0 (0)  
    Diarrhea 3 (6) 1 (3) 2 (11)  
    None 2 (4) 1 (3) 1 (5)  
Sites involved in disease (clinical and/or radiologic) 
    Lymph nodes 38 (78) 23 (77) 15 (79)  
    Waldeyer ring 21 (43) 16 (53) 5 (26)  
    Liver 16 (33) 9 (30) 7 (37)  
    Lung 18 (36) 14 (47) 4 (21)  
    Gastrointestinal tract 14 (29) 8 (26) 6 (31)  
    Spleen 7 (14) 4 (13) 3 (16)  
    CNS/spine 6 (12) 4 (13) 2 (11)  
    Pancreas 1 (2) 1 (3) 0 (0)  
    Thyroid 1 (2) 1 (3) 0 (0)  
    Larynx 1 (2) 1 (3) 0 (0)  
Median time to EBV-LPD after HSCT, d (IQR) 102.5 (64-247) 113 (84-138) 91 (64-247)  

Values in parenthesis are in raw percentages unless stated otherwise.

MMREL indicates mismatch related; MMUREL, mismatch unrelated; MREL, match related; and MUR, match unrelated.

*

Calcineurin inhibitor and/or steroids.

Headache/hemiparesis/lethargy/dysphasia.

Clinical features at diagnosis of EBV-LPD

As summarized in Table 1, the EBV-LPD emerged between 2 and 8 months after transplantation; the median time to diagnosis was 113 days for the DLI group and 91 days for the EBV-CTL group (P = NS). The median time from diagnosis of posttransplantation EBV-LPD to initial cell therapy was 9 days for patients treated with DLIs and 20 days for those treated with EBV-CTLs, reflecting the more immediate accessibility of the DLIs (P = nonsignificant).

Fever was the most common presenting symptom, occurring in 61% of the patients. Lymphadenopathy or enlarged tonsils/sore throat were presenting features in 43% and 31% of all patients, respectively. Other presenting symptoms summarized in Table 1 were observed in 4%-16% of cases. However, 4% of all patients were asymptomatic at the time of diagnosis. In these patients, the EBV-LPD was discovered while monitoring for increased levels of EBV DNA in the blood. Overall, an elevated level of EBV DNA was detected in the blood in 29 of 30 (97%) patients tested between diagnosis and initiation of any therapy for EBV-LPD.

Cervical lymph nodes and the lymphoid tissue in the Waldeyer ring were the sites most frequently involved by disease at diagnosis, followed by liver, lung parenchyma, GI tract, spleen, CNS/spine, pancreas, and larynx (Table 1). Sites of involvement were grouped into 5 categories for statistical analysis: CNS, liver, lung, abdomen/intestine, and Waldeyer ring. We included in the abdomen/intestine group patients with intraabdominal and pelvic lymphadenopathy with or without involvement of the spleen or the gastrointestinal tract. The Waldeyer group included patients with involvement of Waldeyer ring as well as cervical, post-/pre-auricular, supraclavicular, or intrathoracic lymph nodes without involvement of the lung parenchyma. Parenchymal involvement of the liver and lung were each grouped separately.

EBV-LPDs involving the CNS included those affecting the brain and/or the spinal cord.

Pathologic characteristics of posttransplantation EBV-LPD

Histopathologic properties of the EBV+ lymphomas are described in Table 2 and were similar in both treatment groups. Of the 49 patients, 45 (92%) presented with DLBCL. In the EBV-CTL group, 1 patient each had focal lymphoid hyperplasia, anaplastic large cell lymphoma, or EBV-LPD of the Hodgkin type. In the DLI group, 1 patient had polymorphic lymphoplasmacytic–type lymphoma. The lymphoma was monoclonal in 30 of 33 patients adequately tested and was composed of cells exclusively of donor origin in 24 of 30 patients (80%) or a mixture of cells of mostly donor origin in 4 of 30 patients (13%) tested; only 2 of 30 were of host origin (Table 2). The EBV genome was clonal in all but 2 of the 25 tumors adequately evaluated.

Table 2

Histopathologic characteristics of EBV-LPD

Characteristic EBV-CTL recipients (n = 19) DLI recipients (n = 30) 
Time to EBV-LPD after HSCT, d, median (IQR) 91 (64-247) 114 (84-138) 
Histology 
    DLBL 16 29 
    FLH 
    PLPL 
    NHL 
    HD 
Origin 
    Host 
    Donor 10 14 
    Mixed 
    Unknown 12 
Clonality 
    EBV clonal 7 of 7 tested 16 of 18 tested 
    Ig clonal 11 of 11 tested 19 of 22 tested 
Characteristic EBV-CTL recipients (n = 19) DLI recipients (n = 30) 
Time to EBV-LPD after HSCT, d, median (IQR) 91 (64-247) 114 (84-138) 
Histology 
    DLBL 16 29 
    FLH 
    PLPL 
    NHL 
    HD 
Origin 
    Host 
    Donor 10 14 
    Mixed 
    Unknown 12 
Clonality 
    EBV clonal 7 of 7 tested 16 of 18 tested 
    Ig clonal 11 of 11 tested 19 of 22 tested 

FLH indicates focal lymphoid hyperplasia; PLPC, polymorphic lymphoplasmocytic lymphoma; NHL, nonHodgkin lymphoma; and HD, Hodgkin disease.

Characterization of EBV-CTLs and DLIs infused

The populations of EBV-CTLs generated in vitro were 90% CD3+ T cells and contained < 5% CD3CD56+ NK cells and < 1% CD19+ B cells (Figure 1A). Although the majority of EBV-CTL lines contained more than 90% CD8+ T cells, 3 CTL lines comprised mostly CD4+ T cells (> 90% of the cell population). All T-cell lines, including those predominantly containing CD4+ T cells, demonstrated EBV-specific cytotoxic activity against autologous EBV-BLCLs and did not kill NK cell–sensitive targets (K562), EBV-negative autologous or recipient-derived PHA blasts, or HLA-mismatched EBV-BLCLs (Figure 1B). In limiting dilution assays, the EBV-CTLs generated in vitro for 28-35 days contained high frequencies of EBV-CTLps (median, 1156 EBV-CTLps/106 cells; range, 66-6578 EBV-CTLps/106 cells), but low or undetectable alloreactive CTLps (median, 5.6; range, < 1.28-29 allo CTLps/106 cells; Figure 1C). In contrast, DLIs contained higher frequencies of major alloantigen–reactive CTLps (median, 97; range, 2.8-223 allo CTLps/106 cells) and much lower frequencies of the EBV-CTLps (median, 17; range, 5.7-33 EBV-CTLps/106 cells).

Figure 1

Characterization of the EBV specific T-cell lines. (A) Phenotype of the EBV specific T-cell lines used for the treatment of EBV-LPD in patients who responded (closed symbols) and did not respond (open symbols) to the T-cell therapy. All EBV-CTL lines infused contained more than 90% of CD3+ cells with predominance of CD8+ T cells. However, 3 CTL lines contained predominantly CD4+ T cells. Infusion of the CD4+ T cell–predominant lines achieved CR in 2 of the 3 patients infused. All T-cell lines were equally depleted of NK and B cells. (B) Cytotoxic activity of the EBV-CTL lines used for the treatment of EBV-LPD of those patients who responded (closed symbols) and those who did not respond (open symbols) to the T-cell therapy. (C) Frequencies of EBV-specific T cells (black symbols) and alloreactive (gray symbols) detected by limiting dilution analysis in EBV-specific T-cell lines and DLI products before their use for the treatment of EBV-LPD demonstrate higher frequencies of EBV-specific T cells and lower frequencies of alloreactive T cells in the EBV-specific T-cell lines than in the unstimulated donor leukocytes. There were no differences in the frequencies of EBV-specific and alloreactive cells between the cell products used in responders and nonresponders. (D) All EBV-CTL lines infused exhibited exclusively EBV-specific cytotoxicity without any activity against recipient PHA-activated blasts, mismatched EBV-BLCLs, and K562 (a target for NK cells).

Figure 1

Characterization of the EBV specific T-cell lines. (A) Phenotype of the EBV specific T-cell lines used for the treatment of EBV-LPD in patients who responded (closed symbols) and did not respond (open symbols) to the T-cell therapy. All EBV-CTL lines infused contained more than 90% of CD3+ cells with predominance of CD8+ T cells. However, 3 CTL lines contained predominantly CD4+ T cells. Infusion of the CD4+ T cell–predominant lines achieved CR in 2 of the 3 patients infused. All T-cell lines were equally depleted of NK and B cells. (B) Cytotoxic activity of the EBV-CTL lines used for the treatment of EBV-LPD of those patients who responded (closed symbols) and those who did not respond (open symbols) to the T-cell therapy. (C) Frequencies of EBV-specific T cells (black symbols) and alloreactive (gray symbols) detected by limiting dilution analysis in EBV-specific T-cell lines and DLI products before their use for the treatment of EBV-LPD demonstrate higher frequencies of EBV-specific T cells and lower frequencies of alloreactive T cells in the EBV-specific T-cell lines than in the unstimulated donor leukocytes. There were no differences in the frequencies of EBV-specific and alloreactive cells between the cell products used in responders and nonresponders. (D) All EBV-CTL lines infused exhibited exclusively EBV-specific cytotoxicity without any activity against recipient PHA-activated blasts, mismatched EBV-BLCLs, and K562 (a target for NK cells).

Clinical effects of EBV-CTLs and DLIs

The individual patients, their treatment before cell therapy, and the primary cell therapy given, together with their clinical outcomes and survival, are summarized in Table 3. Response to therapy was classified as CR, partial remission (PR), stable disease (SD), or progressive disease (PD) using the International Workshop Criteria for assessing response to treatment in nonHodgkin lymphoma.36  Of 30 patients primarily treated with DLIs, 17 achieved a CR and 1 a stable PR (73% CR or PR). This includes 2 patients in our original series (patients 47 and 48) who died of interstitial pneumonia antedating their treatment with DLIs 7 and 17 days after treatment. In both cases, at autopsy, no residual lymphoma was detected.11  An additional patient (patient 44) had stabilization of CNS disease. Of 19 patients primarily treated with EBV-CTLs, 13 (68%) achieved CR. Therefore, the rate of response (CR + PR) for each group were equivalent. Four patients with multiorgan involvement died soon after initial cell infusion before the cells could exert any effects. These included 1 patient in the EBV-CTL group who died of disease progression 8 days after EBV-CTL infusion and 3 patients in the DLI group who died of disease progression, GVHD predating DLIs, or complications of a lung biopsy at 4, 5, and 7 days after cell infusion. Among those patients surviving ≥ 8 days, the rates of response were 74% for those treated with DLIs and 72% for those treated with EBV-CTLs (P = NS).

Table 3

Patient, treatment, and outcome specifics

Patient no. Donor Primary diagnosis Sites involved in EBV-LPD Histology of EBV-LPD Origin of LPD Tumor clonal Rituximab prior to primary T-cell therapy Response to rituximab Time to primary T-cell therapy, d Primary T-cell therapy T-cell origin T-cell dose, CD3+ × 106/kg Infusions, no. Response to primary T-cell therapy Survival after T-cell therapy, d Status Cause of death 
10/10MREL ALL WR,A/I FLH N/A N/A PR 16 CTL 3rd 1.0 CR 357 Alive N/A 
6/10MMUR AA Li,Lu DLBL N/A N/A Failed 163 CTL SC 1.0 CR 127 Dead GVHD predated CTL 
10/10MREL ALPS type IA,HD Li,A/I HD N/A CTL SC 1.0 NE Dead Relapsed HD 
8/10MMREL ALL Li,A/I ALCL N/A N/A CTL SC 0.1 PD 16 Dead Sepsis 
            0.4     
10/10MUR AML WR,A/I DLBL PR 39 CTL SC 1.0 CR 187 Alive N/A 
4/6MMUR AML WR, LI, Lu, CNS, A/I DLBL N/A Failed 110 CTL 3rd 1.0 CR 203 Alive N/A 
9/10MMUR AML WR DLBL GIR 69 CTL SC 1.0 CR 290 Dead Relapsed AML 
5/10MMREL EBV+ HLH A/I DLBL Failed 1 (89)* CTL 3rd 1.0 CR 217 Alive N/A 
3/6MMREL AML WR,A/I DLBL N/A GIR 312 CTL SC 1.0 CR 2066 Alive N/A 
10 6/6MUR CML CNS DLBL N/A N/A N/A 22 CTL SC 1.0 CR 4848 Alive N/A 
11 7/10MMREL FA WR DLBL N/A Failed CTL SC 1.0 CR 3569 Alive N/A 
12 8/10MMUR AML WR, A/I DLBL N/A Failed 17 CTL SC 0.7 PD 17 Dead EBV-LPD 
            0.96     
13 5/6MMUR MLD A/I,WR DLBL Failed 161 CTL 3rd 1.0 PD 24 Dead EBV-LPD 
14 7/10MMREL ALL WR, Li, Lu DLBL GIR 26 CTL SC 0.5 PD 29 Dead EBV-LPD 
            1.0;1.0;1.0     
15 4/4MUR AA WR DLBL N/A N/A N/A CTL SC 1.0 CR 719 Dead Primary Graft Failure 
16 8/10MMUR AML WR DLBL N/A N/A CTL SC 1.0 CR 2006 Alive N/A 
17 8/10MMUR T-cell NHL WR, A/I, Lu DLBL N/A Failed 29 CTL 3rd 0.85 CR 476 Alive N/A 
            1.0     
18 10/10MREL MDS/AML WR,A/I DLBL Failed 70 DLI SC 0.5 PD 17 Dead EBV-LPD 
19 10/10MREL CML WR,A/I DLBL N/A DLI SC 1.0 CR 4970 Alive N/A 
20 8/10MMUR AML Li,Lu DLBL Failed 28 DLI SC 0.095 NE Dead GVHD 
21 8/8MREL ABL WR,A/I DLBL N/A N/A DLI SC 1.0 CR 188 Dead Interstitial pneumonia 
22 5/6MMREL CML WR, Lu DLBL N/A N/A 16 DLI SC 0.5 CR 2742 Alive N/A 
23 6/6MUR AML WR DLBL N/A DLI SC 0.5 CR 4573 Alive N/A 
24 10/10MREL HD WR, Lu DLBL N/A DLI SC 1.0 CR 2143 Dead Relapsed HD 
25 10/10MREL AML Li,A/I DLBL N/A DLI SC 0.5 CR 3045 Alive N/A 
26 10/10MREL CML WR, A/I DLBL N/A DLI SC 1.0 CR 3310 Dead Relapsed CML 
27 6/6MUR ALL Li, A/I, Lu DLBL N/A N/A DLI SC 0.5 CR 487 Dead Relapsed ALL 
28 8/8MREL AML WR,Li,Lu DLBL N/A N/A 46 DLI SC 1.0 CR 4203 Alive N/A 
29 10/10MREL SecAML WR, A/I PLPL GIR DLI SC 0.1 CR 887 Alive N/A 
30 6/6MUR CML A/I DLBL N/A N/A N/A 14 DLI SC 0.5 CR 801 Dead GVHD 
31 6/6MREL CML WR, Lu DLBL N/A N/A DLI SC 0.5 NE Dead Hemothorax post lung biopsy 
32 6/6MUR CML WR, A/I, Li DLBL N/A 40 DLI SC 0.5 PD 15 Dead Sepsis 
33 6/6MUR CML CNS, Lu DLBL N/A N/A N/A DLI SC 0.21 CR 407 Dead Relapsed CML 
            0.37     
34 10/10MREL MDS/RAEB WR DLBL N/A N/A Failed 31 DLI SC 0.5 CR 60 Dead Fungemia 
35 10/10MUR AML WR, Li, A/I, Lu DLBL N/A Failed 70 DLI SC 0.5 PD 14 Dead EBV-LPD 
36 6/6MUR ALL Lu DLBL N/A Failed 13 DLI SC 0.5 CR 183 Dead Sepsis 
37 6/6MREL AML WR DLBL N/A N/A N/A DLI SC 0.5 CR 5007 Alive N/A 
38 10/10MREL CML A/I DLBL N/A N/A N/A DLI SC 0.79 CR 21 Alive N/A 
39 10/10MREL AML Li, A/I DLBL N/A DLI SC 0.5 NE Dead EBV-LPD 
            1.0     
40 6/6MUR CML WR DLBL N/A DLI SC 0.33 CR 606 Dead Relapsed CML 
41 10/10MREL XLPD WR, A/I, Li DLBL N/A CTL SC 1.0 PD 47 Dead EBV-LPD 
42 9/10MMREL NHL Li, A/I DLBL N/A 20 CTL SC 1.0 CR 3190 Alive N/A 
43 8/10MMREL ALL WR, CNS DLBL Failed 32 DLI SC 0.5 PD 593 Dead Sepsis 
44 10/10MREL AML WR, CNS DLBL N/A GIR 63 DLI SC 0.5 SD 85 Dead EBV-LPD 
45 10/10MUR ALL WR, A/I, CNS DLBL Failed 17 DLI SC 0.1 PD 144 Dead EBV-LPD 
46 10/10MREL ALL WR, Lu, Li, A DLBL N/A N/A N/A 15 DLI SC 0.55 CR 6149 Alive N/A 
0.35 
0.12 
47 10/10MREL CML Lu,WR,A DLBL N/A 13 DLI SC 1.0 CR Dead Interstitial pneumonia 
48 10/10MREL ANLL WR,Lu DLBL N/A DLI SC 2.0 CR 17 Dead Interstitial pneumonia 
49 10/10MREL ANLL WR DLBL N/A N/A 13 DLI SC 1.0 CR    
Patient no. Donor Primary diagnosis Sites involved in EBV-LPD Histology of EBV-LPD Origin of LPD Tumor clonal Rituximab prior to primary T-cell therapy Response to rituximab Time to primary T-cell therapy, d Primary T-cell therapy T-cell origin T-cell dose, CD3+ × 106/kg Infusions, no. Response to primary T-cell therapy Survival after T-cell therapy, d Status Cause of death 
10/10MREL ALL WR,A/I FLH N/A N/A PR 16 CTL 3rd 1.0 CR 357 Alive N/A 
6/10MMUR AA Li,Lu DLBL N/A N/A Failed 163 CTL SC 1.0 CR 127 Dead GVHD predated CTL 
10/10MREL ALPS type IA,HD Li,A/I HD N/A CTL SC 1.0 NE Dead Relapsed HD 
8/10MMREL ALL Li,A/I ALCL N/A N/A CTL SC 0.1 PD 16 Dead Sepsis 
            0.4     
10/10MUR AML WR,A/I DLBL PR 39 CTL SC 1.0 CR 187 Alive N/A 
4/6MMUR AML WR, LI, Lu, CNS, A/I DLBL N/A Failed 110 CTL 3rd 1.0 CR 203 Alive N/A 
9/10MMUR AML WR DLBL GIR 69 CTL SC 1.0 CR 290 Dead Relapsed AML 
5/10MMREL EBV+ HLH A/I DLBL Failed 1 (89)* CTL 3rd 1.0 CR 217 Alive N/A 
3/6MMREL AML WR,A/I DLBL N/A GIR 312 CTL SC 1.0 CR 2066 Alive N/A 
10 6/6MUR CML CNS DLBL N/A N/A N/A 22 CTL SC 1.0 CR 4848 Alive N/A 
11 7/10MMREL FA WR DLBL N/A Failed CTL SC 1.0 CR 3569 Alive N/A 
12 8/10MMUR AML WR, A/I DLBL N/A Failed 17 CTL SC 0.7 PD 17 Dead EBV-LPD 
            0.96     
13 5/6MMUR MLD A/I,WR DLBL Failed 161 CTL 3rd 1.0 PD 24 Dead EBV-LPD 
14 7/10MMREL ALL WR, Li, Lu DLBL GIR 26 CTL SC 0.5 PD 29 Dead EBV-LPD 
            1.0;1.0;1.0     
15 4/4MUR AA WR DLBL N/A N/A N/A CTL SC 1.0 CR 719 Dead Primary Graft Failure 
16 8/10MMUR AML WR DLBL N/A N/A CTL SC 1.0 CR 2006 Alive N/A 
17 8/10MMUR T-cell NHL WR, A/I, Lu DLBL N/A Failed 29 CTL 3rd 0.85 CR 476 Alive N/A 
            1.0     
18 10/10MREL MDS/AML WR,A/I DLBL Failed 70 DLI SC 0.5 PD 17 Dead EBV-LPD 
19 10/10MREL CML WR,A/I DLBL N/A DLI SC 1.0 CR 4970 Alive N/A 
20 8/10MMUR AML Li,Lu DLBL Failed 28 DLI SC 0.095 NE Dead GVHD 
21 8/8MREL ABL WR,A/I DLBL N/A N/A DLI SC 1.0 CR 188 Dead Interstitial pneumonia 
22 5/6MMREL CML WR, Lu DLBL N/A N/A 16 DLI SC 0.5 CR 2742 Alive N/A 
23 6/6MUR AML WR DLBL N/A DLI SC 0.5 CR 4573 Alive N/A 
24 10/10MREL HD WR, Lu DLBL N/A DLI SC 1.0 CR 2143 Dead Relapsed HD 
25 10/10MREL AML Li,A/I DLBL N/A DLI SC 0.5 CR 3045 Alive N/A 
26 10/10MREL CML WR, A/I DLBL N/A DLI SC 1.0 CR 3310 Dead Relapsed CML 
27 6/6MUR ALL Li, A/I, Lu DLBL N/A N/A DLI SC 0.5 CR 487 Dead Relapsed ALL 
28 8/8MREL AML WR,Li,Lu DLBL N/A N/A 46 DLI SC 1.0 CR 4203 Alive N/A 
29 10/10MREL SecAML WR, A/I PLPL GIR DLI SC 0.1 CR 887 Alive N/A 
30 6/6MUR CML A/I DLBL N/A N/A N/A 14 DLI SC 0.5 CR 801 Dead GVHD 
31 6/6MREL CML WR, Lu DLBL N/A N/A DLI SC 0.5 NE Dead Hemothorax post lung biopsy 
32 6/6MUR CML WR, A/I, Li DLBL N/A 40 DLI SC 0.5 PD 15 Dead Sepsis 
33 6/6MUR CML CNS, Lu DLBL N/A N/A N/A DLI SC 0.21 CR 407 Dead Relapsed CML 
            0.37     
34 10/10MREL MDS/RAEB WR DLBL N/A N/A Failed 31 DLI SC 0.5 CR 60 Dead Fungemia 
35 10/10MUR AML WR, Li, A/I, Lu DLBL N/A Failed 70 DLI SC 0.5 PD 14 Dead EBV-LPD 
36 6/6MUR ALL Lu DLBL N/A Failed 13 DLI SC 0.5 CR 183 Dead Sepsis 
37 6/6MREL AML WR DLBL N/A N/A N/A DLI SC 0.5 CR 5007 Alive N/A 
38 10/10MREL CML A/I DLBL N/A N/A N/A DLI SC 0.79 CR 21 Alive N/A 
39 10/10MREL AML Li, A/I DLBL N/A DLI SC 0.5 NE Dead EBV-LPD 
            1.0     
40 6/6MUR CML WR DLBL N/A DLI SC 0.33 CR 606 Dead Relapsed CML 
41 10/10MREL XLPD WR, A/I, Li DLBL N/A CTL SC 1.0 PD 47 Dead EBV-LPD 
42 9/10MMREL NHL Li, A/I DLBL N/A 20 CTL SC 1.0 CR 3190 Alive N/A 
43 8/10MMREL ALL WR, CNS DLBL Failed 32 DLI SC 0.5 PD 593 Dead Sepsis 
44 10/10MREL AML WR, CNS DLBL N/A GIR 63 DLI SC 0.5 SD 85 Dead EBV-LPD 
45 10/10MUR ALL WR, A/I, CNS DLBL Failed 17 DLI SC 0.1 PD 144 Dead EBV-LPD 
46 10/10MREL ALL WR, Lu, Li, A DLBL N/A N/A N/A 15 DLI SC 0.55 CR 6149 Alive N/A 
0.35 
0.12 
47 10/10MREL CML Lu,WR,A DLBL N/A 13 DLI SC 1.0 CR Dead Interstitial pneumonia 
48 10/10MREL ANLL WR,Lu DLBL N/A DLI SC 2.0 CR 17 Dead Interstitial pneumonia 
49 10/10MREL ANLL WR DLBL N/A N/A 13 DLI SC 1.0 CR    

Survival after T-cell therapy is the duration between the primary T-cell therapy and day of last visit or day of death.

Sec indicates secondary; MREL, matched related; MMREL, mismatched related; MUR, matched unrelated; MMUR, mismatched unrelated; HD, Hodgkin disease; NHL, nonHodgkin lymphoma; ALPS, autoimmune lymphoproliferative syndrome; AA, aplastic anemia; FA, Fanconi anemia; HLH, hemophagocytic lymphohistiocytosis; MLD, metachromatic leukodystrophy; MDS, myelodysplastic syndrome; RAEB, refractory anemia with excess blasts; WR, Waldeyer ring; A/I, abdomen/intestine; Li, liver; Lu, lung; NE, not evaluable; GIR, good initial response but short lived; N/A, not applicable; FLH, focal lymphoid hyperplasia; ALCL, anaplastic large cell lymphoma; PLPL, polymorphic lymphoplasmocytic lymphoma; Y, yes; D, donor; H, host; M, mixed (host and donor); N/A, not available; SC, stem cell donor; and 3rd, third-party donor.

*

This patient received EBV-CTLs from 2 different donors. The first set of cells was infused 1 day after the EBV-LPD diagnosis, and the patient failed to respond. The second set of cells was infused 89 days after failure. The patient achieved a CR with the second EBV-CTL therapy.

Five patients who initially received DLIs or EBV-CTLs subsequently received the alternative cell therapy 27-91 days thereafter in an attempt to halt disease progression. Of 2 patients who received EBV-CTLs followed by DLIs, 1 (patient 41) died of PD, and the other (patient 42) achieved CR. This patient was given DLIs 20 days after 3 infusions of predominantly CD4+ EBV-CTLs because imaging studies suggested that the disease was not responding to the EBV-CTLs. Testing of blood samples drawn immediately before infusion of the DLIs, however, demonstrated an increase in the frequency of EBV-CTLps together with a sharp reduction in EBV DNA copy number. Three patients received DLIs followed by EBV-CTLs. One patient (patient 43) achieved CR and a second (patient 45) died of PD. The third patient (patient 44) initially achieved SD after DLIs for an EBV-LPD involving the basal ganglia of the brain. Treatment with EBV-CTLs did not induce further improvement, and this patient ultimately died of neurologic complications of the EBV-LPD without evidence of progression.

Despite the higher initial doses of EBV-CTLps administered, the times to achieve clinical responses to EBV-CTL infusions did not differ significantly from those after DLIs. Fever resolved within 5-14 days after cell therapy if there were no other intercurrent infections. EBV DNA levels decreased significantly within 3-10 days. Clinical improvements, including shrinkage of palpable lymph nodes, reduction of organomegaly, and resolution of pain or intestinal bleeding, were first detected 8-15 days after infusion. Improvements in radiologic/endoscopic findings were documented by approximately 3 weeks after therapy with complete resolution of radiologic findings in patients who achieved CR by 3-6 months after therapy.

In contrast, patients who failed to respond showed persistence of fever with continued clinical deterioration and/or worsening of radiologic findings. In patients with PD who had not been previously treated with rituximab, circulating EBV DNA levels were unchanged or higher when tested 7 days or more after cell infusions. However, EBV DNA levels in the blood were undetectable before and after T-cell infusions in 6 of 17 patients previously treated with rituximab despite clinical and/or radiologic evidence of progression of disease.

Median followup has reached 80 months. All deaths attributable to EBV-LPD occurred within 4.3 months of initiation of T-cell therapy (supplemental Figure 1A, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). The cumulative incidences of EBV-specific mortality at 12 months were 24% and 21% for patients treated with DLIs and EBV-CTLs, respectively (supplemental Figure 1A; P = .93). No patient exhibited a late recurrence of disease.

Immunologic effects of the EBV-CTLs and DLIs

EBV-CTL and DLI infusions resulted in increased blood levels of EBV-CTLps only in those patients who subsequently responded to adoptive cell therapy (Figure 2A-D). Infusions of EBV-CTLs resulted in much higher EBV-CTLp levels (up to 2200 cells/mL), which were sustained in the circulation for longer periods of time (2-3 months) than those detected after DLIs (up to 600 cells/mL for ∼ 2-3 weeks, respectively). The overall expansion of EBV-CTLps after infusions of EBV-CTL therapy was up to 6 log10-fold (from < 0.001-12 EBV-CTLps/mL to 120-2200 EBV-CTLps/mL) and after DLIs up to 2 log10-fold (from 1.3-690 EBV-CTLps/mL). EBV-CTLp frequencies initially increased by 7-14 days after the initial infusion and coincided with decreases in EBV DNA levels, (Figure 2C,G). In contrast, in patients who failed to respond to DLIs or EBV-CTLs, EBV-CTLp frequencies did not increase (Figure 2D,H).

Figure 2

Monitoring of EBV-specific T cells in the circulation of patients with EBV-LPD treated with EBV-specific T cells or unstimulated DLIs. (A) Frequencies of EBV-specific T cells observed over a period of 12 weeks after initiation of T-cell therapy in the peripheral blood of patients who responded to treatment with EBV-specific T cells obtained from their stem cell donors (n = 8 patients evaluated). (B) Frequencies of EBV-specific T cells observed in peripheral blood of the patients with EBV-LPD who did not respond to treatment with EBV-specific T cells (n = 3) generated from PBMCs of their stem cell donors. (C) Monitoring of the EBV DNA levels (purple line) and EBV-CTLps (green line) of patient 15, who responded to treatment with EBV-specific T cells derived from the stem cell donor. EBV-CTLs were infused weekly for 3 consecutive weeks at doses of 1 × 106 cells/kg/infusion. After infusions of T cells, EBV-CTLps increased in frequency. Concurrently, EBV DNA levels in the peripheral blood of the patient decreased. (D) Monitoring of the EBV DNA levels (purple line) and EBV-CTLps (green line) of patient 41 who did not respond to treatment with EBV-specific T cells derived from the stem cell donor. EBV-CTLs were infused weekly for 3 consecutive weeks at doses of 1 × 106 cells/kg/infusion. No increase in EBV-CTLp frequency was observed over the period of 6 weeks of observation and the levels of EBV DNA remained high. (E) EBV-CTLps in patients who responded to therapy with DLIs from their HLA-matched stem cell donors (n = 3 patients evaluated). (F) EBV-CTLps in patients who did not respond to the therapy with DLIs (patients 43 and 45). (G-H) EBV DNA levels (purple line) and EBV-specific CTLps (green line) in the peripheral blood of patient 21, who responded to DLI treatment (G) and patient 45, who did not respond to the infusion of DLIs (H). (J) Monitoring of T-cell responses in the peripheral blood of patients with EBV-LPD who received EBV-specific T cells generated from third-party healthy donors (n = 5). The arrows indicate the times of the T-cell infusions for each of 3 patients treated and are presented in the same color as the line demonstrating the CTLps frequencies. Patients 1 and 6 achieved CR; patient 13 did not respond and died of PD.

Figure 2

Monitoring of EBV-specific T cells in the circulation of patients with EBV-LPD treated with EBV-specific T cells or unstimulated DLIs. (A) Frequencies of EBV-specific T cells observed over a period of 12 weeks after initiation of T-cell therapy in the peripheral blood of patients who responded to treatment with EBV-specific T cells obtained from their stem cell donors (n = 8 patients evaluated). (B) Frequencies of EBV-specific T cells observed in peripheral blood of the patients with EBV-LPD who did not respond to treatment with EBV-specific T cells (n = 3) generated from PBMCs of their stem cell donors. (C) Monitoring of the EBV DNA levels (purple line) and EBV-CTLps (green line) of patient 15, who responded to treatment with EBV-specific T cells derived from the stem cell donor. EBV-CTLs were infused weekly for 3 consecutive weeks at doses of 1 × 106 cells/kg/infusion. After infusions of T cells, EBV-CTLps increased in frequency. Concurrently, EBV DNA levels in the peripheral blood of the patient decreased. (D) Monitoring of the EBV DNA levels (purple line) and EBV-CTLps (green line) of patient 41 who did not respond to treatment with EBV-specific T cells derived from the stem cell donor. EBV-CTLs were infused weekly for 3 consecutive weeks at doses of 1 × 106 cells/kg/infusion. No increase in EBV-CTLp frequency was observed over the period of 6 weeks of observation and the levels of EBV DNA remained high. (E) EBV-CTLps in patients who responded to therapy with DLIs from their HLA-matched stem cell donors (n = 3 patients evaluated). (F) EBV-CTLps in patients who did not respond to the therapy with DLIs (patients 43 and 45). (G-H) EBV DNA levels (purple line) and EBV-specific CTLps (green line) in the peripheral blood of patient 21, who responded to DLI treatment (G) and patient 45, who did not respond to the infusion of DLIs (H). (J) Monitoring of T-cell responses in the peripheral blood of patients with EBV-LPD who received EBV-specific T cells generated from third-party healthy donors (n = 5). The arrows indicate the times of the T-cell infusions for each of 3 patients treated and are presented in the same color as the line demonstrating the CTLps frequencies. Patients 1 and 6 achieved CR; patient 13 did not respond and died of PD.

Clinical variables affecting outcome

As shown in Table 4, time of EBV-LPD onset after transplantation did not affect the probability of achieving CR after DLI or EBV-CTL therapy. The extent of disease at presentation, however, was inversely correlated with response rate. All patients with disease limited to 1 site achieved CR, compared with 60% for those with 2 sites and 50% for those with 3 or more involved sites (P < .01). This was true for patients treated with either DLIs or EBV-CTLs. However, all sites of involvement responded to treatment with either DLIs or EBV-CTLs, including the CNS. Indeed, of the 6 patients with clinical and radiologic evidence of CNS involvement, 4 achieved CR and 1 a prolonged stabilization of disease.

Table 4

Variables associated with response to cell therapy

Variable Response
 
P 
Patients, n CR/SD/PR PD NE 
Overall association of number of sites involved and response (n = 49) 
    1 site 14 14 (100%) 0 (0%) 0 (0%) .01, .01* 
    2 sites 21 13 (60%) 4 (20%) 4 (20%) 
    3+ sites 14 7 (50%) 6 (50%) 1 (0%) 
Overall association of rituximab use and response to cell therapy (n = 49) 
    Patients failing Rituxan 19 11 (58%) 7 (37%) 1 (5%) .07, .06* 
    Patient w/o prior Rituxan or in PR after Rituxan 30 23 (77%) 3 (11%) 4 (11%) 
Overall association of rituximab use and response (n = 49) 
    Patients failing Rituxan by treatment 19 11  
        DLI 4 (44%) 4 (44%) 1 (11%) .47, .63* 
        EBV-CTL 10 7 (70%) 3 (30%) 0 (0%) 
    Patients without prior Rituxan or in PR after Rituxan by treatment 30 23  
        DLI 21 17 (82%) 1 (6%) 3 (12%) .32, .22* 
        EBV CTL 6 (67%) 2 (22%) 1 (11%) 
Overall association of steroid use and response (n = 45) 
    No steroid use 40 29 (72%) 8 (20%) 3 (7%) .85, .99* 
    Steroid use 6 (66%) 2 (22%) 1 (11%) 
Overall association of use of steroids and/or cyclosporine or sirolimus (n = 49) 
    No steroid use 35 26 .76, .44* 
    Steroid use 14 
Variable Response
 
P 
Patients, n CR/SD/PR PD NE 
Overall association of number of sites involved and response (n = 49) 
    1 site 14 14 (100%) 0 (0%) 0 (0%) .01, .01* 
    2 sites 21 13 (60%) 4 (20%) 4 (20%) 
    3+ sites 14 7 (50%) 6 (50%) 1 (0%) 
Overall association of rituximab use and response to cell therapy (n = 49) 
    Patients failing Rituxan 19 11 (58%) 7 (37%) 1 (5%) .07, .06* 
    Patient w/o prior Rituxan or in PR after Rituxan 30 23 (77%) 3 (11%) 4 (11%) 
Overall association of rituximab use and response (n = 49) 
    Patients failing Rituxan by treatment 19 11  
        DLI 4 (44%) 4 (44%) 1 (11%) .47, .63* 
        EBV-CTL 10 7 (70%) 3 (30%) 0 (0%) 
    Patients without prior Rituxan or in PR after Rituxan by treatment 30 23  
        DLI 21 17 (82%) 1 (6%) 3 (12%) .32, .22* 
        EBV CTL 6 (67%) 2 (22%) 1 (11%) 
Overall association of steroid use and response (n = 45) 
    No steroid use 40 29 (72%) 8 (20%) 3 (7%) .85, .99* 
    Steroid use 6 (66%) 2 (22%) 1 (11%) 
Overall association of use of steroids and/or cyclosporine or sirolimus (n = 49) 
    No steroid use 35 26 .76, .44* 
    Steroid use 14 

NE indicates not evaluated (patients dying within 8 days of initiated T-cell infusion who were not evaluated for response to T-cell therapy).

*

P excluding patients who were not evaluable; numbers in parentheses are raw percentages.

Response rates to DLIs and EBV-CTLs did not differ significantly among patients initially treated with cells or those initially treated with rituximab. However, patients who failed to respond to rituximab or relapsed after an initial response were somewhat less likely to respond to treatment with either DLIs or EBV-CTLs (58% vs 77%, P = .07).

We also examined responses in 14 patients receiving immunosuppressive drugs at the time of cell therapy, including steroids alone (n = 6) or steroids in combination with therapeutic levels of cyclosporine or tacrolimus (n = 3), sirolimus alone (n = 4), or cyclosporine alone (n = 1). Of these 14 patients, 9 achieved CR (64%) compared with 26 of 35 (74%) patients not receiving immunosuppressives (P = .76). Specific analysis of those who were or were not receiving systemic steroids also revealed no significant difference in response rate (P = .85). However, by the time cell infusions began, daily doses had been reduced to 0.07-0.0375 mg/kg of prednisone (median 0.14 mg/kg) or its equivalent. When we analyzed T-cell responses in these patients, we could not ascribe any clear effect of these agents on the expansion of EBV-CTLps in vivo. Therefore, among the responders analyzed in Figure 2, patients 2, 6, 16, 21, and 23, who were receiving sirolimus or cyclosporine, responses were comparable to the other patients who were not.

Because the EBV lymphomas were predominantly of donor origin, it is not surprising that the overall probabilities of achieving a CR after treatment with donor-derived DLIs or EBV-CTLs were equivalent for recipients of HLA-matched related, HLA-matched unrelated, or HLA-disparate related or unrelated transplantations. As detailed in the next section, however, in those instances where the donor of cells used for adoptive therapy differed from the origin of the EBV-LPD, HLA disparities between the cell donor and the EBV-LPD likely contributed to treatment failures.

Viral and immunologic variables affecting outcome

Because treatment failures were consistently correlated with a lack of EBV-CTL proliferation in vivo, we investigated whether the EBV-CTLs generated in vitro were able to recognize the EBV lymphomas in vivo. We succeeded in growing spontaneous EBV-transformed B cells from the blood or biopsied tumors of 11 recipients of HLA-matched HCT, who were subsequently treated with EBV-CTLs from the same donor. Each of these spontaneous transformants was of donor origin. In 8 patients who achieved CR, EBV-CTLp frequencies increased after T-cell infusion. The other 3 patients, whose CTLps frequencies remained low or undetectable, failed to respond and died of progressive disease. In all 11 cases, the donor's EBV-CTLs, sensitized with autologous B cells transformed with the B95.8 strain of EBV, lysed EBV B95.8 strain–transformed autologous EBV-BLCLs. As shown in Figure 3A, the EBV-CTLs from the 8 treatment responders also lysed B-cell lines transformed with the patient's endogenous EBV. In contrast, the EBV-CTLs administered to the 3 patients who failed to respond did not lyse the donor-type EBV-BLCLs transformed with endogenous virus. However, when T cells from these 3 donors were sensitized with spontaneous EBV-BLCL transformants grown from the patient's blood or tumor, they were able to lyse both the donor-derived B cells bearing endogenous EBV and the transformed B95.8 strain of EBV (Figure 3B).

Figure 3

Basis for nonresponse: donor-derived EBV-specific T cells that failed to induce a clinical response did not exhibit cytotoxic activity against the spontaneously transformed EBV-BLCLs generated from the patient's tissues. (A) Cytotoxicity of donor EBV–specific CTLs. There were 8 responders (closed symbols) and 3 nonresponders (open symbols; n = 11) sensitized with autologous B95.8-transformed B cells against EBV B95.8–transformed donor–derived autologous BLCL and against donor-type spontaneous EBV-transformed BLCLs cultured from the patient blood or EBV-LPD. The B95.8-sensitized EBV-specific CTLs used in nonresponding patients lysed EBV B95.8 transformed BLCL but did not lyse the spontaneous EBV transformants of donor origin cultured from the patient. (B) Cytotoxicity of donor-derived EBV-CTLs stimulated with spontaneously transformed EBV-BLCLs of donor origin cultured from the patient. The same T cells as in panel A were stimulated in vitro with the spontaneously transformed EBV-BLCLs and were able to kill both the stimulating B-cell line and the donor-derived B95.8 transformant. These data confirm that the EBV-LPD is sensitive to lysis by donor-derived EBV-CTLs if the T cells are sensitized with the endogenous strain of EBV. (C) HLA restriction of EBV-CTLs generated from an HLA haplotype–matched donor for patient 13, who developed an EBV lymphoma in cord blood–derived B cells; these EBV-CTLs were selectively restricted by an HLA DRB, 0401, not shared by the cord blood cells and did not lyse cord blood donor-derived spontaneous EBV-BLCL generated from the tumor.

Figure 3

Basis for nonresponse: donor-derived EBV-specific T cells that failed to induce a clinical response did not exhibit cytotoxic activity against the spontaneously transformed EBV-BLCLs generated from the patient's tissues. (A) Cytotoxicity of donor EBV–specific CTLs. There were 8 responders (closed symbols) and 3 nonresponders (open symbols; n = 11) sensitized with autologous B95.8-transformed B cells against EBV B95.8–transformed donor–derived autologous BLCL and against donor-type spontaneous EBV-transformed BLCLs cultured from the patient blood or EBV-LPD. The B95.8-sensitized EBV-specific CTLs used in nonresponding patients lysed EBV B95.8 transformed BLCL but did not lyse the spontaneous EBV transformants of donor origin cultured from the patient. (B) Cytotoxicity of donor-derived EBV-CTLs stimulated with spontaneously transformed EBV-BLCLs of donor origin cultured from the patient. The same T cells as in panel A were stimulated in vitro with the spontaneously transformed EBV-BLCLs and were able to kill both the stimulating B-cell line and the donor-derived B95.8 transformant. These data confirm that the EBV-LPD is sensitive to lysis by donor-derived EBV-CTLs if the T cells are sensitized with the endogenous strain of EBV. (C) HLA restriction of EBV-CTLs generated from an HLA haplotype–matched donor for patient 13, who developed an EBV lymphoma in cord blood–derived B cells; these EBV-CTLs were selectively restricted by an HLA DRB, 0401, not shared by the cord blood cells and did not lyse cord blood donor-derived spontaneous EBV-BLCL generated from the tumor.

The importance of ascertaining the origin of the EBV-LPD and the HLA restriction of the EBV-CTLs generated in vitro was demonstrated in 2 other patients. Patient 13, with a rituximab-unresponsive EBV+ lymphoma of UCB origin, was treated with EBV-CTLs from an HLA-haplotype–matched related donor. Subsequent analysis demonstrated that these EBV-CTLs were selectively restricted by a class II HLA allele not shared by the UCB donor and were unable to lyse spontaneously EBV-transformed EBV-BLCLs of UCB origin generated from the patient's tumor (Figure 3C).

The second patient, (patient 8) had received a 7 of 10 HLA allele–matched related TCD-HCT for chemotherapy-resistant EBV+ hemophagocytic lymphohistiocytosis. Shortly after engraftment, EBV reactivation was documented by rising levels of EBV DNA in the blood. The patient was immediately treated with EBV-CTLs previously generated from the donor. Despite this treatment, he continued to have fever, high EBV DNA levels, and worsening abdominal pain. EBV-CTLp frequencies did not increase. Subsequent PET/CT scans revealed progressively enlarging masses involving the gastric wall and mesenteric nodes. Biopsy of the gastric lesion revealed an EBV+ DLBCL of host rather than donor origin. Analysis of the donor's EBV-CTLs revealed that they were selectively restricted by HLA*A1101 (Figure 4B), an allele not shared with the patient (Figure 4A). Based on this finding, we treated the patient with EBV-CTLs from a partially mismatched third-party donor, which were restricted by an HLA*A2601 allele shared by the EBV-LPD. Ten days thereafter, EBV-CTLp frequencies increased (Figure 4C). By 14 days, EBV DNA levels and fever began to decline and abdominal pain lessened. By day 25, radiologic evidence of disease had markedly improved (Figure 4D). This patient subsequently achieved a CR, and remains in CR for both EBV-LPD and EBV+ HLH > 18 months later.

Figure 4

Basis for nonresponse: donor-derived EBV-specific T cells did not recognize host origin EBV-LPD (patient 8). (A) HLA type of the stem cell donor (mother), third-party EBV-CTLs donor 1, and third-party EBV-CTL donor 2. (B) HLA-restriction analysis of EBV-specific T cells generated from the HSCT donor (mother; blue bars) or from the third-party donor 1 (brown bars) or third-party donor 2 (yellow bars) tested in a Cr51-release assay against a panel of allogeneic EBV-BLCLs, each matching 1 HLA allele of each of the T-cell donors' HLA types. (C) Monitoring of the EBV DNA levels (red line) and EBV-specific T cells (green line) after infusions of EBV-CTLs derived from the stem cell donor (blue arrows), third-party donor 1 (purple arrow), and third-party donor 2 (red arrows) at the doses of 1 × 106 cells/kg/infusion. Treatment with Rituxan (375mg/m2; orange arrows), as well as injections of EBV-CTLs derived from the HSCT donor that were restricted by the donor-unique HLA A1101 allele, did not affect high levels EBV DNA, whereas the administration of the EBV-specific T cells from the third-party donors 1 and 2, both restricted by the A2601 HLA allele presented on the EBV+ tumor cells of patient origin, resulted in a rapid decrease of the EBV DNA in the circulation. (D) Sequential PET scans demonstrating no response to the transplant donor's T cells with rapid development of an EBV lymphoma of the gastric wall and adjacent lymph nodes. By 3 weeks after the first infusion of third-party T cells (d97), gastric lymphoma was no longer detected.

Figure 4

Basis for nonresponse: donor-derived EBV-specific T cells did not recognize host origin EBV-LPD (patient 8). (A) HLA type of the stem cell donor (mother), third-party EBV-CTLs donor 1, and third-party EBV-CTL donor 2. (B) HLA-restriction analysis of EBV-specific T cells generated from the HSCT donor (mother; blue bars) or from the third-party donor 1 (brown bars) or third-party donor 2 (yellow bars) tested in a Cr51-release assay against a panel of allogeneic EBV-BLCLs, each matching 1 HLA allele of each of the T-cell donors' HLA types. (C) Monitoring of the EBV DNA levels (red line) and EBV-specific T cells (green line) after infusions of EBV-CTLs derived from the stem cell donor (blue arrows), third-party donor 1 (purple arrow), and third-party donor 2 (red arrows) at the doses of 1 × 106 cells/kg/infusion. Treatment with Rituxan (375mg/m2; orange arrows), as well as injections of EBV-CTLs derived from the HSCT donor that were restricted by the donor-unique HLA A1101 allele, did not affect high levels EBV DNA, whereas the administration of the EBV-specific T cells from the third-party donors 1 and 2, both restricted by the A2601 HLA allele presented on the EBV+ tumor cells of patient origin, resulted in a rapid decrease of the EBV DNA in the circulation. (D) Sequential PET scans demonstrating no response to the transplant donor's T cells with rapid development of an EBV lymphoma of the gastric wall and adjacent lymph nodes. By 3 weeks after the first infusion of third-party T cells (d97), gastric lymphoma was no longer detected.

Complications of the EBV-CTL and DLI therapies

There were no immediate adverse reactions observed due to either of the cell therapies. Seven of 26 patients (27%) in the DLI group and 7 of 19 patients (36%) in the EBV-CTL group had documented GVHD before infusion. In the DLI group, 3 patients developed acute grade 2-3 GVHD de novo, 1 patient had a grade 2-3 flare of acute skin GVHD, and 3 patients developed chronic GVHD de novo (supplemental Figure 2A-B). Although most EBV-CTL donors were not HLA matched with the recipient, no patient in the EBV-CTL group developed de novo acute or chronic GVHD or a flare of preexisting GVHD. All patients with acute and all but 1 of the patients with chronic GVHD de novo cleared with therapy. The cumulative incidences of acute and chronic GVHD attributable to DLIs at 1 year after transplantation are 14% and 14%, respectively.

Discussion

This study details the effects of donor-derived DLIs or EBV-CTLs in the treatment of 49 allogeneic HCT recipients with pathologically confirmed EBV-LPD. These EBV-LPD cases presented as rapidly growing DLBCL in 45 of 49 patients, which were clonal in 95% and exclusively of donor origin in 24 of 30 (80%) tumors tested. Four other tumors containing predominantly donor cells were likely also of donor origin, but were infiltrated with residual host lymphocytes or stromal cell elements. These characteristics are consistent with prior published descriptions of EBV-LPD after allogeneic HCT.6,14 

In this series, 73% of the patients series treated with DLIs and 68% of those treated with EBV-CTLs achieved a sustained CR, including 76% in the DLI group and 72% in the CTL group, who survived ≥ 8 days after initiation of adoptive immunotherapy, the minimum time required for expansion of EBV-CTLs in vivo. The doses of EBV-CTLs required to induce remissions of bulky multiorgan disease were modest. In patients treated with unselected DLIs, the administered doses of EBV-CTLps ranged from 2.8-223 EBV-CTLps/kg, or 196-15 610 total CTLps for a 70-kg adult. These doses are consistent with our previous study documenting a CR in a patient who received a total dose of approximately 800 EBV-CTLps.3  The total doses of EBV-CTLps provided by the 3 infusions of EBV-CTLs were approximately 90-fold higher. We previously found that the frequency of EBV-specific IFNγ+ T cells may be 100-fold greater than that of EBV-CTLps25 ; however, given that a 1-cm3  tumor nodule is estimated to contain 108-109 tumor cells,37  the EBV-specific T-cell doses administered were still extremely small compared with tumor burdens in the patients.

Because of the numeric disparity between the effector T cells administered and resident populations of growing EBV lymphoma cells, we hypothesized that: (1) expansion of adoptively transferred EBV-specific T cells in vivo would be essential to the induction of tumor regressions and the reestablishment of a normal equilibrium between virus-specific T cells and residual EBV transformed cells in the host; and (2) patients with more extensive disease at the initiation of cellular therapy might be less likely to achieve a CR. Extent of disease did in fact adversely affect outcome. Furthermore, if the DLIs or EBV-CTLs infused failed to expand in vivo, the patient did not respond. In contrast, in those patients responding to DLIs or EBV-CTLs, EBV-CTLp frequencies consistently increased by 2-3 log10. In patients responding to cells derived from their transplantation donor, increased frequencies of EBV-CTLps were usually sustained for periods of at least 4-8 weeks after infusion, by which time clinical and radiologic regression of disease was evident. Even among those patients responding to third party–derived EBV-CTLs, which only transiently engraft, such increments in circulating levels of EBV-CTLps were observed for at least 14-21 days after each infusion. In contrast, expansion of the EBV-CTLp populations was never observed in the patients whose disease progressed despite adoptive cell therapy.

The consistent failure of transferred EBV-CTLs to proliferate in nonresponding patients suggested at least 2 possibilities: (1) the transferred T cells were unable to recognize or effectively respond to the EBV+ lymphoma cells; or (2) they were actively prevented from doing so either by the tumor and its products or by the host environment. Consistent with the first possibility are our findings in 3 patients who failed treatment with HLA-matched, donor-derived EBV-CTLs. These EBV-CTLs, which had been sensitized with autologous BLCLs transformed with EBV strain B95.8, did not lyse spontaneous EBV-BLCLs of donor type isolated from the patient's blood or biopsied EBV lymphoma. Gottshalk et al have described a similar case in which the donor's EBV-CTLs, sensitized with EBV-B95.8–transformed autologous BLCLs, failed to lyse spontaneous tranformants derived from the patient.38  In that patient, the endogenous EBV strain had a deletion in the EBNA 3b protein that resulted in the elimination of 2 epitopes presented by HLA A11 that were specifically targeted by the transferred T cells. Whether such mutations in endogenous strains of EBV are also responsible for such treatment failures in our patient cohort is currently under study. Donor T cells, sensitized with these donor-type spontaneous BLCL transformants, could nevertheless lyse autologous B cells transformed with either the endogenous or the B95.8 strain. This indicates that the failure of the B95.8-sensitized T cells to lyse the endogenous transformants is not based on the transformants' intrinsic resistance to T-cell effector activity. In patients like these, treatment with EBV-CTLs from a second donor restricted by a different shared HLA allele should be considered, because such T cells would be expected to target a different peptide epitope that might not be mutated in the endogenous EBV strain.

In 2 other patients who failed to respond, the transferred donor-derived EBV-CTLs were selectively reactive against immunodominant epitopes of EBV presented by HLA alleles not expressed by an EBV+ lymphoma of host origin. One of these patients was later treated successfully with partially HLA-matched third-party EBV-CTLs restricted by an HLA allele expressed by the patient's EBV-LPD, again suggesting that an inability of the T cells to recognize the tumor rather than an intrinsic tumor resistance contributed to the initial treatment failure.

Our studies also indicate that both DLIs and EBV-CTLs can induce durable CRs in patients failing rituximab therapy. Such patients responded less consistently, however, than patients treated initially with cellular immunotherapy, even though the differences were not significant. The possibility that prior treatment with rituximab or the interval of time that rituximab is administered permits selection of more resistant EBV lymphoma cells must nevertheless be considered.

Because of the rapid progression of EBV lymphomas emerging after HCT and the protracted time required to generate EBV-CTLs (60 days), the equivalent therapeutic activity and immediate accessibility of DLIs constitute major advantages. However, DLIs also incurs the risk of GVHD, particularly if the transplantation donor is not HLA matched. To reduce this risk, we limited DLIs to patients with EBV-LPD arising after an HLA-matched or a single, donor-unique HLA allele–disparate HCT. The highest dose of T cells administered, 1 × 106 CD3+ T cells/kg, is 10-fold higher than the 105 CD3+ T-cell/kg reported as the threshold for GVHD at the time of HCT using BM stem cells,39,40  but 10-fold lower than the 107 CD3+ T-cell/kg doses that we have shown can induce molecular remissions of chronic myeloid leukemia with low risk of acute or chronic GVHD when administered ≥ 9 months after HCT.41  In our series, cumulative risks for acute and chronic GVHD attributable to DLIs administered from 2-9 months after HCT were 14% and 14% at 12 months, respectively. In all but 1 case, GVHD cleared with treatment.

In contrast to DLIs, no patient developed either acute or chronic GVHD or a flare of preexisting GVHD after infusion of HCT donor–derived EBV-CTLs, irrespective of their level of HLA disparity. Heslop et al have documented a similarly low risk of GVHD in more than 100 patients given EBV-CTLs to prevent EBV-LPD after HCT.16  These findings reflect the depletion of alloresponsive T cells achieved by extended in vitro sensitization with autologous EBV-BLCLs (Figure 1B).

Until recently, the time required for generating such EBV-CTLs had necessitated their production for all high-risk patients before disease onset to be able to rapidly treat the 4%-6% who develop EBV-LPD. However, new techniques permitting rapid selection of virus-specific T cells may accelerate access to HCT-donor–derived EBV-CTLs.42,43  In addition, as recently reported by Haque et al44  and our group18  and further demonstrated in the present study, EBV-CTLs selected from preestablished banks of normal third-party donor–derived EBV-CTLs on the basis of partial HLA matching and appropriate HLA restriction can provide an immediately accessible source of effector cells for the treatment of EBV-LPD after transplantation.

There is an Inside Blood commentary on this article in this issue.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgments

This work was supported in part by the National Institutes of Health (grants CA23766, CA59350, and CA08748), the Clair L. Tow Foundation, the Larry H. Smead Fund, the Aubrey Fund for Pediatric Cancer Research, the Ryan E. McGeough Charitable Gift Fund, the Major Family Fund for Cancer Research, and the Laura Rosenberg Foundation.

National Institutes of Health

Authorship

Contribution: E.D., B.O.-S., and R.J.O. performed the research, analyzed the clinical, virologic, and immune responses, interpreted the data, and wrote the manuscript; J.T.-F., C.H., and D.G. analyzed the pathologic, immunohistologic, and molecular features of each EBV-LPD; S.A. analyzed and interpreted all radiologic responses; G.H. and J.F.C. assisted with the study design and performed the biostatistical analyses; R.K. assisted in data management and analysis; and S.E.P., N.A.K., J.N.B., F.B., H.C.-M., A.J., G.K, E.B.P., A.S., T.N.S., and J.W.Y. managed the patients clinically, performed their sequential evaluations, and helped write the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Richard J. O'Reilly, MD, Department of Pediatrics and Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10065; e-mail: oreillyr@mskcc.org.

References

References
1
Martin
PJ
Shulman
HM
Schubach
WH
, et al. 
Fatal Epstein-Barr-virus-associated proliferation of donor B cells after treatment of acute graft-versus-host disease with a murine anti-T-cell antibody.
Ann Intern Med
1984
, vol. 
101
 
3
(pg. 
310
-
315
)
2
Peres
E
Savasan
S
Klein
J
Abidi
M
Dansey
R
Abella
E
High fatality rate of Epstein-Barr virus-associated lymphoproliferative disorder occurring after bone marrow transplantation with rabbit antithymocyte globulin conditioning regimens.
J Clin Microbiol
2005
, vol. 
43
 
7
(pg. 
3540
-
3543
)
3
Lucas
KG
Small
TN
Heller
G
Dupont
B
O'Reilly
RJ
The development of cellular immunity to Epstein-Barr virus after allogeneic bone marrow transplantation.
Blood
1996
, vol. 
87
 
6
(pg. 
2594
-
2603
)
4
Meijer
E
Cornelissen
JJ
Epstein-Barr virus-associated lymphoproliferative disease after allogeneic haematopoietic stem cell transplantation: molecular monitoring and early treatment of high-risk patients.
Curr Opin Hematol
2008
, vol. 
15
 
6
(pg. 
576
-
585
)
5
Brunstein
CG
Weisdorf
DJ
DeFor
T
, et al. 
Marked increased risk of Epstein-Barr virus-related complications with the addition of antithymocyte globulin to a nonmyeloablative conditioning prior to unrelated umbilical cord blood transplantation.
Blood
2006
, vol. 
108
 
8
(pg. 
2874
-
2880
)
6
Curtis
RE
Travis
LB
Rowlings
PA
, et al. 
Risk of lymphoproliferative disorders after bone marrow transplantation: a multi-institutional study.
Blood
1999
, vol. 
94
 
7
(pg. 
2208
-
2216
)
7
Elstrom
RL
Andreadis
C
Aqui
NA
, et al. 
Treatment of PTLD with rituximab or chemotherapy.
Am J Transplant
2006
, vol. 
6
 
3
(pg. 
569
-
576
)
8
Gross
TG
Bucuvalas
JC
Park
JR
, et al. 
Low-dose chemotherapy for Epstein-Barr virus-positive post-transplantation lymphoproliferative disease in children after solid organ transplantation.
J Clin Oncol
2005
, vol. 
23
 
27
(pg. 
6481
-
6488
)
9
van Esser
JW
Niesters
HG
van der Holt
B
, et al. 
Prevention of Epstein-Barr virus-lymphoproliferative disease by molecular monitoring and preemptive rituximab in high-risk patients after allogeneic stem cell transplantation.
Blood
2002
, vol. 
99
 
12
(pg. 
4364
-
4369
)
10
Choquet
S
Leblond
V
Herbrecht
R
, et al. 
Efficacy and safety of rituximab in B-cell post-transplantation lymphoproliferative disorders: results of a prospective multicenter phase 2 study.
Blood
2006
, vol. 
107
 
8
(pg. 
3053
-
3057
)
11
Papadopoulos
EB
Ladanyi
M
Emanuel
D
, et al. 
Infusions of donor leukocytes to treat Epstein-Barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation.
N Engl J Med
1994
, vol. 
330
 
17
(pg. 
1185
-
1191
)
12
Bonini
C
Bondanza
A
Perna
SK
, et al. 
The suicide gene therapy challenge: how to improve a successful gene therapy approach.
Mol Ther
2007
, vol. 
15
 
7
(pg. 
1248
-
1252
)
13
Ocheni
S
Kroeger
N
Zabelina
T
, et al. 
EBV reactivation and post transplant lymphoproliferative disorders following allogeneic SCT.
Bone Marrow Transplant
2008
, vol. 
42
 
3
(pg. 
181
-
186
)
14
Styczynski
J
Einsele
H
Gil
L
Ljungman
P
Outcome of treatment of Epstein-Barr virus-related post-transplant lymphoproliferative disorder in hematopoietic stem cell recipients: a comprehensive review of reported cases.
Transpl Infect Dis
2009
, vol. 
11
 
5
(pg. 
383
-
392
)
15
Rooney
CM
Smith
CA
Ng
CY
, et al. 
Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation.
Lancet
1995
, vol. 
345
 
8941
(pg. 
9
-
13
)
16
Heslop
HE
Slobod
KS
Pule
MA
, et al. 
Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients.
Blood
2010
, vol. 
115
 
5
(pg. 
925
-
935
)
17
Merlo
A
Turrini
R
Dolcetti
R
Zanovello
P
Amadori
A
Rosato
A
Adoptive cell therapy against EBV-related malignancies: a survey of clinical results.
Expert Opin Biol Ther
2008
, vol. 
8
 
9
(pg. 
1265
-
1294
)
18
Barker
JN
Doubrovina
E
Sauter
C
, et al. 
Successful treatment of EBV-associated posttransplantation lymphoma after cord blood transplantation using third-party EBV-specific cytotoxic T lymphocytes.
Blood
2010
, vol. 
116
 
23
(pg. 
5045
-
5049
)
19
Knowles
DM
Cesarman
E
Chadburn
A
, et al. 
Correlative morphologic and molecular genetic analysis demonstrates three distinct categories of posttransplantation lymphoproliferative disorders.
Blood
1995
, vol. 
85
 
2
(pg. 
552
-
565
)
20
Kingma
DW
Shad
A
Tsokos
M
, et al. 
Epstein-Barr virus (EBV)-associated smooth-muscle tumor arising in a post-transplant patient treated successfully for two PT-EBV-associated large-cell lymphomas. Case report.
Am J Surg Pathol
1996
, vol. 
20
 
12
(pg. 
1511
-
1519
)
21
Quintanilla-Martinez
L
Kumar
S
Fend
F
, et al. 
Fulminant EBV(+) T-cell lymphoproliferative disorder following acute/chronic EBV infection: a distinct clinicopathologic syndrome.
Blood
2000
, vol. 
96
 
2
(pg. 
443
-
451
)
22
Mackinnon
S
Barnett
L
Bourhis
JH
Black
P
Heller
G
O'Reilly
RJ
Myeloid and lymphoid chimerism after T-cell-depleted bone marrow transplantation: evaluation of conditioning regimens using the polymerase chain reaction to amplify human minisatellite regions of genomic DNA.
Blood
1992
, vol. 
80
 
12
(pg. 
3235
-
3241
)
23
Cleary
ML
Nalesnik
MA
Shearer
WT
Sklar
J
Clonal analysis of transplant-associated lymphoproliferations based on the structure of the genomic termini of the Epstein-Barr virus.
Blood
1988
, vol. 
72
 
1
(pg. 
349
-
352
)
24
Raab-Traub
N
Flynn
K
The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation.
Cell
1986
, vol. 
47
 
6
(pg. 
883
-
889
)
25
Koehne
G
Smith
KM
Ferguson
TL
, et al. 
Quantitation, selection, and functional characterization of Epstein-Barr virus-specific and alloreactive T cells detected by intracellular interferon-gamma production and growth of cytotoxic precursors.
Blood
2002
, vol. 
99
 
5
(pg. 
1730
-
1740
)
26
Roskrow
MA
Suzuki
N
Gan
Y
, et al. 
Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes for the treatment of patients with EBV-positive relapsed Hodgkin's disease.
Blood
1998
, vol. 
91
 
8
(pg. 
2925
-
2934
)
27
Trivedi
D
Williams
RY
O'Reilly
RJ
Koehne
G
Generation of CMV-specific T lymphocytes using protein-spanning pools of pp65-derived overlapping pentadecapeptides for adoptive immunotherapy.
Blood
2005
, vol. 
105
 
7
(pg. 
2793
-
2801
)
28
Bourgault
I
Gomez
A
Gomard
E
Levy
JP
Limiting-dilution analysis of the HLA restriction of anti-Epstein-Barr virus-specific cytolytic T lymphocytes.
Clin Exp Immunol
1991
, vol. 
84
 
3
(pg. 
501
-
507
)
29
Rickinson
AB
Rowe
M
Hart
IJ
, et al. 
T-cell-mediated regression of “spontaneous” and of Epstein-Barr virus-induced B-cell transformation in vitro: studies with cyclosporin A.
Cell Immunol
1984
, vol. 
87
 
2
(pg. 
646
-
658
)
30
MacDonald
JD
Haller
DG
Mayer
RJ
Manual of Oncology Therapeutics
1991
Philadelphia, PA
Lippincott Williams and Wilkins
31
Glucksberg
H
Storb
R
Fefer
A
, et al. 
Clinical manifestations of graft-versus-host disease in human recipients of marrow from HL-A-matched sibling donors.
Transplantation
1974
, vol. 
18
 
4
(pg. 
295
-
304
)
32
Lucas
KG
Burton
RL
Zimmerman
SE
, et al. 
Semiquantitative Epstein-Barr virus (EBV) polymerase chain reaction for the determination of patients at risk for EBV-induced lymphoproliferative disease after stem cell transplantation.
Blood
1998
, vol. 
91
 
10
(pg. 
3654
-
3661
)
33
Niesters
HG
van Esser
J
Fries
E
Wolthers
KC
Cornelissen
J
Osterhaus
AD
Development of a real-time quantitative assay for detection of Epstein-Barr virus.
J Clin Microbiol
2000
, vol. 
38
 
2
(pg. 
712
-
715
)
34
Prentice
RL
Kalbfleisch
JD
Peterson
AV
Jr
Flournoy
N
Farewell
VT
Breslow
NE
The analysis of failure times in the presence of competing risks.
Biometrics
1978
, vol. 
34
 
4
(pg. 
541
-
554
)
35
Gray
RJ
A class of K-sample tests for comparing the cumulative incidence of a competing risk.
Ann Stat
1988
, vol. 
16
 
3
(pg. 
1141
-
1154
)
36
Cheson
BD
Pfistner
B
Juweid
ME
, et al. 
Revised response criteria for malignant lymphoma.
J Clin Oncol
2007
, vol. 
25
 
5
(pg. 
579
-
586
)
37
Del Monte
U
Does the cell number 10(9) still really fit one gram of tumor tissue?
Cell Cycle
2009
, vol. 
8
 
3
(pg. 
505
-
506
)
38
Gottschalk
S
Ng
CY
Perez
M
, et al. 
An Epstein-Barr virus deletion mutant associated with fatal lymphoproliferative disease unresponsive to therapy with virus-specific CTLs.
Blood
2001
, vol. 
97
 
4
(pg. 
835
-
843
)
39
Kernan
NA
Collins
NH
Juliano
L
Cartagena
T
Dupont
B
O'Reilly
RJ
Clonable T lymphocytes in T cell-depleted bone marrow transplants correlate with development of graft-v-host disease.
Blood
1986
, vol. 
68
 
3
(pg. 
770
-
773
)
40
Urbano-Ispizua
A
Rozman
C
Pimentel
P
, et al. 
Risk factors for acute graft-versus-host disease in patients undergoing transplantation with CD34+ selected blood cells from HLA-identical siblings.
Blood
2002
, vol. 
100
 
2
(pg. 
724
-
727
)
41
Mackinnon
S
Papadopoulos
EB
Carabasi
MH
, et al. 
Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic myeloid leukemia after bone marrow transplantation: separation of graft-versus-leukemia responses from graft-versus-host disease.
Blood
1995
, vol. 
86
 
4
(pg. 
1261
-
1268
)
42
Moss
P
Rickinson
A
Cellular immunotherapy for viral infection after HSC transplantation.
Nat Rev Immunol
2005
, vol. 
5
 
1
(pg. 
9
-
20
)
43
Peggs
KS
Thomson
K
Samuel
E
, et al. 
Directly selected cytomegalovirus-reactive donor T cells confer rapid and safe systemic reconstitution of virus-specific immunity following stem cell transplantation.
Clin Infect Dis
2011
, vol. 
52
 
1
(pg. 
49
-
57
)
44
Haque
T
Wilkie
GM
Jones
MM
, et al. 
Allogeneic cytotoxic T-cell therapy for EBV-positive posttransplantation lymphoproliferative disease: results of a phase 2 multicenter clinical trial.
Blood
2007
, vol. 
110
 
4
(pg. 
1123
-
1131
)

Author notes

*

E.D. and B.O.-S. contributed equally to this work.