Besides relapse of malignancy, the two main causes of death after allogeneic hematopoietic stem-cell transplantation (HSCT) remain graft-versus-host disease (GVHD) and infection. Among opportunistic viral infections, cytomegalovirus was the most dreadful until prophylactic and preemptive strategies were developed after the approval of modern antiviral agents, strategies that are now recommended with a “IA” grade by most scientific societies (1).
Another viral agent, Epstein-Barr virus (EBV), causes a rarer but lethal neoplastic complication in HSCT as well as in solid organ transplant recipients: EBV-associated posttransplant lymphoproliferative disease (PTLD).
The incidence of PTLD in solid organ transplant adult recipients is a major concern and depends on the type of transplant: 1–2.3% of kidney transplants, 1–2.8% of liver transplants, 1–6.3% of heart transplants, 2.4–5.8% of heart-lung transplants, and 4.2–10% of lung transplants and up to 20% of small bowel transplants (2). In HSCT, the incidence can reach 18% of patients (3) and overall mortality of PTLD has been estimated as 80% (4). Children are more at risk due to EBV-seronegativity before HSCT, whereas known risk factors for developing PTLD in adult HSCT recipients are T-cell depletion (including administration of antithymocyte globulin [ATG]) and human leukocyte antigen (HLA)-mismatched transplants (5), both of which are more and more performed with the advent of cord blood as stem-cell source, reduced-intensity conditioning regimens, and haploidentical HSCT.
Natural history of PTLD commonly begins in the first year posttransplant and tumor progression is often rapidly fatal. Symptoms and signs resemble those of infectious mononucleosis and lymphoma, although in some cases occult disease and extranodal involvement may occur. Diagnosis is made by biopsy of involved nodal or extranodal sites showing polyclonal or monoclonal B-cell lymphoproliferation.
The optimal strategy for prevention and treatment of PTLD is still a matter of discussion in the transplant field. Various approaches have been attempted to avoid morbidity and mortality associated with PTLD. Treatment may include the reduction or discontinuation of immunosuppressive drugs, surgery, radiotherapy, antiviral drugs, chemotherapy, immunotherapy with interferon or monoclonal antibodies, and cell therapy. Unfortunately, clinical outcomes are described only in case reports or limited series of patients, and to our knowledge, until now, no controlled trial with therapeutic intervention has been performed. At all events, overall survival rates are poor as cited earlier. The recent introduction of monoclonal antibody against CD20, rituximab, has provided a relatively simple and safe treatment modality for PTLD (6–8). Rituximab is also used in the treatment of de novo B-cell non-Hodgkin's lymphomas since the early 2000s based on many randomized controlled phase III trials and has been universally acclaimed as a breakthrough in the lymphoma field.
Because of the high mortality of PTLD, preventive approaches are also being developed mainly in two directions: immune-cell treatment or adoptive immunotherapy, and B-cell depletion with monoclonal antibodies. The former technique uses ex vivo generation and expansion of EBV-specific cytotoxic T lymphocytes from donor origin and reinfusion to the patient after engraftment. It has shown excellent responses without toxicity in a series of 39 high-risk patients, with no patient developing PTLD at 1 year posttransplant whereas historic incidence in similar patients was 12% (9). The other modality uses rituximab before PTLD development. Rituximab requires much less technical burden than generation of cytotoxic T lymphocytes and has shown little toxicity in its large-scale use to treat lymphomas and rheumatoid arthritis, above all no increase in the incidence of infections (10, 11).
Thus preemptive approaches, that is, before clinical disease, are being developed to stop the replication of EBV-infected B cells with rituximab when viral load increases after transplantation (12–15). Quantitative measurement of EBV DNA has been made possible by various polymerase chain reaction (PCR) techniques (16) and data support a correlation between viral load and risk of PTLD (17, 18).
An effective preemptive therapeutic approach in a selected population needs three key elements: a reliable diagnostic test, a treatment modality with a low risk-benefit ratio (rituximab), and a defined cutoff point for treatment initiation—without which the “benefit” in the above-mentioned ratio is insignificant. We report our experience with this type of approach in our HSCT population, comparing our data with the published literature.
As many other HSCT teams (12–15), we developed a preemptive attitude based on qPCR monitoring and rituximab administration. We retrospectively reviewed our patient files to determine the effectiveness of this approach.
PATIENTS AND METHODS
Patients
We monitored 115 consecutive patients transplanted between August 2000 and September 2006. Clinical evaluation and EBV PCR were performed weekly during the first 3 months after transplantation, fortnightly thereafter during 3 months and then monthly until 1 year posttransplant. In case of a positive qPCR, the patient was monitored weekly until normalization. Patients were considered as being at high risk for PTLD according to Curtis et al. (5) as described earlier. Characteristics of the study population are listed in Tables 1 and 2. Transplant and rituximab administration protocols were approved by the local Ethical Review Board and patients signed consent forms.
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TABLE 1: Characteristics of transplant population (n=115)
TABLE 2: Characteristics of treated patients (n=19)
Transplants
We used HLA-identical sibling donors, 8 of 8 HLA-matched unrelated donors, or haploidentical-related donors. Conditioning regimens were myeloablative (cyclophosphamide plus busulfan or 12 Gy total-body irradiation), or nonmyeloablative (fludarabine, cyclophosphamide). Haploidentical donors required a specific conditioning including fludarabine, melphalan and cyclophosphamide or 10 Gy total-body irradiation. GVHD prophylaxis was cyclosporine plus methotrexate in myeloablative transplants, and pretransplant ATG followed by cyclosporine plus mycophenolate mofetil in nonmyeloablative transplants. Haploidentical transplants used ex vivo T-cell depleted grafts, and ATG was given to the recipient as part of the conditioning regimen. Stem-cell source was peripheral blood in all patients.
qPCR
The blood samples were collected on edetate (EDTA) and DNA was extracted using the QIAamp DNA blood Kit (Qiagen, Hilden, Germany). Each sample was analyzed in duplicate. The EBV-specific primers and a fluorogenic probe were selected to conserved sequences in the BALF5 gene encoding the EBV DNA polymerase (16). All of these oligonucleotides were synthesized by Applied Biosystems. We used a real-time PCR TaqMan assay (ABI Prism 7500 Sequence Detection System, Perkin Elmer Applied Biosystems) in a total volume of 25 μL, including 12.5 μL TaqMan Universal Master Mix (Applied Biosystems), 0.2 μM of each primer, 0.1 μM of probe, and 5-μL aliquot of sample DNA. Thermal cycling conditions consisted of heating at 94°C for 10 min, which preceded a two-stage temperature profile of 30 sec at 95°C and 1 min and 30 sec at 62°C for 40 cycles. Reactions are characterized by the time during cycling when a threshold of baseline fluorescence (CT) is exceeded. In each reaction run, a standard EBV (B95-8 viral DNA; Tebu-bio, Belgium) was serially diluted over a range of 5 logs (106–101 copies) to enable the generation of a standard curve. The latter is generated by plotting the CT values versus log 10 (N), where N is the concentration of the standard. The EBV DNA level in each blood sample is determined by locating its CT on the standard curve. Negative controls were tested by using the same PCR reaction mixture under the amplification conditions described above but without template DNA. Viral load on whole blood was expressed in genome copies per milliliter (gCop/mL).
Rituximab
Rituximab was administered at the dose of 375 mg/m2 as soon as possible after a positive PCR result above study cutoffs and reduction of immunosuppressant drugs if possible. Treatment was discontinued in case of a severe side effect according to attending clinician's appraisal, and as soon as a negative PCR result was available. The product was provided in a “medical need program” by nv Roche sa (Brussels, Belgium) after approval of our Ethical Review Board.
Cutoffs for Treatment
EBV reactivations with qPCR levels above 40,000 gCop/mL or two consecutive rising viral loads above 10,000 gCop/mL were treated with the above-mentioned protocol. This second threshold value has been previously described for T-cell-depleted transplants to show a positive predictive value of 50% and a negative predictive value of 96% for PTLD (17).
Diagnosis of PTLD
Patients were assessed clinically at each follow-up visit for symptoms and signs suggestive of PTLD, as mentioned above. Imaging studies including computerized tomography and biopsies were performed whenever deemed necessary by the attending physician.
Endpoints
Objectives included incidence of EBV reactivation, toxicity of the procedure, and incidence of PTLD in our treated population. Feasibility of this preemptive approach and evaluation of mortality related to EBV were also assessed. Paired analysis with historic controls was controverted due to the whole new approaches used since the early 2000s, numerical data are thus compared with published figures.
RESULTS
One hundred fifteen patients underwent HSCT during the 74-month period (Table 1). Thirty-six HSCTs were ex vivo and in vivo T-cell depleted, and 49 patients received ATG alone during conditioning (n=46) or for refractory GVHD (n=3), which brings the proportion of patients at high risk for PTLD at 69%.
EBV reactivation requiring preemptive treatment according to qPCR thresholds described above occurred in 19 (16.5%) patients, 12 (63%) of whom were in the high-risk group (P=0.38). Characteristics of treated patients are given in Table 2.
Median time from transplantation to EBV reactivation was 86 (range, 29–304) days and median viral load at initiation of therapy was 42,748 (range, 12,480–268,118) gCop/mL. Preemptive therapy was started after a median of 6.5 (range, 1–13) days of qPCR result. The median number of weekly treatment cycles was 3 (range, 1–4).
Seventeen of those 19 (89%) patients sustained negative viral loads after therapy. The two other patients had a rapidly unfortunate outcome. One patient died from acute GVHD before achieving qPCR negativity, the other presented a serious immediate adverse reaction to rituximab, which was discontinued, and he later developed PTLD and died from acute GVHD.
Seven patients (37%) obtained clearance of viral load after a single dose of therapy. Incidence of severe adverse event after rituximab therapy was 1 in 19 patients (5%). Incidence of PTLD in our population using our preemptive approach was thus 0.8% and when looking only to the high-risk population reached 1.2%, whereas PTLD-related mortality was abolished. All-cause mortality was 72% in the surveyed population and 68% in the treated population.
Median duration of follow-up from transplant to death or last contact was 354 days (range, 49–1853 days).
DISCUSSION
Since the type of transplants used has been changing rapidly during the last 10 years, we could not perform any reliable matched-pair analysis. The growing use of nonmyeloablative regimens and mismatched donors requires profound immunosuppression, by the means of ex vivo T-cell purging or in vivo administration of ATG, could not allow us to use historic controls, which would have mainly been standard HLA-matched T-cell replete myeloablative transplants.
Compared with the higher risk of PTLD with the use of ATG, some authors (unpublished) reported a lower incidence with alemtuzumab, an anti-CD52 monoclonal antibody targeting T cells, and also EBV-hosting B cells. Furthermore, alemtuzumab has recently been demonstrated to deplete naïve T cells more efficiently than memory T cells potentially sparing T cells involved in established protective EBV immunity (19). Data on alemtuzumab in HSCT are lacking but in a recent study of 59,560 kidney recipients, T-cell depletion with ATG was associated with a significantly increased PTLD risk, as opposed to alemtuzumab, basiliximab, and daclizumab (20).
The other known risk factors for PTLD in adult HSCT is the use of HLA-mismatched donors (5), but since this type of transplants often requires ATG, it is probably a surrogate for in vivo T-cell depletion.
Two thirds of our study population underwent such newer preparative regimens, which correspond to the current trends in HSCT, and have been described as being at high risk for PTLD (5). However, the incidence of EBV reactivation meeting our criteria for treatment was the same in that high-risk population compared with the standard-risk group. This probably correlates with an overestimated risk for PTLD with our cutoffs for treatment.
Indeed two main pitfalls with qPCR-guided therapy have to be solved before we can test the procedure in randomized control trials.
First, routine techniques vary from one laboratory to another, as noticed by the different units used to express qPCR results. We have used whole blood to perform the analyses, which better reflects viral presence in the cellular compartment of blood. Serum samples of PTLD patients are known to be low in EBV genome copies despite elevated EBV DNA levels in simultaneously obtained unfractionated whole blood samples. This indicates that the elevated EBV DNA loads are associated with the cellular blood compartment, that is, the EBV-infected B cell originating from the PTLD itself or from aberrant expansion of the infected B-cell pool in the circulation in the absence of lytic viral replication (18). On the other hand, qPCR on mononuclear cells alone underestimates this lytic viral replication.
Currently, there is no consensus concerning the best blood compartment to be analyzed for EBV-load monitoring, for the diagnosis of EBV-associated disorders and assessment of the efficacy of therapeutic intervention. Recently, the quantification of EBV in the whole blood has been demonstrated to be at least comparable with peripheral blood mononuclear cells for diagnosis and patient care (21). The use of whole blood seems to offer potential advantages as the specimen of choice for qPCR. Compared with peripheral blood mononuclear cells, it requires less sample volume and fewer processing steps.
Second, threshold values predictive of PTLD remain uncertain. We have used values that were described to lead to PTLD in more than 50% of cases, but these numbers were drawn from plasma-based analyses. Exact predictive value of whole-blood based techniques remains to be appraised and prospective trials should probably consider higher thresholds for treatment initiation.
A review from Wagner et al. (22) even advocates a prompt intervention based on clinical grounds and qPCR results, instead of a truly preemptive attitude as ours. The main problem with this method is the unspecific clinical presentation of PTLD, their treated patients predominantly had fever as the only clinical abnormality. Others have shown that PTLD may remain asymptomatic until full-blown progressive neoplasia.
Fortunately, despite likely overestimation of PTLD risk in our population our approach led to the logical consequence that incidence of the disease was low, even in this high-risk group, and moreover, PTLD-related mortality has been abolished.
If patients are on immunosuppressant drugs at the time of EBV reactivation, reducing the doses or discontinuation may be an option. But haploidentical transplants do not use posttransplant immunosuppression, and the risk of GVHD can be a significant concern (Fig. 1).
FIGURE 1.:
Three different patterns of the impact of therapy on viral load. Patients A and C had haploidentical transplants, that is, they were free of immunosuppressive drugs after transplantation.
The optimal dose of rituximab for the prevention of PTLD is not yet defined, and we have chosen the most used regimen for the treatment of lymphoproliferative disorders. Duration of treatment was short, a median of 3 weekly cycles were sufficient to obtain negative qPCR results. Some authors (22) have even shown abolition of PTLD-related mortality with a single dose of rituximab. This could allow us to design a therapeutic model with only one or two rituximab infusions.
The procedure was shown to be relatively safe, we observed one case of respiratory distress and hypotension that prompted us to stop the preemptive therapy. Immunoglobulin production impairment and clinically relevant infectious complications are already well known to be insignificant, even with long-term use of rituximab (10).Unfortunately, we cannot draw conclusions on immunoglobulin levels or B-cell reconstitution due to lack of date, but we were not stricken by an unusual pattern of infectious complications in our treated population.
Causes of death in both groups were balanced mainly between treatment-related mortality and relapse, adding up to a high mortality rate, mainly due to our transplant eligibility criteria that include refractory and relapsing diseases. Furthermore, as a pilot center for haploidentical transplantation, we included those patients in the analysis.
Finally, practical clinical management seems feasible with a readily available molecular diagnostic laboratory. Cost-effectiveness issues remain to be solved after prospective controlled trials have evaluated the clinical impact of PCR-guided approaches. Although regarding the overall cost of a transplant procedure and the high mortality of the diseases treated with HSCT it is unlikely to be immoderate.
In conclusion, we developed a qPCR-guided and rituximab-based preemptive approach to avoid PTLD after allogeneic hematopoietic stem-cell transplantation. Our approach was feasible but probably overtreats patients. Other similar strategies are being developed (12, 15) to find the correct refinements that allow us to clear the patients' way from posttransplant lymphoma. Prospective controlled trials are strongly needed, and should use uniform PCR techniques and probably consider higher threshold values for treatment initiation.
REFERENCES
1. Centers for Disease Control and Prevention; Infectious Disease Society of America; American Society of Blood and Marrow Transplantation. Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients.
MMWR Recomm Rep 2000; 49(RR-10): 1, CE1.
2. Taylor AL, Marcus R, Bradley JA. Post-transplant lymphoproliferative disorders (PTLD) after solid organ transplantation.
Crit Rev Oncol Hematol 2005; 56: 155.
3. Gerritsen EJ, Stam ED, Hermans J, et al. Risk factors for developing EBV-related B cell lymphoproliferative disorders (BLPD) after non-HLA-identical BMT in children.
Bone Marrow Transplant 1996; 18: 377.
4. Harris NL, Swerdlow SH, Frizzera G, et al. Post-transplant lymphoproliferative disorders In: Jaffe ES, Harris NL, Stein H, Vardiman JW, eds. World Health Organization classification of tumours. Pathology and genetics of tumours of haematopoietic and lymphoid tissues. Lyon, IARC Press 2001, p 264.
5. Curtis RE, Travis LB, Rowlings PA, et al. Risk of lymphoproliferative disorders after bone marrow transplantation: A multi-institutional study.
Blood 1999; 94: 2208.
6. Blaes AH, Peterson BA, Bartlett N, et al. Rituximab therapy is effective for posttransplant lymphoproliferative disorders after solid organ transplantation: results of a phase II trial.
Cancer 2005; 104: 1661.
7. Trappe RU, Choquet S, Reinke P, et al. Salvage therapy for relapsed posttransplant lymphoproliferative disorders (PTLD) with a second progression of PTLD after Upfront chemotherapy: The role of single-agent rituximab.
Transplantation 2007; 84: 1708.
8. 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; 107: 3053.
9. Rooney CM, Smith CA, Ng CY, et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients.
Blood 1998; 92: 1549.
10. Popa C, Leandro MJ, Cambridge G, et al. Repeated B lymphocyte depletion with rituximab in rheumatoid arthritis over 7 yrs.
Rheumatology (Oxford) 2007; 46: 626.
11. Looney RJ, Srinivasan R, Calabrese LH. The effects of rituximab on immunocompetency in patients with autoimmune disease.
Arthritis Rheum 2008; 58: 5.
12. 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; 99: 4364.
13. Gärtner BC, Schäfer H, Marggraff K, et al. Evaluation of use of Epstein-Barr viral load in patients after allogeneic stem cell transplantation to diagnose and monitor posttransplant lymphoproliferative disease.
J Clin Microbiol 2002; 40: 351.
14. Weinstock DM, Ambrossi GG, Brennan C, et al. Preemptive diagnosis and treatment of Epstein-Barr virus-associated post transplant lymphoproliferative disorder after hematopoietic stem cell transplant: An approach in development.
Bone Marrow Transplant 2006; 37: 539.
15. Meerbach A, Wutzler P, Häfer R, et al. Monitoring of Epstein-Barr virus load after hematopoietic stem cell transplantation for early intervention in post-transplant lymphoproliferative disease.
J Med Virol 2008; 80: 441.
16. Kimura H, Morita M, Yabuta Y, et al. Quantitative analysis of Epstein-Barr virus load by using a real-time PCR assay.
J Clin Microbiol 1999; 37: 132.
17. van Esser JW, van der Holt B, Meijer E, et al. Epstein-Barr virus (EBV) reactivation is a frequent event after allogeneic stem cell transplantation (SCT) and quantitatively predicts EBV-lymphoproliferative disease following T-cell–depleted SCT.
Blood 2001; 98: 972.
18. Stevens SJ, Verschuuren EA, Pronk I, et al. Frequent monitoring of Epstein-Barr virus DNA load in unfractionated whole blood is essential for early detection of posttransplant lymphoproliferative disease in high-risk patients.
Blood 2001;97: 1165.
19. Pearl JP, Parris J, Hale DA, et al. Immunocompetent T-cells with a memory-like phenotype are the dominant cell type following antibody-mediated T-cell depletion.
Am J Transplant 2005; 5: 465.
20. Kirk AD, Cherikh WS, Ring M, et al. Dissociation of depletional induction and posttransplant lymphoproliferative disease in kidney recipients treated with alemtuzumab.
Am J Transplant 2007; 7: 2619.
21. Hakim H, Gibson C, Pan J, et al. Comparison of various blood compartments and reporting units for the detection and quantification of Epstein-Barr virus in peripheral blood.
J Clin Microbiol 2007; 45: 2151.
22. Wagner HJ, Cheng YC, Huls MH, et al. Prompt versus preemptive intervention for EBV lymphoproliferative disease.
Blood 2004; 103: 3979.
