• lymphoproliferative disorder;
  • Epstein–Barr virus load;
  • anti-thymocyte globulin;
  • haematopoietic stem cell transplantation


  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Epstein-Barr virus (EBV)-related lymphoproliferative disorder (LPD) is a serious complication of haematopoietic stem cell transplantation (HSCT). To clarify the frequency, natural course and risk factors for LPD, we prospectively monitored 38 allogeneic (allo)-HSCT patients, focusing on the use of anti-thymocyte globulin (ATG). We used a recently developed real-time polymerase chain reaction assay to monitor EBV genome load. The subjects consisted of 19 patients given ATG for conditioning and 19 patients not given ATG. Of the 19 patients given ATG, 47·4% (nine patients) had a significant increase in EBV genome load (102·5 copies/µg DNA). Of these nine patients, two developed LPD. Therefore, 10·5% of the patients receiving allo-HSCT with ATG developed LPD. In contrast, none of the 19 patients without ATG had a significantly increased EBV load. The increases in viral load were observed in the second or third month after HSCT. We found that the peak viral loads of LPD patients were > 104·0 copies/µg DNA. On the other hand, the viral loads of most patients with no symptoms were < 102·5 copies/µg DNA. In conclusion, routine monitoring of EBV load during the second and third months after transplantation may benefit patients undergoing HSCT with ATG. We propose that an EBV load > 102·5 copies/µg DNA is the reactivation of EBV, and that an EBV load > 104·0 copies/µg DNA is indicative of developing LPD.

Epstein–Barr virus (EBV) is a ubiquitous virus, and most seropositive-individuals harbour the virus for life. EBV infects and immortalizes B cells, which proliferate in the peripheral blood and lymph nodes. After the emergence of EBV-specific immunity, virus-infected B cells are controlled by cytotoxic T lymphocytes. EBV often reactivates under immunosuppressive conditions such as acquired immunodeficiency syndrome (AIDS) and transplantation (Rickinson & Kieff, 1996). Lymphoproliferative disorder (LPD) is caused by the proliferation of EBV-infected B cells, and is one of the most serious complications of organ and haematopoietic stem cell transplantation (HSCT). Once it develops, it rapidly progresses and is sometimes fatal.

In HSCT, various factors are considered as risk for LPD, including T-cell depletion, the use of anti-thymocyte globulin (ATG), unrelated donors and mismatched human leucocyte antigen (HLA) (Shapiro et al, 1988; Hale & Waldmann, 1998; Curtis et al, 1999; Gross et al, 1999; Small et al, 1999). ATG is commonly included as a conditioning regimen of HSCT in patients with severe aplastic anaemia (SAA). We, and colleagues, have reported cases of LPD in patients who received unmanipulated bone marrow transplantation with ATG for conditioning (Small et al, 1997; Hoshino et al, 2000; Kuzushima et al, 2000). However, the frequency and natural course of EBV infection and reactivation (including LPD) are not well known in HSCT with ATG for conditioning. Recent studies have reported successful prevention and treatment of LPD by adoptive transfer of EBV-specific T cells, which requires considerable labour and is technically difficult (Rooney et al, 1995a; Heslop et al, 1996; Kuzushima et al, 1996; Rooney et al, 1998). To optimize the management of EBV-related LPD, it is essential to clarify the frequency, natural course, and risk factors of LPD.

In this study, we prospectively monitored EBV load using a real-time polymerase chain reaction (PCR) assay in HSCT patients, focusing on the use of ATG for conditioning. Of the HSCT patients given ATG, nearly half showed a significant increase in EBV genome load, usually during the second or third month. Overall, 10% of patients who received ATG for conditioning developed LPD. Interestingly, some patients had transient unexplained thrombocytopenia and febrile episodes associated with the increased EBV load, although they did not develop LPD. Furthermore, we propose diagnostic criteria for virus reactivation and LPD based on a real-time PCR method. We hope that this will facilitate the diagnosis of LPD and aid in the process of determining a therapeutic strategy for LPD.

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patients Thirty-eight patients were enrolled for prospective monitoring of EBV load; they were selected based on the use of ATG for conditioning. The subjects consisted of 19 patients given ATG for conditioning and 19 patients not given ATG. All subjects received allogeneic-HSCT between July 1998 and July 2000 for severe aplastic anaemia, haematopoietic malignancies, solid tumours, congenital immunodeficiencies or metabolic disease. The characteristics of these patients are summarized in Table I. The clinical course and EBV-specific immune responses of three patients (patients 1, 2 and 40) have been described elsewhere (Hoshino et al, 2000; Kuzushima et al, 2000). Most of the patients were < 20 years old, the mean age was 8·6 years (range 5 months to 35 years) and the number of men was 23 and women 15.

Table I.   Characteristics of prospectively monitored 38 patients.
Patient Age /sex Underlying diseaseSource of stem cellsNumber of mismatch HLA Conditioning regimen GVHD prophylaxisEBV load (copies/ µg DNA) Diagnosis/ symptom
  • WAS; Wiskott–Aldrich syndrome; SAA, severe aplastic anaemia; CML, chronic myeloid leukaemia; ALL, acute lymphoblastic leukaemia; ML, malignant lymphoma; HLH, haemophgocytic lymphohistiocytosis; ALD; adrenoleucodystorophy; AML, acute myelogenous leukaemia; SCID, severe combined immunodeficiency; NB, neuroblastoma; UR, unrelated; R, related; BM, bone marrow; PBSC, peripheral blood stem cell; CB, cord blood; TBI, total body irradiation; TLI, total lymph-node irradiation; CY, cyclophosphamide; BU; busulphan; Ara-C, cytarabine; L-PAM. melphalan; CyA, cyclosporin A; FK, tacrolimus; MTX, methotrexate; mPLS, methylprednisolone; Plt, thrombocytopenia; n.d., not detected; bolded numbers indicate significant increase of EB viral load.

  • *

    CD34-positive selection.


Five additional patients who had significantly high EBV genome loads after HSCT were included in the analysis. Although these five patients were not prospectively monitored, they were included for showing time course and peak load of viral genome. Their viral loads were serially quantified because of symptoms suggesting LPD. Among these five patients, three developed LPD. Two had histologically confirmed LPD, and one was diagnosed clinically with enlarged lymph nodes at the hilum of the left kidney. The other two patients had fevers of unknown origin without any recognizable enlarged lymph nodes. The characteristics of these five patients are summarized in Table II.

Table II.   Characteristics of additional five patients who were suspected LPD.
Patient Age (years) /sex Disease Source of stem cellNumber of mismatch HLA Conditioning regimen GVHD prophylaxisEBV load (copies/µg DNA) Diagnosis/ symptom DLT
  1. Abbreviations are the same as Table I. DLT, donor lymphocyte transfusion.


Stem cell transplantation procedures The source of stem cell, HLA disparity, conditioning regimens and method of graft-versus-host disease (GVHD) prophylaxis of each patient are listed in Tables I and II. Conditioning regimens varied according to the disease and clinical status. In patients with malignant diseases, conditioning regimens included melphalan (140–210 mg/m2) or cyclophosphamide (120–200 mg/kg) combined with busulphan (16/kg or 56 mg/m2) or total body irradiation (TBI, 12Gy). Busulphan or cytosine arabinoside was added in high-risk patients. In patients transplanted from HLA-matched siblings, the conditioning regimen consisted of cyclophosphamide and ATG (Thymoglobulin, 2·5 mg/kg/d × 4, IMTIX, Lyon, France). Radiation was added to cyclophosphamide and ATG in two heavily transfused patients. Combination of ATG, cyclophosphamide and TBI was used in AA patients transplanted from an unrelated donor or HLA-mismatched related donor (Kojima et al, 2000). Two patients with severe combined immunodeficiency underwent transplantation without conditioning regimen. ATG was included in conditioning regimen for to prevent graft rejection. To prevent GVHD, all patients were given either cyclosporin A (CyA; 3 mg/m2/d from d −1) or tacrolimus (0·02–0·05 mg/kg/d from d −1) with or without methotrexate (15 mg/m2 d 1, followed by 10 mg/m2 on d 3, 6, 11) or methylprednisolone (1 mg/kg/d from d −1).

Blood sampling Sample collection was started after engraftment was confirmed. In general, sample collection started the second or third week after transplantation. Thereafter, whole peripheral blood was obtained every other week. The blood was examined weekly if any symptoms suggesting LPD appeared or if a high EBV genome load was detected. When no signs or symptoms suggesting LPD were observed after 3 months, routine examinations were stopped. These studies were performed after informed consent was obtained from either the patients or their parents.

Real-time quantitative PCR. To quantify EBV load, a real-time quantitative PCR method was used as previously described (Kimura et al, 1999). Briefly, mononuclear cells (MNCs) from EDTA-treated peripheral blood were collected from a Ficoll–Hypaque gradient. DNA was extracted from 106 MNCs using a QIAamp Blood Kit. The PCR reaction was carried out using a TaqMan PCR kit and a model 7700 Sequence Detector. The copy number was calculated and corrected for the quantity of DNA, and the final results were expressed as copies of EBV genome per µg of DNA. The detection limit of this assay was approximately four copies/µg DNA.

Statistical analysis The association of clinical factors with activation of EBV was examined by Fisher's exact probability test (Table III). The following factors were evaluated for a relationship to increase in EBV-DNA; use of ATG, use of irradiation, method of GVHD prophylaxis (all patients received either CyA or tacrolimus), type of HSCT and HLA mismatching (defined as one or more serologically disparate class I or II HLAs). The additional five patients who were not prospectively monitored were not included in the statistical analysis.

Table III.   Statistical analysis of risk factors.
Risk FactorsIncidenceP-value
  • *

    Including total lymph node irradiation; ATG, anti-thymocyte globulin.

Use of ATG
Total Body Irradiation
Prophylaxis for GVHD
 Cyclosporin A4/17 
Type of donor
 HLA-matched sibling1/60·5592
 Alternative donor8/32 
HLA-matching among alternative donor


  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We prospectively monitored the EBV loads of 38 patients, 19 of whom received ATG and 19 of whom did not. The peak viral load of each patient is shown in Table I. Previously, we reported that more than 102·5 (= 316) copies/µg DNA was detected in patients with symptoms of EBV infection (Kimura et al, 1999). In this study, we defined an EBV genome load of more than 102·5 copies/µg DNA as meaningful. Among the 38 patients, nine had a significantly elevated EBV load. Each of these nine patients had received ATG for conditioning. Among the patients with high viral loads, two developed LPD, three had unexplained, transient thrombocytopenia, one had febrile symptoms and three had no recognizable symptoms. The diagnosis of LPD in two patients was confirmed histologically. The LPD of one patient regressed spontaneously after reducing the dose of immunosuppressive drugs. The LPD of the other patient regressed after donor lymphocyte transfusion (DLT). The viral load and symptoms of the other patients regressed spontaneously with or without reducing the dose of the immunosuppressive drugs. From these results, we estimated that the incidence of a significantly elevated EBV load was 47·4% (9/19) in ATG+ HSCT and 0% (0/19) in ATG HSCT. Two of the nine patients (22·2%) with significant viral loads developed LPD. Overall, 10·5% of patients with ATG+ HSCT developed LPD. The risk factors of significant increases in EBV load were examined using multivariate analysis. In this study, only the use of ATG for conditioning was statistically significant among the examined risk factors. Irradiation, use of tacrolimus, use of alternative donor and mismatch HLA were not identified as risk factors for a significant increase in EBV load. The Cox proportional hazards model was used to confirm the risk factors of significant increase in EBV-DNA. It should be noted that 74% (14/19) of ATG+ patients had SAA. In contrast, none of the SAA patients were ATG patients. Because ATG and SAA were closely correlated (0·76), we could not analyse the diagnosis of underlying disease as a potential predictor in multivariate analysis.

Figure 1 shows the time course of EBV genome load in all 43 serially examined patients. Because there were only two LPD patients among the prospectively monitored patients, five more patients with significantly increased EBV genome loads were included for further analysis. The EBV genome was detectable throughout the observation period, but most genome loads were < 102·5 copies/µg DNA. Development of LPD and significant increases in viral genome load were seen in the second or third month. The EBV load of the LPD patients rapidly increased within 2 weeks from the time of first detection and subsequently exceeded 104·0 copies/µg DNA, indicating a rapid multiplication of EBV-infected cells. The viral load of patients whose LPD regressed rapidly, decreased. In contrast, the viral load of patient 41, who died from LPD, remained at a high level and did not decrease prior to his death.


Figure 1.  Time course of EBV genome loads for all serially sampled patients. Each line indicates the viral load of an individual patient. Bolded lines indicate LPD patients. †Indicates death (patient 41). The dashed line indicates the detection limit of the PCR assay. The solid line indicates the threshold of a significant increase in EBV load (102·5 copies/µg DNA).

Download figure to PowerPoint

Figure 2 shows the peak EBV genome load of each patient. The patients were grouped into four categories: LPD patients, thrombocytopenic patients, patients with fever of unknown origin and asymptomatic patients. The peak viral loads of all LPD patients > 104·0 copies/µg DNA. Of the six patients who had more than 104·0 copies/µg DNA, five (83%) had LPD. On the other hand, most asymptomatic patients had EBV loads < 102·5 copies/µg DNA. None of the patients who had < 102·5 copies/µg DNA had any recognizable symptoms associated with EBV. Most of the patients with episodes of fever of unknown origin and thrombocytopenia coincident with increases in EBV genome had viral copy loads between 102·5 and 104.° copies/µg DNA. These results confirm our previous report that showed that an EBV genome load > 102·5 copies/µg DNA is associated with symptomatic EBV infection (Kimura et al, 1999).


Figure 2.  Peak viral load of each patient. The LPD cases included four histologically confirmed cases and one case clinically diagnosed with enlarged lymph nodes. Thrombocytopenia occurred as an unexplained transient platelet decrease lasting more than 1 week that was associated with an elevated EBV genome load. Fever indicates patients with an episode of fever of unknown origin (> 38°C) without any evidence of LPD or enlarged lymph nodes that coincided with an elevated EBV genome load. Asymptom indicates asymptomatic patients. The dashed lines indicate 102·5 copies and 104·0 copies/µg DNA respectively.

Download figure to PowerPoint


  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We prospectively monitored EBV-DNA in 38 HSCT patients using a real-time PCR assay. Our results confirmed that inclusion of ATG for conditioning regimen is a risk factor for EBV reactivation. On the other hand, factors such as irradiation, use of tacrolimus, use of unrelated donor and mismatching HLA, which were previously reported as risk factors for LPD, were not identified such (Hale & Waldmann, 1998; Shapiro et al, 1988; Curtis et al, 1999; Gross et al, 1999; Small et al, 1999). Our data indicate that the use of ATG had the strongest effect on EBV burden, but other factors might have some effects that allow the proliferation of EBV-infected cells, resulting in development of LPD. In fact, all five patients with full-blown LPD were conditioned with cyclophosphamide, ATG and TBI, and had received unmanipulated bone marrow from an unrelated donor. In the current study, most of the patients with SAA received ATG as pre-conditioning. In contrast, only a few patients with other underling diseases received ATG. Because the use of ATG is closely correlated with diagnosis of SAA, we could not analyse the diagnosis of underling disease as a potential risk factor. However, no previous studied identified diagnosis of SAA as a risk factor for developing LPD. In our study, a patient with Wiskott–Aldrich syndrome who was conditioned with cyclophosphamide, ATG and TBI developed LPD. These findings suggest that diagnosis of SAA is probably not a risk factor for developing LPD.

In our study, 11 of 14 patients with elevated EBV genome levels decreased without any manipulation. All of these patients received ATG for conditioning. LPD that occurs after transplantation with T-cell-depleted bone marrow usually requires adoptive immunotherapy. This suggests that specific immunity for EBV could develop endogenously before LPD progresses to the full-blown stage in most patients with elevated EBV load in the setting of ATG therapy. There are many reports that indicate the importance of immune reconstitution in controlling EBV (Papadopoulos et al, 1994; Lucas et al, 1996, 1998). It has been postulated that the existence of EBV-specific CD8 T cells is important for the regression of LPD (Khatri et al, 1999; Hoshino et al, 2000; Kuzushima et al, 2000). It should also be noted that overall there was no increase in the instance of EBV-associated LPD in T-cell-depleted unrelated donor or haploidentical transplantation with the use of CAMPATH, which depletes T cells, B cells, NK cells and monocytes (Curtis et al, 1999). Presumably, this suggests the importance of the relative quantity of T cells against EBV-infected B cells. In this context, frequent monitoring of the EBV load as well as lowering immunosuppressive agents to promote an appropriate immune response will probably be beneficial in the treatment of LPD.

Our data demonstrate that the EBV genome was often detectable after HSCT, but most detectable EBV levels were subclinical and did not always indicate the diagnosis of LPD, which is compatible with another report (Esser et al, 1999). Therefore, quantitative analysis is necessary for the diagnosis of LPD. Various methods have been developed for monitoring the EBV loads of transplant patients (Savoie et al, 1994; Rooney et al, 1995b; Rowe et al, 1997; Drachenberg et al, 1998; Egawa et al, 1998; Kimura et al, 1999; Niesters et al, 2000; Orii et al, 2000). A recent report showed the dynamics of Epstein–Barr virus infection after transplantation using semiquantitative PCR (Gustafsson et al, 2000). The real-time PCR method has several advantages compared with other methods as this assay is fast, accurate and reproducible (Niesters et al, 2000; Orii et al, 2000). Previously, we showed that 102·5 copies/µg DNA is associated with symptomatic EBV infection (Kimura et al, 1999). Here, we propose diagnostic criteria and a therapeutic strategy based on EBV load to classify the severity of EBV infection in HSCT. First, an EBV genome level of > 102·5 copies/µg DNA is considered without activity and no intervention is required. Second, > 102·5 copies/µg DNA is a warning sign of activation of EBV infection, and patients with symptoms should be treated by the decrease or removal of immunosuppressive drugs. Third, viral loads > 104·0 copies/µg DNA in association with ATG in conditioning therapy would represent LPD and could be treated either with removal of immunosuppression or DLT. In cases that are difficult to biopsy or in cases without recognizably enlarged lymph nodes, an EBV genome load of > 104·0 copies/µg DNA would be helpful for diagnosis and therapeutic decision-making. Moreover, serial measuring of EBV load is essential for evaluating the intervention and prediction of disease progression in patients with suspected LPD.

In this study, we encountered three patients with fever of unknown origin coincident with elevations in EBV load. Fever is a typical preceding sign of LPD (Shapiro et al, 1988). Although these three patients lacked recognizable lymphadenopathy or other symptoms suggestive of LPD, they might have had subclinical disease caused by reactivation of EBV. The viral load of these patients > 102·5 copies/µg DNA. We also experienced three patients with unexplained transient thrombocytopenia coincident with an increased EBV load. It is unclear whether the thrombocytopenia was caused by the activation of EBV. It is unlikely that EBV causes a direct inhibition of platelet production; possibly, inflammatory cytokines induced by EBV infection could indirectly suppress the production of platelets and cause thrombocytopenia. To the best of our knowledge, there is no report that suggests association of EBV with fever of unknown of origin or thrombocytopenia in the second or third month after transplantation. Further studies are necessary to clarify the incidence and mechanism of the febrile episode and the thrombocytopenia.

In conclusion, routine monitoring of EBV load during the second and third month after transplantation may be useful for patients with a high risk of developing LPD. Prospective monitoring during this period is especially recommended when ATG is used for pre-conditioning. More than 102·5 copies/µg DNA suggest a meaningful increase in EBV genome, and > 104·0 copies/µg DNA indicates the risk of progression to LPD.


  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This work was supported by a grant from the Japanese Society for the Promotion of Science (JSPS-RFTFL00703). We thank Dr Tetsuya Nishida and Dr Seitaro Terakura, of the Japanese Red Cross First Hospital; Dr Hidefumi Kaku, of the Tokyo Metropolitan Komagome General Hospital; Naoko Kinugawa, of the Chiba Children's Hospital; Megumi Oda, of the Okayama University Medical School; and Masahiro Sako, and the Osaka City General Hospital, for providing blood samples and patient information. We also thank Dr Kazuyoshi Watanabe, of the Department of Paediatrics, Nagoya University School of Medicine, for helpful suggestions and advice.


  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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