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MAJOR ARTICLE Clinical and Virological Characteristics of 15 Patients with Chronic Active Epstein-Barr Virus Infection Treated with Hematopoietic Stem Cell Transplantation Kensei Gotoh, 1 Yoshinori Ito, 1 Yukiko Shibata-Watanabe, 1 Jun-ichi Kawada, 1 Yoshiyuki Takahashi, 1 Hiroshi Yagasaki, 1 Seiji Kojima, 1 Yukihiro Nishiyama, 2 and Hiroshi Kimura 2 Departments of 1 Pediatrics and 2 Virology, Nagoya University Graduate School of Medicine, Nagoya, Japan (See the editorial commentary by Gottschalk on pages 1535 6) Background. Chronic active Epstein-Barr virus (EBV) infection is characterized by recurrent infectious mononucleosis like symptoms, and infected patients have high viral loads in their peripheral blood. Standard therapy for the disease has not yet been established. Recently, hematopoietic stem cell transplantation (HSCT) has been introduced and has the potential to become a standard treatment, although guidelines for HSCT to treat chronic active EBV infection have not yet been proposed. Methods. Fifteen patients were retrospectively analyzed, both clinically and virologically, to investigate the factors associated with prognosis of chronic active EBV infection treated with HSCT. Results. After HSCT, 7 patients died after survival periods that ranged from 1 to 16 months (mean duration of survival after HSCT, 5 months). Three patients were considered to have died of transplantation-related complications. The duration between infection onset and diagnosis was significantly longer in patients who died than in those who survived. Five of the 7 patients who died experienced 3 life-threatening complications. The plasma concentrations of interferon-g, interleukin-10, thrombomodulin, and soluble E-selectin did not differ significantly between the groups of patients. With regard to sequence variations in the EBV latent membrane protein 1 gene, no specific patterns were found in the patients who died. Importantly, the plasma EBV load at diagnosis was significantly higher in patients who died than in living patients. Moreover, plasma viral load was shown to be an important factor to monitor during follow-up for patients after HSCT. Conclusions. The number of life-threatening complications and plasma viral load are indicative of the stage of disease progression and may be useful factors for predicting the outcome of HSCT. Epstein-Barr virus (EBV) is a ubiquitous virus that infects most humans during early adulthood. Primary EBV infection is usually asymptomatic, but sometimes it results in infectious mononucleosis, which resolves spontaneously after the emergence of EBV-specific immunity [1]. EBV can cause chronic infections in apparently immunocompetent persons. Chronic active EBV (CAEBV) infection is characterized by chronic or Received 16 September 2007; accepted 20 December 2007; electronically published 4 April 2008. Reprints or correspondence: Dr. Hiroshi Kimura, Dept. of Virology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan (hkimura@med.nagoya-u.ac.jp). Clinical Infectious Diseases 2008; 46:1525 34 2008 by the Infectious Diseases Society of America. All rights reserved. 1058-4838/2008/4610-0007$15.00 DOI: 10.1086/587671 recurrent infectious mononucleosis-like symptoms, such as fever, swelling of lymph nodes, and hepatosplenomegaly. CAEBV infection is associated with high mortality and morbidity, with various life-threatening complications, such as virus-associated hemophagocytic syndrome, interstitial pneumonia, lymphoma, coronary artery aneurysm, and CNS involvement [2 4]. Patients with CAEBV infection also have an unusual pattern of EBV-related antibodies and high viral loads in their peripheral blood [5 8]. Recent studies have indicated that clonal expansion of EBV-infected T cells and natural killer (NK) cells plays a central role in the pathogenesis of CAEBV infection [7, 9 12], although CAEBV infection in Western countries may not always be associated with the expansion of EBV-infected T or NK cells [13]. CAEBV with Stem Cell Transplantation CID 2008:46 (15 May) 1525

Standard therapy for CAEBV infection has not yet been established. Antiviral, anticancer, and immunomodulating agents have been administered, although they seem to have only a transient effect and are unable to control CAEBV infection [13]. Recently, hematopoietic stem cell transplantation (HSCT) has been introduced as a curative therapy for CAEBV infection. Bone marrow [14], cord blood [15 17], and peripheral blood [18 20] have been reported as stem cell sources. HSCT is not a standard therapy, even after successful cases have been reported, because patients with CAEBV infection are at high risk for treatment-related toxicity attributable to multiple organ dysfunction [12, 13]. In the present study, 15 patients with CAEBV infection treated with HSCT were investigated to establish which factors affect treatment outcome. Clinical characteristics were compared between living patients and patients who died. Viral loads in PBMCs and plasma, the plasma concentrations of cytokines and vascular endothelial cell associated molecules, and sequence variations in EBV isolated from samples from patients with CAEBV infection were compared. PATIENTS AND METHODS Patients. Fifteen patients treated with allogeneic HSCT during the period 1999 2006 were enrolled in this study. These patients were referred to the Nagoya University School of Medicine (Nagoya, Japan) with CAEBV infection. Informed consent was obtained from all patients or their parents. All patients fulfilled the following diagnostic criteria [7]: (1) EBV-related illness or symptoms for 16 months, including fever, persistent hepatitis, extensive lymphadenopathy, hepatosplenomegaly, pancytopenia, uveitis, interstitial pneumonia, hydroa vacciniforme, or hypersensitivity to mosquito bites; (2) increased quantity of EBV in either affected tissues or peripheral blood (the amount of EBV was defined as increased when 1 ofthe following criteria were met: EBV DNA detected in tissue or peripheral blood samples by Southern blot hybridization; EBVencoded small RNA1 positive cells detected in tissue or peripheral blood samples; or an EBV DNA level 110 2.5 copies/mg of DNA detected in PBMCs [21]); and (3) no evidence of any prior immunologic abnormalities or of any other recent infection that might explain the condition. The time of diagnosis was defined as the time when the patient was found to meet the above criteria. Serial blood samples were obtained before and after transplantation. Clinical assessments, such as standard cardiac and neurological tests, were performed to evaluate lifethreatening complications. Determination of EBV-infected cells. To determine which cells harbored EBV, PBMCs were fractionated into CD3 +, CD4 +, CD8 +, CD16 +, CD19 +, and CD56 + cells with use of Dynabeads (Invitrogen). Patients were defined as having T cell type infection when CD3 + cells were the major group of cells that harbored EBV. Patients were defined as having NK cell type infection when their CD16 + or CD56 + cells were the major group of cells infected with EBV [7, 22]. Plasma concentrations of cytokines and vascular endothelial cell associated molecules. Plasma concentrations of IFNg, IL-6, IL-10, thrombomodulin, and soluble E-selectin were measured using ELISA kits (for IFN-g and IL-10, BioSource Europe; for IL-6, Fuji Rebio; for thrombomodulin, Daiichi Fine Chemicals; for soluble E-selectin, R&D Systems), according to the manufacturers instructions. Quantification of EBV DNA. DNA was extracted from either 1 10 6 PBMCs or 200 ml of plasma. A real-time quantitative PCR assay was performed, as described elsewhere [21]. The amount of EBV DNA was calculated as the number of virus copies per microgram of PBMC DNA or per milliliter of plasma. The lower limits of detection in this assay were 10 copies/mg of DNA for PBMCs and 50 copies/ml for plasma. Expression of the EBV latent membrane protein 1 (LMP1) gene in PBMCs. To detect expression of the LMP1 gene, RNA was extracted from the PBMCs of 12 patients at diagnosis and was used for a nested RT PCR assay, as described elsewhere [22]. Sequence analysis of the C-terminal region of the LMP1 gene. DNA was extracted from PBMCs at diagnosis and was used for sequencing. Sequences of the C-terminal region of the LMP1 gene were obtained using the direct sequencing method as described elsewhere [23]. PCR products were not obtained from the PBMCs of some patients. In these cases, a seminested PCR was performed using the primers described elsewhere [24]. The sequence data obtained were checked for homology in BLAST [25]. The sequences of the C-terminal region of the LMP1 genes of patients 3, 5, 8, and 11 have been reported elsewhere [26]. Statistical analysis. Statistical analysis was conducted using StatView software, version 5.0 (SAS Institute). Either Fisher s exact test or the x 2 test was used for the comparison of clinical data. The Mann Whitney U test was used to compare viral loads and plasma concentrations of IFN-g, IL-6, IL-10, thrombomodulin, and soluble E-selectin. Multivariate analysis was also performed using a multiple regression model to analyze factors identified as statistically significant. P!.05 was considered to be statistically significant. RESULTS Clinical characteristics of patients with CAEBV infection treated with HSCT. The clinical features and follow-up results of all 15 patients are shown in table 1, and the life-threatening complications encountered by each patient are listed in table 2. There were 6 male and 9 female patients, and the age at onset of disease ranged from 1 to 26 years (median age as disease onset, 4.2 years). Seven patients were defined as having 1526 CID 2008:46 (15 May) Gotoh et al.

Table 1. Clinical characteristics of 15 patients with chronic active Epstein-Barr virus (EBV) infection treated with stem cell transplantation. Patient number Sex Age, years EBVpositive Stem cell transplant characteristic Disease LMP1 Therapy before HLA onset Diagnosis Transplantation cells expression transplantation Cells Conditioning Source matched Outcome Cause of death 1 M 1 5 8 T ACV, IFN-a, IL-2, AraA PBSC FLU, LPAM Mother 6/6 Alive 2 F 1 1 2 T None BM FLU, CY, TBI Unrelated 5/6 Alive 3 F 2 4 6 NK + ACV, Bet BM FLU, CY, TBI Unrelated 6/6 Alive 4 M 2 13 20 NK + PSL, Dex, CyA, VP16, ICE BM FLU, LPAM, TBI Sibling 6/6 Dead Relapse 5 F 3 12 15 NK + AraA, MZR, PSL BM VP16, CY, TBI Sibling 6/6 Dead VOD, sepsis, intracranial bleeding 6 F 4 31 31 NA ND Dex, VP16, CyA BM VP16, CY, TBI Sibling 6/6 Dead DIC, acute pancreatitis 7 M 4 4 9 T + mpsl, CyA, MZR BM VP16, CY, TBI Sibling 6/6 Rejection 8 M 4 4 9 NK ND PSL, CyA, VP16, CY, ACV BM VP16, CY, TBI Unrelated 5/6 Alive 9 M 10 11 16 NK AraA, PSL, GCV, ACV, VP16 BM FLU, CY, TBI Unrelated 5/6 Alive 10 M 12 15 19 NK + None BM FLU, CY, TBI Unrelated 5/6 Alive 11 F 13 24 24 T ND PSL, CyA BM FLU, BUS, TBI Unrelated 6/6 Dead Relapse 12 F 14 17 18 T + PSL, CyA, MTX/AraC/HDC, CTL PBSC VP16, BUS, TBI Sibling 6/6 Dead Intracranial bleeding 13 F 14 14 15 NK + PSL, VP16, CyA, CHOP, ICE BM FLU, TBI Sibling 6/6 Dead Relapse 14 F 19 23 24 T Dex, VP16, CyA BM FLU, LPAM Unrelated 5/6 Dead ADEM 15 F 26 26 27 T + CHOP, HDCA BM FLU, BUS Unrelated 6/6 Alive NOTE. ACV, acyclovir; ADEM, acute disseminated encephalomyelitis; AraA, adenine-arabinoside; AraC, cytosine-arabinoside; Bet, betamethasone; BM, bone marrow; BUS, busulfan; CHOP, cyclophosphamide,daunorubicin, vincristine, and prednisolone; CTL, cytotoxic T lymphocyte therapy; CY, cyclophosphamide; CyA, cyclosporin A; Dex, dexamethasone; DIC, disseminated intravascular coagulation; FLU, fludarabine; GCV, ganciclovir; HDC, hydrocortisone; HDCA, high-dose cytosine-arabinoside; ICE, ifosfamide, carboplatin, and etoposide; LMP1, latent membrane protein 1; LPAM, melphalan; mpsl, methylprednisolone; MTX, methotrexate; MZR, mizoribine; NK, natural killer; NA, not available; ND, not done; PBSC, peripheral blood stem cell; PSL, prednisolone; TBI, total body irradiation; VOD, veno-occlusive disease; VP16, etoposide.

Table 2. Life-threatening complications for 15 patients with chronic active Epstein-Barr virus infection treated with stem cell transplantation. Patient number Malignant lymphoma HPS DIC Myocarditis Life-threatening complication Interstitial pneumonia Intestinal perforation CNS Hepatic failure Hypersplenism No. of complications Outcome (no. of months after transplantation) 1 0 Alive (67) 2 0 Alive (23) 3 0 Alive (13) 4 + + + 3 Dead (2) 5 + + + + 4 Dead (1) 6 + + + + 4 Dead (67) 7 0 Rejected (49) 8 0 Alive (12) 9 + + + + 4 Alive (57) 10 0 Alive (9) 11 0 Dead (10) 12 + + + + 4 Dead (1) 13 + + + + 4 Dead (16) 14 0 Dead (4) 15 + 1 Alive (46) NOTE. DIC, disseminated intravascular coagulation; HPS, hemophagocytic syndrome. T cell type infection, and 7 patients had NK cells predominantly infected with EBV. Seven patients died after survival periods that ranged from 1 to 16 months (median period of survival, 3 months) after HSCT. Each cause of death is shown in table 1. Three patients who experienced reappearance of clinical symptoms and increased viral load after undergoing HSCT were considered to have experienced relapse. Three patients (patients 5, 6, and 12) died of transplantation-related complications. One patient (patient 14) died of acute disseminated encephalomyelitis. Transplantation-related complications were defined as those that occurred within 60 days after transplantation and that were considered to be associated with transplantation rather than the disease itself. Eight patients were alive after a follow-up period of 9 67 months (median followup period, 40 months). One of the 8 patients was excluded from our analysis because of graft rejection. Fourteen patients achieved myeloid engraftment (absolute neutrophil count, 1500 neutrophils/ml) in a median period of 18 days (range, 12 26 days). Persistent complete donor chimerism within 70 days after transplantation was achieved in 9 of 12 patients. The number of recipient cells increased after complete donor chimerism was achieved in 1 patient who experienced relapse (patient 4). Two patients (patients 7 and 14) did not achieve complete donor chimerism. Chimerism was not evaluated in 3 patients who died of transplantation-related complications. We did not evaluate the relationship between the EBV status of the donors and patient outcome after transplantation because of the limited availability of information. Comparison of clinical features between living patients and patients who died. Fourteen patients were divided into living patients and patients who died (7 patients each), and the clinical manifestations of these groups were compared (table 3). One of 8 living patients (patient 7), who had been alive for 5 years after transplantation, was excluded from this comparison. This patient had recently experienced full relapse of disease and received a second transplantation. The age at diagnosis was significantly older for patients who died than for living patients (table 3). The interval between disease onset and diagnosis was also significantly longer for patients who died than for living patients. Ten patients underwent nonmyeloablative HSCT to avoid transplantation-related complications. Six of 7 living patients were treated with a nonmyeloablative regimen. There were no statistically significant differences between patients who lived and patients who died with regard to the other factors listed (table 3). The relationship between individual outcome and the number of life-threatening complications before HSCT is shown in table 2. Five of 7 patients who died experienced 3 life-threatening complications, whereas only 1 of the 7 surviving patients experienced 3 life-threatening complications ( P p.051). Five of 6 patients with complicated hematologic disorders, such as malignant lymphoma ( n p 3) and hemophagocytic syndrome ( n p 4), died. Plasma concentrations of IFN-g, IL-6, IL-10, thrombomodulin, and soluble E-selectin. To investigate whether the levels of cytokines known to be elevated in patients with CAEBV infection [22, 27 29] affect the mortality rate, the plasma concentrations of IFN-g, IL-6, and IL-10 at a time when patients were treatment-free before HSCT were compared between living patients and patients who died. Moreover, thrombomodulin and soluble E-selectin concentrations were measured, because some patients with CAEBV infection develop vascular and car- 1528 CID 2008:46 (15 May) Gotoh et al.

Table 3. Comparisons of clinical data between living patients and patients who died. Characteristic Living patients (n p 7) Patients who died (n p 7) P a Sex.13 Male 4 1 Female 3 6 Age at disease onset, mean years SD 8.0 9.0 9.8 6.8.28 Age at diagnosis, mean years SD 9.6 8.6 19.3 6.9.048 Interval from disease onset to diagnosis, mean years SD 1.7 1.7 9.5 9.0.033 Interval from diagnosis to transplantation, mean years SD 2.9 1.8 1.8 2.4.33 Conditioning.28 Myeloablative 1 3 Nonmyeloablative 6 4 Stem cell source.051 Related 1 5 Unrelated 6 2 Achievement time to myeloid engraftment, mean days SD 15.0 2.8 21.5 3.8.085 Main cell type infected.28 T 3 3 NK 4 3 Unclassified 0 1 NOTE. Data are no. of patients, unless otherwise indicated. Values in boldface font indicate statistically significant results. NK, natural killer. a Fisher s exact test, Mann-Whitney U test, or x 2 test was used to compare factors. diac complications [12]. The concentrations of IFN-g, IL-6, and IL-10 in plasma specimens from one-half of the patients with CAEBV infection increased, compared with normal levels in healthy persons, although there were no statistically significant differences between the 2 groups (table 4). The mean levels of thrombomodulin and soluble E-selectin were not elevated in either group of patients. Viral load in the peripheral blood. Viral load was measured by real-time quantitative PCR in consecutive samples obtained from each patient. First, the amounts of EBV DNA in PBMCs and plasma were compared between living patients and patients who died (figure 1). Interestingly, the number of copies of EBV DNA in the plasma at diagnosis was significantly higher in patients who died than in living patients. Of note, all of the patients who had a plasma viral load 10 3.8 copies/ ml at diagnosis died after undergoing HSCT (figure 1). On the other hand, the plasma viral load before HSCT was similar between living patients and patients who died. The plasma EBV load at diagnosis and 2 other clinical factors that differed significantly (age at diagnosis and the interval between disease onset and diagnosis) (table 3) were analyzed using a multivariate analysis. Only plasma viral load at diagnosis was confirmed to be significantly higher in patients who died than in living patients ( P p.007). Second, viral load was measured longitudinally after HSCT. Figure 2 shows the time course of EBV DNA load in 12 of the patients. Two patients, for whom the number of samples was not sufficient to see the time course, and patient 7, who experienced graft rejection, were excluded from the longitudinal study. In living patients, viral load decreased in both PBMCs and plasma after HSCT (figure 2A). None of these patients showed any symptoms associated with CAEBV infection after HSCT. There was good agreement between the disappearance of clinical symptoms and decreased viral load after HSCT. Moreover, viral DNA was not detected in plasma 70 days after HSCT. Viral load also decreased after HSCT in 3 patients who died of transplantation-related complications and in 1 patient Table 4. Plasma concentrations of IFN-g, IL-6, IL-10, thrombomodulin, and soluble E-selectin in patients who lived and patients who died after hematopoietic stem cell transplantation. Cytokine or vascular endothelial cell associated molecule concentration Patients who lived (n p 7) Patients who died (n p 7) P a IFN-g, IU/mL 1.4 2.2 1.3 1.8.89 IL-6, pg/ml 10.2 10.9 228.1 520.6.39 IL-10, pg/ml 14.2 29.8 3.5 2.4.39 Thrombomodulin, FU/mL 3.2 1.1 3.2 1.2.94 Soluble E-selectin, ng/ml 34.4 19.8 23.2 7.2.28 NOTE. Data are mean values SDs. The concentrations of all molecules were measured before conditioning treatment before transplantation. Normal plasma concentrations of these molecules in healthy persons are as follows: IFN-g concentration,!0.1 IU/mL; IL-6 concentration,!4 pg/ml; IL-10 concentration,!5 pg/ml; thrombomodulin concentration, 2.0 4.0 FU/mL; and soluble E-selectin concentration, 29.1 63.4 ng/ml. a P values were determined using the Mann-Whitney U test. CAEBV with Stem Cell Transplantation CID 2008:46 (15 May) 1529

who died of a neurological complication (figure 2B). In contrast, the viral load increased or did not decrease in patients who experienced relapse. Importantly, plasma EBV reappeared in these patients after they underwent HSCT (figure 2C). Expression of LMP1 in PBMCs and sequence variation in the LMP1 gene. Expression of the LMP1 gene and specific sequence variation in the C-terminal region were investigated to determine whether these factors were associated with disease outcome. First, the expression of LMP1 in PBMCs was observed in 8 of 12 patients, including 3 of 6 living patients, 4 of 5 patients who died, and 1 patient who experienced graft rejection (table 1). There was no statistically significant difference between the groups of patients who had PBMCs with LMP1 expression and those who did not. Next, partial LMP1 nucleotide sequences were obtained from 12 of 15 patients. The nucleotide variations are shown in figure 3. No specific patterns were associated with patients who died, compared with living patients. In addition, the LMP1 sequences before and after transplantation in 1 patient who experienced relapse (patient 4) and had increased viral load after undergoing HSCT were compared. In this case, the sequence before HSCT was identical to that after HSCT (data not shown). Figure 1. Comparison of viral loads in PBMCs or plasma samples from living patients with chronic active Epstein-Barr virus (EBV) infection and patients with such infection who died. EBV load was measured using realtime PCR. A, EBV load at diagnosis. B, EBV load before hematopoietic stem cell transplantation. Samples were collected 7 50 days (median, 10 days) before conditioning for transplantation. Dashed lines represent detection limits. The Mann-Whitney U test was used to compare viral loads. DISCUSSION Recently, HSCT has been used to treat CAEBV infection. Transplantation not only can eliminate EBV-infected cells but can also reconstitute EBV-specific cellular immunity. However, patients with CAEBV infection may have a higher risk of transplantation-related complications, because they often experience multiple organ failure and life-threatening complications [14 20]. More recently, allogeneic nonmyeloablative stem cell transplantation was shown to result in the elimination of disease in 1 patient with CAEBV infection and multiple organ dysfunction [20]. HSCT with a nonmyeloablative regimen has the advantage of reducing regimen-related toxicity and may be sufficient to control EBV-infected cells. In the present study, 10 patients with CAEBV infection were treated with nonmyeloablative transplantation. Six of 7 living patients had nonmyeloablative conditioning. Further studies of patients with CAEBV infection treated with HSCT are needed to determine the ideal transplantation regimens. Because the impact of HSCT in the treatment of CAEBV infection is not conclusive, other therapies, such as antiviral, anticancer, and immunomodulating agents, should be improved. Cytotoxic T lymphocytes specific for EBV antigens expressed in CAEBV infection also have potential for controlling EBV-infected cells [30]. To clarify the factors associated with the mortality rate of CAEBV infection after HSCT, we investigated clinical characteristics. Five of 6 patients who experienced 3 life-threatening complications died, although statistical significance was marginal ( P p.051). Age at diagnosis was significantly older for patients who died than for living patients, whereas age at disease onset was similar. The interval between disease onset and diagnosis was also significantly longer in patients who died than in living patients. These results suggest that delayed diagnosis affects patient outcome. In patients with CAEBV infection, the viral load in PBMCs is useful not only as a diagnostic factor but also as a predictor of therapeutic efficacy [7]. Regarding EBV DNA in plasma, the viral load has been shown to reflect disease stage and prognosis in EBV-related diseases [31 34]. In addition, viral load in the plasma of persons with CAEBV infection has been reported to be higher when the patient s clinical status deteriorates [35, 36]. In the present study, the EBV DNA level in PBMCs and plasma was measured using real-time PCR. The plasma viral load at diagnosis was significantly higher in patients who died than in living patients. Regarding EBV DNA detected in plasma, an experiment using DNase revealed that the presence of EBV DNA in plasma is attributable to cell-free nucleic acids that are 1530 CID 2008:46 (15 May) Gotoh et al.

Figure 2. Dynamics of viral load in patients with chronic active Epstein-Barr virus (EBV) infection treated with hematopoietic stem cell transplantation (HSCT). A, EBV loads in PBMCs and plasma samples from living patients (patients 1 3, 8 10, and 15). The number of plasma samples from patient 10 was insufficient to establish the time course of plasma viral load measurements. B, EBV loads in patients who died without experiencing relapse (patients 5, 6, 12, and 14). Patients 5, 6, and 12 died of transplantation-related complications. Patient 14 died of acute disseminated encephalomyelitis. C, EBV loads in patients who died after experiencing relapse (patients 4 and 11). EBV load was measured using real-time PCR. Dotted lines represent detection limits., Death.

Figure 3. Alignment of the DNA sequences of the C-terminal regions of the LMP1 gene from patients (Pts) with chronic active Epstein-Barr virus infection before hematopoietic stem cell transplantation. DNA sequence differences from the B95-8 strain are shown for each sequence pattern. Pt numbers are the same as those listed in table 1. Dots indicate that the nucleotide sequence is the same as the above sequence. Dashes represent deletions. likely released from dead or damaged cells [22]. The same mechanism has been reported for nasopharyngeal carcinoma and lymphoma [37]. The plasma viral load at diagnosis is an indicator of the amount of EBV-infected cells infiltrating organs, such as the liver and spleen, and may reflect multiple organ failure, which causes transplantation-related complications. By contrast, the PBMC viral load may reflect the amount of EBV-infected T and/or NK cells in blood and may not be related to multiple organ failure. As mentioned earlier, older age at diagnosis and the interval between disease onset and diagnosis may affect disease outcome. When these results are taken together, HSCT before life-threatening complications or hematologic malignancy have developed, even in the early stage of the disease, may be a rational choice. 1532 CID 2008:46 (15 May) Gotoh et al.

There are no definite criteria for remission of CAEBV infection, whereas, for example, the percentage of blast cells in bone marrow is used as a remission criterion for leukemia. EBV load can be used as an indicator of remission of CAEBV infection. In the living patient group, viral load obviously decreased in both the PBMCs and plasma after HSCT, as shown in figure 2A, and there was excellent agreement between clinical symptoms and plasma viral load after HSCT, which suggests that the patients were experiencing remission when the viral DNA was not being detected. In addition, the titers of serum IgG antibodies to the viral capsid antigen were clearly decreased after HSCT in the living patient group (data not shown). In patients who experienced relapse, plasma EBV DNA reappeared along with clinical deterioration. These data suggest that plasma viral load is a useful marker to monitor during the follow-up of CAEBV infection in patients after they have undergone HSCT. LMP1 contains some of the functional domains and epitopes of cytotoxic T lymphocytes [38 40]; therefore, expression and sequence variations may be associated with increased tumorigenicity and reduced cytotoxic T lymphocyte responses. Sequence heterogeneity among isolates has been reported for the C-terminal region of the LMP1 gene [39 41]. In this study, the expression of LMP1 in PBMCs was confirmed in 66% of patients, and there were no statistically significant differences between living patients and patients who died. There were no specific patterns of genomic sequence in the 2 groups, which suggests that no specific virus strain influenced disease outcome. In conclusion, 15 patients with CAEBV infection treated with HSCT were analyzed, and clinical and virological factors were compared between living patients and patients who died. Most patients who died appeared to have experienced more lifethreatening complications than did living patients. Plasma EBV load at diagnosis was significantly higher in patients who died than in living patients. Moreover, plasma viral load is thought to be an important factor to monitor during follow-up for patients after HSCT. The number of life-threatening complications and plasma viral load may be important in establishing the status of CAEBV infection and predicting the outcome of HSCT. Acknowledgments We thank the following for their contributions to this study: Yoshitoyo Kagami (Aichi Cancer Center); Kazushi Tanimoto (Ehime Prefectural Central Hospital); Masaki Yasukawa (Ehime University); Masaki Ito, Mitsuaki Hosoya, and Atsushi Kikuta (Fukushima Medical University); Hitoshi Kiyoi, Tomohiro Kinoshita, and Tomoki Naoe (Nagoya University); Motoko Koyama, Nobuharu Fujii, and Takanori Teshima (Okayama University); Misako Ueda and Yasunobu Takeoka (Osaka City University); Hirokazu Kanegane (Toyama University); Tsuyoshi Ito (Toyohashi City Hospital); Masahiro Tsuchida (Ibaraki Children s Hospital); and Chikako Kanazawa (Yamagata University). We thank F. Ando for secretarial assistance. Financial support. Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (18591212 to Y.I. and 19591247 to H.K.). Potential conflicts of interest. All authors: no conflicts. References 1. Cohen JI. Epstein-Barr virus infection. 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