ORIGINAL ARTICLE LIVER TRANSPLANTATION 19: , Additional Supporting Information may be found in the online version of this article.

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1 LIVER TRANSPLANTATION 19: , 2013 ORIGINAL ARTICLE Decreasing Incidence of Symptomatic Epstein-Barr Virus Disease and Posttransplant Lymphoproliferative Disorder in Pediatric Liver Transplant Recipients: Report of the Studies of Pediatric Liver Transplantation Experience Michael R. Narkewicz, 1,2 Michael Green, 3,4,5 Stephen Dunn, 6,7 Michael Millis, 8 Susan McDiarmid, 9,10,11 George Mazariegos, 3,5 Ravinder Anand, 12 and Wanrong Yin, 12 for the Studies of Pediatric Liver Transplantation Research Group 1 Section of Pediatric Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO; 2 Children s Hospital Colorado, Aurora, CO; Departments of 3 Surgery and 4 Pediatrics, Thomas E. Starzl Transplantation Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA; 5 Children s Hospital, Pittsburgh, PA; 6 Divison of Pediatric Solid Organ Transplantation, Department of Surgery, Thomas Jefferson University School of Medicine, Wilmington, DE; 7 Nemours Children s Hospital, Wilmington, DE; 8 Section of Transplantation, Department of Surgery, University of Chicago Medical Center, Chicago, IL; Departments of 9 Pediatrics and 10 Surgery, University of California Los Angeles School of Medicine, Los Angeles, CA; 11 Mattel Children s Hospital, Los Angeles, CA; and 12 Emmes Corporation, Rockville, MD Posttransplant lymphoproliferative disorder (PTLD) causes significant morbidity and mortality in pediatric recipients of liver transplantation (LT). Objective: Describe the incidence of PTLD and symptomatic Epstein-Barr virus (SEBV) disease in a large multicenter cohort of children who underwent LT with a focus on the risk factors and changes in incidence over time. SEBV and PTLD were prospectively determined in 2283 subjects who had undergone LT for the first time with at least 1 year of follow-up in the Studies of Pediatric Liver Transplantation database. SEBV was defined with specific criteria, and PTLD required tissue confirmation. The incidence of SEBV and PTLD was determined with a Kaplan-Meier analysis. Univariate and multivariate modeling of risk factors was performed with standard methods. SEBV occurred in 199 patients; 174 (87.4%) were EBV negative at LT. Seventy-five patients developed PTLD, and 64 (85.3%) of these patients were EBV-negative at LT. Among the 2048 patients with at least 2 years of follow-up, 8.3% developed SEBV by the second year after LT, and 2.8% developed PTLD. There were lower rates of SEBV (5.9% versus 11.3%, P < 0.001) and PTLD (1.7% versus 4.2%, P ) in versus In , tacrolimus and cyclosporine trough blood levels in the first year after LT were significantly lower, and fewer children were receiving steroids. Biliary atresia, and recipient EBV status were correlated. In a multivariate analysis, era of LT, recipient EBV status, and frequent rejection episodes were associated Additional Supporting Information may be found in the online version of this article. Abbreviations: CI, confidence interval; EBV, Epstein-Barr virus; LT, liver transplantation; PELD, Pediatric End-Stage Liver Disease; PTLD, posttransplant lymphoproliferative disorder; SD, standard deviation; SEBV, symptomatic Epstein-Barr virus; SPLIT, Studies of Pediatric Liver Transplantation. Studies of Pediatric Liver Transplantation is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (grant U01-DK A1) and by Astellas Pharma US, Inc., and Roche Laboratories (an unrestricted grant). The centers, investigators, and coordinators composing the Studies of Pediatric Liver Transplantation Research Group are listed in the supporting information. Address reprint requests to Michael R. Narkewicz, M.D., Children s Hospital Colorado, East 16th Avenue, B290, Aurora, CO Telephone: ; FAX: ; michael.narkewicz@childrenscolorado.org DOI /lt View this article online at wileyonlinelibrary.com. LIVER TRANSPLANTATION.DOI /lt. Published on behalf of the American Association for the Study of Liver Diseases VC 2013 American Association for the Study of Liver Diseases.

2 LIVER TRANSPLANTATION, Vol. 19, No. 7, 2013 NARKEWICZ ET AL. 731 with SEBV and PTLD. The incidence of SEBV and PTLD is decreasing in pediatric LT recipients concomitantly with a reduction in immunosuppression. Younger recipients and those with multiple rejections remain at higher risk for SEBV and PTLD. Liver Transpl 19: , VC 2013 AASLD. Received September 7, 2012; accepted April 12, Posttransplant lymphoproliferative disorder (PTLD) is a significant source of morbidity and mortality in pediatric recipients of liver transplantation (LT) with a reported incidence of 6% to 20% and a mortality rate of 12% to 60%. 1-6 PTLD was first recognized in the late 1960s; most cases are associated with infections with Epstein-Barr virus (EBV) and represent a spectrum of diseases ranging from polyclonal PTLD to lymphoma. 7 PTLD related to EBV infections is most frequent in the first 1 to 2 years following LT, with non EBV-related PTLD more frequent later. 8 Risk factors for PTLD have been reported to include recipient EBV seronegativity, 9 recipient age, 1,2 older donors, high levels of immunosuppression, 1 immunosuppression regimens, 2,10 and anti-lymphocyte therapies. 11 These risk factors have been reported in single-center experiences and may reflect center effects. 12 There have been significant changes in the management of pediatric LT recipients since the early reports on PTLD. Some of these changes, such as increases in the use of newer and/or potentially more potent immunosuppressive agents 10,13 and in the use of partial grafts from either split livers or living donors (with consequently older donors leading to a higher risk of EBV-positive donors for younger pediatric recipients, who are more likely to be EBV-naive), may increase the risk for PTLD. In contrast, some changes, such as the development of molecular monitoring for EBV infections 14 and subsequent responses to this information (eg, the reduction of immunosuppression and the use of as yet unproven preventative and preemptive interventions such as antiviral therapy), might be expected to reduce the risk for PTLD Using standardized definitions, we report the incidence of symptomatic Epstein-Barr virus (SEBV) infections and histologically confirmed PTLD in 2283 pediatric LT recipients from 1995 to 2008 who were prospectively enrolled and followed for at least 1 year as part of the Studies of Pediatric Liver Transplantation (SPLIT). The goal of this study was to report the incidence of PTLD and SEBV and describe factors that may be related to longitudinal changes in PTLD and SEBV in a large, diverse pediatric population of LT recipients. PATIENTS AND METHODS The SPLIT data registry is a multicenter data registry for children undergoing LT in the United States and Canada. As of May 31, 2008, the SPLIT registry database contained data on 2283 children who had undergone LT alone for the first time at 1 of 45 SPLIT centers with follow-up for at least 1 year. As reported previously, all of the participating SPLIT centers had institutional review board and/or research ethics board approval for data collection and analysis. 18 Informed consent was obtained on an individual basis from parents and/or guardians. For an analysis of changes in prevalence over time, we a priori designated 2 time periods: [before the initiation of the Pediatric End-Stage Liver Disease (PELD) system] and (after the initiation of the PELD system). Data Collection Detailed data were collected at listing, at transplant, and during follow-up. After LT, follow-up data were submitted by each participating center to the SPLIT data coordinating center every 6 months for 2 years and annually thereafter until the subject s 18th birthday. One-year follow-up was defined as a follow-up visit months after transplantation. These regular follow-up forms requested data elements on demographics, blood chemistry, hospitalizations, school status and performance, infections, posttransplant complications, immunosuppression, and other medication regimens. Specific events, including death, retransplantation, allograft rejection, SEBV infections, and histologically confirmed PTLD, were reported on separate forms. Missing data indicate that nothing was entered onto the data forms, and unknown data indicate that the testing was not done or the result was not known. For the EBV status, missing data were treated as unknown for analyses. SEBV was determined locally and was defined as seroconversion (the development of a new positive viral capsid antigen immunoglobulin M finding in a patient previously documented to be negative) or the development of a positive viral load with either histological evidence of an EBV infection (as determined by the local pathologist) or symptoms associated with an EBV infection (fever, leukopenia, atypical lymphocytosis, exudative tonsillitis and/or adenopathy, or hepatitis). PTLD was defined locally on the basis of the minimum requirement of a tissue biopsy sample considered diagnostic of PTLD by the local pathologist according to the published classification scheme. 19 EBV polymerase chain reaction was determined with the method in place at the local site. For patients with SEBV or PTLD after LT, the following information was requested with a specific EBV/ PTLD form and reviewed for inclusion in the database: EBV/PTLD symptoms, diagnostic criteria, immunosuppression before the diagnosis, initial treatment of EBV disease or PTLD, and use of monoclonal/polyclonal antibody therapy for induction or rejection. Each new diagnosis of SEBV or PTLD required a new enrollment. After a diagnosis of SEBV or PTLD, data were

3 732 NARKEWICZ ET AL. LIVER TRANSPLANTATION, July 2013 TABLE 1. Baseline Characteristics of Patients With SEBV and PTLD Disease All Patients SEBV PTLD n % n % P Value* n % P Value* Total Sex Missing Male Female EBV status: recipient/donor Unknown/unknown < <0.001 Positive/positive Positive/negative Positive/unknown Negative/positive Negative/negative Negative/unknown Unknown/positive Unknown/negative Immunosuppression at transplant Missing Cyclosporine A base Tacrolimus base Other Age at transplant (years) < <0.001 Period from transplant to SEBV/PTLD (months) NOTE: Bolded values are significant. *For comparisons between SEBV and non-sebv subjects or PTLD and non-ptld subjects. Unknown indicates that the recipient or donor did not have EBV testing or that the data were missing. The data are presented as means and SDs (instead of percentages). captured every 3 months until resolution (up to a maximum of 12 months after the diagnosis), exit, or death. The date of follow-up or resolution, immunosuppression, and other treatments were queried in the follow-up assessments. In the data analysis of this study, for patients with multiple enrollments, only the first diagnosis with associated follow-up visits was considered. Because maternal EBV antibodies can cross the placenta and remain with the infant until approximately 12 months of age, for the purposes of this analysis, the EBV status of recipients and donors was classified by serology for those > 12 months of age and was classified as negative for those 12 months of age. For the purposes of this study, the SEBV group included those patients with or without PTLD, and the PTLD patients were, therefore, a subset of the SEBV group. Statistical Methods Kaplan-Meier estimates were used to calculate and plot the times to the first development of SEBV or PTLD. Univariate statistical analyses included v 2 tests or Fisher s exact tests for comparisons of baseline categorical variables between SEBV/PTLD patients and non-sebv/ptld patients and Wilcoxon rank-sum tests for comparisons of means of continuous factors. Hazard ratios and P values for risk factors for the time to the first development of SEBV or PTLD were estimated with univariate and multivariate Cox proportional hazards models. Demographic factors, such as the transplant era, age, sex, race, primary diagnosis, and donor age, and baseline characteristics, including the recipient and donor EBV status, PELD score, organ type, patient hospitalization status at the time of LT, recipient cytomegalovirus status, medical treatments, and rejection, were assessed in the univariate model. To develop the multivariate model, we included factors significant at the 0.10 level in the univariate analyses in the initial multivariate model. We investigated the correlation between univariate factors, including the age at transplant, primary diagnosis, and recipient EBV status. The final multivariate model was derived with the stepwise backward elimination procedure. Factors remaining significant at P 0.05 were kept in the final model. The multivariate analysis was performed with separate models excluding 2 of the 3 individual correlated factors (age, recipient EBV status, and primary diagnosis). The n values, means, medians, and standard deviations (SDs) were calculated, and P values were estimated with the Wilcoxon rank-sum test to compare the trough levels of calcineurin inhibitors between the eras of

4 LIVER TRANSPLANTATION, Vol. 19, No. 7, 2013 NARKEWICZ ET AL. 733 Figure 1. (A) Kaplan-Meier estimates of the time from transplantation to the first development of SEBV disease with 95% CIs and (B) Kaplan-Meier analysis of the time to the first incidence of PTLD with 95% CIs. transplantation. All statistical analyses were performed with SAS for Windows (version 9.2, SAS Institute, Cary, NC). The number of rejections was analyzed as a time-varying covariate. In such analyses, a patient s rejection status varied over time, and rejections occurring after the outcome of interest were not included. RESULTS In all, 2283 children undergoing LT since 1995 with at least 1 year of follow-up were included in this study. The baseline data for all subjects and subjects with SEBV or PTLD are shown in Table 1. The donor EBV status was unknown for 37.9% of all subjects and for 43.2% of those with SEBV. This was in part due to sites not performing EBV screening. There was a difference between eras 1 and 2, with 45.4% unknown in era 1 and 32.7% unknown in era 2 for all patients and with 46.3% unknown in era 1 and 38.5% unknown in era 2 for patients with SEBV. In contrast, the recipient EBV status was unknown for only 3.9% of all subjects and for 3.0% of those with SEBV. SEBV occurred in 199 subjects after transplantation; 174 of these patients (87.4%) were EBV-negative at the time of transplantation. One hundred twenty-four had SEBV without PTLD; 88 of these patients (71%) developed SEBV within 12 months of transplantation. Seventy-five of the 199 subjects with SEBV (37.7%) developed PTLD, and 46 of these patients (61.3%) developed PTLD within 12 months of transplantation. Among those who developed PTLD, 64 (85.3%) were EBV-negative at the time of transplantation, and the rate was higher than that (1524/ 2208 or 69%) for those who did not develop PTLD (P < 0.001). Similarly to those who developed SEBV, 174 patients (87.4%) were EBV-negative at the time of transplantation, and the rate was higher than that (1414/2084 or 67.9%) for those who did not develop SEBV (P < 0.001). Fifty-two percent of the EBV-negative patients developing PTLD received an organ from a donor with an unknown EBV status. Patients with SEBV and PTLD were younger than the overall cohort. As can be seen in Fig. 1, the majority of the patients developed SEBV disease (Fig. 1A) or PTLD (Fig. 1B) in the first 24 months after transplantation. Among the 2283 patients with at least 1 year of follow-up and among the 2048 patients with at least 2 years of follow-up, 5.9% and 8.3% developed SEBV and 2.0% and 2.8% developed PTLD within the first 1 and 2 years after transplantation, respectively. There was an effect of era on the incidence of SEBV and PTLD, with significantly higher 1- and 2-year prevalences of both SEBV and PTLD in patients undergoing transplantation from 1995 to 2001 versus those undergoing transplantation from 2002 to 2007 (Table 2 and Fig. 2A,B). We next performed a univariate analysis of the time to SEBV and PTLD, which is presented in Table 3. Factors found to be significant for both SEBV and PTLD included the transplant era, recipient EBV status, age at transplant, primary diagnosis, and number of rejection episodes. Because the number of patients with 4 or more rejection episodes was very small, we combined patients with 3 or more rejection episodes into 1 group. None of the following oft cited factors were associated with a risk for PTLD: donor EBV status, recipient cytomegalovirus status, antiviral treatments or intravenous immunoglobulin, early rejection, or any rejection episode. Importantly, the

5 734 NARKEWICZ ET AL. LIVER TRANSPLANTATION, July 2013 TABLE 2. Incidence of SEBV and PTLD Patients With at Least 2 Years of Follow-Up Patients With at Least 1 Year of Follow-Up PTLD in the First 2 Years After Transplantation EBV in the First 2 Years After Transplantation PTLD in the First Year After Transplantation EBV in the First Year After Transplantation Incidence Total Incidence Incidence (%) P Value (n) n (%) P Value n (%) P Value Incidence Total (n) n (%) P Value n Total Transplant era < < NOTE: Bolded values are significant. Figure 2. (A) Kaplan-Meier estimates of the time from transplantation to the first development of SEBV disease by the era of transplantation and (B) Kaplan-Meier analysis of the time from transplantation to the first development of PTLD by the era of transplantation. donor EBV status was unknown for 43.2% of the SEBV patients and for 49.3% of the PTLD cases, and this likely affected the outcome of the role of this factor in the univariate and multivariate analyses. There was a significant relationship between the primary diagnosis, age at transplant, and recipient EBV status, so the multivariate model was performed with only 1 of the 3 factors (age at transplant, primary diagnosis, or recipient EBV status). There were no differences in the other resultant factors, so the multivariate analysis results using the recipient EBV status are shown in Table 4. The factors associated with an increased

6 LIVER TRANSPLANTATION, Vol. 19, No. 7, 2013 NARKEWICZ ET AL. 735 TABLE 3. Univariate Survival Analysis of the Time to EBV and PTLD (n ) Outcome EBV (n 5 199) PTLD (n 5 75) Reference Factor Comparison Group(s) Group Hazard Ratio P Value Overall P Value Hazard Ratio P Value Overall P Value Transplant era <0.001 < <0.001 <0.001 Sex Female Male Organ type Living Cadaveric Monoclonal antibody No Yes use at transplant Antiviral use at transplant Yes No Intravenous immunoglobulin Yes No use at transplant Cytomegalovirus at transplant Yes No EBV status of recipient Negative Positive 3.58 <0.001 < Unknown EBV status of donor Positive Negative Unknown Age at transplant 0-11 months 13 years 5.47 <0.001 < to <5 years 4.15 < to <13 years Race Black White Hispanic Other Primary diagnosis Biliary atresia Other chronic liver disease Intensive care unit status Continuous hospitalization Immunosuppression at transplant 1.99 <0.001 < <0.001 <0.001 Fulminant liver failure Continuous medical care Intensive care unit Cyclosporine A base Tacrolimus base Other Donor age 0-11 months 18 years years Calculated PELD score < to< to < Rejection status Yes No Number of rejection episodes < NOTE: Bolded values are significant.

7 736 NARKEWICZ ET AL. LIVER TRANSPLANTATION, July 2013 TABLE 4. Multivariate Analysis of the Time to SEBV and PTLD EBV (n , Cases 5 197) PTLD (n , Cases 5 74) Factor Comparison Group(s) Reference Group Hazard Ratio P Value Overall P Value Hazard Ratio P Value Overall P Value Transplant era < Number of rejection episodes EBV status of recipient Negative Positive 3.51 <0.001 < Unknown NOTE: The factors in the initial model included the transplant era, age at transplant, monoclonal antibody use at transplant, recipient EBV status, and number of rejection episodes. The primary diagnosis and the age at transplant were not included because of their correlation with the recipient EBV status. TABLE 5. Trough Levels of Calcineurin Inhibitors by the Transplant Era Time After Transplantation Day 7 Month 1 Month 6 Month 12 Patients Patients Medication (n) Mean SD Median (n) Mean SD Median P Value Cyclosporine <0.001 Tacrolimus <0.001 Cyclosporine Tacrolimus Cyclosporine Tacrolimus <0.001 Cyclosporine Tacrolimus <0.001 risk of SEBV and PTLD were limited to the era of transplantation, more than 1 rejection episode, and the recipient EBV status (negative) at transplant. We sought to explore factors that might account for the reduced incidence of SEBV and PTLD in the 2 eras. Overall, the immunosuppression regimens did change between the 2 eras. The proportion of subjects with cyclosporine-based immunosuppression decreased from 34.0% in era 1 to 8.5% in era 2, and tacrolimus use increased from 54.6% to 80.5%. Induction therapy was used in 11.7% in era 1 and in 27.4% in era 2. Steroid use in the regimens did not change. There was no difference in the percentage of recipients who were EBV-negative at the time of transplantation between the 2 eras (71.2% versus 68.4%, P ). In the earlier era, donors were slightly older, and there were more living donors. In the later era, there were more cadaveric split liver donors. To assess the relative intensity of immunosuppression, we compared the mean and median trough levels of tacrolimus and cyclosporine at 7 days and 1, 6, and 12 months after transplantation. Among all evaluable subjects in the database, there were significantly lower trough levels of tacrolimus at 7 days (P < 0.001) and 1 (P ), 6 (P < 0.001), and 12 months (P < 0.001) and significantly lower trough levels of cyclosporine at 7 days (P < 0.001) and 1 month (P ) in versus the earlier era (Table 5). There were significantly lower percentages of patients receiving steroids 1 (P ), 6 (P < 0.001), and 12 months (P < 0.001) after transplantation in versus the earlier era. There were no differences in the mean or median trough levels of tacrolimus or cyclosporine before 12 months after

8 LIVER TRANSPLANTATION, Vol. 19, No. 7, 2013 NARKEWICZ ET AL. 737 TABLE 6. Number of Rejection Episodes by the Transplant Era and the Recipient EBV Status EBV-Positive/ Unknown Recipients EBV-Negative Recipients EBV-Positive/ Unknown Recipients EBV-Negative Recipients Total Number of rejection episodes n % n % P Value n % n % P Value transplantation between the patients who were EBVnegative at the time of transplantation and the patients who were EBV-positive or whose status was unknown in either era. There were significantly more rejection episodes in the earlier era versus the more recent era, and there were more rejection episodes in EBV-positive/unknown recipients in the earlier era (Table 6). EBV monitoring and treatment paradigms based on circulating EBV viral loads have evolved in the last 10 years. We did not begin to systematically collect these data until We examined the use of EBV monitoring for the diagnosis of SEBV, and this was not significantly different between the 2 eras. Outcomes The outcomes for patients with SEBV and PTLD clearance of disease, death, and graft loss are shown in Table 7. One hundred forty-two of the 199 SEBV cases (71.4%) and 60 of the 75 PTLD cases (80.0%) were resolved with mean times to resolution of 5.8 and 5.7 months, respectively. Twelve patients (6.0%) with SEBV and 5 patients (6.7%) with PTLD died with unresolved SEBV and PTLD, respectively. Twenty-one patients (10.6%) with SEBV and 7 patients (9.3%) with PTLD experienced graft loss (death or rejection after an SEBV/PTLD diagnosis). Similar rates of resolution, death, and graft loss were seen in the subset of patients with SEBV who did not have PTLD in comparison with the larger SEBV group and patients with PTLD. There was no significant era effect on outcomes. DISCUSSION PTLD is a multifactorial process in children undergoing LT that is related to the interplay between immunosuppression and an EBV infection. As previously described and as expected, this study has demonstrated that children who are EBV-naive at the time of transplantation and those who have frequent episodes of rejection are at the highest risk for the development of SEBV and PTLD. Several important observations have been made in this large multicenter study. There was a significant reduction in the risk of the development of SEBV and PTLD during the first 2 years after LT in the more recent era ( ) versus the earlier era ( ) with equal numbers of patients and risks. This suggests that some factor or factors in management have changed the natural history of the development of PTLD. This study provides strong evidence that the primary reason for the reduction in the incidence of SEBV and PTLD is that there has been a general reduction in the overall level of immunosuppression in pediatric patients. There were significantly lower early levels of tacrolimus and cyclosporine and less steroid use in the patients in the more recent era, and this is consistent with an overall reduction in immunosuppression during the recent era. Despite these lower levels, the frequency of rejection in all patients was lower in the more recent era with an apparent reduction in the level of immunosuppression. Recent data from the SPLIT registry have suggested that the major mortality associated with pediatric LT is related to infections, and this implicates potentially excessive immunosuppression. 20 This is reinforced by our finding that the risk of both SEBV disease and PTLD was greater in patients with more than 1 rejection episode in the first year after transplantation. These data indicate a relationship between the effect of increased levels of immunosuppression and the risk for SEBV and PTLD. With the knowledge from previous studies of the importance of the recipient EBV status (specifically the increased risk in an EBVnaive recipient), selective changes in the immunosuppressive management of these patients likely contributed to the decreasing incidence of both SEBV disease and PTLD. There was a slight reduction in the use of older donors in the more recent era, which was also reflected in fewer living donors. This is another factor

9 738 NARKEWICZ ET AL. LIVER TRANSPLANTATION, July 2013 TABLE 7. Morbidity, Graft Loss, Recurrence of Disease, and Recovery After a Diagnosis of SEBV or PTLD by the Transplant Era All SEBV PTLD Total Total n % n % n % P Value n % n % n % P Value Total Recovery* Resolved (log rank) (log rank) Unresolved, <12 months of follow-up Unresolved, 12 months of follow-up Death before resolution Death No (log rank) (log rank) Yes No (log rank) (log rank) Graft loss status Yes Recurrence of disease No (v 2 ) (v 2 ) Yes Follow-up time (years) (Kruskal <0.001 Wallis)* <0.001 (Kruskal- Wallis)* *Nonparametric. Death or retransplantation after a diagnosis of EBV or PTLD. The data are presented as means and SD (instead of percentages).

10 LIVER TRANSPLANTATION, Vol. 19, No. 7, 2013 NARKEWICZ ET AL. 739 that may contribute to the reduced incidence of PTLD, but because most of the recipients were EBV-naive, immunosuppression is still likely the major factor for the decline in PTLD. Another potential factor is the use of techniques to monitor the EBV burden and specifically to take measurements of EBV viral loads in blood to identify transplant recipients who may be at increased risk for the development of disease. Unfortunately, most of our data come from a period before we prospectively determined EBV monitoring and management resulting from that monitoring. This is an area that needs further study. However, we speculate that EBV molecular monitoring leads to changes in immunosuppression before the development of symptomatic disease that may in part account for the decreasing incidence of both SEBV disease and PTLD in the recent era. Many centers have developed strategies for either prophylactic (antiviral treatments or alterations in the immunosuppression regimen in higher risk patients) or preemptive strategies (antiviral treatments or alterations in the immunosuppression regimen in patients with asymptomatic elevated EBV viral loads). 15,21 The use of these strategies has increased with the increased availability of EBV monitoring. However, antiviral use at the time of transplantation (a surrogate for prophylactic treatment) was not found to be a significant factor in the univariate analysis. This may be complicated by the overlap between cytomegalovirus prophylaxis and EBV prophylaxis, but it does suggest that antiviral prophylaxis in this group did not alter the risk of either SEBV disease or PTLD independently of the level of immunosuppression. Unfortunately, although the incidence of PTLD decreased in the recent era, the outcomes for those who developed PTLD in this study did not significantly improve over time. A possible implication of this finding is that PTLD represents a threshold beyond which the outcome is already determined. It may also indicate that our treatment modalities have potentially improved the resolution of PTLD or SEBV but lead to an imbalance in graft survival. An alternative explanation is that the presence of SEBV or PTLD indicates a level of immunosuppression imbalance that leads to complications of infections, as we have previously reported. 20 The primary risk factors for SEBV and PTLD in the multivariate analysis were the recipient EBV status (negative), transplant era, and number of rejection episodes. The transplant era and the number of rejection episodes are strong indicators of the level of immunosuppression. The EBV status of the recipient is tightly correlated with a younger age at transplant and a diagnosis of biliary atresia, and this may explain the variations in these risk factors in other studies. This large study of pediatric LT recipients has demonstrated that the incidence of SEBV and PTLD clearly decreased in the more recent era. The reasons behind this decline are likely multifactorial and complex. However, the primary factors are more attention to the level of immunosuppression and a global decrease in the level of immunosuppression in the more recent era. Future research should focus on the management of patients with subclinical EBV and target younger patients, EBV-naive recipients, and those with more rejection episodes, who seem to be at the highest risk for SEBV and PTLD. REFERENCES 1. Cox KL, Lawrence-Miyasaki LS, Garcia-Kennedy R, Lennette ET, Martinez OM, Krams SM, et al. An increased incidence of Epstein-Barr virus infection and lymphoproliferative disorder in young children on FK506 after liver transplantation. Transplantation 1995;59: Guthery SL, Heubi JE, Bucuvalas JC, Gross TG, Ryckman FC, Alonso MH, et al. Determination of risk factors for Epstein-Barr virus-associated posttransplant lymphoproliferative disorder in pediatric liver transplant recipients using objective case ascertainment. Transplantation 2003;75: Sokal EM, Antunes H, Beguin C, Bodeus M, Wallemacq P, de Ville de Goyet J, et al. Early signs and risk factors for the increased incidence of Epstein-Barr virus-related posttransplant lymphoproliferative diseases in pediatric liver transplant recipients treated with tacrolimus. Transplantation 1997;64: Newell KA, Alonso EM, Whitington PF, Bruce DS, Millis JM, Piper JB, et al. Posttransplant lymphoproliferative disease in pediatric liver transplantation. Interplay between primary Epstein-Barr virus infection and immunosuppression. Transplantation 1996;62: Cacciarelli TV, Reyes J, Jaffe R, Mazariegos GV, Jain A, Fung JJ, Green M. Primary tacrolimus (FK506) therapy and the long-term risk of post-transplant lymphoproliferative disease in pediatric liver transplant recipients. Pediatr Transplant 2001;5: Younes BS, McDiarmid SV, Martin MG, Vargas JH, Goss JA, Busuttil RW, Ament ME. The effect of immunosuppression on posttransplant lymphoproliferative disease in pediatric liver transplant patients. Transplantation 2000;70: Tsao L, Hsi ED. The clinicopathologic spectrum of posttransplantation lymphoproliferative disorders. Arch Pathol Lab Med 2007;131: Kremers WK, Devarbhavi HC, Wiesner RH, Krom RA, Macon WR, Habermann TM. Post-transplant lymphoproliferative disorders following liver transplantation: incidence, risk factors and survival. Am J Transplant 2006;6(pt 1): Ho M, Jaffe R, Miller G, Breinig MK, Dummer JS, Makowka L, et al. The frequency of Epstein-Barr virus infection and associated lymphoproliferative syndrome after transplantation and its manifestations in children. Transplantation 1988;45: Cao S, Cox KL, Berquist W, Hayashi M, Concepcion W, Hammes GB, et al. Long-term outcomes in pediatric liver recipients: comparison between cyclosporin A and tacrolimus. Pediatr Transplant 1999;3: Swinnen LJ, Costanzo-Nordin MR, Fisher SG, O Sullivan EJ, Johnson MR, Heroux AL, et al. Increased incidence of lymphoproliferative disorder after immunosuppression with the monoclonal antibody OKT3 in cardiac-transplant recipients. N Engl J Med 1990;323: Holmes RD, Sokol RJ. Epstein-Barr virus and posttransplant lymphoproliferative disease. Pediatr Transplant 2002;6: Otte JB. History of pediatric liver transplantation. Where are we coming from? Where do we stand? Pediatr Transplant 2002;6:

11 740 NARKEWICZ ET AL. LIVER TRANSPLANTATION, July Gridelli B, Spada M, Riva S, Colledan M, Segalin A, Lucianetti A, et al. Circulating Epstein-Barr virus DNA to monitor lymphoproliferative disease following pediatric liver transplantation. Transpl Int 2000;13(suppl 1):S399- S McDiarmid SV, Jordan S, Kim GS, Toyoda M, Goss JA, Vargas JH, et al. Prevention and preemptive therapy of posttransplant lymphoproliferative disease in pediatric liver recipients. Transplantation 1998;66: Green M, Michaels MG, Katz BZ, Burroughs M, Gerber D, Shneider BL, et al. CMV-IVIG for prevention of Epstein Barr virus disease and posttransplant lymphoproliferative disease in pediatric liver transplant recipients. Am J Transplant 2006;6: Lee TC, Savoldo B, Rooney CM, Heslop HE, Gee AP, Caldwell Y, et al. Quantitative EBV viral loads and immunosuppression alterations can decrease PTLD incidence in pediatric liver transplant recipients. Am J Transplant 2005;5: SPLIT Research Group. Studies of Pediatric Liver Transplantation (SPLIT): year 2000 outcomes. Transplantation 2001;72: Harris NL, Swerdlow SH, Frizzera G, Knowles DM. Posttransplant lymphoproliferative disorders. In: Jaffe ES, Harris NL, Stein H, Vardiman JW, eds. Pathology and Genetics: Tumours of Haematopoietic and Lymphoid Tissues (World Health Organization Classification of Tumors). Lyon, France: IARC Press; 2001: Shepherd RW, Turmelle Y, Nadler M, Lowell JA, Narkewicz MR, McDiarmid SV, et al.; for SPLIT Research Group. Risk factors for rejection and infection in pediatric liver transplantation. Am J Transplant 2008;8: Holmes RD, Orban-Eller K, Karrer FR, Rowe DT, Narkewicz MR, Sokol RJ. Response of elevated Epstein-Barr virus DNA levels to therapeutic changes in pediatric liver transplant patients: 56-month follow up and outcome. Transplantation 2002;74: